Showing papers in "Applied Immunohistochemistry & Molecular Morphology in 2010"

Comparison of thyroid transcription factor-1 expression by 2 monoclonal antibodies in pulmonary and nonpulmonary primary tumors.

Abstract: Thyroid transcription factor-1 (TTF-1) is a transcription factor that plays a role in the development and physiology of the thyroid and lungs. Expression of TTF-1 is used as a marker of lung and thyroid clinically. Commercially available clones of TTF-1 monoclonal antibodies, 8G7G3/1 and SPT24, have been reported to have different sensitivities for the detection of neoplasms of different origins. Although they are used extensively in daily practice, a comprehensive comparative study of these antibodies in a wide variety of neoplasms is lacking. We examined TTF-1 expression in primary tumors of the lung, prostrate, pancreas, stomach, salivary glands, breast, bladder, colon, and squamous cell carcinoma of the head and neck and compared the results obtained with both TTF-1 clones. The SPT24 clone detected more primary lung tumors of all histologic subtypes. Importantly, the SPT24 clone detected a significantly higher number of squamous cell carcinomas and carcinoid tumors of the lung. Among nonpulmonary primary tumors, a significant number of invasive urothelial carcinoma of the bladder (5.1%) was TTF-1 positive. In addition, a small proportion of prostate (1.2%), stomach (0.9%), salivary gland (1.8%), and colon (2.5%) carcinomas were positive with both clones. Of note, both clones stained the same nonpulmonary tumors with similar intensity and distribution. Carcinomas of the pancreas, breast, and squamous cell carcinomas of the head and neck were negative with both clones. In summary, the SPT24 clone detected a higher number of pulmonary non-small cell tumors of all histologic subtypes whereas both clones stained a similar proportion of nonpulmonary tumors.

The use of Cytokeratin 19 (CK19) immunohistochemistry in lesions of the pancreas, gastrointestinal tract, and liver.

Abstract: Cytokeratin immunostaining forms the bedrock of the immunohistochemical evaluation of tumors. Cytokeratin 19 (CK19) belongs to a family of keratins, which are normally expressed in the lining of the gastroenteropancreatic and hepatobiliary tracts. CK19 immunohistochemistry has been used successfully in thyroid tumors to recognize papillary carcinomas for some time. However, its use in the pancreas, liver, and gastrointestinal tract (GIT) has only recently come to the fore. The purpose of this review is to look at the use of CK19 immunohistochemistry in tumors occurring at these sites. CK19 has been shown to be an independent prognostic factor for pancreatic neuroendocrine tumors, especially the insulin-negative tumors. CK19 positive tumors are associated with poor outcome irrespective of the established pathologic parameters such as size, mitoses, lymphovascular invasion, and necrosis. It is recommended that CK19 be part of the immunohistochemical panel in the work-up of pancreatic endocrine tumors. CK19 is positive in the most of neuroendocrine tumors occurring in the rest of the GIT, except rectal tumors, which are negative. In the liver, CK19 is of prognostic value in hepatocellular carcinomas and is of use in distinguishing cholangiocarcinoma from hepatocellular carcinomas. It can also be used to highlight native ductules in the liver and helps separate conditions such as focal nodular hyperplasia from hepatic adenoma. The vast majority of adenocarcinomas in the GIT and pancreas are CK19 positive.

Intratumoral heterogeneity of immunohistochemical marker expression in breast carcinoma: a tissue microarray-based study.

Abstract: Core needle biopsies of breast carcinomas provide diagnostic, prognostic, and predictive information before neoadjuvant therapy. Possible intratumoral heterogeneity of biomarker expression questions the validity of core needle biopsy interpretation in small biopsy specimens. Using tissue microarray (TMA) technology, we studied intratumoral hetero- geneity of 7 immunomarkers. Five TMAs were constructed from 44 breast carcinomas and 5 normal breast tissues, each represented by 1-mm cores in triplicate from each of 3 foci. TMAs were immunostained for monoclonal estrogen receptor (ER), monoclonal progesterone receptor (PR), polyclonal human epidermal growth factor receptor 2 (HER2), monoclonal E-cadherin (E-cad), monoclonal epidermal growth factor receptor (EGFR), monoclonal p53, and monoclonal MIB-1. Expression was quantified visually by light microscopy and by image cytometry as intensity, percentage of cells positive, and score. Using intraclass correlation coefficient, heterogeneity in the expression of the immunomarkers within subjects was compared with the overall variance. Intratumoral heterogeneity was seen with 5 immunomarkers: ER, PR, HER2, p53, and MIB-1. E-cad and EGFR failed to show intratumoral hetero- geneity. Intratumoral heterogeneity in ER, PR, HER2, p53, and MIB-1 indicates their problematic interpretation in small biopsy specimens as indicative of the status of the entire tumor. A negative result does not exclude the expression of these markers in the remainder of the tumor. E-cad (positive in ductal carcinomas) and EGFR lacked heterogeneity.

