Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene.

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T(0) and T(1) generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T(4) generation in an agronomically elite 'Grasslands Sustain' genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T(4) generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.

Alpha-methylacyl-CoA racemase: a multi-institutional study of a new prostate cancer marker.

AIM: To test whether alpha-methylacyl-CoA racemase (AMACR) is a sensitive and specific marker of prostate cancer. METHODS AND RESULTS: The expression levels of AMACR mRNA were measured by real-time polymerase chain reaction. A total of 807 prostatic specimens were further examined by immunohistochemistry specific for AMACR. Quantitative immunostaining analyses were carried out by using the ChromaVision Automated Cellular Imaging System and the Ariol SL-50 Imaging System, respectively. AMACR mRNA levels measured in prostatic adenocarcinoma were 55 times higher than those in benign prostate tissue. Of 454 cases of prostatic adenocarcinoma, 441 were positive for AMACR, while 254 of 277 cases of benign prostate were negative for AMACR. The sensitivity and specificity of AMACR immunodetection of prostatic adenocarcinomas were 97% and 92%, respectively. Both positive and negative predictive values were 95%. By automatic imaging analyses, the AMACR immunostaining intensity and percentage in prostatic adenocarcinomas were also significantly higher than those in benign prostatic tissue (105.9 versus 16.1 for intensity, 45.7% versus 0.02% and 35.03% versus 4.64% for percentage, respectively). CONCLUSIONS: We have demonstrated the promising features of AMACR as a biomarker for prostate cancer in this large series and the potential to develop automated quantitative diagnostic tests.

Keratin expression in human tissues and neoplasms.

Keratin expression in human tissues and neoplasms Keratin filaments constitute type I and type II intermediate filaments (IFs), with at least 20 subtypes named keratin 1-20. Since certain keratin subtypes are only expressed in some normal human tissues but not others, and vice versa, various tissues have been subclassified according to the pattern of keratin staining. Simple epithelia generally express the simple epithelial keratins 7, 18, 19, and 20, while complex epithelia express complex epithelial keratins 5/6, 10, 14, and 15. When an epithelium undergoes malignant transformation, its keratin profile usually remains constant. The constitution and expression patterns of keratin filaments in human epithelial neoplasms are complex and often distinctive. In this article, we first briefly review the molecular and cell biology of keratin filaments. We then focus on the expression patterns of keratin filaments in various human neoplasms.

Cytokeratin 14 immunoreactivity distinguishes oncocytic tumour from its renal mimics: an immunohistochemical study of 63 cases.

AIMS: The cytokeratin 14 (CK14) expression in oncocytomas or oncocytic tumours of various tissue origins has not been established. We have studied CK14 expression in 30 cases of oncocytic tumours of various tissue origins and 33 cases of renal cell carcinoma with overlapping features (mimics) by immunohistochemistry. METHODS AND RESULTS: Immunohistochemistry (ABC-HRP method) was performed for detection of CK14 in 30 cases of oncocytic tumour and 33 cases of renal mimics. To demonstrate CK14 specificity and sensitivity in oncocytic tumours, mES-13 (an anti-mitochondrial monoclonal antibody) immunohistochemistry was also performed in 20 of 30 cases on oncocytic tumour and all 33 cases of renal mimics. We found that all 30 cases of oncocytic tumour showed cytoplasmic CK14 positivity. All 20 cases of oncocytic tumour studied with mES-13 were positive. CK14 immunoreactivity was identified in only four cases of renal cell carcinoma (one conventional renal cell carcinoma with granular cytoplasm and three chromophobe renal cell carcinomas with eosinophilic cytoplasm). In contrast, all 33 cases of renal cell carcinoma were positive for mES-13 to varying degrees. CONCLUSION: The homogeneous, cytoplasmic, and granular CK14 immunoreactivity is sensitive and specific for oncocytic tumours, whereas CK14 immunoreactivity in renal mimics is light and sporadic with peripheral accentuation.

