Peritumoral immune infiltrates in primary tumours are not associated with the presence of axillary lymph node metastasis in breast cancer: a retrospective cohort study.

BACKGROUND: The axillary lymph nodes (ALNs) in breast cancer patients are the body regions to where tumoral cells most often first disseminate. The tumour immune response is important for breast cancer patient outcome, and some studies have evaluated its involvement in ALN metastasis development. Most studies have focused on the intratumoral immune response, but very few have evaluated the peritumoral immune response. The aim of the present article is to evaluate the immune infiltrates of the peritumoral area and their association with the presence of ALN metastases. METHODS: The concentration of 11 immune markers in the peritumoral areas was studied in 149 patients diagnosed with invasive breast carcinoma of no special type (half of whom had ALN metastasis at diagnosis) using tissue microarrays, immunohistochemistry and digital image analysis procedures. The differences in the concentration of the immune response of peritumoral areas between patients diagnosed with and without metastasis in their ALNs were evaluated. A multivariate logistic regression model was developed to identify the clinical-pathological variables and the peritumoral immune markers independently associated with having or not having ALN metastases at diagnosis. RESULTS: No statistically significant differences were found in the concentrations of the 11 immune markers between patients diagnosed with or without ALN metastases. Patients with metastases in their ALNs had a higher histological grade, more lymphovascular and perineural invasion and larger-diameter tumours. The multivariate analysis, after validation by bootstrap simulation, revealed that only tumour diameter (OR = 1.04; 95% CI [1.00-1.07]; p = 0.026), lymphovascular invasion (OR = 25.42; 95% CI [9.57-67.55]; p < 0.001) and histological grades 2 (OR = 3.84; 95% CI [1.11-13.28]; p = 0.033) and 3 (OR = 5.18; 95% CI [1.40-19.17]; p = 0.014) were associated with the presence of ALN metastases at diagnosis. This study is one of the first to study the association of the peritumoral immune response with ALN metastasis. We did not find any association of peritumoral immune infiltrates with the presence of ALN metastasis. Nevertheless, this does not rule out the possibility that other peritumoral immune populations are associated with ALN metastasis. This matter needs to be examined in greater depth, broadening the types of peritumoral immune cells studied, and including new peritumoral areas, such as the germinal centres of the peritumoral tertiary lymphoid structures found in extensively infiltrated neoplastic lesions.

The Immune Response in Nonmetastatic Axillary Lymph Nodes Is Associated with the Presence of Axillary Metastasis and Breast Cancer Patient Outcome.

Tumor cells can modify the immune response in primary tumors and in the axillary lymph nodes with metastasis (ALN+) in breast cancer (BC), influencing patient outcome. We investigated whether patterns of immune cells in the primary tumor and in the axillary lymph nodes without metastasis (ALN-) differed between patients diagnosed without ALN+ (diagnosed-ALN-) and with ALN+ (diagnosed-ALN+) and the implications for clinical outcome. Eleven immune markers were studied using immunohistochemistry, tissue microarray, and digital image analysis in 141 BC patient samples (75 diagnosed-ALN+ and 66 diagnosed-ALN-). Two logistic regression models were derived to identify the clinical, pathologic, and immunologic variables associated with the presence of ALN+ at diagnosis. There are immune patterns in the ALN- associated with the presence of ALN+ at diagnosis. The regression models revealed a small subgroup of diagnosed-ALN+ with ALN- immune patterns that were more similar to those of the ALN- of the diagnosed-ALN-. This small subgroup also showed similar clinical behavior to that of the diagnosed-ALN-. Another small subgroup of diagnosed-ALN- with ALN- immune patterns was found whose members were more similar to those of the ALN- of the diagnosed-ALN+. This small subgroup had similar clinical behavior to the diagnosed-ALN+. These data suggest that the immune response present in ALN- at diagnosis could influence the clinical outcome of BC patients.

Development of automated quantification methodologies of immunohistochemical markers to determine patterns of immune response in breast cancer: a retrospective cohort study.

INTRODUCTION: Lymph nodes are one of the main sites where an effective immune response develops. Normally, axillary nodes are the first place where breast cancer produces metastases. Several studies have demonstrated the importance of immune cells, especially dendritic cells, in the evolution of breast cancer. The goal of the project is to identify differences in the patterns of immune infiltrates, with particular emphasis on dendritic cells, in tumour and axillary node biopsies between patients with and without metastases in the axillary nodes at the time of diagnosis. It is expected that these differences will be able to explain differences in survival, relapse and clinicopathological variables between the two groups. METHODS AND ANALYSIS: The study will involve 100 patients diagnosed with invasive breast cancer between 2000 and 2007, 50% of whom have metastases in the axillary lymph node at diagnosis. In selected patients, two cylinders from biopsies of representative areas of tumour and axillary nodes (with and without metastasis) will be selected and organised in tissue microarrays. Samples will be stained using immunohistochemical techniques for different markers of immune response and dendritic cells. Two images of each cylinder will be captured under standardised conditions for each marker. Each marker will be quantified automatically by digital image procedures using Image-Pro Plus and Image-J software. Associations of survival, relapse and other clinicopathological variables with the automatically quantified levels of immune infiltrates in patients with and without axillary node metastasis will be sought. ETHICS AND DISSEMINATION: The present project has been approved by the Clinical Research Ethics Committee of the Hospital Universitari Joan XXIII (Ref: 22p/2011). Those patients whose biopsies and clinical data are to be used will give their signed informed consent. Results will be published in peer-reviewed journals.

An automated system for whole microscopic image acquisition and analysis.

The field of anatomic pathology has experienced major changes over the last decade. Virtual microscopy (VM) systems have allowed experts in pathology and other biomedical areas to work in a safer and more collaborative way. VMs are automated systems capable of digitizing microscopic samples that were traditionally examined one by one. The possibility of having digital copies reduces the risk of damaging original samples, and also makes it easier to distribute copies among other pathologists. This article describes the development of an automated high-resolution whole slide imaging (WSI) system tailored to the needs and problems encountered in digital imaging for pathology, from hardware control to the full digitization of samples. The system has been built with an additional digital monochromatic camera together with the color camera by default and LED transmitted illumination (RGB). Monochrome cameras are the preferred method of acquisition for fluorescence microscopy. The system is able to digitize correctly and form large high resolution microscope images for both brightfield and fluorescence. The quality of the digital images has been quantified using three metrics based on sharpness, contrast and focus. It has been proved on 150 tissue samples of brain autopsies, prostate biopsies and lung cytologies, at five magnifications: 2.5×, 10×, 20×, 40×, and 63×. The article is focused on the hardware set-up and the acquisition software, although results of the implemented image processing techniques included in the software and applied to the different tissue samples are also presented.

Emerging trends: grid technology in pathology.

Grid technology has enabled clustering and access to, and interaction among, a wide variety of geographically distributed resources such as supercomputers, storage systems, data sources, instruments as well as special devices and services, realizing network-centric operations. Their main applications include large scale computational and data intensive problems in science and engineering. Grids are likely to have a deep impact on health related applications. Moreover, they seem to be suitable for tissue-based diagnosis. They offer a powerful tool to deal with current challenges in many biomedical domains involving complex anatomical and physiological modeling of structures from images or large image databases assembling and analysis. This chapter analyzes the general structures and functions of a Grid environment implemented for tissue-based diagnosis on digital images. Moreover, it presents a Grid middleware implemented by the authors for diagnostic pathology applications. The chapter is a review of the work done as part of the European COST project EUROTELEPATH.