A model for the impact of FFPE section thickness on gene copy number measurement by FISH.

Fluorescent in situ hybridization (FISH) assays to detect gene amplification such as HER2 or MET in tumors are used for prognosis evaluation and selection of targeted therapies. Although FISH guidelines recommended 4~6 µm FFPE sections, many laboratories use 2~3 µm sections, which is a common practice for H&E staining and immunohistochemistry. A former study concluded that section thickness did not affect FISH results. We found, however, that thinner FFPE sections may lead to false negative results for gene amplification. A mathematic model was constructed and cell-line based controls with known gene copy number were prepared, and the model had a reasonable fit with the experimental data. The model revealed that even when counting the apparently full-sized nuclear images, many of them have partial volumes, which leads to under-estimation of gene copy number. Therefore, improperly thinner sections are prone to give false negative results, and thicker sections give a better approximation to the true value. The discrepancy between this and the former study was discussed. In summary, the model applies generally to FISH/ISH detection of gene copy number, and section thickness is an important parameter to control for precision medicine research, assay development, clinical trials and daily practice in pathology laboratory.

Interventional bleeding, hematoma and scar-formation after vacuum-biopsy under stereotactic guidance: Mammotome(®)-system 11 g/8 g vs. ATEC(®)-system 12 g/9 g.

PURPOSE: To evaluate prospectively the correlation of scar-formations after vacuum-assisted biopsy with different systems and needle-sizes and interventional bleeding/post-interventional hematoma. METHODS AND MATERIALS: Between 01/2008 and 12/2009, 479 patients underwent vacuum-assisted biopsy under stereotactic-guidance, using the Mammotome(®)-system with 11/8-gauge and ATEC(®)-system with 12/9-gauge, whereas in 178 cases with representative benign histology no surgical-biopsy after vacuum-assisted biopsy was performed and at least a 2-plane-follow-up-mammogram after 6 month post-vacuum-assisted biopsy was available. Bleeding during intervention, hematoma post-intervention and scar-tissue was scored as minimal and moderate/severe. Statistical analysis included Chi-Square-trend-test, p-value <0.05 was considered to be significant. RESULTS: Significantly more bleedings and post-interventional hematomas for 8-gauge-Mammotome(®)-system vs. 11-gauge-Mammotome(®)-system (41.9% vs. 8.4%, p<0.001/35.5% vs. 16.7%, p=0.029), no significant-differences for the ATEC(®)-systems 9-gauge vs. 12-gauge (26.9% vs. 29.7%, p=0.799/42.3% vs. 43.2%, p=0.596). 11-gauge-Mammotome(®)-system vs. ATEC(®)-12-gauge-system revealed significantly less bleedings/hematomas (8.4% vs. 29.7%, p=0.015/16.7% vs. 43.2%, p=0.001), no significant differences for the large-systems (p=0.135/p=0.352). Follow-up of Mammotome(®)-11/8-gauge-system system has shown 13.1/16.1% minimal scar-formation and 1.2/3.2% moderate/severe scars, whereas ATEC(®)-12/9-gauge-system has shown 10.8/3.8% minimal scar-formation and 0/11.5% moderate/severe scars, no significant differences. No significant difference was found when comparing Mammotome(®)-11/8-g-systems vs. ATEC(®)-12/9-g-systems (p=0.609/p=0.823). There was also no correlation between risk of scar-formation after occurrence of bleeding or hematoma with any examined VAB-system or any needle size in this study (p=0.800). CONCLUSION: Using larger needle-sizes significantly (Mammotome(®))/not significant for ATEC(®)) more interventional bleedings and post-interventional hematomas were detected, only a tendency concerning scar-formation.

Comparative analysis of size estimation by mapping and counting number of blocks with ductal carcinoma in situ in breast excision specimens.