Sox2 Expression in Pulmonary Non-small Cell and Neuroendocrine Carcinomas

Abstract: Sox2 is a transcription factor that regulates embryonic stem cell pluripotency and drives commitment of airway precursor cells to basal-type and neuroendocrine cells in the developing lung. In cancer, Sox2 has been associated with a "stemness" phenotype that predicts for poor outcomes. We examined Sox2 expression in pulmonary neoplasms with respect to tumor type and differentiation, in comparison with conventional markers. Immunohistochemistry for Sox2, p63, CK5/6, and thyroid transcription factor-1 was performed on 121 tumors, including 34 adenocarcinomas (ACA), 32 squamous cell carcinomas (SCC), 14 typical carcinoids, 12 atypical carcinoids, 14 large cell neuroendocrine carcinomas, and 15 small cell carcinomas. Sox2 was strongly, diffusely expressed in 91% of SCC and 21% of ACA. Ninety-four percent of SCC coexpressed Sox2 and p63; 1 case was only focally positive for p63 but diffusely positive for Sox2. Twenty-nine percent of ACA were at least focally p63+; 12% were Sox2+/p63+. All of the ACA diffusely positive for Sox2 were p63 negative. Among non-small cell lung carcinoma overall, there was a significant association between Sox2+/p63- expression and high-grade histology (P = 0.02). Strong Sox2 expression was detected in 23% of low-grade and 72% of high-grade neuroendocrine carcinomas (P = 0.0004). Sox2 is highly expressed in concert with p63 in most SCC, but may also influence tumor differentiation in both non-small cell lung carcinomas and pulmonary neuroendocrine tumors.

DOG1 for the diagnosis of gastrointestinal stromal tumor (GIST): Comparison between 2 different antibodies.

Abstract: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. Discovered on GIST-1 (DOG1) is a recently described protein expressed in GISTs irrespective of mutation status. The aim of this study was to investigate the immunohistochemical expression of DOG1 using 2 different monoclonal antibodies (DOG1.1 and the commercially available K9 antibody) in 668 GIST cases and to compare the results with the expression of KIT. DOG1 and KIT expression also were studied in most human normal tissues and several nonmesenchymal and mesenchymal tumors other than GIST. KIT was expressed in 643 (96.3%) GISTs. DOG1.1 and K9 were positive in 538 (80.5%) and 642 (96.1%) GIST cases, respectively. In 25 (3.7%) KIT-negative GIST cases, DOG1 was expressed in 5 (20.0%) and 19 (76.0%) using DOG1.1 and K9 antibodies, respectively. Only 0.9% of GISTs were negative for KIT, DOG1.1, and K9. Most normal human tissues did not reveal KIT and DOG1 expression. DOG1.1 was positive in only 2 of 57 synovial sarcomas and 1 of 61 soft tissue leiomyosarcomas. K9 was positive in 5 of 57 synovial sarcomas, 1 of 14 angiosarcomas, 1 of 61 soft tissue leiomyosarcomas, 3 of 4 adenoid cystic carcinomas of the head and neck, and in myoepithelial cells of 9 of 11 fibroadenomas of the breast. In conclusion, the commercially available K9 is of great utility for the diagnosis of most KIT-negative GISTs, and the combination of both KIT and K9 antibody in a panel of immunohistochemistry can define the diagnosis of GIST in more than 99% of cases.

Determining tissue of origin for metastatic cancers: meta-analysis and literature review of immunohistochemistry performance.

Abstract: BackgroundPathologists use various panels of immunohistochemical (IHC) stains to identify the site of tissue of origin for metastatic tumors, particularly poorly or undifferentiated cancers of unknown or uncertain origin. Although clinicians believe that immunostains contribute greatly to determinin

PAX-2 expression in non-neoplastic, primary neoplastic, and metastatic neoplastic tissue: A comprehensive immunohistochemical study.

Abstract: PAX-2 is a transcription factor that controls the development of the kidney, organs deriving from the mesonephric (Wolffian) duct, and those related to the Mullerian duct. Although PAX-2 is shown to be a sensitive marker for tumors derived from these organs, but whether it is specific, that is, whether other tumor types also express PAX-2, has not been systematically evaluated in either primary or metastatic tumors. Tissue sections from 937 normal or reactive tissue samples, 759 primary neoplasms, and 332 metastatic neoplasms were submitted to PAX-2 immunostain. Among the non-neoplastic tissue, PAX-2 was expressed in glomerular parietal epithelial cells, renal collecting duct cells, atrophic renal tubular cells, epithelial cells of ovarian surface, fallopian tube, endocervix, endometrium, seminal vesicle, and lymphocytes. Among the primary neoplasms, PAX-2 was noted in 104/122 (85%) of renal cell carcinoma, 31/95 carcinomas of Mullerian origin, 17/17 (100%) lymphomas, 4/4 (100%) nephrogenic adenomas, and 1/16 (6%) benign parathyroid tumors, but was negative in 477 other tumors. Among the metastatic tumors, PAX-2 was noted in 70/95 (74%) metastatic renal cell carcinomas, 14/20 (70%) metastatic tumors of Mullerian origin, 1/20 (5%) metastatic colon carcinoma of lymph nodes, 1/62 (2%) metastatic breast carcinoma of lymph nodes, but was not seen in the remaining 247 metastatic tumors. PAX-2 expression in non-neoplastic mature tissue is limited to the organs whose embryonic development depends on this transcription factor. PAX-2 is a sensitive and specific marker for tumors of renal or Mullerian origin in both primary and metastatic contexts.

