Evaluation of automated silver-enhanced in situ hybridization (SISH) for detection of HER2 gene amplification in breast carcinoma excision and core biopsy specimens.

AIMS: To validate the use of the silver-enhanced in situ hybridization (SISH) technique in assessing HER2 status of breast carcinoma in excision biopsy specimens, and to assess its reliability in determining HER2 status in core biopsy specimens. METHODS AND RESULTS: Routinely processed paraffin sections of 65 excised breast carcinomas and 56 available preoperative core biopsy specimens from the same patients were selected from the archives for testing with the SISH technique using the automated Ventana Benchmark XT machine. For each case, two sections were used, one for the assessment of HER2 gene amplification and the other for assessment of chromosome 17. Of the 65 excision specimens tested, sections of 53 cases were also available for fluorescence in situ hybridization (FISH) examination. HER2 gene amplification was detected by SISH in 14 (21%) out of 65 excision specimens and in eight (14%) out of 56 core biopsy specimens. The results of SISH and FISH were identical in 50 (94%) out of the 53 excision cases examined by the two techniques. Two cases were SISH-, FISH+, and one case was the other way round. SISH results of core biopsy specimens and corresponding excision biopsy specimens were identical in 50 (89%) out of 56 cases. Four cases (7%) were SISH- in cores but positive in excision specimens, whereas two cases were the other way round. CONCLUSIONS: The results validate the use of the SISH technique for assessing HER2 status of excised breast carcinoma tissue sections. The results are comparable to those obtained with FISH, but SISH has the advantage of having a permanent end result that can be visualized by an ordinary light microscope. There is a reasonable 89% concordance between SISH results obtained in core and excision biopsy specimens. However, it may be prudent to postpone doing SISH, if possible, until sections of the resected specimen are available, as these seem to be more reliable.

FGF-2 is bound to perlecan in the pericellular matrix of articular cartilage, where it acts as a chondrocyte mechanotransducer.

OBJECTIVE: We have shown previously that cutting or loading articular cartilage resulted in a fibroblast growth factor-2 (FGF-2) dependent activation of the extracellularly regulated kinase (ERK), and induction of a number of chondrocyte regulatory proteins including tissue inhibitor of metalloproteinase-1 and matrix metalloproteinases 1 and 3. An extracellular matrix-bound pool of FGF-2 was apparent, which could be liberated from the tissue by heparitinase (Vincent et al., Proc Natl Acad Sci U S A 2002;99(12):8259-64, Vincent et al., Arthritis Rheum 2004 Feb;50(2):526-33). Our objectives were to determine where FGF-2 was stored in articular cartilage, to which proteoglycan it was bound, and to elucidate its role in chondrocyte mechanotransduction. METHODS: Immunohistochemistry and confocal microscopy were used to localise FGF-2 in the tissue. In vitro binding studies were performed using IASYS surface plasmon resonance. To study the role of pericellular FGF-2 in mechanotransduction cartilage explants or articular chondrocytes encapsulated in alginate were loaded using an in house loading rig. The loading response was assessed by the activation of ERK, in the presence or absence of a specific FGFR inhibitor. RESULTS: Here we have identified perlecan as the heparan sulphate proteoglycan that sequesters FGF-2 in articular cartilage. Perlecan and FGF-2 co-localised within the type VI collagen-rich pericellular matrix of porcine and human articular cartilage. Chondrocytes encapsulated in alginate were able to accumulate pericellular perlecan and FGF-2 in culture, and deliver an FGF-dependent activation of ERK when loaded. CONCLUSION: Loading-induced ERK activation was dependent upon the presence and concentration of pericellular FGF-2, suggesting a functional role for this matrix-bound growth factor in chondrocyte mechanotransduction.

Phosphorylation of ERalpha at serine 118 in primary breast cancer and in tamoxifen-resistant tumours is indicative of a complex role for ERalpha phosphorylation in breast cancer progression.

Oestrogen receptor-alpha (ERalpha) is an important prognostic marker in breast cancer and endocrine therapies are designed to inhibit or prevent ERalpha activity. In vitro studies have indicated that phosphorylation of ERalpha, in particular on serine 118 (S118), can result in activation in a ligand-independent manner, thereby potentially contributing to resistance to endocrine agents, such as tamoxifen and aromatase inhibitors. Here we report the immunohistochemistry (IHC) of S118 phosphorylation in 301 primary breast tumour biopsies. Surprisingly, this analysis shows that S118 phosphorylation is higher in more differentiated tumours, suggesting that phosphorylation at this site is associated with a good prognosis in patients not previously treated with endocrine agents. However, we also report that S118 phosphorylation was elevated in tumour biopsies taken from patients who had relapsed following tamoxifen treatment, when compared to pre-treatment biopsies. Taken together, these data are consistent with the view that S118 phosphorylation is a feature of normal ERalpha function and that increases in levels of phosphorylation at this site may play a key role in the emergence of endocrine resistance in breast cancer.