CONTEXT: The size of ductal carcinoma in situ (DCIS) is a significant predictor of local tumor recurrence and is used for selection of patients for conservative versus aggressive therapy. A standardized method for size assessment is lacking. OBJECTIVE: To evaluate 2 commonly used methods for measurement of DCIS extent: one based on the distribution of the lesion in sequential series of sections (mapping method) and the other on the number of sections with DCIS (block method). DESIGN: Ninety-eight consecutive cases of DCIS, measuring at least 1.0 cm, were retrieved from our files. All specimens were serially sectioned along the long axis. The size of DCIS was calculated for each case by 2 different methods: (1) mapping method (average thickness of each slice x number of consecutive slices with DCIS) and (2) block method (number of blocks with DCIS x 0.3 cm). Additional calculations were performed by using 0.35, 0.4, and 0.5 cm as multiplication factors for the block method in order to improve concordance. RESULTS: The block method underestimated the size in 71 cases (72%) by 4.5% to 81.3% (mean, 33%). Using 0.4 cm as the multiplication factor improved concordance, while multiplying by 0.5 cm led to an overestimation of size. CONCLUSIONS: Assessment of DCIS size by the block method is inaccurate and underestimates size in most cases (72%), with an average reduction of 33%. Using 0.4 cm as the multiplication factor improves concordance. A standardized method for size estimation is necessary for effective patient management.

Prediction of the variance of stereological volume estimates from systematic sections using computer-intensive methods.

The Cavalieri method is an unbiased estimator of the total volume of a body from its transectional areas on systematic sections. The coefficient of error (CE) of the Cavalieri estimator was predicted by a computer-intensive method. The method is based on polynomial regression of area values on section number and simulation of systematic sectioning. The measurement function is modelled as a quadratic polynomial, with an error term superimposed. The relative influence of the trend and the error component is estimated by techniques of analysis of variance. This predictor was compared with two established short-cut estimators of the CE based on transitive theory. First, all predictors were applied to data sets from six deterministic models with analytically known CE. For these models, the CE was best predicted by the older short-cut estimator and by the computer-intensive approach, if the measurement function had finite jumps. The best prediction was provided by the newer short-cut estimator when the measurement function was continuous. The predictors were also applied to published empirical datasets. The first data set consisted of 10 series of areas of systematically sectioned rat hearts with 10-13 items, the second data set consisted of 13 series of systematically sampled transectional areas of various biological structures with 38-90 items. On the whole, similar mean values for the predicted CE were obtained with the older short-cut estimator and the computer-intensive method. These ranged in the same order of magnitude as resampling estimates of the CE from the empirical data sets, which were used as a cross-check. The mean values according to the newer short-cut CE estimator ranged distinctly lower than the resampling estimates. However, for individual data sets, it happened that the closest prediction as compared to the cross-check value could be provided by any of the three methods. This finding is discussed in terms of the statistical variability of the resampling estimate itself.

A note on the stereological implications of irregular spacing of sections.

Stereological methods for serial sections traditionally assume that the sections are exactly equally spaced. In reality, the spacing and thickness of sections can be quite irregular. This may affect the validity and accuracy of stereological techniques, especially the Cavalieri estimator of volume. We present a new formula for the accuracy of the Cavalieri estimator that includes the effect of random variability in section spacing. A modest amount of variability in section spacing can cause a substantial increase in estimator variance.

Intraoperative cytologic diagnosis of sentinel node metastases in breast cancer.

OBJECTIVE: To clarify the usefulness of imprint cytology for intraoperative investigations of sentinel lymph nodes in breast cancer, comparing the results with those of examinations using frozen and permanent sections. STUDY DESIGN: The material consisted of 303 sentinel lymph nodes from 124 cases of clinically node negative breast cancer. Touch imprint cytologic slides and frozen sections were obtained from the same cut surface of the sentinel nodes. Correlations with the final histopathologic results in paraffin sections were evaluated. RESULTS: The sensitivity, specificity and accuracy of imprint cytology were 70.3%, 99.6% and 96.0%, and those of frozen sections were 83.8%, 100%, 98.0%, respectively. The values were improved when the 2 methods were combined (89.2%, 99.6%, 98.3%), though the concordance between imprint cytology and frozen section was 91.9%. CONCLUSION: Both imprint cytology and frozen section are useful for evaluating sentinel lymph node status in breast cancer. However, the 2 techniques should be combined to improve the diagnostic sensitivity.

Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.

Organotypic slice cultures of rat hippocampus are widely used as experimental preparations for the study of synaptic plasticity, but their degree of correspondence with intact brain is not fully known. Here, using postembedding immunogold labelling, we describe the ultrastructural distribution of AMPA-type glutamate receptors (GluR1-4) in CA3 stratum radiatum of organotypic hippocampal slice cultures at 10 days to 11 weeks in vitro and compare the labelling with intact brain of corresponding age. In both types of preparation, the 11-week-old samples contained the highest proportion of AMPA receptor-like immunoreactive synapses. The incidence of labelled synapses, however, was higher in vivo (49%) than in vitro (24%). The intensity of labelling (number of gold particles per labelled synapse) also increased with age and was also higher in vivo than in vitro. In both organotypic cultures and intact brain, labelling was frequently found at presynaptic sites, often attached to vesicular structures. The specificity of these findings was supported both by light microscopic immunolabelling of GluR2/3 subunits and by electron microscopic double labelling of different epitopes of the GluR2 subunit. The vesicular localization of AMPA receptors was supported by Western blot analysis of subcellular fractions. Morphological evidence of presynaptic excitatory innervation of glutamatergic neurons supports a functional role for presynaptically located AMPA receptors. Our results therefore suggest that AMPA receptors occur in both pre- and postsynaptic profiles and that the distribution of AMPA receptors in cultured brain slices is fundamentally similar to intact brain, but that synaptic maturation may be retarded in vitro.

Application of the van nuys prognostic index in a retrospective series of 367 ductal carcinomas in situ of the breast examined by serial macroscopic sectioning: practical considerations.

UNLABELLED: The Van Nuys prognostic index (VNPI) was thought to be useful for predicting response to radiotherapy and local recurrence of ductal carcinoma in situ (DCIS). We applied the VNPI under the conditions defined by Silverstein et al., in 367 retrospective DCIS entirely sectioned into serial macroscopic 2 mm slices (155 patients had radiotherapy, median follow-up 71 months). The percentage of positive blocks with DCIS was also estimated for each specimen with cut-offs at 30% and 60% to obtain three scores. One hundred and ninety five lesions had a low VNPI, 152 an intermediate VNPI, and 20 a high VNPI. There were 9% of local recurrences (half invasive, all in the group without radiotherapy) in the low VNPI group. The local recurrence rate increased with size (p = 0.001), with reduction of distance to margins (p = 0.05), with histologic grade (p = 0.02), with percentage of positive blocks (p = 0.0003) and with VNPI score (p = 0.03). The percentage of positive blocks was the only independent predictor for local recurrence (p = 0.0001). CONCLUSION: (1) The VNPI was a local recurrence rate predictor between the low and the intermediate groups but in our series the low VNPI group had a surprisingly high local recurrence rate. (2) Only prospective studies will assess the importance of margin width and the role of radiotherapy in maintaining local control. (3) Estimation of the percentage of positive blocks is simple, may be an alternative when measurement of DCIS is difficult and should be taken into account.

Valor de la biopsia rápida en el diagnóstico de cancer / The accuracy of quick biopsy for cancer diagnosis

El objetivo fue estudiar estadísticamente el valor de la biopsia rápida para diagnosticar malignidad, global y específicamente en los diversos órganos y sistemas. Se consideró como presente la malignidad cuando la biopsia diferida así lo concluyó. Se realizó un estudio retropectivo de 659 biopsia rápida y sus correspondientes biopsias diferidas, efectuadas enel Servicio de Anatomía Patológica del Hosp. Dr. G. Fricke entre Septiembre de 1995 y Febrero de 1999.Seaplicó un test de screening para el análisis de los resultados al universo de muestras y por órganos y sistemas, excluyendo las biopsias rápidas no concluyentes