MUM1/IRF4: A Review.

Abstract: MUM1/IRF4 protein is a member of the interferon regulatory factor (IRF) family of transcriptional factors initially described as downstream regulators of interferon signaling. The quantity of this factor varies within the hematopoietic system in a lineage and stage-specific way. It is considered to be a key regulator of several steps in lymphoid, myeloid, and dendritic cell differentiation and maturation. MUM1/IRF4 expression is observed in many lymphoid and myeloid malignancies, and may be a promising target for the treatment of some of these neoplasms. We reviewed the literature on MUM1/IRF4, with emphasis on the pathologic aspects of this marker in reactive and malignant hematologic and nonhematologic conditions.

A High Number of CD8+ T Cells Infiltrated in NSCLC Tissues is Associated With a Favorable Prognosis

Abstract: We investigated the number of intratumoral tumor-infiltrating lymphocytes (TILs), including CD4(+), CD8(+), and CD25(+) T cells, in nonsmall cell lung cancers (NSCLC) and their correlation with patient survival time. Tumor specimens from 30 NSCLC patients were consecutively obtained during surgery. Patient survival status was monitored. Based on survival time, patients were divided into 2 groups: 5-year survival group and 5-year nonsurvival group. CD4(+), CD8(+), and CD25(+) T cells that infiltrated tumors were detected and counted by immunohistochemistry. Patients with a lower number of TILs and CD8(+) T cells showed significantly shorter survival time compared with those with a higher number (P 0.05). These data demonstrate that high numbers of CD8(+) T cells among TILs is a strong indicator for a favorable clinical outcome.

Utility of Flow Cytometry Immunophenotyping in Fine-needle Aspirate Cytologic Diagnosis of Non-Hodgkin Lymphoma A Series of 252 Cases and Review of the Literature

Abstract: Flow cytometry (FC) immunophenotyping of fine-needle aspiration (FNA) has been reported to be useful in the diagnosis of non-Hodgkin lymphomas (NHL) The authors reviewed their 5-year experience to assess the ability that FC has in improving the diagnostic capacity of cytomorphology in the diagnosis and subclassification of NHL according to the World Health Organization's classification FC was performed on 252 FNA specimens These included 123 cases of NHL (89 primary and 34 recurrent lymphomas) The FC immunophenotyping included CD3, CD4, CD8, CD10, CD19, CD20, CD45, and kappa/lambda antibodies combinations in the screening panel and additional panels for B or T lineage in the presence of positivity for lymphoma after the screening An immunologic diagnosis was obtained by FC in 90% (111/123) of cases identified as NHL FC was able to improve the total number of NHL detected in 8 cases where cytomorphology had failed to do so In 7% (9/123) of cases, FC failed to formulate a diagnostic hypothesis owing to the sample inadequacy; 2 cases (2%) were not identified as lymphomas by FC (1 of them considered only "suggestive" also by cytomorphology); 1 case was not identified neither by FC, nor by cytomorphology In cases having a histologic follow-up, levels of diagnostic sensitivity and specificity of the combination cytomorphology/FC were 97% and 94%, respectively FC applied to FNA enhanced the diagnostic potential of cytologic diagnosis and subclassification of NHL, thus avoiding the need for invasive surgical biopsies in many cases

Immunohistochemical expression of estrogen receptor in adenocarcinomas of the lung: the antibody factor.

Abstract: BackgroundImmunohistochemistry for estrogen receptor may be used to distinguish metastatic breast cancers from adenocarcinomas of other sites, including those of the lung. The estrogen receptor exists as 2 subtypes, α and β. Estrogen receptor α is the predominant subtype expressed by more than two-t

Protocol for PTEN expression by immunohistochemistry in formalin-fixed paraffin-embedded human breast carcinoma.

Abstract: The PI3K/PTEN pathway plays a major role in carcinogenesis. Dysregulation of this pathway occurs frequently in breast cancer, and loss of PTEN expression is emerging as a potentially important mechanism of resistance to the widely used anti-HER2 therapy, trastuzumab. However, assays for loss of PTEN expression have suffered from lack of consistency. Here, we describe an automated and reliable protocol for PTEN protein expression by immunohistochemistry in formalin-fixed paraffin-embedded tissue that can be easily incorporated into clinical trials.

Clinical utility of immunohistochemistry in the diagnoses of urinary bladder neoplasia.

Abstract: Urothelial carcinomas demonstrate diverse morphologic and immunologic features that frequently lead to diagnostic challenges. Recent advances have identified a number of immunohistochemical stains that, when used in the context of a panel, can be a valuable tool in properly classifying primary urothelial carcinoma and carcinomas secondarily involving the urinary bladder. In addition, new biomarkers prove helpful in the staging of bladder carcinoma. In this article, we review the clinical utility of immunohistochemistry in a series of diagnostic scenarios, including flat urothelial lesions with atypia, rare variants of urothelial carcinoma, primary adenocarcinoma versus secondary colorectal tumors, distinguishing prostate from urothelial carcinoma, and the utility of smoothelin in staging bladder carcinoma. Emphasis is placed on panels of commonly used biomarkers to establish diagnoses.

Estrogen receptor, progesterone receptor, and glucocorticoid receptor expression in normal breast tissue, breast in situ carcinoma, and invasive breast cancer.