CD10 immunostaining of bile canaliculi in liver biopsies: change of staining pattern with the development of cirrhosis.

AIMS: This study was prompted by published observations concerning the absence of normal bile canalicular CD10 staining in some cases of primary liver cell carcinoma. Our aim was to investigate the possibility that this loss of staining occurs prior to the development of cancer. METHODS AND RESULTS: The study comprised 164 liver biopsies, including 96 from patients with hepatitis C infection of various grades and stages including nine cases with cirrhosis, 24 other cases of cirrhosis of other aetiology, five cases of primary liver carcinoma, 12 cases of metastatic carcinoma, as well as biopsies with a variety of other liver diseases. CD10 was demonstrated in paraffin sections using the avidin-biotin immunoperoxidase technique. In hepatitis C cases, a significant loss of the canalicular pattern was seen in four out of 41 (10%) biopsies with stages 0-1 compared with 25 out of 55 (45%) with stages 2-6 (P < 0.001). There was also a significant difference (P < 0.001) between biopsies with stage 2-3 and those with stage 4-6, where marked pattern loss was seen in 9/37 (24%) and 16/18 (89%), respectively. Marked loss of the pattern was also seen in 16 out of the 24 (67%) other cirrhotic biopsies, as well as in cases with severe lobular inflammation and cholestasis and liver cell dysplasia and carcinoma. In hepatitis C biopsies, no relationship was noted between the reduction in the canalicular pattern and the necroinflammatory score. CONCLUSIONS: CD10-stained bile canalicular pattern in liver biopsies is preserved in cases with mild fibrosis and inflammation, but it becomes increasingly reduced with the advance of fibrosis or the presence of severe lobular inflammation or extensive metastases. Further investigations into the relationship between the changes in CD10 staining pattern and liver function tests may be useful in explaining test results.

Immunocytochemical analysis of AE1/AE3, CK 14, Ki-67 and p53 expression in benign, premalignant and malignant oral tissue to establish putative markers for progression of oral carcinoma.

Squamous cell carcinoma (SCC) is the most common form of oral malignancy and is often preceded by premalignant lesions, some of which are more likely to progress to carcinoma than others. In this study, a panel of monoclonal antibodies (AE1/AE3, cytokeratin [CK] 14, Ki-67 and p53) is applied to 10 cases of human oral tissue in each of six categories to establish staining patterns indicative of which lesions are more likely to progress to malignancy. The six tissue categories are normal tissue; abnormal benign lesions; mild, moderate and severe dysplasia; and SCC. A statistical analysis of Ki-67 and p53 immunoexpression is performed. The results showed that AE1/AE3 and CK 14 expression was reduced as a late event in oral carcinogenesis, particularly in poorly differentiated SCC. Expression of Ki-67 and p53 proved to be a weak but statistically significant predictor of malignant progression in oral tissue.

p57(KIP2) immunohistochemical staining of gestational trophoblastic tumours does not identify the type of the causative pregnancy.

AIM: To determine whether immunohistochemical staining for p57(KIP2), the product of the maternally expressed gene CDKN1C, can be used to differentiate between gestational trophoblastic tumours arising from a complete hydatidiform mole and those originating from non-molar pregnancies. METHODS: The immunohistochemical expression of p57(KIP2) was investigated in 23 cases of choriocarcinoma and 17 placental site trophoblastic tumours. Fourteen of the tumours examined were shown by DNA analysis to have arisen from complete hydatidiform moles and 26 from non-molar pregnancies. RESULTS: Five of 11 (45%) post-complete hydatidiform mole choriocarcinomas and two of three (67%) post-complete hydatidiform mole placental site trophoblastic tumours were found to be p57(KIP2)+ and showed similar immunostaining characteristics to tumours that developed from non-molar pregnancies. Although there was a statistically significant reduction in the proportion of cases showing positive p57(KIP2) staining in post-complete hydatidiform mole tumours compared with those originating in non-molar pregnancies [proportion difference 0.35 [95% confidence interval (CI) 0.05, 0.61], P = 0.02], immunostaining did not provide diagnostically useful information to differentiate between these tumours in clinical practice. There was no significant difference between the extent of staining in choriocarcinoma versus placental site trophoblastic tumours [proportion difference 0.17 (95% CI - 12, 42), P = 0.19]. The majority of both types of gestational trophoblastic tumour were positive for the presence of the p57(KIP2) protein irrespective of their genetic origin. CONCLUSION: Immunostaining for p57(KIP2) fails to discriminate between gestational trophoblastic tumours that have arisen from complete hydatidiform moles and those that have originated from other types of pregnancy.

