Edwin A. Dublin

Immunohistochemical detection of p53 protein in mammary carcinoma: An important new independent indicator of prognosis?

In an immunohistochemical pilot study of 195 primary breast cancer patients with a 10-year median follow-up we found that patients with carcinomas who express p53 protein in the majority of their tumor cells (19% of the cases) have a considerably worse prognosis than those who do not. The effect of the presence of the protein is seen on disease-free interval (chi-square, 11.69; P < .001), overall survival (chi-square, 19.68; P < .001), and survival after relapse (chi-square, 4.93; P < .02), and is seen in node-negative (chi-square, 6.99; P < .009) and node-positive (chi-square, 13.05; P < .001) patients. Furthermore, the effect is most apparent in patients with infiltrating lobular and grade II infiltrating ductal carcinomas (chi-square, 27.97; P < .001) that have a rather heterogeneous clinical behaviour and are difficult to subdivide on the basis of currently available markers. Cox multivariate analysis shows that p53 majority staining is second only to node status in significance of effect on overall survival.

Invasive lobular and invasive ductal carcinoma of the breast show distinct patterns of vascular endothelial growth factor expression and angiogenesis

Angiogenesis is essential for tumour growth and important in tumour metastasis and prognosis. Vascular endothelial growth factor (VEGF) stimulates endothelial proliferation in vitro and angiogenesis in vivo. VEGF expression has been correlated with high vascularity in tumours, including carcinoma of the breast. This study investigated VEGF expression and vascularity of invasive lobular (n=10) and invasive ductal carcinoma (n=28), and pure ductal carcinoma in situ of the breast (n=33). VEGF protein expression was studied with immunohistochemistry and VEGF mRNA with in situ hybridization. Vascular density was assessed on sections stained for von Willebrand factor. There was more expression of both VEGF protein (P=0·006) and mRNA (P=0·002) in invasive ductal than in invasive lobular carcinoma. VEGF protein (rs=0·32, P=0·047) and mRNA (rs=0·56, P=0·04) correlated with vascular density in invasive ductal carcinoma. In invasive lobular carcinoma, vascular density did not correlate with VEGF mRNA (rs=0·15, P=0·35) and was inversely related to VEGF protein (rs=−0·57, P=0·04). There were no significant differences in vascular density between the two types of invasive carcinoma, suggesting that VEGF is important in angiogenesis in invasive ductal carcinoma, but that other angiogenic factors are important in invasive lobular carcinoma. Although VEGF protein was frequently expressed in ductal carcinoma in situ, no relationship was found between VEGF and the two patterns of angiogenesis previously described.

Retinoblastoma and p16 proteins in mammary carcinoma: Their relationship to cyclin D1 and histopathological parameters

The cell cycle‐associated retinoblastoma protein (pRb) and p16 protein were demonstrated using immuno‐histochemistry on paraffin sections from 192 cases of invasive breast carcinoma. Abnormal expression of pRb was defined as negative staining and was seen in 17% of tumours. Such abnormal expression was significantly more frequent in tumours with negative oestrogen receptor (ER) status. There was also a trend for tumours which were negative for pRb to be grade III ductal carcinomas. There was no association between p16 staining and any histopathological parameter, though, surprisingly, log‐rank analysis showed that strong staining was associated with a poor outcome. There was a significant inverse relationship between pRb and p16 expression and a significant positive association between pRb and cyclin D1. In a Cox multivariate analysis, which included cyclin D1, neither pRb nor p16 was an independent predictor of patient outcome. Int. J. Cancer (Pred. Oncol.) 79:71–75, 1998.

Aberrant expression of p53 tumour‐suppressor protein in non‐melanoma skin cancer

Expression of the cellular p53 tumour‐suppressor protein was examined in 78 epidermal tumours, including basal and squamous cell carcinomas, keratoacanthomas, solar keratoses, Bowens disease and viral warts. An immunohistochemical study was employed using the antibody CM‐1, raised against recombinant human p55 protein. Positive staining for p53, not detectable in normal cells because wild‐type p53 is rapidly degraded, reflects abnormal stabilization of p53 protein, and in many cases suggests p53 gene mutation.

