Comparison of the efficiency of transgelin, smooth muscle myosin, and CD31 antibodies for the assessment of vascular tumor invasion and free tumor deposits in gastric, pancreatic, and colorectal adenocarcinomas.

BACKGROUND: This study aimed to compare CD31, smooth muscle myosin (SMM), and transgelin antibodies for their efficiency in detecting venous invasion (VI) and the nature of free tumor deposits (TDs) in gastric, pancreatic, and colorectal adenocarcinomas. MATERIALS AND METHODS: Eleven Whipple, 5 gastrectomy, and 3 colectomy specimens and 1 low anterior resection specimen were reviewed and examined, revealing 254 probable foci. Foci were reviewed and divided into 3 types: Type A, the "orphan artery" pattern; Type F, free TDs in the periorgan adipose and connective tissue without an unaccompanied artery; and Type X, a focus that could be detected only with the immunohistochemical procedures mentioned. RESULTS: No foci were positive for CD31. Transgelin staining was more sensitive than SMM staining in all focus types, Type A only and Type F only (P < 0.001, P = 0.001, and P = 0.10, respectively). In free TDs (Type F), 35.7% of the samples were negative for all four stains, and 64.2% of the samples were positive for SMM and transgelin. We did not make the distinction between a metastatic lymph node and VI in positive foci. CONCLUSION: We conclude that hematoxylin and eosin (H and E) staining is inadequate and that smooth muscle markers, such as transgelin and/or SMM, are more effective than endothelial markers, such as CD31, in revealing VI and lymph node/large extramural invasion.

A direct immunohistochemistry (IHC) method improves the intraoperative diagnosis of breast papillary lesions including breast cancer.

Tumors with the attributes of rapid growth, infiltration, and metastasis are the leading causes of death among cancer patients. Angiogenesis is essential to tumor nutrition support and tumor progression. Endoglin is a glycoprotein highly expressed on the endothelial cell membrane and is regarded as the most reliable marker of tumor vascular proliferation. In this review, we summarize recent advances in targeting endoglin for the imaging of cancer angiogenesis and the development of monoclonal antibodies and vaccines to inhibit cancer angiogenesis. In addition, we forecast the future promise of endoglin as a novel target for the diagnosis and treatment of malignant tumors.

Discriminating the earliest stages of mammary carcinoma using myoepithelial and proliferative markers.

Mammographic screening has led to increased detection of breast cancer at a pre-invasive state, hence modelling the earliest stages of breast cancer invasion is important in defining candidate biomarkers to predict risk of relapse. Discrimination of pre-invasive from invasive lesions is critically important for such studies. Myoepithelial cells are the barrier between epithelial cells and the surrounding stroma in the breast ductal system. A number of myoepithelial immunohistochemistry markers have been identified and validated in human tissue for use by pathologists as diagnostic tools to distinguish in situ carcinoma from invasive breast cancer. However, robust myoepithelial markers for mouse mammary tissue have been largely under-utilised. Here, we investigated the utility of the myoepithelial markers smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), cytokeratin-14 (CK14) and p63 to discriminate mammary intraepithelial neoplasia (MIN) from invasive disease in the C57BL/6J MMTV-PyMT transgenic model of mammary carcinoma. We identified that SMMHC and CK14 are retained in early in situ neoplasia and are appropriate markers for distinguishing MIN from invasive disease in this model. Additionally, the proliferation marker Ki67 is a superior marker for differentiating between normal and hyperplastic ducts, prior to the development of MIN. Based on this, we developed a scoring matrix for discriminating normal, hyperplasia, MIN and invasive lesions in this spontaneous mammary tumorigenesis model. This study demonstrates heterogeneous expression of myoepithelial proteins throughout tumour development, and highlights the need to characterise the most appropriate markers in other models of early breast cancer to allow accurate classification of disease state.

Morphologic, molecular and microenvironment factors associated with stromal invasion in breast ductal carcinoma in situ: Role of myoepithelial cells.