Head and neck cancer in patients with human immunodeficiency virus-1 infection: incidence, outcome and association with Epstein-Barr virus.

Head and neck cancers have been described in patients with human immunodeficiency virus-1 (HIV-1) infection. However the incidence, aetiology and clinical features of the disease remain unclear. Patients with head and neck cancer and HIV were identified from a large HIV centre. The incidence and clinical features were recorded, and the tumours were stained for Epstein-Barr virus (EBV). Head and neck cancer occurred more frequently than in an age-matched control group (1.66 vs 0.55/10,000 patient years respectively p < 0.05). Highly active anti-retroviral therapy has not significantly altered the incidence of the disease. All of the tumours tested were positive for EBV. Patients were moderately immunosuppressed at diagnosis and had aggressive tumours. All but one of the patients died of cancer with a median survival of 28 months. Head and neck cancer occurs more frequently in HIV. It is an aggressive disease and EBV may play a role in its pathogenesis.

Expression of prolactin receptors in normal, benign, and malignant breast tissue: an immunohistological study.

AIMS: Prolactin plays an important role in the proliferation and differentiation of normal breast epithelium, and possibly in the development of breast carcinoma. The effects of prolactin are mediated by its receptor; thus, alteration in the expression of this receptor could be important in studying the biology of breast cancer. This investigation was aimed at comparing the expression of prolactin receptors in normal, benign, and malignant breast tissue. MATERIAL/METHODS: The expression of prolactin receptors was studied in paraffin wax embedded sections of 102 breast biopsies (93 female and nine male), using the monoclonal antibody B6.2, and the avidin-biotin immunoperoxidase technique. Six biopsies were normal, 34 had benign lesions, and 62 were malignant. RESULTS: In normal cases, prolactin receptor positivity was seen only on the luminal borders of the epithelial cells lining ducts and acini. In most benign lesions, variable degrees of luminal and cytoplasmic staining were seen. Cells showing apocrine metaplasia and florid regular ductal epithelial hyperplasia were mostly negative. In malignant cases, the staining pattern was mostly cytoplasmic and heterogeneous. Forty one of the 59 carcinomas in women showed a degree of positivity involving 10-100% of the tumour cells. A significant direct correlation was found between prolactin receptor and oestrogen receptor staining when only cases that scored more than 100/300 for the latter receptor, using the H scoring system, were considered (p = 0.0207). No correlation was found between prolactin receptors and progesterone receptors, patient's age, tumour size, tumour grade, or axillary lymph node status. CONCLUSIONS: Prolactin receptors seem to be expressed at different cellular sites in normal, benign, and malignant breast epithelial cells. The receptor is expressed in more than two thirds of female breast carcinomas, suggesting that it may play a role in the pathogenesis of the disease. The positivity is correlated with moderate and strong staining for oestrogen receptors in tissue sections, but not with other prognostic factors.

Provocation of experimental aortic inflammation and dilatation by inflammatory mediators and Chlamydia pneumoniae.