Abstract: Glucocorticoids (GCs) are used in cancer treatment to induce programmed cell death in transformed cells of the hematopoietic system and to lessen side effects. Moreover, GCs have been described not only as inhibitors of some chemotherapy or radiation-induced apoptosis, but also as inhibitors of cancer progression by down-regulation or up-regulation of different gene expressions. Recently, it has been suggested that GCs can attenuate estrogen responses through induction of expression and activity of the sulfotransferase. The presence or absence of glucocorticoid receptor (GR) in normal and abnormal breast tissue is thus interesting, and the aim of this study was to analyze the expression of GR during the progression of breast tissue. We tested by immunohistochemistry the expression status of estrogen receptor (ER), progesterone receptor (PR), and GR in normal breast parenchyma (n=49), ductal intraepithelial neoplasia (DIN) 1a (n=9), DIN 1b-1c (n=15), DIN 2-3 (n=21), and invasive breast carcinoma (n=39). The evaluation of GR expression was made by using the Allred score. All the normal parenchyma, DIN 1a, DIN 1b, and DIN 1c were ER-positive (ER+) and PR-positive (PR+). Seventeen of 21 DIN 2-3 and 30 of 39 invasive carcinomas were ER+/PR+. The other samples were ER-negative (ER-) and PR-negative (PR-). Moreover, all the ER-/PR- samples were GR-negative. Interestingly, we found a significant correlation between the histologic grade and the GR-negative tumors, and a percentage of positive patients presented with nuclear immunoreaction to GR, which decreases significantly with tumor histologic grade. Understanding the role of GCs in breast carcinoma is thus essential before continuing the widespread use of GCs combined with antineoplastic drugs or agents in the clinical management of women with breast cancer.

Breast cancer molecular class ERBB2: preponderance of tumors with apocrine differentiation and expression of basal phenotype markers CK5, CK5/6, and EGFR.

Abstract: This is a study of 205 consecutive invasive breast carcinomas. The principal aim was to identify morphologic and immunohistochemical features of tumors that belong to the molecular class ERBB2. The invasive breast carcinomas were classified using semiquantitative immunohistochemical results for estrogen receptor (ER), progesterone receptor, (PR), and HER2 into following classes: Luminal A (strong ER+, HER2 negative), Luminal B (weak to moderate ER/PR+, HER2 negative), Triple Negative (TN; ER/PR negative, HER2 negative), ERBB2 (ER/PR negative, HER2 positive), Luminal A-HER2 Hybrid (strong ER+, HER2 positive), Luminal B-HER2 Hybrid (weak to moderate ER/PR+, HER2 positive). Of the 205 tumors, 113 (55%) were classified as Luminal A, 34 (17%) as Luminal B, 32 (15%) as TN, 8 (4%) as ERBB2, 10 (5%) as Luminal A-HER2 Hybrid, and 8 (4%) as Luminal B-HER2 Hybrid. Majority of the ERBB2 tumors were high grade as expected, with average Nottingham score of 8. Moderate lymphoid infiltrate (constituting 25% to 50% of the tumor) was seen in 5 of 8 (63%) cases and necrosis in 3 of 8 (38%) cases. The most striking morphologic feature associated with ERBB2 tumors was the presence of apocrine differentiation seen in 7 of 8 (88%) cases. CK5 immunoreactivity was seen in 5 of 8 cases (63%). Epidermal growth factor receptor staining with 2+ or 3+ score was also seen in 5 cases (63%). Due to low prevalence of ERBB2 tumors, additional data set of 191 cases enriched in ERBB2 and TN tumors was used for confirmation of morphologic findings. We conclude that tumors with apocrine differentiation are most often of ERBB2 type. ERBB2 tumors demonstrate some features classically ascribed to TN basal-like tumors. Epidermal growth factor receptor overexpression in ERBB2 tumors may have additional predictive value.

Novel monoclonal antibodies against the proximal (carboxy-terminal) portions of MUC16.

Abstract: The CA125 antigen, recognized by the OC125 antibody, is a tissue-specific circulating antigen expressed in ovarian cancer. The CA125 antigen is encoded by the MUC16 gene cloned by Yin and Lloyd. The full-length gene describes a complex tethered mucin protein present primarily in a variety of gynecologic tissues, especially neoplasms. OC125 and other related antibodies react with glycosylation-dependent antigens present exclusively in the cleaved portion of the molecule. These antibodies are not useful as screening tools, nor can they detect the proximal residual MUC16 protein fragment after cleavage. This has limited its diagnostic and therapeutic applications. Using synthetic peptides, we raised novel-specific antibodies to the carboxy-terminal portion of MUC16 retained by the cell proximal to the putative cleavage site. These antibodies were characterized using fluorescence-activated cell-sorting analysis, enzyme-linked immunoassay, Western blot analysis, and immunohistochemistry. Each of the selected monoclonal antibodies was reactive against recombinant GST-ΔMUC16 protein and the MUC16-transfected SKOV3 cell line. Three antibodies, 4H11, 9C9, and 4A5 antibodies showed high affinities by Western blot analysis and saturation-binding studies of transfected-SKOV3 cells and displayed antibody internalization. Immunohistochemical positivity with novel antibody 4H11 was similar to OC125 but with important differences, including diffuse positivity in lobular breast cancer and a small percentage of OC125-negative ovarian carcinomas that showed intense and diffuse 4H11. Development of such antibodies may be useful for the characterization of MUC16 biology and allow for future studies in targeted therapy and diagnostics.