No direct role for Epstein-Barr virus in American hepatocellular carcinoma.

Epstein-Barr virus (EBV) was recently linked to hepatocellular carcinogenesis in Japanese patients. It is not clear whether EBV infection is also associated with hepatocellular carcinoma (HCC) occurring in American patients. We studied 41 cases of HCC from the Los Angeles area for evidence of EBV infection by in situ hybridization, immunohistochemistry, and polymerase chain reaction methods. Of 41 cases, 16 were seropositive for hepatitis B virus surface antigen (39%), 9 of 29 tested were seropositive for hepatitis C virus antibody (31%); in total, 22 cases were seropositive for hepatitis B virus and/or hepatitis C virus (53%). Of 41 cases, 1 was positive for EBV-encoded small nonpolyadenylated RNA (EBER)-1 (2%) by in situ hybridization. By immunohistochemistry, two cases were positive for EBV nuclear antigen (EBNA)-1 (5%), one was positive for the transactivating immediate early BZLF1 (ZEBRA) (2%), and none was positive for latent membrane protein-1. None of the 41 cases was positive for latent membrane protein-1 and EBV nuclear antigen (EBNA)-4 DNAs by polymerase chain reaction assay. All four positive cases showed rare EBER-1-, ZEBRA-, or EBNA-1- positive cells (<0.1%); in none of these cases was there expression of any other EBV viral genes. In the one case each that was positive for EBER-1 and ZEBRA, both of which occurred in patients of non-Asian ethnicity, the staining was limited to infiltrating small lymphocytes, and tumor cells were negative. In the two cases that were positive for EBNA-1, both of which occurred in patients of Asian ethnicity, the staining was limited to tumor cells, and infiltrating small lymphocytes were negative. Our study indicates that rare cases of American HCC may contain EBV-infected cells, but it is unlikely that EBV plays a major role in the carcinogenesis of HCC.

No significant association of Epstein-Barr virus infection with invasive breast carcinoma.

We studied 48 cases of invasive breast carcinoma for evidence of Epstein-Barr virus (EBV), which is associated with many human malignancies. In situ hybridization studies to detect the presence of EBV-encoded small nonpolyadenylated RNA (EBER)-1 were performed in paraffin sections. Immunohistochemical studies to detect EBV nuclear antigen (EBNA)-1, latent membrane protein (LMP)-1, and the transactivating immediate-early BZLF1 (ZEBRA) protein were also performed in paraffin sections. The presence of EBV genomic DNA was studied by polymerase chain reaction (PCR) amplification using sets of primers flanking the EBNA-4 and the EBV-LMP-1 genes in frozen tissues. Southern blot analysis using a probe flanking the EBV terminal repeat region was then attempted in cases that were PCR-positive. Five of 48 cases (10%) of breast carcinoma showed focal EBER-positive tumor cells. Twelve cases (25%) were positive for EBNA-1 by immunohistochemistry, all but one different from the EBER-positive cases. None of the cases were positive for LMP-1 or ZEBRA protein by immunohistochemistry. PCR studies for EBNA-4 and LMP-1 were each positive in five cases (including three cases in common). However, Southern blot studies successfully performed in all but one of the PCR-positive cases were completely negative. The identification of EBV by any methodology was not correlated with tumor size, grade, or lymph node status. This study demonstrated evidence of EBV infection in tissues involved by invasive breast carcinomas in a significant subset of cases. However, the lack of localization of EBV infection to a significant population of the tumor cells in any case, the negativity by Southern blot hybridization, and the lack of expression of multiple antigens in any case strongly argue against a significant role for EBV in the pathogenesis of breast carcinoma.

Cytokeratin 14 expression in epithelial neoplasms: a survey of 435 cases with emphasis on its value in differentiating squamous cell carcinomas from other epithelial tumours.

