Cross-flow microfiltration for isolation, selective capture and release of liposarcoma extracellular vesicles.

We present a resource-efficient approach to fabricate and operate a micro-nanofluidic device that uses cross-flow filtration to isolate and capture liposarcoma derived extracellular vesicles (EVs). The isolated extracellular vesicles were captured using EV-specific protein markers to obtain vesicle enriched media, which was then eluted for further analysis. Therefore, the micro-nanofluidic device integrates the unit operations of size-based separation with CD63 antibody immunoaffinity-based capture of extracellular vesicles in the same device to evaluate EV-cargo content for liposarcoma. The eluted media collected showed ∼76% extracellular vesicle recovery from the liposarcoma cell conditioned media and ∼32% extracellular vesicle recovery from dedifferentiated liposarcoma patient serum when compared against state-of-art extracellular vesicle isolation and subsequent quantification by ultracentrifugation. The results reported here also show a five-fold increase in amount of critical liposarcoma-relevant extracellular vesicle cargo obtained in 30 min presenting a significant advance over existing state-of-art.

[Fourth edition of WHO classification tumours of soft tissue]. / Quatrième édition de la classification OMS des tumeurs des tissus mous.

The new World Health Organization (WHO) classification of soft tissue tumours was published in 2013, 11years after the previous edition. This new classification includes several changes: newly included sections (gastrointestinal stromal tumors…), newly recognized entities (pseudomiogenic haemangioendothelioma, haemosiderotic fibrolipomatous tumour…), and new genetic and molecular data leading to better understanding and definition of tumours, and are useful as diagnostic tools. This brief review summarizes changes in this new edition of the WHO classification of tumours of soft tissue.

Diagnostic utility of p16, CDK4, and MDM2 as an immunohistochemical panel in distinguishing well-differentiated and dedifferentiated liposarcomas from other adipocytic tumors.

Adipocytic tumors are the most common type of soft tissue neoplasms. Distinguishing atypical lipomatous tumor-well-differentiated liposarcoma (WDL) from benign adipocytic neoplasms and dedifferentiated liposarcoma (DDL) from pleomorphic or myxoid liposarcoma (LPS) can be difficult. WDL and DDL characteristically harbor amplifications of the MDM2 and CDK4 cell cycle oncogenes with protein overexpression and can also overexpress the cell cycle regulator p16. We assessed the utility of immunohistochemistry for CDK4, MDM2, and p16 in the routine histopathologic diagnosis of WDL/DDL from other adipocytic tumors. Immunohistochemistry for the trio of markers was performed on 216 adipocytic neoplasms (31 WDLs, 57 DDLs, 11 myxoid LPS, 2 pleomorphic LPS, 91 lipomas (including intramuscular, fibro, angio, and ossifying subtypes), 18 spindle/pleomorphic lipomas, and 6 hibernomas. Sixty-eight percent of WDLs and 72% of DDLs expressed all 3 antigens, whereas 100% of WDLs and 93% of DDLs expressed at least 2 antigens. The sensitivity and specificity of the trio for detecting WDLs/DDLs were 71% and 98%, respectively. The sensitivity and specificity of CDK4 for detecting WDLs/DDLs were 86% and 89%, those of MDM2 were 86% and 74%, and those of p16 were 93% and 92%, respectively. The immunohistochemical trio of CDK4, MDM2, and p16 is a useful ancillary diagnostic tool that provides strong support in distinguishing WDLs and DDLs from other adipocytic neoplasms and is potentially more sensitive than previously assessed combinations of CDK4 and MDM2. p16 was the most sensitive and specific marker for detecting WDL/DDL, and the combination of CDK4 and p16 is of more discriminatory value than the combination of either with MDM2, the least sensitive and specific of the 3 markers.

p16 immunohistochemistry as an alternative marker to distinguish atypical lipomatous tumor from deep-seated lipoma.

Atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDLPS) is a locally aggressive malignant mesenchymal neoplasm, resembling ordinary lipoma in many clinical aspects. This study investigates the value of expression of p16, an important cell cycle regulator, alone or in combination with MDM2, to distinguish the 2 entities. Fifty cases of lipomatous neoplasms, with cytogenetic results, from 45 patients were collected from the archives in Department of Pathology, University of Medicine and Dentistry of New Jersey/New Jersey Medical School during 1998 to 2006. These include 18 cases of deep-seated lipoma, 1 hibernoma, 1 lipoblastoma, and 30 cases of ALT/WDLPS. p16 was detected in 25/30 (83.3%) of ALT/WDLPS, and none (0/18) of the deep-seated lipomas (P<0.0000001, Fisher exact test). MDM2 was detected in 18/30 (60%) of ALT/WDLPS, and was negative in 0/18 of the deep-seated lipomas (P<0.0001, Fisher exact test). Combined together, 27/30 (90%) of ALT/WDLPS showed positive staining of either p16, MDM2, or both, whereas no staining was observed in all the deep-seated lipomas (P<0.0000001, Fisher exact test). The single case of hibernoma and lipoblastoma revealed p16+MDM2- phenotype. These results indicated that p16 is yet another marker which seems to be a valuable marker to differentiate ALT/WDLPS from deep-seated lipomas.

Adenomyolipoma of the uterus: a case report.