BACKGROUND: Ductal carcinoma in situ is the last step preceding invasive ductal carcinoma in breast carcinogenesis. OBJECTIVE: We investigated the role of myoepithelial cells and epithelium characteristics as predictors of the risk of stromal invasion. METHODS: We selected 236 cases with initial diagnosis of DCIS followed by surgical ressection distributed in groups 1 (without invasion) and 2 (with invasive carcinoma). RESULTS: The risk of stromal invasion after a DCIS diagnosis in biopsy was associated to triple-negative profile and loss of CD10 expression by myoepithelial cells, and inversely associated with CK5/6 expression by neoplastic cells and high expression of Smooth Muscle Myosin Heavy Chain (SMMHC) by myoepithelial cells. CONCLUSIONS: A combination of characteristics of epithelial and myoepithelial cells in DCIS in biopsy specimens is related to the risk of stromal invasion.

Derivation and maturation of synthetic and contractile vascular smooth muscle cells from human pluripotent stem cells.

AIMS: Embryonic vascular smooth muscle cells (vSMCs) have a synthetic phenotype; in adults, they commit to the mature contractile phenotype. Research shows that human pluripotent stem cells (hPSCs) differentiate into vSMCs, but nobody has yet documented their maturation into the synthetic or contractile phenotypes. This study sought to control the fate decisions of hPSC derivatives to guide their maturation towards a desired phenotype. METHODS AND RESULTS: The long-term differentiation of hPSCs, including the integration-free-induced PSC line, in high serum with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-ß1, allowed us to induce the synthetic vSMC (Syn-vSMC) phenotype with increased extracellular matrix (ECM) protein expression and reduced expression of contractile proteins. By monitoring the expression of two contractile proteins, smooth muscle myosin heavy chain (SMMHC) and elastin, we show that serum starvation and PDGF-BB deprivation caused maturation towards the contractile vSMC (Con-vSMC) phenotype. Con-vSMCs differ distinctively from Syn-vSMC derivatives in their condensed morphology, prominent filamentous arrangement of cytoskeleton proteins, production and assembly of elastin, low proliferation, numerous and active caveolae, enlarged endoplasmic reticulum, and ample stress fibres and bundles, as well as their high contractility. When transplanted subcutaneously into nude mice, the human Con-vSMCs aligned next to the host's growing functional vasculature, with occasional circumferential wrapping and vascular tube narrowing. CONCLUSION: We control hPSC differentiation into synthetic or contractile phenotypes by using appropriate concentrations of relevant factors. Deriving Con-vSMCs from an integration-free hiPSC line may prove useful for regenerative therapy involving blood vessel differentiation and stabilization.

Immunohistochemical staining characteristics of low-grade adenosquamous carcinoma of the breast.

BACKGROUND: Low-grade adenosquamous carcinoma (LGASC) is an uncommon variant of metaplastic carcinomas of the breast. The immunohistochemical profile of this entity has not been well characterized and is likely because of its seemingly inconsistent staining patterns when commonly used immunohistochemical stains are employed. We set out to further elucidate the immunohistochemical profile of this uncommon entity in a sizable cohort of patients. MATERIALS AND METHODS: Thirty cases of LGASC were identified in our files. Commonly used immunohistochemical stains such as myoepithelial and cytokeratin markers used to evaluate a small glandular proliferation in the breast (the differential diagnosis of which includes LGASC) were utilized. The pattern and location of immunoreactivity were recorded in each case. Results were compared for staining trends. RESULTS: All cases of LGASC demonstrated variable staining in both lesional glands and stromal cells for myoepithelial (p63, smooth muscle myosin, smooth muscle actin, CD10, calponin) and cytokeratin (CK AE1/3, CK5/6, CK7, CK 34ßE12, Cam 5.2) markers. Within a single case, circumferential staining using myoepithelial markers was complete, discontinuous, and absent in over a third of the studied cases. A minority of cases showed either complete circumferential staining (or complete absence) by any single immunohistochemical stain. Lamellar staining of stromal cells surrounding glands was best highlighted using smooth muscle myosin heavy chain or calponin. Using cytokeratin stains, core staining (luminal glandular cells demonstrating distinctly stronger staining intensity than the basally located cells in the same gland) was observed in approximately half of the studied cases. These lesional stromal cells were negative for all cytokeratins, with the exception of 1, which was focally positive for 1 cytokeratin immunostain (CK7) while being negative for 3 others. CONCLUSIONS: LGASC consistently stains in an inconsistent manner using commonly used immunohistochemical stains. In addition, we found lamellar staining and core staining using myoepithelial and cytokeratin stains, respectively, to be distinctive and therefore diagnostically valuable.

