Osteosarcoma microenvironment: whole-slide imaging and optimized antigen detection overcome major limitations in immunohistochemical quantification.

BACKGROUND: In osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment. METHODS: Immunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis. RESULTS: More than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35-65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method. CONCLUSION: Standard staining and quantification procedures are not applicable in decalcified formalin-fixed and paraffin-embedded samples for major parameters of the immunovascular microenvironment in osteosarcoma. Whole-slide imaging and optimized antigen retrieval overcome these limitations

A short-term in vivo model for giant cell tumor of bone

<p>Abstract</p> <p>Background</p> <p>Because of the lack of suitable <it>in vivo </it>models of giant cell tumor of bone (GCT), little is known about its underlying fundamental pro-tumoral events, such as tumor growth, invasion, angiogenesis and metastasis. There is no existing cell line that contains all the cell and tissue tumor components of GCT and thus <it>in vitro </it>testing of anti-tumor agents on GCT is not possible. In this study we have characterized a new method of growing a GCT tumor on a chick chorio-allantoic membrane (CAM) for this purpose.</p> <p>Methods</p> <p>Fresh tumor tissue was obtained from 10 patients and homogenized. The suspension was grafted onto the CAM at day 10 of development. The growth process was monitored by daily observation and photo documentation using <it>in vivo </it>biomicroscopy. After 6 days, samples were fixed and further analyzed using standard histology (hematoxylin and eosin stains), Ki67 staining and fluorescence <it>in situ </it>hybridization (FISH).</p> <p>Results</p> <p>The suspension of all 10 patients formed solid tumors when grafted on the CAM. <it>In vivo </it>microscopy and standard histology revealed a rich vascularization of the tumors. The tumors were composed of the typical components of GCT, including (CD51+/CD68+) multinucleated giant cells whichwere generally less numerous and contained fewer nuclei than in the original tumors. Ki67 staining revealed a very low proliferation rate. The FISH demonstrated that the tumors were composed of human cells interspersed with chick-derived capillaries.</p> <p>Conclusions</p> <p>A reliable protocol for grafting of human GCT onto the chick chorio-allantoic membrane is established. This is the first <it>in vivo </it>model for giant cell tumors of bone which opens new perspectives to study this disease and to test new therapeutical agents.</p

Prognostic impact of reduced connexin43 expression and gap junction coupling of neoplastic stromal cells in giant cell tumor of bone

Missense mutations of the GJA1 gene encoding the gap junction channel protein connexin43 (Cx43) cause bone malformations resulting in oculodentodigital dysplasia (ODDD), while GJA1 null and ODDD mutant mice develop osteopenia. In this study we investigated Cx43 expression and channel functions in giant cell tumor of bone (GCTB), a locally aggressive osteolytic lesion with uncertain progression. Cx43 protein levels assessed by immunohistochemistry were correlated with GCTB cell types, clinico-radiological stages and progression free survival in tissue microarrays of 89 primary and 34 recurrent GCTB cases. Cx43 expression, phosphorylation, subcellular distribution and gap junction coupling was also investigated and compared between cultured neoplastic GCTB stromal cells and bone marow stromal cells or HDFa fibroblasts as a control. In GCTB tissues, most Cx43 was produced by CD163 negative neoplastic stromal cells and less by CD163 positive reactive monocytes/macrophages or by giant cells. Significantly less Cx43 was detected in alpha-smooth muscle actin positive than alpha-smooth muscle actin negative stromal cells and in osteoclast-rich tumor nests than in the adjacent reactive stroma. Progressively reduced Cx43 production in GCTB was significantly linked to advanced clinico-radiological stages and worse progression free survival. In neoplastic GCTB stromal cell cultures most Cx43 protein was localized in the paranuclear-Golgi region, while it was concentrated in the cell membranes both in bone marrow stromal cells and HDFa fibroblasts. In Western blots, alkaline phosphatase sensitive bands, linked to serine residues (Ser369, Ser372 or Ser373) detected in control cells, were missing in GCTB stromal cells. Defective cell membrane localization of Cx43 channels was in line with the significantly reduced transfer of the 622 Da fluorescing calcein dye between GCTB stromal cells. Our results show that significant downregulation of Cx43 expression and gap junction coupling in neoplastic stromal cells are associated with the clinical progression and worse prognosis in GCTB

Centrosome abnormalities in giant cell tumour of bone: possible association with chromosomal instability.