Adenomyolipoma of the uterus is a rare, benign, polypoid lesion considered to be of hamartomatous origin or represent an unusual type of benign Müllerian mixed tumour with a heterologous element. The authors present a case of uterine adenomyolipoma and discuss its pathogenesis. A 62-year-old woman complained of lower abdominal pain and postmenopausal bleeding. Imaging techniques revealed a solid ovarian mass and a polypoid intrauterine lesion. The frozen section diagnosis of the ovarian mass was a thecoma. A total hysterectomy and bilateral salpingo-oophorectomy were performed. On gross examination a pedunculated, polypoid lesion of 7x4.5x3cm was found in the uterine cavity. Microscopically, the polypoid lesion contained both epithelial and mesenchymal elements. The epithelial elements were endometrial glands of various size, formed by proliferative endometrial cells. The mesenchymal elements were composed of endometrial stroma, smooth muscle and mature adipocytes. Both the epithelial and the mesenchymal elements showed a benign appearance, were intermingled with each other and periglandular stromal condensation was absent. The lesion had an irregular surface. Microscopic diagnosis was an adenomyolipoma. The peculiar shape and microscopic features of this lesion suggested that it was a variant of benign Müllerian mixed tumour.

Structure of the supernumerary ring and giant rod chromosomes in adipose tissue tumors.

Supernumerary ring or giant rod marker chromosomes are a characteristic of well-differentiated liposarcomas (WDLPS) and atypical lipomas (ALP) and are often observed as the sole cytogenetic abnormality, but are rare in lipomas. Using a combination of different methods, we extensively investigated the structure and composition of rings and giant rods in a series of 17 WDLPS-ALP samples and three intra- or intermuscular lipomas (IMLP), revealing a unique combination of particular features strikingly related to these tumors. Although the rings and rods displayed in vitro and in vivo stability, the presence of alpha-satellites could not be detected on these supernumerary structures. Comparative genomic hybridization analysis, in combination with fluorescence in situ hybridization, identified the chromosomal regions contributing to the formation of these chromosomes: in WDLPS-ALP, all carried amplifications of 12q 14-15 and the MDM2 gene, with variable other noncontiguous regions. In the three IMLP, the rings consistently carried amplifications of 12q15-21 and 1q21, but increased copies of MDM2 were found in only one case. Other genes located more proximal in 12q14-15 were amplified in several WDLPS-ALP, but showed a normal copy number in IMLP. Furthermore, the immunohistochemical expression of the MDM2 protein was detected in most (12/14) WDLPS-ALP, in 1-30% of the cells, but never in IMLP. These supernumerary chromosomes represent a peculiar kind of amplification structure, midway between double minute chromosomes and homogeneously staining regions, but the mechanisms underlying the formation of these structures remain obscure.

Gradient, high-resolution, magic-angle spinning nuclear magnetic resonance spectroscopy of human adipocyte tissue.

The recently developed technique of gradient, high-resolution magic-angle spinning NMR (g-hr-MAS-NMR) spectroscopy was applied to the study of ex vivo human lipoma and liposarcoma tissue. Compared with conventional 1H-NMR, the g-hr-MAS method yielded a large improvement in spectral resolution and permitted the detection of metabolite resonance's in a well-differentiated liposarcoma that was not observed in spectra of similar samples obtained using nonspinning NMR methods. These findings suggest that g-hr-MAS-NMR spectroscopy provides a key improvement in spectral quality for ex vivo lipoma and liposarcoma tissue thereby permitting a more precise determination of tissue metabolite composition than conventional nonspinning NMR methods.

Distribution of basement membrane components in normal adipose tissue and in benign and malignant tumors of lipomatous origin.

Normal adipose tissue as well as 13 benign and 17 malignant lipomatous tumors (lipomas, hibernomas, lipoblastomas, and liposarcomas) were immunohistochemically analyzed for their expression of the basement membrane components collagen IV, laminin, heparan sulfate proteoglycan, and fibronectin. Monovacuolar cells in normal white fat tissue and in lipomas generally exhibited a distinctive pericellular basement membrane composed of collagen IV and laminin, whereas heparan sulfate proteoglycan and fibronectin were almost completely missing. In brown fat tissue and hibernomas the characteristic multivacuolated cells differed from the monovacuolated white fat cells by the additional content of heparan sulfate proteoglycan and fibronectin and the more intensive staining for the other components tested. In contrast, multi-/monovacuolated cells in lipoblastomas exhibited no characteristic immunohistochemical feature because they reacted irregularly and only faintly for collagen IV, laminin, and heparan sulfate proteoglycan. Spindle cell areas in benign lipomatous tumors displayed more fibronectin than laminin and heparan sulfate proteoglycan indicating a "preadipose" fibroblast-like cellular differentiation. In liposarcomas, only well-differentiated lipoma-like neoplasms revealed a basement membrane pattern resembling that of white fat tissue. Otherwise, in nonlipoma-like liposarcomas, a marked decrease particularly of collagen IV staining was evident. Poorly differentiated liposarcomas mostly failed to express any of the basement membrane components, but showed a relative increase of fibronectin. Our results provide evidence that the staining pattern of basement membrane components parallels the histogenetic derivation of benign lipomatous tumors from either brown or white adipose tissue and, additionally, may reflect such a derivation in liposarcomas.

Immunohistochemical identification of tumours of adipocytic differentiation using an antibody to aP2 protein.

AIM: To determine whether aP2 expression is a useful diagnostic marker in soft tissue tumour pathology. METHODS: A polyclonal antibody to aP2 was used to investigate the immunohistochemical expression of this protein in benign and malignant tumours of adipocytic differentiation and a wide variety of other neoplasms. RESULTS: aP2 was only expressed by lipoblasts (in all types of liposarcoma and in lipoblastomatosis) and by brown fat cells (in both hibernomas and normal periadrenal fetal fat). Other benign adipose tissue tumours and malignant connective tissue or epithelial tumours were distinguished from liposarcoma by the absence of staining for aP2. CONCLUSION: Identification of lipoblasts using markers of aP2 expression is of value in the differential diagnosis of benign and malignant adipose tissue tumours and in distinguishing liposarcomas from other malignant mesenchymal and epithelial neoplasms, some of which contain cells that morphologically resemble lipoblasts.