Immunohistochemical Expression of uPA, uPAR, and PAI-1 in Breast Carcinoma: Fibroblastic Expression Has Strong Associations with Tumor Pathology

The urokinase-type plasminogen activator (uPA) system has been implicated in tumor spread. We have used immunohistochemistry to examine three components of this system, ie, uPA, uPA receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1), in a pilot study on 142 cases of breast carcinoma. We wished to determine whether there were any relationships between expression of the proteins in either tumor cells or fibroblasts and clinical and pathological features. Strong uPA expression in each cell type was significantly related to high tumor grade (P = 0.013 and 0.008, respectively), and was more common in invasive than in in situ carcinomas (P < 0.0001). Fibroblastic expression of uPAR was only related to the presence of invasion (P < 0.0001). Strong PAI-1 expression in both cell types was seen in high-grade tumors (tumor cells, P = 0.012; fibroblasts, P < 0.001), but only fibroblastic expression was related to the presence of invasion (P = 0.042). Fibroblastic expression of both uPA and uPAR were positively correlated with tumor size. Although patients with strong fibroblastic expression of uPA showed a tendency toward a shorter time to relapse, none of the plasminogen activator proteins were significantly associated with relapse-free survival. These results suggest that strong expression of uPA, uPAR, and PAI-1 in fibroblasts rather than in tumor cells have the most impact on the clinical behavior of breast cancer. Larger prospective studies are needed to confirm these findings.

Primary mucinous carcinomas of the skin express TFF1, TFF3, estrogen receptor, and progesterone receptors.

Mucinous carcinoma may present at various sites, including the breast and the gastrointestinal tract. Rarely, such tumors arise within the skin. Comparatively, breast lesions are relatively common and usually associated with a good prognosis. When pure, they are typically estrogen (ER) and progesterone receptor (PR) positive and responsive to tamoxifen. The authors studied 12 mucinous carcinomas of the skin and compared the morphology with that of typical mammary lesions. The authors also evaluated for expression of estrogen receptor, progesterone receptor, and the mucus-associated peptides of the trefoil factor family (TFF), TFF1 (formerly pS2) and TFF2 (formerly SP), using immunohistochemistry. The localization of mRNAs for TFF1, TFF2, and TFF3 (formally ITF) was also studied in a subset of three tumors, using in-situ hybridization with S35 labeled riboprobes. The Grimelius stain was used to look for evidence of neuroendocrine differentiation. Eight resembled type A mucinous carcinomas of the breast, two resembled type B, and one had composite features. The 12th was a papillary neoplasm. The two type B tumors exhibited argyrophilia. All showed strong nuclear staining with the estrogen receptor antibody but a more varied pattern with antibodies to progesterone receptor and TFF1. None labeled for TFF2. The detection of TFF1 in mammalian skin is a novel finding. Cutaneous mucinous carcinoma shows strong similarities to its mammary counterpart, including expression of estrogen receptor, TFF1, and TFF3 mRNA. These observations suggest that some mucinous carcinomas of the skin might respond to antiestrogenic therapies.

p53 immunoreactivity in human malignant melanoma and dysplastic naevi.

Expression of the tumour suppressor protein, p53, was determined in 77 cutaneous melanocytic lesions, and in five lymph node metastases from malignant melanoma, in an immunohistochemical study employing CM‐1, an antiserum raised against recombinant human p53 protein. Because wildtype p53 protein is rapidly degraded in normal cells, p53 immunoreactivity suggests the presence of an abnormally stable p53 protein. This may occur through either post‐translational mechanisms or gene mutation.