FLI-1 distinguishes Ewing sarcoma from small cell osteosarcoma and mesenchymal chondrosarcoma.

Abstract: Small cell osteosarcoma and mesenchymal chondrosarcoma are 2 primary bone tumors with a small round blue cell component, which can mimic the appearance of Ewing sarcoma. Distinguishing these tumors from each other on biopsy material is important clinically, as optimal therapy differs according to the tumor type. However, separating these entities on morphology alone can be challenging. FLI-1 has been described to be a useful marker for Ewing sarcoma, particularly when hematolymphoid markers are negative. In small cell osteosarcoma and mesenchymal chondrosarcoma, the FLI-1 staining pattern has not been adequately characterized. Using a monoclonal FLI-1 antibody, nuclear immunoreactivity in tumor cells was evaluated in 10 small cell osteosarcomas, 10 mesenchymal chondrosarcomas, and 8 Ewing sarcomas, together with a number of other small, round, blue cell tumors. None of the small cell osteosarcomas or mesenchymal chondrosarcomas exhibited FLI-1 staining in the tumor cells, in contrast to the positive nuclear FLI-1 staining in the stromal endothelial cells. In comparison, 6 of the 8 Ewing sarcomas showed moderate-to-strong nuclear FLI-1 staining of the tumor cells in addition to strong staining of the stromal endothelial cell nuclei. With the exception of lymphoblastic lymphomas, FLI-1 positivity was not seen in the other small round blue cell tumors examined. These findings show that, in contrast to Ewing sarcoma, small cell osteosarcoma and mesenchymal chondrosarcoma lack FLI-1 immunoreactivity. FLI-1 is therefore useful in the differential diagnosis of small round blue cell tumors of the bone.

BCL6, MUM1, and CD10 expression in mantle cell lymphoma.

Abstract: Mantle cell lymphoma (MCL) characteristically express CD20, CD5, and cyclin-D1, carries the translocation t(11;14) (q13;q32) and typically has no expression of germinal center cell markers. So-called aberrant phenotypes such as CD5 negative and cyclin-D1-negative MCL have been described. Also few cases with CD10 and/or BCL-6 protein expression have been reported. We analyzed 127 MCL looking for the frequency of aberrant immunophenotype, CD10, BCL-6, and MUM1 expression. All cases were CD20 and cyclin-D1 positive, 96% expressed CD5, and 98% showed the t(11;14). BCL-6 expression was observed in 12% of the cases and MUM1 in 35%. No one case showed CD10 positivity in 30% or more neoplastic cells. Only 3 cases showed 10% to 20% of tumoral cells positive for CD10. MUM1 expression was observed in 67% of the BCL-6 positive cases. Thirty-two percent of the cases showed a MUM1+/BCL-6-/CD10- phenotype and 56% had a triple-negative-pattern. Aberrant phenotype is infrequent but not rare, and does not rule out a diagnosis of MCL in an otherwise typical case.

The Role of Cytotoxic and Regulatory T-Cells in Relapsed/Refractory Hodgkin Lymphoma

Abstract: Recent data suggests the presence of cytotoxic (TIA-1 and granzyme B+) and regulatory T-cells (FOXP3+) in classical Hodgkin lymphoma (cHL) tissues has been shown to correlate with poor overall survival in mainly diagnostic biopsies. By tissue microarray analyses, we extend this observation to a cohort of relapsed/refractory cHL tissue biopsies and analyze immunohistochemical expression of FOXP3, TIA-1, and granzyme B in the inflammatory background and the tumor microenvironment. High expression of TIA-1 (>50%) correlated with poor overall survival (P 50%) and low FOXP3 (<25%) correlated with poor overall survival (P<0.0001). Expression of cytotoxic and regulatory T-cells shows prognostic significance in the relapsed/refractory clinical setting of cHL.

Podoplanin Expression in Basal and Myoepithelial Cells: Utility and Potential Pitfalls

Abstract: Podoplanin, D2-40, is often used for highlighting lymphatics. However, it has been described in a variety of normal and neoplastic tissues. We evaluated the expression of D2-40 in breast, salivary gland, skin, and mucosa, all organs rich in myoepithelial cells and basal cells. This study assessed the utility of using D2-40 to highlight basal/myoepithelial cells and to identify potential pitfalls in misinterpretation of lymphatic invasion. Our results showed that myoepithelial cells in breast and salivary gland and basal cells in prostate consistently demonstrate D2-40 immunoreactivity, but typically less intensely than lymphatics. Cutaneous and mucosal-based basal cells also have D2-40 expression, but often in a patchy, focal manner. In addition, many salivary gland tumors and squamous cell carcinomas had strong D2-40 expression, sometimes making distinction of lymphatics versus tumor edge staining difficult. D2-40 is excellent for assessing lymphatic invasion in breast, prostate, and cervix as long as the pathologist is aware of the expression in myoepithelial cells/basal cells to avoid misinterpretation of ductal carcinoma in situ or normal prostate glands or tumor stroma retraction as tumor lymphatic invasion. Given the patchy positivity in basal cells of skin and mucosa and the reactivity in squamous cell carcinoma, D2-40 was not helpful in assessing for microinvasion of squamous cell carcinoma.