AIMS: The tissue distribution of cytokeratin 14 (CK14) in epithelial neoplasms is not well defined. We have evaluated 435 cases of epithelial neoplasm of various origins with cytokeratin 14 monoclonal antibody with special attention to possible use in differential diagnosis. METHODS AND RESULTS: Immunohistochemistry (ABC-HRP method) was performed for detection of CK14. We found that the expression of cytokeratin 14 was generally restricted to: (i) the majority of cases of squamous cell carcinoma regardless of origin (67/74) and degree of differentiation; (ii) neoplasms with focal squamous differentiation, including endometrial, and ovarian adenocarcinoma, malignant mesothelioma and transitional cell carcinoma; (iii) thymoma (8/8); (iv) myoepithelial components of salivary gland pleomorphic adenoma (3/4); and (v) oncocytic neoplasms, including thyroid Hurthle cell adenoma (1/1) and salivary gland Warthin's tumour (2/2). CONCLUSION: CK14 protein is a useful marker in differential diagnosis of squamous cell carcinomas.

CD79: a review.

CD79 is composed of CD79a and CD79b components expressed almost exclusively on B cells and B-cell neoplasms. CD79a and CD79b expression precedes immunoglobulin (Ig) heavy-chain gene rearrangement and CD20 expression during B-cell ontogeny and disappears later than CD20 in the late (plasma cell) stage of B-cell differentiation. Therefore, antibodies to CD79a and CD79b are useful in the differential diagnosis of B-cell neoplasms from T-cell neoplasms or myeloid neoplasms, or L and H lymphocyte predominance Hodgkin's lymphoma from classic Hodgkin's lymphoma. In addition, CD79a and CD79b antibodies are useful markers in the diagnosis of precursor B-acute lymphoblastic leukemia (pre-B-ALL) because many of these tumors are negative for other B-cell markers, such as CD20 and CD45RA. Furthermore, for B-cell neoplasms, wherein CD20 expression is aberrantly lost, such as in diffuse large B-cell lymphoma, or for B-cell neoplasms after CD20-antibody therapy, CD79a may be used as a first-line B-cell marker for the diagnosis. In this review, the authors discuss the molecular biology of CD79 and the frequency and usefulness of CD79 expression in these neoplasms.

Utility of CD10 in distinguishing between endometrial stromal sarcoma and uterine smooth muscle tumors: an immunohistochemical comparison of 34 cases.

Endometrial stromal sarcoma (ESS), uterine cellular leiomyoma (UCL), and uterine leiomyosarcoma (ULS) are composed mainly of spindle cells that express similar antigens such as desmin, smooth muscle actin (SMA), and muscle-specific actin (MSA). The differential diagnosis of an ESS versus a uterine smooth muscle tumor or an extrauterine spindle cell sarcoma can be problematic based solely on clinical presentation, histologic assessment, or routine immunohistochemistry. Recently, we reported that normal endometrium, but not myometrium, as well as five cases of ESS, were positive for CD10. We now report the results of CD10 immunohistochemistry in an additional 11 cases of ESS (total 16 cases), 10 cases of UCL, and nine cases of ULS. CD10 immunoreactivity was detected in 16 of 16 cases of ESS (100%) as compared to only 2 of 10 cases of UCL (20%) and none of nine cases of ULS (0%). We compared the utility of CD10 immunoreactivity with that of desmin, SMA, MSA, estrogen receptor (ER), and inhibin in these tumors. Although the majority of cases of UCL and ULS were positive for SMA, MSA, and desmin, a substantial portion of cases of ESS were also positive for SMA, MSA, and desmin. We conclude that in combination with SMA, MSA, and desmin, CD10 is a useful immunohistochemical marker in the differential diagnosis of ESS versus UCL or ULS.

Immunohistochemical detection of CD10 in paraffin sections of hematopoietic neoplasms: a comparison with flow cytometry detection in 56 cases.