Immunohistochemical staining for smoothelin in the duplicated versus the true muscularis mucosae of Barrett esophagus.

BACKGROUND: The muscularis mucosa underlying the metaplastic mucosa of Barrett esophagus is frequently duplicated, with an intervening layer of lamina propria between the superficial or neomuscularis mucosa (NMM) and the deep/true muscularis mucosa (TMM). This duplication causes difficulties with accurate staging of superficially invasive carcinoma in biopsy specimens and endoscopic mucosal resections (EMRs), as invasion underneath the superficial muscle layers may be mistaken for submucosal invasion. Mucosal resections or other ablative nonsurgical therapies can be curative in patients with esophageal intramucosal carcinoma, whereas patients with submucosal invasion are recommended for esophagectomy. Therefore, the accurate staging of such specimens is crucial. Smoothelin is a novel smooth muscle protein expressed only by fully differentiated smooth muscle cells and not by proliferative or noncontractile smooth muscle cells and fibroblasts. It has been suggested that in the bladder, immunohistochemistry for smoothelin may help separate hyperplastic muscularis mucosa from the true muscularis propria. We hypothesized that in the esophagus, immunohistochemistry for smoothelin would differentiate the NMM from the TMM. DESIGN: Thirty cases of EMRs for Barrett esophagus-related neoplasia were retrieved from the archives of the pathology department, St Michael's Hospital. Immunohistochemical staining for smoothelin was performed to evaluate differential staining in the TMM versus NMM. Fifteen cases were stained for smooth muscle actin and smooth muscle myosin. The staining score was evaluated on a scale from 0 to 3 according to the percentage and intensity of staining. RESULTS: Immunohistochemical staining results for smoothelin were as follows: the NMM showed weak focal staining (+1) in 23 of 30 cases (82%), and moderate staining (+2) in 7 of 30 cases (12%), and the TMM showed very strong and diffuse staining (+3) in 30 of 30 cases. No cases showed negative (0) staining in the NMM. With smooth muscle actin and myosin, strong and diffuse staining was observed with similar intensity in both the TMM and NMM in 15 of 15 cases. CONCLUSIONS: In our study, smoothelin staining in the NMM is significantly weaker than that seen in the true/deep muscularis mucosa. This pattern is similar to that reported for the muscularis mucosae of the urinary bladder. Although smoothelin can readily distinguish the 2 layers, its value might be limited by the need to simultaneously compare the 2 layers. Although this might be of use in EMR specimens in which both layers are visible, use in biopsies may be limited as the residual staining in the NMM may inhibit definitive evaluation. This issue may be resolved by the use of appropriate standard controls, individual optimization of the antibody, and the use of an automated digital assessment.

Differential expression of smooth muscle myosin, smooth muscle actin, h-caldesmon, and calponin in the diagnosis of myofibroblastic and smooth muscle lesions of skin and soft tissue.