E-cadherin and β-catenin Loss of Expression Related to Bone Metastasis in Prostate Cancer

Abstract: OBJECTIVES E-cadherin and beta-catenin are adhesion molecules responsible for the maintenance of normal epithelial cell phenotype. A disturbance in epithelial cell adhesion, which leads to a more invasive and metastatic phenotype, is a hallmark of tumor progression. Several immunohistochemical studies have reported a strong correlation between loss of their expression to higher stage and grade in prostate carcinoma, but their influence in metastatic process is not yet known. The aim of this study is to verify the role of adhesion molecules in the progression of prostate cancer (PC), assessing the expression of E-cadherin and beta-catenin in bone metastasis. MATERIALS AND METHODS Twenty-eight bone metastases of prostate carcinoma were submitted to immunohistochemistry analysis for E-cadherin and beta-catenin expression. In 6 patients, we were able to assess the expression of the adhesion molecules in the primary tumors and their respective metastases. The definition of normal expression for both antibodies was strong and diffuse expression in more than 70% of tumor cells. RESULTS In bone metastases, there was loss of expression of E-cadherin and beta-catenin in 86% and 82%, respectively. Among the primary tumors, E-cadherin and beta-catenin expression was normal in 83% and 50% cases, respectively. Considering the 6 patients with paired primary and bone metastasis, we found loss of expression for both E-cadherin and beta-catenin in most of the cases. CONCLUSIONS Comparing primary PC and its metastasis, we showed persistent loss of E-cadherin and beta-catenin expression. This phenomenon may be related to metastatic potential in PC, because we have shown underexpression for E-cadherin and beta-catenin in 86% and 82% of bone metastases.

Expression of CD133 in synovial sarcoma

Abstract: The development of synovial sarcoma, a translocationally defined soft tissue tumor of unknown histogenesis with considerable resistance to systemic therapy and a poor prognosis, may involve cancer stem-like cells. Recent studies suggest that synovial sarcoma arises from a primitive progenitor-type cell and have shown that synovial sarcomas contain subpopulations with enhanced tumorigenic potential; however, little is known about cancer stem-like cells in synovial sarcoma. Histologic and gene expression studies have reported features of synovial sarcoma that are reminiscent of neural development, suggesting that a neural cancer stem-like cell marker, such as CD133, may mark cancer stem-like cells in synovial sarcoma, allowing for further characterization. Here, the immunohistochemical expression of CD133 in primary synovial sarcoma tumor tissue and synovial sarcoma cell lines is determined. Subpopulations of CD133 expressing cells are present in all primary synovial sarcomas (5/5) and synovial sarcoma cell lines (3/3) examined. Histologically, CD133 positive cells are dispersed and seem to have dendritic processes. This study demonstrates the presence of CD133 expressing cells in synovial sarcoma for the first time and validates 3 synovial sarcoma cell lines as models for further study of the CD133+ subpopulation. The relationship between CD133 expression and cancer stem-like cells suggests that CD133 expressing synovial sarcoma cells may represent cancer stem-like cells in synovial sarcoma, which has significant implications for understanding the pathogenesis of this tumor and developing effective therapies.

Expression of Thyroid Transcription Factor-1 in Colorectal Carcinoma

Abstract: Thyroid transcription factor-1 (TTF-1) is a member of the homeodomain transcription family expressed in epithelial cells of the thyroid and lung. Although nuclear TTF-1 is generally considered a specific marker for lung and thyroid neoplasms, it has been reported to be positive in other types of tumors including colorectal carcinoma (CRC). During metastatic adenocarcinoma workup for patients who had a history of CRC, we identified 4 positive TTF-1 cases using clone 8G7G3/1. Three of the 4 corresponding primary carcinomas were also positive for TTF-1. Therefore, we sought to retrospectively investigate the expression of TTF-1 in 100 CRC cases constructed in tissue microarray blocks and whole tissue sections of the 4 primary tumors corresponding to the 4 positive metastases. In tissue microarray cases, all cases had negative nuclear staining. Our results suggest that during immunohistochemical workup for adenocarcinoma, especially when the differential diagnosis includes the lung and CRC, TTF-1 results should be interpreted with caution as a small percentage of CRC expresses this marker. Positive nuclear TTF-1 in a metastatic carcinoma does not rule out CRC primary. Clinicopathologic correlation, tumor morphology, and a panel of immunohistochemical markers are essential to render the correct diagnosis.

The mTOR pathway is frequently activated in pancreatic ductal adenocarcinoma and chronic pancreatitis.