Giant cell tumour of bone, a benign but potentially aggressive neoplasm, shows an increasing rate of chromosomal aneusomy that correlates with clinical course. Mechanisms that generate chromosomal instability in giant cell tumour of bone are poorly understood. One possible cause of chromosomal instability is an error in mitotic segregation due to numeric and/or functional abnormalities of centrosomes. Centrosome alteration is a common phenomenon in many cancers and has a major role in the development of chromosomal instability in cancer cells. To gain an insight into the possible mechanism for the generation of chromosomal instability in giant cell tumour of bone, we analysed 100 cases, including 57 primary nonrecurrent, 35 recurrent and 8 malignant giant cell tumour of bone cases. gamma-Tubulin immunohistochemistry was performed on tissue microarrays of 59 formalin-fixed paraffin-embedded cases, whereas pericentrin and gamma-tubulin fluorescent immunocytochemistry was carried out on 41 frozen smears. Fluorescent in situ hybridization was performed on 23 cases of pericentrin immunostained smears, allowing the simultaneous analysis of centrosomes and chromosome aberrations. Centrosome amplification was significantly higher in recurrent and malignant giant cell tumour of bones compared with nonrecurrent tumours (P&lt;0.001). A comparison of the percentage of aneusomic cells with a normal centrosome content (4.7%) with that of aneusomic cells with centrosome amplification (6.4%) revealed no significant association between chromosome number alterations and centrosome aberrations (P=0.31). These findings indicate that centrosome alteration and frequency of aneusomy correlate with clinical behaviour; the lack of an association between centrosome amplification and chromosome number alteration suggests that alternative causative mechanisms produce genetic instability in giant cell tumour of bone

The impact of distinct triple-negative breast cancer subtypes on misdiagnosis and diagnostic delay

BACKGROUND Triple-negative breast cancer (TNBC) includes mostly aggressive types of breast cancer with poor prognosis. Due to its growth pattern, misinterpretation in clinical imaging is more frequent than in non-TNBC. As the group of TNBC contains heterogeneous types of tumors, marker expression-based subtypes have recently been established. We analyzed clinical features and false-negative imaging findings that could potentially lead to diagnostic delay within the subtypes. METHODS An exploratory analysis compared the imaging features across the a priori defined subtypes and related these findings to molecular subtype, disease stage, potential diagnostic delay, and patient outcome. RESULTS TNBC cases were categorized into basal-like (BL; 38.6%), mesenchymal-like (ML; 19.9%), luminal androgen receptor (LAR; 28.3%), and immunomodulatory (IM; 13.3%) subtype. In almost every third patient, malignant classification was missed in at least one imaging method. Misclassification in mammogram was more frequent in ML, while benign ultrasound features were reported more often in the BL subtype. Diagnostic delay due to misclassification in imaging led to tumor growth and/or upgrading of the tumor stage in 8.9% of BL tumors, which had the lowest overall survivals. Despite misclassification rate was higher in the ML subtype it showed better outcomes. Misdiagnosis of axillary lymph node metastasis was higher in LAR; however, this subtype showed a higher percentage of affected axillary lymph nodes. CONCLUSION TNBC subtypes have different clinical features, benign appearances, and diagnostic delay, which can lead to tumor stage upgrade. Future clinical studies on TNBC outcomes might consider the confounder of clinical delay in the subtypes

Prognostic impact of PIK3CA protein expression in triple negative breast cancer and its subtypes