The diagnosis of low-grade and pseudosarcomatous spindle cell lesions of skin and soft tissue can sometimes be problematic; in particular, distinction between fibroblastic, myofibroblastic, and smooth muscle proliferations can occasionally pose difficulties on routine histologic examination. We have applied a panel of immunohistochemical markers to a series of spindle cell lesions of skin and soft tissue to assess the utility of the differential expression of smooth muscle and myofibroblastic-associated markers. Twenty-eight cases of nodular fasciitis, 42 cases of fibromatosis, and 3 cases of myofibroblastic sarcoma were stained with antibodies against smooth muscle actin (SMA), smooth muscle myosin (SMMS), calponin, and high-molecular weight caldesmon (h-caldesmon). For comparison, 12 cases of cutaneous leiomyoma and 8 cases of leiomyosarcomas involving superficial soft tissues and fascia were studied with the same panel of antibodies. Thirty-eight of 42 cases of fibromatosis were positive for SMA, 42/42 cases were positive for calponin, 39/42 cases were negative for SMMS, and all cases were negative for h-caldesmon. All cases of nodular fasciitis were positive for SMA and calponin, and all were negative for h-caldesmon and SMMS. All cases of myofibroblastic sarcoma were positive for SMA and 2/3 cases for calponin, and were negative for SMMS and h-caldesmon. All cases of cutaneous leiomyoma and leiomyosarcoma were positive for all 4 markers tested. Our results demonstrate a remarkably consistent pattern of reactivity of muscle and myofibroblastic-associated markers in lesions predominantly composed of myofibroblastic spindle cells, characterized by positive staining for SMA and calponin and absence of reactivity for SMMS and h-caldesmon. Application of this panel of stains may be of aid in the differential diagnosis of low-grade myofibroblastic lesions such as nodular fasciitis and fibromatosis from smooth muscle tumors of skin and soft tissue. This panel may additionally be of assistance in the diagnosis of myofibroblastic sarcoma.

Immunostaining patterns of myoepithelial cells in breast lesions: a comparison of CD10 and smooth muscle myosin heavy chain.

BACKGROUND: Recent studies have reported CD10 expression in myoepithelial cells (MEC) of the breast, supporting its use as a marker to help distinguish invasive breast carcinoma (IC) from ductal carcinoma in situ (DCIS). AIM: To compare the effectiveness of CD10 with smooth muscle myosin heavy chain (SMMHC) in the detection of MEC in benign and malignant breast lesions. METHODS: Histological material from 25 patients with DCIS and 21 with IC were immunostained for CD10 and SMMHC. Staining was scored on a scale of 0 to 3+ (0, no staining; 3+, intense) and the staining distribution was documented as focal, partial, or circumferential. RESULTS: Uniform, 3+ circumferential CD10 and SMMHC staining of MEC was seen in normal breast ducts and lobules, and in ducts and acini involved in sclerosing adenosis and apocrine metaplasia. In an analysis of total ducts involved by DCIS, 3+ circumferential staining was seen in 65 of 366 ducts (17.7%) stained for CD10 versus 190 of 396 ducts (48%) stained for SMMHC. MEC were not detected immunohistochemically in 116 of 366 ducts (31.7%) with anti-CD10 and 50 of 396 (12.7%) with anti-SMMHC. In contrast, all ICs were negative for both CD10 and SMMHC. Focal background staining of stromal myofibroblasts was seen with both CD10 and SMMHC, but CD10 showed a higher rate of non-specific staining of epithelial cells. CONCLUSION: Although CD10 can aid in the distinction between IC and DCIS, SMMHC is a more sensitive and specific marker of MEC and shows less heterogeneity of immunostaining patterns.

Non-contractile cells with thin processes resembling interstitial cells of Cajal found in the wall of guinea-pig mesenteric arteries.

Arterial interstitial cells of Cajal (ICC)-like cells (AIL cells) with a multipolar, irregular, elongated shape and with numerous thin (often less than 1 microm), sometimes branching, processes with lengths up to approximately 60 microm were isolated enzymatically from 1st to 7th order branches of guinea-pig mesenteric artery. Some of the processes of AIL cells were growing (average speed approximately 0.15 microm min-1) and their growth was blocked by 10 microM latrunculin B, an inhibitor of actin polymerisation. Staining with BODIPY phalloidin, a fluorescent dye selective for F-actin, showed the presence of F-actin in the processes of AIL cells. Voltage clamp of single AIL cells revealed an inward current that was four times more dense than in myocytes and was abolished by 10 microM nicardipine, and an outward current carried exclusively by potassium ions that was reduced by 1 mM 4-aminopyridine and/or 100 nM iberiotoxin but unaffected by 10 nM dendrotoxin-K. Imaging of intracellular ionised calcium with fluo-4 using a laser scanning confocal microscope showed local or global calcium transients lasting several seconds in approximately 28 % of AIL cells. When membrane current was recorded simultaneously, the calcium transients were found to correspond to long-lasting transient outward currents, which occurred at potentials positive to -40 mV. Unlike myocytes, AIL cells did not contract in response to 1 mM caffeine or 5 microM noradrenaline, although they responded with a [Ca2+]i increase. The segments of intact arteries did not stain for c-kit, a marker of ICCs. Single AIL cells stained positive for vimentin, desmin and smooth muscle myosin. The presence of ICC-like cells is demonstrated for the first time in the media of resistance arteries.