Paraffin-section immunohistochemistry with heat-induced epitope retrieval using a newly characterized monoclonal antibody (clone 56C6) against the CD10 antigen was performed on 56 hematopoietic tumors previously studied for CD10 expression by flow cytometry. The cases included 33 precursor B-lymphoblastic leukemias, 10 acute myeloid leukemias, five precursor T-lymphoblastic leukemias, five follicular lymphomas, and three Burkitt cell leukemias. Forty of the 56 cases were CD10 positive by flow cytometry studies, including all five follicular lymphomas (100%); 30 of 33 (91%) cases of precursor B-lymphoblastic leukemias, two of three (66%) cases of Burkitt cell leukemias, two of five (40%) cases of precursor T-lymphoblastic leukemias, and none of the 10 cases of acute myeloid leukemia. Thirty-nine of the 40 (97%) flow cytometric CD10-positive cases also expressed CD10 by immunohistochemistry in formalin- or B5-fixed, paraffin-embedded tissue, with only one case of precursor B-lymphoblastic leukemia being positive by flow cytometry and negative by immunohistochemistry. The 16 CD10-negative flow cytometry specimens were all also negative by immunohistochemistry. Thirty-seven CD10 immunohistochemistry positive cases showed a diffuse membranous staining pattern and two cases demonstrated a Golgi staining pattern. The fixation methods (10% neutral buffered formalin versus B5) and decalcification did not affect the CD10 immunostaining results. This study demonstrates that the new CD10 monoclonal antibody clone 56C6 is a reliable marker for detection of CD10 antigen expression in formalin-and B5-fixed paraffin-embedded tissue after heat-induced epitope retrieval when compared with flow cytometry detection of fresh tissue samples.

Immunophenotyping of hematopoietic neoplasms.

Advances in the staging and treatment of hematopoietic neoplasms have necessitated a high degree of accuracy in the diagnosis and classification of these tumors. A greater degree of diagnostic precision has resulted from recent advances in immunophenotyping and genotyping of hematopoietic neoplasms. This review discusses several new immunohistochemical reagents, many of which are derived from results of molecular studies.

Nasal glomus tumors: report of two cases with emphasis on immunohistochemical features and differential diagnosis.

We describe 2 cases of nasal glomus tumor that presented as nasal polyps. Grossly, each of the polypectomy specimens consisted of small fragments of polypoid soft tissue with glistening mucosa. Histopathological examination of each of the specimens showed sheets and nests of monomorphic round cells intimately associated with capillary-sized blood vessels. The tumor cells were strongly cytoplasmic positive for vimentin, smooth-muscle specific actin, muscle-specific actin, and CD34. Collagen IV showed pericellular positivity. Nasal glomus tumors are extremely rare and represent less than 0.5% of nasal nonepithelial tumors. Nasal polyps are common surgical pathological specimens, with the majority of nasal polyps being inflammatory polyps or a respiratory epithelial proliferation. Histologically, many nasal polyps show vascular proliferation with an inflammatory cell infiltrate, which may be confused with the rare glomus tumor. In addition, other nasal vascular tumors, in particular nasal hemangiopericytoma and neural tumors, may histologically mimic nasal glomus tumors.

Epstein-Barr virus (EBV) nuclear antigen (EBNA)-4 mutation in EBV-associated malignancies in three different populations.

