Modulation of hyaluronan production by CD44 positive glioma cells.

This study examines the functional relationship between glioma cell production of hyaluronan (HA), known to play a role in glioma invasion, expression of its CD44 receptor, and glioma cell viability. Production of HA by CD44 positive mouse G26 and human U373 glioma cell lines was evaluated and compared to that of a CD44 positive mouse fibroblast-like L929 cell line. We found that both G26 and U373 MG glioma cells, but not L929 fibroblast-like cells, synthesized HA. The synthesis of HA by glioma cells was found during the proliferative phase as well as post-confluency, as detected by fluorophore-assisted carbohydrate electrophoresis. Eighty to ninety percent of the HA synthesized was secreted into the medium and 10-20% remained associated with the cells. To examine a possible mechanistic link between the CD44-HA interaction and endogenous HA production, glioma cells were treated with either anti-CD44 antibodies (clones KM201 or IM7) or HA oligosaccharides (hexamer oligoHA-6 or decamer oligoHA-10). We found that oligoHA-10, which was previously shown to compete effectively with the CD44-HA interaction, enhanced glioma HA synthesis by approximately 1.5-fold, without affecting cell viability. IM7 treatment of human U373 glioma cells resulted in over 50% decrease of HA production, which was associated with changes in cell size and apoptosis. Taken together, these data show that CD44 specific ligands, such as the IM7 antibody or oligoHA-10 could down-regulate or up-regulate glioma HA production, respectively. Our results suggest that interference with CD44/HA may lead to the discovery and development of new treatment modalities for glioma.

Rapid frozen section immunostaining of melanocytes by microphthalmia-associated transcription factor.

Mohs micrographic surgery (MMS) has increasingly become an accepted therapy for melanoma in situ on chronically sun damaged skin (CSDS). However, melanocytes are difficult to locate in frozen material on hematoxylin and eosin. In addition, determining the cut-off between the melanoma and the "atypical melanocytic hyperplasia" in CSDS can be challenging in frozen or formalin-fixed paraffin-embedded sections, with or without immunohistochemistry (IHC). In this article, we report the use of a rapid, 35-minute protocol using microphthalmia-associated transcription factor (MITF) IHC for identifying melanocytes in frozen tissue for its potential use in MMS. In contrast to melanoma antigen recognized by T cells (MART-1), MITF is a nuclear stain, which simplifies identification of melanocytes and quantification of melanocytic parameters. In this study, MITF IHC in frozen sections yielded equivalent melanocyte nuclear diameter and density measurements compared with formalin-fixed paraffin-embedded sections. Nuclear diameter measurements obtained with MITF were similar to that previously reported with MART-1, but the melanocyte density figures were lower. Reliable labeling of melanocytes in frozen sections required the use of diaminobenzidine (DAB) chromogen with Giemsa counterstaining and a buffer devoid of surfactant. Our experience with MITF IHC indicates that it is a dependable immunostain in frozen sections, and may prove to be useful in MMS as an adjunct to hematoxylin and eosin and MART-1 IHC for interpretation of margins for melanoma in situ on CSDS.

Mohs surgery for melanoma in situ: how we do it.

The advent of rapid immunostains has made the use of Mohs surgery to treat melanoma more practical. Mohs surgery is especially useful in the management of lentigo maligna, which often has indistinct clinical margins. The authors describe their technique for treating melanoma in situ with Mohs surgery.

Comparison of MART-1 frozen sections to permanent sections using a rapid 19-minute protocol.

BACKGROUND: The use of melanoma-associated antigen recognized by T cells (MART-1) immunostain has been proposed as a useful adjunct to overcome the inherent difficulties in the use of frozen sections during Mohs surgery for the treatment of melanoma, but no studies have compared MART-1 frozen sections with MART-1 permanent sections. Current MART-1 1-hour protocols add significant time to the procedure. OBJECTIVE: To determine whether there is a significant difference between frozen and permanent MART-1 immunostained sections using a rapid 19-minute protocol. METHODS: Frozen and permanent sections stained with MART-1 were made from dog-ears excised during 25 reconstructions. A rapid 19-minute protocol was used to stain the frozen tissue. The sections were examined blinded, and statistical analysis was performed to analyze the data. RESULTS: No significant difference was found in number of keratinocytes, nuclear diameter of keratinocytes, number of melanocytes, melanocytic nuclear diameter, confluence, pagetoid spread, melanocytic nesting, or atypical melanocytes. CONCLUSIONS: The 19-minute protocol is a rapid and effective MART-1 immunostain. Frozen sections stained with MART-1 provide information equivalent to that obtained from MART-1 stained permanent sections. Mohs surgeons using MART-1 can feel confident that they have the same information as they would have obtained using permanent sections using the slow Mohs method.