Isolation of bone marrow stromal cell-derived smooth muscle cells by a human SM22alpha promoter: in vitro differentiation of putative smooth muscle progenitor cells of bone marrow.

BACKGROUND: Bone marrow stromal cells (BMSCs) have many characteristics of mesenchymal stem cells that can differentiate into smooth muscle cells (SMCs). However, there have been few studies closely following the cell development of smooth muscle lineage among BMSCs. METHODS AND RESULTS: To investigate the possible existence of a cell population committed to the SMC lineage among bone marrow adhesion cells, we tried to detect and follow the in vitro differentiation of such a cell type by using a promoter-sorting method with a human SM22alpha promoter (-480 bp)/green fluorescent protein (GFP) construct. The construct was transfected to adhesion cells that appeared 5 days after the seeding of mononuclear cells from bone marrow. GFP was first detectable 5 days after the transfection in a cell population [Ad(G) cells], which expressed PDGF-beta but neither mature (calponin) nor immature (SMemb) SMC-specific proteins at that time. However, the cells were eventually grown into individual clones that expressed SMC-specific proteins (alpha-smooth muscle actin, calponin, and SM-1), suggesting that Ad(G) cells have partly at least progenitor properties. Because early studies have reported that PDGF-beta signaling plays pivotal roles in the differentiation of mesenchymal smooth muscle progenitor cells, Ad(G) cells might be putative mesenchymal smooth muscle progenitors expressing PDGF-beta. CONCLUSIONS: We demonstrated the presence of a cell population fated to become SMCs and followed their differentiation into SMCs among BMSCs.

Immunohistochemical distinction of invasive from noninvasive breast lesions: a comparative study of p63 versus calponin and smooth muscle myosin heavy chain.

Identification of myoepithelial cells using antibodies to cytoskeletal proteins, such as smooth muscle myosin heavy chain (SMM-HC) and calponin, can play an important role in distinguishing invasive carcinoma from its histologic mimics. However, antibodies to these proteins may also cross-react with stromal myofibroblasts and vascular smooth muscle cells. It has recently been demonstrated that myoepithelial cells express the nuclear protein, p63, a member of the p53 gene family. We compared the patterns of reactivity of antibodies with p63, calponin, and SMM-HC on 85 breast lesions, including 11 cases of sclerosing adenosis, 33 cases of ductal carcinoma in situ, including 10 that showed microinvasion, 6 cases of lobular carcinoma in situ, and 35 cases of infiltrating ductal carcinoma. All three antibodies were positive on the vast majority of myoepithelial cells in all cases. A small minority of cases showed focal gaps in the revealed myoepithelial cell layer, reflected in discontinuous positive immunostaining around noninvasive epithelial nests (including ductal carcinoma in situ). No case showed p63 expression by myofibroblasts or vascular smooth muscle cells, whereas myofibroblasts expressed, in 8% and 76% of cases, SMM-HC and calponin, respectively. Although no tumor cell reactivity was noted with antibodies to calponin or SMM-HC, tumor cells in 11% of cases showed at least focal p63 expression. And although antibodies to p63 offer excellent sensitivity and increased specificity for myoepithelial detection relative to antibodies to calponin and SMM-HC, they have the following diagnostic limitations: 1) they occasionally demonstrate an apparently discontinuous myoepithelial layer, particularly around ductal carcinoma in situ, and 2) they react with a small but significant subset of breast carcinoma tumor cells. p63 may represent a myoepithelial marker that can complement or replace SMM-HC and/or calponin in the analysis of difficult breast lesions.