BACKGROUND: The macrophage appears to have a key role in the inflammation and proteolysis associated with the growth and development of abdominal aortic aneurysms. The role of inflammatory mediators and Chlamydia pneumoniae in stimulating the influx of macrophages and dilatation of the abdominal aorta was investigated in an experimental model. METHODS: Periaortic application of calcium chloride solution (and monocyte chemoattractant protein (MCP) 1, a cocktail of cytokines or C. pneumoniae) to the abdominal aorta of New Zealand White rabbits was performed at laparotomy. Some animals were fed a cholesterol-rich diet. The diameter of the aorta was measured by ultrasonography and after perfusion fixation, 3 weeks after laparotomy. Aortic sections were stained with RAM-11 to identify macrophages for counting. The presence of C. pneumoniae DNA was confirmed using the polymerase chain reaction. RESULTS: Aortic macrophage influx in response to MCP-1, thioglycollate or C. pneumoniae was more than doubled in the cholesterol-fed animals. In response to human recombinant MCP-1 (1 microg) the mean(s.d.) macrophage count increased from 79(19) to 340(215) per unit area (P < 0.02). Even in cholesterol-fed animals, application of MCP-1 (recombinant human or rabbit form) was not associated with aortic dilatation. Application of thioglycollate 0.1 mol/l, or live or formalin-inactivated C. pneumoniae (0.5 x 108 organisms), was associated with a similar increase in macrophages to that caused by MCP-1 and a significant (approximately twofold) increase in aortic diameter after 3 weeks. CONCLUSION: Macrophage influx into rabbit abdominal aorta, without macrophage activation, is insufficient to cause experimental aortic dilatation. C. pneumoniae antigens appeared to stimulate aortic dilatation, probably by specific activation of macrophages.

Comparison of immunoperoxidase staining of 3 different types of CD5 antibodies in a spectrum of breast lesions.

CONTEXT: We recently described a patient with chronic lymphocytic leukemia who presented with a breast carcinoma that stained positive for CD5 using a commercially available antibody (CD5-4C7, Novocastra, Newcastle upon Tyne, UK). OBJECTIVES: To study the distribution of CD5 immunoreactivity in tissue sections of a variety of benign and malignant breast lesions using the antibody CD5-4C7 and to compare the results with those obtained with 2 other commercially available CD5 antibodies (CD5/54/F6, Dako, Ely, Cambridgeshire, UK, and CD5/54/B4, Novocastra). DESIGN: Paraffin sections of 102 breast biopsy specimens with various diagnoses were examined using the avidin-biotin immunoperoxidase complex technique. SETTING: The histopathology department of a tertiary referral teaching hospital. RESULTS: The staining results obtained with CD5-4C7 were different from those obtained with the other 2 antibodies. With 4C7, the normal and benign biopsy specimens showed varying numbers of positive epithelial cells and lymphocytes. Heterogeneous positive staining was also present in 47 (78%) of 60 invasive female breast carcinomas and in all 3 male breast carcinomas examined. A statistically significant correlation was found between CD5 positivity and tumor grade, with grade 3 tumors being less likely to be CD5 positive than grades 1 and 2 (P =.0035). No correlation was found between CD5 positivity and patient's age, tumor histologic type, axillary lymph node status, or progesterone receptors. On the other hand, the CD5/54/F6 and CD5/54/B4 antibodies only stained lymphocytes and occasional normal breast ducts, mostly those showing apocrine metaplasia. All other normal benign and malignant epithelial cells were negative. CONCLUSIONS: Positive staining for CD5 using the antibody 4C7 is seen in normal and benign breast tissue and 78% of invasive breast carcinomas. The positivity is more common in low-grade tumors. No significant staining was seen with the 2 other CD5 clones used in this study. The significance of the positive staining obtained with CD5-4C7 is not obvious, but this clone may be more sensitive than the others, or it may be recognizing an epitope shared by another antigen.

CD9 immunohistochemical staining of breast carcinoma: unlikely to provide useful prognostic information for routine use.

AIMS: CD9, a cell membrane glycoprotein, is found in a variety of tumour cells and is believed to regulate cell motility and possibly cell growth. It has been reported that the absence of CD9 is associated with increased aggressiveness of breast carcinoma, but no detailed studies of the distribution of CD9 in normal and abnormal breast tissue are available. This investigation was aimed at studying the distribution of CD9 in a wide variety of breast biopsies including normal, benign, and malignant cases, and assessing its usefulness as a prognostic marker in breast cancer. METHODS AND RESULTS: Sections of 113 breast biopsies from female and male patients including 10 normal, 23 benign, and 80 malignant cases were examined. The monoclonal antibody CD9 (Novacastra Ltd, Newcastle-upon-Tyne, UK) was used with the avidin-biotin complex immunoperoxidase technique. The results were assessed semiquantitatively using a four scale system of 3+, 2+, 1+ and negative. All normal and benign epithelial cells were strongly stained (3+). In female breast carcinomas, 40% were 3+, 49% were 2+, and 11% were 1+. Both cases of male breast carcinomas scored 3+. For female breast cancers, the results were then correlated to tumour grade, the presence or absence of lymph node metastases, and oestrogen and progesterone receptor status. No significant statistical correlation was found with any of these parameters. We then examined 11 axillary lymph nodes with metastases from some of the above cases. Three of these cases had a CD9 score of 3+, seven were 2+, and one was 1+. The metastatic tumours in all 11 cases were strongly stained (3+). CONCLUSIONS: Immunostaining for CD9 is unlikely to provide any useful additional prognostic information for clinical purposes.