Abstract: INTRODUCTION Mammalian target of rapamycin (mTOR) is a serine/threonine kinase critical to cell growth and proliferation through its effects on protein translation. Activation of the phosphatidylinositol 3-kinase/Akt/mTOR pathway has been described in various tumor types. Earlier studies have demonstrated loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) function in some pancreatic ductal adenocarcinomas (PDAs). We performed immunohistochemistry for PTEN and p-RPS6 (major downstream mTOR effector) in a group of PDAs. An assessment of chronic pancreatitis (CP) and normal pancreas (NL) was performed in parallel. MATERIALS AND METHODS Tissue microarrays were constructed from 49 PDA, 27 CP, and 12 NL. Cases were scored as follows: PTEN (intact: ≥ 5% staining and lost: < 5%) and p-RPS6 (0, 1+: modest intensity in ≥ 5% of cells and 2+: strong intensity ≥ 5% of cells). RESULTS Forty-one percent of PDAs demonstrated loss of PTEN, and 75% demonstrated p-RPS6 immunoreactivity (1+ in 22 and 2+ in 3). PTEN was uniformly intact in NL and CP. Although p-RPS6 immunoreactivity was only noted in 1 NL control (8%), 1+ positivity was observed in 62% of CP. CONCLUSIONS mTOR pathway activation, as evidenced by p-RPS6 immunoreactivity, is frequent in PDA. p-RPS6 expression was also frequent in CP, highlighting the importance of this pathway in both neoplastic and inflammatory processes. Given evidence of pathway activation and the existence of specific anti-mTOR therapeutics, mTOR represents a logical target for directed biologic therapy.

Clear cell meningioma: a clinicopathologic study of 18 tumors and examination of the use of CD10, CA9, and RCC antibodies to distinguish between clear cell meningioma and metastatic clear cell renal cell carcinoma.

Abstract: Clear cell meningiomas (CCM) can be difficult to distinguish from metastatic clear cell renal cell carcinomas by standard light microscopy. Distinction is important in deciding patient management and establishing prognosis. The purpose of this study is to evaluate the use of immunomarkers CA9, CD10, and RCC in differentiating between CCM and clear cell renal cell carcinoma. The study retrospectively reviewed the clinicopathologic features of 18 patients with CCM (9 females, 9 males; age range at the time of surgery 16 to 86 y) including immunostaining results with antibodies to CA9, CD10, and RCC. Immunostaining results were compared with those found in 26 cases of clear cell renal cell carcinoma. The most common sites of origin for the CCM included the meninges overlying the frontal lobe (n=7), cavernous sinus (n=3), and cerebellopontine angle/posterior fossa (n=2). All tumors had at least a 10% clear cell component (mean 41%). All tumors showed a sheet-like growth pattern. Other commonly observed morphologic features included increased cellularity (n=12), nucleolation (n=8), small cell change (n=6), microcalcifications (n=5), and necrosis (n=5). A mean of 1.9 mitotic figures per 10 high-power fields and a mean Ki-67 labeling index of 12.1% were observed. Seven tumors (38.9%) showed CA9 immunoreactivity, 5 tumors (27.8%) CD10 staining, and 0 cases showed RCC staining. Immunostaining results observed in the clear cell renal cell carcinoma group included 93.8% CA9 staining (15/16 cases evaluated), 100% CD10 staining (15/15 cases), and 36.4% RCC staining (4/11 cases). Follow-up was available in 16 CCM patients (mean follow-up of 58.9 mo); 10 patients (62.5%) developed at least 1 recurrence requiring surgical intervention. In conclusion, meningiomas with at least a 10% clear cell component tend to behave in a more aggressive fashion with increased risk of recurrence. Immunohistochemical staining with antibodies to CA9, CD10, and RCC are potentially useful in differentiating CCM from metastatic renal cell carcinoma. In the majority of cases in which immunostaining was observed in meningiomas, staining was focal (involving <5% of neoplastic cells) in comparison with CA9 and CA10 immunostaining in renal cell carcinomas in which more than 50% of tumor cells stained the majority of cases.

A tissue microarray study of osteosarcoma: histopathologic and immunohistochemical validation of xenotransplanted tumors as preclinical models.

Abstract: Background: Osteosarcomas (OS) are aggressive neoplasms with a wide range of morphologic patterns. Materials and Methods: OS cases (primary and xenotransplanted) with para!n blocks available were collected and included in tissue microarrays (TMAs). A morphologic evaluation including the di"erent passages in mice was carried out according to the new WHO criteria. In addition, TMAs were analyzed with a wide panel of immunohistochemical (IHC) markers (osteonectin, osteocalcin,cytokeratin, S100, Sox-9, Ki-67, Bcl-2, p53, p16, survivin, CD99, and caveolin-1).

Concordance between semiquantitative immunohistochemical assay and oncotype DX RT-PCR assay for estrogen and progesterone receptors.

Abstract: Estrogen receptor (ER) status is a strong predictor of response to hormonal therapy in women with breast cancer. Not only presence of ER, but also level of expression has been shown to correlate with time to recurrence in patients undergoing therapy with tamoxifen or anastrozole. In addition, risk r

Galectin-3 is overexpressed in various forms of endometriosis.

