CDKN2A loss and PIK3CA mutation in myoepithelial-like metaplastic breast cancer.

Metaplastic breast carcinoma comprises a heterogeneous group of tumours with poorly understood pathogenesis. A subset of metaplastic breast cancers show myoepithelial differentiation and constitute a morphological spectrum with ill-defined borders from fibromatosis-like spindle cell carcinoma to myoepithelial carcinoma. In a series of 34 metaplastic breast cancers with spindle cell and myoepithelial differentiation, we found recurrent genetic aberrations, which set them apart from other metaplastic breast cancers and suggest a unique pathogenesis. The majority of cases (28 of 34 patients; 82.4%) showed distinct chromosomal loss in the 9p21.3 region, including CDKN2A and CDKN2B. Biallelic loss of the CDKN2A/B region was found in 50% of deleted cases. Expression of the cyclin-dependent kinase inhibitor CDKN2A (p16) was missing in all samples affected by 9p21.3 loss. Other genomic alterations frequently occurring in triple-negative and metaplastic breast cancer were absent or found in only a minority of cases. Gains of whole chromosome 5 and chromosomal region 5p were observed in nine cases, and were associated with recurrences (p < 0.001). In 64.3% of cases, 9p21.3 loss was accompanied by concurrent PIK3CA mutation. Both genomic abnormalities were also detectable in adenomyoepitheliomas (4/12), which are considered to represent the precursor lesion of myoepithelial metaplastic breast cancer. In adenomyoepithelioma, PIK3CA mutation was present in both luminal epithelial and myoepithelial cells, whereas p16 loss was found only in the latter. We conclude that 9p21.3 (CDKN2A) loss and PIK3CA mutation characterize a subgroup of metaplastic breast cancers with myoepithelial and spindle cell differentiation. Myoepithelial cells in adenomyoepithelioma may show identical aberrations. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Macrophage-inducible C-type lectin Mincle-expressing dendritic cells contribute to control of splenic Mycobacterium bovis BCG infection in mice.

The macrophage-inducible C-type lectin Mincle has recently been identified to be a pattern recognition receptor sensing mycobacterial infection via recognition of the mycobacterial cell wall component trehalose-6',6-dimycolate (TDM). However, its role in systemic mycobacterial infections has not been examined so far. Mincle-knockout (KO) mice were infected intravenously with Mycobacterium bovis BCG to mimic the systemic spread of mycobacteria under defined experimental conditions. After intravenous infection with M. bovis BCG, Mincle-KO mice responded with significantly higher numbers of mycobacterial CFU in spleen and liver, while reduced granuloma formation was observed only in the spleen. At the same time, reduced Th1 cytokine production and decreased numbers of gamma interferon-producing T cells were observed in the spleens of Mincle-KO mice relative to the numbers in the spleens of wild-type (WT) mice. The effect of adoptive transfer of defined WT leukocyte subsets generated from bone marrow cells of zDC(+/DTR) mice (which bear the human diphtheria toxin receptor [DTR] under the control of the classical dendritic cell-specific zinc finger transcription factor zDC) to specifically deplete Mincle-expressing classical dendritic cells (cDCs) but not macrophages after diphtheria toxin application on the numbers of splenic and hepatic CFU and T cell subsets was then determined. Adoptive transfer experiments revealed that Mincle-expressing splenic cDCs rather than Mincle-expressing macrophages contributed to the reconstitution of attenuated splenic antimycobacterial immune responses in Mincle-KO mice after intravenous challenge with BCG. Collectively, we show that expression of Mincle, particularly by cDCs, contributes to the control of splenic M. bovis BCG infection in mice.

Next-Generation Sequencing Analysis of Laser-Microdissected Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

In recent years, next-generation sequencing (NGS) became widely used in molecular pathology. Comprehensive mutational profiling improved diagnosis and prognosis, as well as the identification of therapeutically relevant genetic alterations. However, the vast majority of studies analyzing tissue samples use DNA extracted from bulk tissue or only manually microdissected specimens. Laser-assisted microdissection offers the possibility of isolating morphologically defined small tissue compartments (like individual glands) or even of single cells for further molecular analysis. Even formalin-fixed paraffin-embedded (FFPE) tissue specimens can be used for laser-assisted microdissection. Combining these two innovative powerful methodological approaches provides invaluable insights into the genetic profile of any cell type and tissue compartment of interest, contributing to a better understanding of fundamental biological processes and disease-specific mechanisms.In this chapter, a detailed protocol is provided for microdissection of human mammary adenomyoepithelioma tissue specimens and subsequent targeted resequencing of a panel of cancer-related genes using IonTorrent/PGM technology.

Biocompatibility of Intensified Decellularized Equine Carotid Arteries in a Rat Subcutaneous Implantation Model and in a Human In Vitro Model.