Adenoid cystic carcinoma of the breast: a tumour commonly devoid of oestrogen receptors and related proteins.

AIM: Adenoid cystic carcinoma (ACC) of the breast is an uncommon well-differentiated tumour with good prognosis, and sometimes difficult to distinguish from in-situ and invasive cribriform carcinoma (ICC) which are relatively more common. Recently, we encountered a case of ACC that proved to be totally oestrogen receptor (ER) negative by immunohistochemistry. We investigated the possibility that this may be a consistent feature that can help in differentiating ACC from ICC which are usually ER positive. METHODS AND RESULTS: The immunoperoxidase technique was used to study the expression of ER and other related proteins in six cases of ACC and two cases of ICC. All ACC cases were negative for oestrogen (ER) and progesterone (PgR) receptors whereas the two ICC were strongly positive for ER and showed a variable degree of PgR positivity. In addition, ACC cases were pS2 negative and showed minimal expression of prolactin receptors (PrlR), while the two ICC showed widespread and strong staining for pS2 and PrlR. The percentages of cells staining positively for Ki67 and p27 were generally lower in ACC than in ICC. Both tumour types were c-erbB-2 negative, but p53 was weakly to moderately positive. CONCLUSIONS: The findings suggest that a negative immunoperoxidase staining for ER would confirm the diagnosis of ACC in contrast to the positive staining which is always seen in ICC. The findings also raise the issue of the presence of a specific class of ER negative breast carcinomas which are negative not because of poor differentiation, but because of their derivation from, or differentiation along, an ER negative cell lineage.

High level expression of p27(kip1) and cyclin D1 in some human breast cancer cells: inverse correlation between the expression of p27(kip1) and degree of malignancy in human breast and colorectal cancers.

The expression of cyclin-dependent kinase inhibitor p27(kip1) in human tumors and normal tissues was investigated using a panel of novel anti-p27(kip1) mAbs. An inverse correlation between expression of p27(kip1) and cell proliferation was generally observed after analyzing its expression in 25 different normal human tissues. In some highly proliferative human breast cancer cells, however, high level p27(kip1) expression was seen, indicating the existence of a mechanism by which some growing tumor cells may tolerate this inhibitor of cell cycle progression. Detailed studies demonstrated a correlation between the high level expression of p27(kip1) and cyclin D1 in human breast cancer cells. There was also an inverse correlation between the expression of p27(kip1) and the degree of tumor malignancy in human breast and colorectal cancers, indicating that p27(kip1) may be a useful prognostic marker in these cancers.

Oestrogen and progesterone receptor assessment in core biopsy specimens of breast carcinoma.

AIMS: To assess the suitability of core biopsy specimens for the immunohistological assessment of oestrogen and progesterone receptors in breast carcinoma. METHODS: Thirty consecutive cases of clinically palpable breast carcinoma, from which both core and excision biopsy specimens were available, were examined. Routinely processed paraffin wax sections were stained using the specific monoclonal antibodies 1D5 (Dako) for oestrogen receptor and NCL-PGR (Novocastra) for progesterone receptor, after an antigen retrieval step using a pressure cooker. Staining results were assessed using the H score system with the results being expressed as negative, weakly positive, moderately positive or strongly positive. RESULTS: Twenty six biopsy specimens contained enough tumour tissue for assessment. Absolute agreement between scoring categories was seen in 19 (73%) cases for oestrogen receptors. However, when all positive categories were added together, agreement between core and excision biopsy specimens increased to 93% (24 cases). Disagreement was seen only in two cases which stained positive in the core biopsy specimens and negative in the excision biopsy specimens. For progesterone receptors, the absolute agreement between all scoring categories was seen only in 11 (42%) cases. When all positive categories were considered together, agreement increased to 69% (18 cases). Five cases were progesterone receptor positive in core but not in excisional biopsy specimens, while three cases were negative in core but positive in excisional biopsy specimens. CONCLUSIONS: The results suggest that core biopsy specimens can be reliably used for oestrogen receptor assessment, but are less reliable for progesterone receptor assessment, probably because of a greater heterogeneity of progesterone receptor staining.