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Human NK lymphocytes are involved in antitumor immunity. The therapeutic potential of this population against cancers has stimulated their study and led to the discovery of several NK cell subsets, each of which is endowed with different immunoregulatory functions. We have previously reported that NK cell functions are profoundly altered in advanced breast cancer patients. In this study, we show that these tumor-mediated alterations also variably affect NK cell subsets. We found that in addition to the known human CD56dimCD16+, CD56brightCD16−, and CD56−CD16+ NK cell subsets, two additional subsets, namely the CD56brightCD16+ and CD56dimCD16− subsets, were increased in the peripheral blood of patients with advanced invasive breast cancers. These subsets corresponded to the main two subsets found at the tumor site. The extensive phenotype of these subsets revealed an “à la carte” pattern of expression for the various NK receptors, functional molecules, adhesion molecules, and chemokine receptors, depending on the subset. We next compared these subsets to known NK cell populations endowed with specific phenotypic characteristics, but also with functional properties. Our data show that advanced breast cancer patients have an increased proportion of more immature and noncytotoxic NK cell subsets in their peripheral blood, which might account for at least part of the low cytotoxic functions observed in these patients. They reveal a major heterogeneity and plasticity of the NK cell compartment, which are both tightly linked to the microenvironment. The identification of NK cell subsets endowed with particular functional capabilities might help monitor residual antitumor NK cell-mediated responses in breast cancer patients.

© F e r r a t a S t o r t i F o u n d a t i o nposed to distinguish FLIS from partial involvement by FL (PFL). In PFL there is greater architectural distortion, and the diagnosis of lymphoma is more evident histologically. However, interestingly, patients with PFL appear to present with low-stage disease, with a relatively low risk of progression and often a very prolonged disease-free interval with limited therapy. 12 Thus, PFL may represent a bona fide early stage in the biological evolution of FL, and not just a limited state of lymph node replacement by a fully developed neoplasm.14 Advances in laser capture microdissection now enable the isolation of even small numbers of cells representative of the clonotypic population, 15 and improvements in genomic methods allow the analysis of genetic aberrations in DNA derived from formalin-fixed, paraffin-embedded tissues. These new technologies now provide the unique opportunity to explore the genetic landscape of these early lesions, and to understand their relationship to overt FL better. Methods SamplesMost cases were selected from formalin-fixed, paraffin-embedded archive specimens submitted to the Hematopathology Section at the National Cancer Institute (NCI) or the Institute of Pathology, at the Medical University of Vienna. Seven cases of FLIS, five cases of PFL and five cases of DFL were identified based on previously published criteria. 11,12 Patients with FLIS and PFL had no other evidence of disease during the period of follow-up. 12 These samples were compared to five cases of FL grade 1-2 and five of FL grade 3A, included as controls of the most frequent alterations occurring in FL. Finally, sections of reactive follicular hyperplasia (RFH, n=2) and laser micro-dissected lymphoid cells from uninvolved areas of FLIS#3 (FLIS background) were also hybridized and used as references for normal cells (Online Supplementary Table S1). Only cases with confirmed t(14;18) were included in the study. The Institutional Review Boards of the NCI and the Medical University of Vienna approved the study. Laser capture microdissection, DNA extraction and quality controlTo obtain sufficient enrichment of lesional cells positive for t(14;18), which was necessary for the genome-wide array analysis, BCL2 + GC were microdissected from the selected FLIS/PFL/DFL/FL grade 1-2 cases. Laser capture microdissection was performed using a Leica LMD6000 (Leica Microsystems, Germany) on hematoxylin-stained slides with BCL2 + immunostained slides as a guide for involved follicles.15 BCL2-negative GC were microdissected from RFH, without evidence of FLIS. Tumor cell DNA was isolated from FL grade 3A without microdissection, given the high tumor cell content (>75%). DNA was extracted using a QIAamp ® DNA FFPE Kit. The amount of DNA collected from the different cases ranged from 506 to 2820 ng. With a calculation of 6.6 pg of DNA/cell this amounts to a minimum of 77,000 cells per case needed for appropriate hybridization. The integrity of the DNA was controlled with an Agilent DNA1...

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