Different ethnic groups with a high human leukocyte antigen (HLA)-A11 prevalence have been shown to experience a high rate of Epstein-Barr virus (EBV) infection, EBV-associated malignancies, and Epstein-Barr nuclear antigen (EBNA)-4 mutations. The epitopes 399-408 and 416-424 of EBNA-4 are major antigenic epitopes that elicit an HLA-A11 cytotoxic T lymphocyte (CTL) response to EBV infection. Mutations selectively involving one or more nucleotide residues in these epitopes affect the antigenicity of EBNA-4, because the mutant EBV strains are not recognized by the HLA-A11-restricted CTLs. To investigate these mutations in common EBV-associated malignancies occurring in different populations, we studied the mutation rate of epitopes 399-408 and 416-424 of EBNA-4 in 25 cases of EBV-associated Hodgkin's disease (HD), nine cases of AIDS-related non-Hodgkin's lymphoma, and 37 cases of EBV-associated gastric carcinoma (GC) from the United States, Brazil, and Japan. We found one or more mutations in these two epitopes in 50% (6/12) of United States HD, 15% (2/13) of Brazilian HD, 50% (6/12) United States GC and 28% (7/25) Japanese GC, and 22% (2/9) of United States AIDS-lymphoma. Similar mutations were found in 30% (3/10) of United States reactive, 0% (0/6) of Brazilian reactive, and 25% (2/8) Japanese reactive tissues. The most frequent amino acid substitutions were virtually identical to those seen in previously reported isolates from EBV-associated nasopharyngeal carcinomas and Burkitt's lymphomas occurring in high prevalence HLA-A11 regions. However, only 2/28 (7%) mutations occurred in HLA-A11-positive patients. Our studies suggest that: 1) EBNA-4 mutations are a common phenomenon in EBV-associated HD, GC, and AIDS-lymphoma; 2) the mutation rate does not vary in these geographic areas and ethnic groups; 3) EBNA-4 mutations in EBV-associated United States and Brazilian HD, United States and Japanese GC, and United States AIDS lymphomas are not related to patients' HLA-A11 status.

Practical applications of immunohistochemistry in hematolymphoid neoplasms.

Immunohistochemistry plays a key role in the diagnosis and classification of hematolymphoid neoplasms. New cell and lineage markers are constantly being discovered and added to the existing long list of antibodies. In this review article we provide general information and new applications of the commonly used hematolymphoid markers. We also discuss the features and applications of some newly discovered markers, such as ALK, fascin, granzyme/perforin, and tryptase. There is no universal "panel" for the diagnosis of hematolymphoid neoplasms. However, in this review article, we provide suggested panels for a given hematolymphoid neoplasm that is based on our experience and that reported in the literature.

Functional inhibition of retinal pigment epithelial cell-substrate adhesion with a monoclonal antibody against the beta 1 subunit of integrin.

In the preceding report, experiments were described which identify the 2A10 antigen of retinal pigment epithelial (RPE) cells as a beta 1 subunit of the cell-surface extracellular matrix receptor protein integrin. In this article, experiments are presented which use the 2A10 and CSAT antibodies, both directed against the beta 1 subunit, to investigate the role of integrin in RPE and fibroblast (FB) cell-substrate adhesion. When added to cultures simultaneously with cells, either the 2A10 or CSAT antibodies inhibit both FB and RPE cell adhesion and spreading on laminin (LM). However, although the 2A10 antibody blocks adhesion and spreading of FB and RPE cells on fibronectin (FN), the CSAT antibody has no effect. The inhibition of the 2A10 antibody is specific for integrin-mediated adhesion; it does not affect FB or RPE cell adhesion and spreading on tissue-culture plastic. When RPE cells are first allowed to attach to and spread on FN and LM and then the 2A10 or CSAT antibody is added to the cultures, both cause detachment and rounding of RPE cells from LM, but neither has any effect on the cells already spread on FN. These results indicate that there are differences in the way FB and RPE cells interact with LM and FN. Furthermore, these results provide the first direct functional demonstration that RPE cell-substrate adhesion is mediated by integrin.

Identification of an adhesion-associated protein of the retinal pigment epithelium.

The normal function of the retinal pigment epithelium (RPE) is dependent on the maintenance of tight adhesions between cells. In order to identify cell surface molecules which may be important for maintaining the integrity of the RPE, we have undertaken a combined functional, biochemical, and immunohistochemical analysis of cell surface proteins of the RPE. These studies have led to the identification of a 100-kD cell surface protein whose presence correlates with the maintenance of calcium-dependent adhesions between RPE cells. In intact RPE tissue the protein is concentrated at the junctions between RPE cells. The properties of the protein suggest that it may be a member of the cadherin family of calcium-dependent cell adhesion proteins.