Immunohistochemical stains in Mohs surgery: a review.

BACKGROUND: During Mohs surgery, there are instances in which residual tumor cells may be difficult to detect, thereby increasing the risk of incomplete excision and tumor recurrence. It is possible to employ immunohistochemical techniques as an adjunct to routine hematoxylin and eosin staining to aid in ensuring negative margins. OBJECTIVE: To review the literature regarding the use of immunostains in Mohs surgery. RESULTS: Various immunostains have proved useful in detecting tumor cells in various malignancies, including melanoma, basal cell carcinoma, squamous cell carcinoma, dermatofibrosarcoma protuberans, extramammary Paget's disease, primary cutaneous mucinous carcinoma, granular cell tumor, and trichilemmal carcinoma. CONCLUSIONS: In this article, we review immunohistochemical stains that have been employed in Mohs micrographic surgery and evaluate their utility in enhancing detection of residual tumors with respect to tumor type, particularly in situations in which detection of residual tumor may be difficult.

Innovative 19-minute rapid cytokeratin immunostaining of nonmelanoma skin cancer in Mohs micrographic surgery.

BACKGROUND: Dense inflammation can obscure nonmelanoma skin cancer (NMSC) on frozen sections, prompting removal of additional layers to ensure negative margins. Cytokeratin (CK) immunostaining in Mohs micrographic surgery (MMS) has been examined and found to be useful but is limited by lengthy 1-hour processing. OBJECTIVE: Our objective was to develop an effective ultrarapid CK frozen section immunostain to be used during MMS in cases of NMSC with dense or perineural inflammation. METHODS: An ultrarapid immunostain with a mixture of AE1/AE3 monoclonal antibodies was performed in 21 MMS cases and compared with permanent sections prepared from the same material. RESULTS: The ultrarapid CK protocol stained all of the cells in each of the 21 examples of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) in frozen tissue in a way equivalent to immunostains being applied to permanent sections. CONCLUSION: The 19-minute CK immunohistochemistry protocol in frozen tissue appears to be as effective at labeling tumor cells of SCC and BCC as methods requiring permanent sections. It is hopeful that this technique may prevent recurrences after MMS and limit the number of Mohs layers required to obtain free margins when inflammation is abundant. It also is effective in uncovering subtle perineural invasion.

CD44 adhesion molecule and neuro-glial proteoglycan NG2 as invasive markers of glioma.

Glioma invasion into the CNS involves the interaction of tumor cells with the host's cells and extracellular matrix (ECM) molecules. In this study, the expression of ECM-associated and cell-associated proteins such as the transmembrane CD44 adhesion molecule and neuro-glial proteoglycan 2 (NG2), a member of the chondroitin sulfate proteoglycan family, were evaluated during glioma progression, in vitro and in vivo, using a model of a highly invasive and aggressive intracerebral mouse G-26 glioma. We found a marked increase in CD44 and NG2 expression in brain tissue containing glioma. The glioma levels of these proteins gradually increased over time to reach 3-15 times the levels in the contralateral control. NG2 and CD44 expression paralleled progression of the glioma, being higher on days 14 and 21 than on day 2 post-glioma implant. In addition, when invading glioma crossed the midline in the advanced tumor stage, levels of each of these proteins in the contralateral tissue were elevated, but were still significantly lower than in the ipsilateral, tumor-bearing hemisphere. Immunohistochemistry of advanced stage G-26 glioma (day 21) showed CD44 expression to be most prominent at the front of the glioma invasion line, sharply separated from normal brain parenchyma which expressed glial fibrillary acidic protein (GFAP). However, single CD44 positive cells that escaped the tumor mass penetrated between the astrocytes that encased the tumor at its periphery. In contrast, NG2 was expressed on nearly all glioma cells within the tumor mass but less so at the leading edge of the tumor. The NG2 positive cells were clearly demarcated and morphologically distinguishable from GFAP positive cells and only sporadic, small groups of NG2 positive cells were seen in the GFAP positive zone of the neuropil. Taken together, these data show that during glioma progression in the brain, the level and pattern of glioma-associated molecules such as CD44 and NG2 may aid in tracing and targeting the invading glioma cells.