Background Triple negative breast cancer (TNBC) harbors a heterogeneous group of carcinomas with poor prognosis and high genetic variability. As a potential aim for targeted therapy, genetic mutations leading to an activation of the phosphoinositide 3-kinase pathway in a catalytic subunit (PIK3CA) in breast cancer have been analyzed currently. Little is known about the clinical impact and prognostic or predictive value of this marker in TNBC subtypes. Methods Samples from 119 TNBC cases were submitted to immunohistochemical PIK3CA protein expression analysis and scored semi-quantitatively as negative, weak (1 +), or strongly expressed (2 +). Expression scores were correlated to patient’s characteristics, imaging features, and TNBC subtypes. TNBC subtypes were categorized into four subtypes: basal like, mesenchymal like, luminal androgen receptor (LAR), and immunomodulatory. Results We did not observe differences in clinical aspects and imaging features between TNBC with and without PIK3CA expression. PIK3CA expression was in general higher in the LAR subtype. The disease-free survival and overall survival were significantly better in TNBC with PIK3CA protein expression, independent of TNBC subtypes. Conclusion Despite conflicting results in the literature, our study clearly shows a better outcome of PIK3CA-expressing TNBC, independent of TNBC subtypes. PIK3CA expression in TNBC is not associated with specific clinical or diagnostic features. Further molecular studies and meta-analysis are warranted to clarify the prognostic and predictive role of PIK3CA protein expression

Comparative analysis of confocal microscopy on fresh breast core needle biopsies and conventional histology

BACKGROUND Evaluation of core needle biopsies (CNB) is a standard procedure for the diagnosis of breast cancer. However, tissue processing and image preparation is a time- consuming procedure and instant on-site availability of high-quality images could substantially improve the efficacy of the diagnostic procedure. Conventional microscopic methods, such as frozen section analysis (FSA) for detection of malignant cells still have clear disadvantages. In the present study, we tested a confocal microscopy scanner on fresh tissue from CNB with intention to develop an alternative device to FSA in clinical practice. PATIENTS AND METHODS In 24 patients with suspicious breast lesions standard of care image-guided biopsies were performed. Confocal images have been obtained using the Histolog™ Scanner and evaluated by two independent pathologists. Hematoxylin-Eosin (H&E) histological sections of the biopsies were routinely processed in a blinded fashion with respect to the confocal images. RESULTS In total 42 confocal images were generated from 24 biopsy specimens, and available for analysis within a few minutes of taking the biopsy. This resulted in 2 × 42 = 84 pathologic evaluations. In four cases, a pathologic diagnosis was not possible with confocal microscopy. An exact correlation based on the B-classification was reached in 41 out of 80 examinations and in another 35 cases in a broader sense of correspondence definition (i.e. malignant vs. benign). CONCLUSIONS As a reliable on-site method, the Histolog™ Scanner provides a visualization of cellular details equivalent to the H&E standards, permitting rapid and accurate diagnosis of malignant and benign breast lesions. Furthermore, this device offers great potential for immediate margin analysis of specimen in breast conserving therapy

Epidermal growth factor receptor signalling contributes to osteoblastic stromal cell proliferation, osteoclastogenesis and disease progression in giant cell tumour of bone.

AIMS: Epidermal growth factor receptor (EGFR) is implicated in bone remodelling. The aim was to determine whether EGFR protein expression contributes to the aggressiveness and recurrence potential of giant cell tumour of bone (GCTB), an osteolytic primary bone tumour that can exhibit markedly variable clinical behaviour. METHODS AND RESULTS: Immunohistochemical analysis on tissue microarrays (TMA) of 231 primary, 97 recurrent, 17 metastatic and 26 malignant GCTBs was performed using TMA analysis software and whole digital slides allowing validated scoring. EGFR expression was restricted to neoplastic stromal cells and was significantly more frequent in recurrent (71 of 92; 77%) than in non-recurrent GCTBs (86 of 162; 53%) (P = 0.002); and in clinicoradiologically aggressive (31 of 43; 72%) than latent (27 of 54; 50%) cases (P = 0.034). Detecting phosphotyrosine epitopes pY1068 and -pY1173 indicated active EGFR signalling, and finding EGFR ligands EGF and transforming growth factor-α restricted to cells of the monocytic lineage suggested paracrine EGFR activation in stromal cells. In functional studies EGF supported proliferation of GCTB stromal cells, and the addition of EGF and macrophage-colony stimulating factor promoted osteoclastogenesis. CONCLUSION: In GCTB, EGFR signalling in neoplastic stromal cells may contribute to disease progression through promoting stromal cell proliferation and osteoclastogenesis