Limited biocompatibility of decellularized scaffolds is an ongoing challenge in tissue engineering. We recently demonstrated that intensified detergent-based decellularization of equine carotid artery (dEACintens) removed residual cellular molecules from the scaffold more efficiently than a conventional decellularization (dEACcon), although this approach did not eliminate its immunogenicity entirely. CCN1 has been shown to improve biocompatibility of dEACcon in a sheep model. In this study, we tested the biocompatibility of dEACintens and dEACcon with or without CCN1 coating after subcutaneous implantation in rats for up to 12 weeks. Explants were assessed by conventional histopathology and immunostaining for infiltrating M2 macrophages. Moreover, human macrophages derived from monocytes (MDM) or THP-1 cells (THP-derived macrophages [TDM]) were seeded onto dEACcon and dEACintens, and activation was assessed either by cytokine expression or matrix metalloprotease 2 and 7 staining. dEACintens showed a significantly reduced inflammatory infiltration (52%; p < 0.0001), as well as an earlier and denser neovascularization (1.4-fold, p < 0.0001) independent of CCN1 coating, which, however, reduced fibrosis exclusively with dEACintens (26-53%; p < 0.05). Human MDM seeded for 48 h onto dEACintens showed higher transcript levels for anti-inflammatory IL-10 (2.3-fold), proinflammatory TNFα (2.2-fold), and macrophage/monocyte recruiting MIP1α (3.5-fold; all p < 0.05) and MCP (2.7-fold; p < 0.01), whereas 1.92-fold more TDM on dEACintens showed staining for MMP2 (p > 0.001). Thus, although being advantageous in regard to fibrosis, CCN1 coating of dEACintens does not appear to be necessary for further improving dEACintens excellent biocompatibility in rats. In humans, the unspecific cellular immune response toward dEACintens seemed to be more complex, but generally comparable to the mild acute inflammatory tissue reaction with high remodeling activity as observed after rat subcutaneous implantation.

Biosynthetic Nanostructured Cellulose Patch for Chest Wall Reconstruction: Five-Month Follow-up in a Porcine Model.

PURPOSE: Ideal approaches and materials for reconstruction of large chest wall defects remain a topic of debate. We sought to explore the suitability of a reinforced nanostructured cellulose (NC) patch for chest wall reconstruction in an animal model. MATERIALS AND METHODS: In four domestic pigs, a standardized 10 × 10 cm chest wall defect was created by resecting three rib segments. Subsequently the defect was reconstructed via a biosynthetic NC patch (16 × 12 cm) reinforced by polytetrafluoroethylene mesh. After 1, 2, 4, and 5 months respectively, gross examination of NC patches was performed following sacrifice of the animals. Specimens of NC patches and surrounding connective tissue underwent histological examinations after staining with Hematoxylin-eosin and Elastica van Gieson. RESULTS: All animals survived their observation period without encountering major adverse events. On gross examination all NC patches were intact and well integrated into the surrounding tissue. Histological examination showed clearly demarked zones of foreign body reaction at the patch/host-tissue interface. After 5 months a slight increase in foreign body reaction, fibrous capsule formation and cellular infiltration were observed. No signs of fibroblast proliferation or neovascularization were seen within NC patches at any point. CONCLUSIONS: Our findings suggest a quick healing process and good overall biocompatibility following NC patch implantation.NC might prove an efficient and suitable biomaterial for complex chest wall reconstruction.

Prognostic factors in the myoepithelial-like spindle cell type of metaplastic breast cancer.

Metaplastic breast carcinoma (MBC) comprises a heterogeneous group of tumors with difficult to predict biological behavior. A subset of MBC, characterized by spindle-shaped tumor cells with a myoepithelial-like immunophenotype, was entered into a retrospective study (n = 42, median follow-up time 43 months). Molecular parameters (DNA sequences of mutation hot spots in AKT1, ALK, APC, BRAF, CDH1, CTNNB1, EGFR, ERBB2, FBXW7, FGFR2, FOXL2, GNAQ, GNAS, KIT, KRAS, MAP2K1, MET, MSH6, NRAS, PDGFRA, PIK3CA, PTEN, SF3B1, SMAD4, SRC, SRSF2, STK11, TP53, and U2AF1; copy numbers for EGFR, c-myc, FGFR, PLAG, c-met) were assessed. None of the patients had axillary lymph node involvement. In 13 cases, local recurrence developed after surgery (30.9 %). Distant metastasis occurred in seven patients (17 %; four after local recurrence). The most frequent genetic alteration was PIK3CA mutation (50 % of cases). None of the pathological parameters (size, grade, stage, Ki-67 labeling index) was significantly associated with disease-free survival (DFS) or overall survival (OS). PIK3CA mutation, especially the H1047R type, tended to adversely affect OS. Type of resection (mastectomy vs. breast-conserving therapy, width of margins) or adjuvant radiotherapy had no influence on DFS or OS, whereas in the group treated with radio-/chemotherapy, no local recurrence or metastasis and no death occurred. We conclude that the spindle cell type of MBC with myoepithelial features exhibits a higher frequency of PIK3CA mutation than other types of metaplastic or basal-like breast cancer and may benefit from combined radio-/chemotherapy. Classical pathological parameters are not helpful in identifying the high-risk tumors among this subgroup of MBC.