Abstract: Endometriosis is an enigmatic disease of unknown etiology and pathogenesis, which is defined as the presence of endometrial glands and stroma outside the uterus The most widely accepted theory to explain endometriosis is probably the transplantation of an endometrial fragment during menstruation to ectopic sites, but the development of endometriosis is extremely complex and includes the adherence to the peritoneal surface and secondary invasion of the underlying tissues In this study, we have investigated the potential role of galectin-3 (gal-3), a member of a group of carbohydrate-binding proteins, which plays a major role in cell adhesion, migration, angiogenesis, and invasion The expression of gal-3 has been carried out by immunohistochemistry, according to the different phases of cycle in 50 cases of endometriosis (peritoneal endometriosis: n=10; ovarian endometriosis: n=10; deeply infiltrating endometriosis: n=30) and in 34 cases of eutopic endometrium (10 without endometriosis and 24 with endometriosis) In the proliferative and secretory phases of the cycle, the nuclear and membranous gal-3 expression was higher, first in each variant of the endometriosis than in the eutopic endometrium (P<005), and second in the eutopic endometrium of women with endometriosis than in eutopic endometrium of women without endometriosis Our data suggest that gal-3 may have a potential role in the development of endometriosis

Evaluation of 5 different labeled polymer immunohistochemical detection systems.

Abstract: Immunohistochemical staining is important for diagnosis and therapeutic decision making but the results may vary when different detection systems are used. To analyze this, 5 different labeled polymer immunohistochemical detection systems, REAL EnVision, EnVision Flex, EnVision Flex+ (Dako, Glostrup, Denmark), NovoLink (Novocastra Laboratories Ltd, Newcastle Upon Tyne, UK) and UltraVision ONE (Thermo Fisher Scientific, Fremont, CA) were tested using 12 different, widely used mouse and rabbit primary antibodies, detecting nuclear, cytoplasmic, and membrane antigens. Serial sections of multitissue blocks containing 4% formaldehyde fixed paraffin embedded material were selected for their weak, moderate, and strong staining for each antibody. Specificity and sensitivity were evaluated by subjective scoring and digital image analysis. At optimal primary antibody dilution, digital image analysis showed that EnVision Flex+ was the most sensitive system (P < 0.005), with means of 8.3, 13.4, 20.2, and 41.8 gray scale values stronger staining than REAL EnVision, EnVision Flex, NovoLink, and UltraVision ONE, respectively. NovoLink was the second most sensitive system for mouse antibodies, but showed low sensitivity for rabbit antibodies. Due to low sensitivity, 2 cases with UltraVision ONE and 1 case with NovoLink stained false negatively. None of the detection systems showed any distinct false positivity, but UltraVision ONE and NovoLink consistently showed weak background staining both in negative controls and at optimal primary antibody dilution. We conclude that there are significant differences in sensitivity, specificity, costs, and total assay time in the immunohistochemical detection systems currently in use.

Triplex-forming MicroRNAs form stable complexes with HIV-1 provirus and inhibit its replication.

Abstract: BACKGROUND One of the most fascinating discoveries in biology in recent years is unquestionably the identification of the family of small, noncoding RNAs known as microRNAs (miRNAs). Each miRNA targets multiple mRNA species through recognition of complementary sequences, typically located at multiple sites within the 3 untranslated region. In animals, single-stranded miRNA binds specific messenger RNA (mRNA) by a mechanism that is yet to be fully characterized. The bound mRNA remains untranslated resulting in reduced levels of the corresponding protein; however, if the sequence match between the miRNA and its target is precise, the bound mRNA can be degraded resulting in reduced levels of the corresponding transcript. Eukaryotic genes are also regulated by triplex formation between double helix and a third small RNA or DNA molecule. Thousands of triplex-forming (TF) islands in human genomes are mapped. However, the role of TF miRNAs within the hairpin structures of miRNA and the target mRNA has not been reported. We have explored TF complexes between human miRNAs (hsa-miR) that are complementary to human immunodeficiency virus (HIV)-1 and their antiviral potential as therapeutic agents. METHODS We downloaded mature miRNA sequences from the human miRBase Sequence Database (http://microrna.sanger.ac.uk/sequences/), and computationally analyzed miRNAs that have significant homologies to HIV-1 genome (pNL 4-3 Accession #AF324493). We developed an algorithm to look for triplex-binding motifs (C+CG and T AT) and selected 4 miRNAs with 3 negative controls. TF stability was tested by using fluorophore-labeled duplexes connected by a single hexaethylene glycol moiety, representing HIV-1 proviral motifs, and black-hole quencher-1 labeled oligonucleotides, representing miRNA. RESULTS Fifty miRNAs were discovered that showed greater than 80% homology to HIV-1, of which 4 hsa-miR that exhibited an ability to form stable triplex with double stranded-HIV-1 sequences were selected. Three negative controls were used. The TF stability of the 4 hsa-miRs and the negative controls were confirmed and measured. Stably transfected Hela-CD4+ cell lines expressing each of the hsa-miR were developed. All 4 miRNAs exhibited a significant inhibition of HIV-1 as measured by HIV-1 p24 enzyme-linked immunosorbent assay (>90%; P>0.001) when compared with the 3 negative controls. By using immunohistochemical staining with triplex binding monoclonal antibodies, significant expression of TF miRNAs was detected in the cell lines, but not in the negative controls (P<0.001). CONCLUSIONS In this study, we demonstrated for the first time that besides the well-established post-transcriptional silencing based on mRNA degradation, miRNAs may be responsible for long-term latency of HIV-1 by TF, a different mechanism. We provide a possible molecular mechanism by which HIV-1 homologous miRNAs may impart resistance to HIV-1 and suggest a new miRNA-based therapeutic strategy for HIV-1.