Combination of microdissection and microarray analysis to identify gene expression changes between differentially located tumour cells in breast cancer

Comparison of gene expression changes between cancer cells at the periphery and in the centre of breast cancers was performed using a combination of microdissection and microarray analysis. Cancer cells from the two areas were pooled separately from five patients with ductal carcinoma in situ and separately from five patients with frankly invasive cancer. Limited total RNA, 100–200 ng, from this microdissected tissue required use of the Atlas SMART™ Probe Amplification Kit to synthesize and amplify cDNA and make 33P-labelled probes. Probes were then hybridized to Atlas Human Cancer 1.2 Arrays containing 1176 known genes. Triplicate analysis revealed that 22 genes changed their expression levels in the periphery relative to the central region: 15 upregulated and seven downregulated (arbitrary threshold of 1.5-fold or greater). Differences in RNA levels were confirmed by quantitative real-time PCR for two of the genes and by changes in protein levels, detected by immunohistochemistry, for a couple of representative gene products. Thus, changes in gene expression associated with variation in microanatomical location of neoplastic cells can be detected within even small developing tumour masses.

BAG-1 expression in human breast cancer: interrelationship between BAG-1 RNA, protein, HSC70 expression and clinico-pathological data

BAG‐1 (BCL‐2 athanogene‐1), a multifunctional protein which associates with steroid hormone receptors (including the oestrogen receptor) and the anti‐apoptotic BCL‐2 protein, regulates steroid hormone‐dependent transcription and apoptosis. Direct interaction with 70 kD heat‐shock proteins, HSC70 and HSP70, may mediate the diverse functions of BAG‐1. Immunohistochemistry was used to examine the expression of BAG‐1 and HSC70 in 160 cases of invasive breast cancer. BAG‐1 was expressed in 92% of cases; most tumours exhibited cytoplasmic BAG‐1, while a smaller proportion also had nuclear immunostaining. There was a significant inverse correlation between histological grade and nuclear BAG‐1 expression, with higher‐grade tumours tending to have reduced nuclear BAG‐1 expression, but there was no association with cytoplasmic BAG‐1. There was also no significant correlation between nuclear or cytoplasmic BAG‐1 expression and oestrogen receptor positivity. Since BAG‐1 may be influenced by hormonal background, the relationship between grade and oestrogen receptor was examined separately in pre‐menopausal and post‐menopausal women. The statistically significant correlation between nuclear BAG‐1 expression and low tumour grade was strong in pre‐menopausal, but not apparent in post‐menopausal women. A statistically significant correlation was observed between cytoplasmic, but not nuclear, BAG‐1 expression and oestrogen receptor status in pre‐menopausal, but not post‐menopausal, women. There was no correlation between BAG‐1 protein expression and RNA, suggesting that important post‐transcriptional mechanisms control BAG‐1 expression in vivo. HSC70 was also detected in the majority (97%) of cases, although expression was not correlated with BAG‐1 levels, oestrogen receptor status or tumour grade. Overall survival in cases with high levels of nuclear BAG‐1 expression was improved, though not significantly. These results are consistent with the hypothesis that BAG‐1 plays an important but variable role in breast cancers developing in pre‐menopausal and post‐menopausal women. Copyright

Posttransplant skin cancer: A possible role for p53 gene mutation but not for oncogenic human papillomaviruses*

BACKGROUND Loss of p53 tumor suppressor function is a critical step in the development of diverse malignancies, including skin cancers in nonimmunosuppressed patients where UV-specific p53 gene mutations have been identified. In tumors associated with human papillomavirus (HPV), such as cervical carcinoma, p53 may be inactivated instead by binding to a viral oncoprotein. OBJECTIVE Our purpose was to examine the hypothesis that HPV may play an analogous role in the development of posttransplant skin cancer. METHODS p53 Immunoreactivity, suggestive of p53 gene mutation, was examined by immunocytochemistry. Oncogenic HPV DNA was detected by polymerase chain reaction. RESULTS Comparable p53 immunoreactivity was seen in skin tumors from both transplant and nontransplant patients. HPV DNA was not demonstrated in any tumor specimen. CONCLUSION Our data do not implicate oncogenic HPV in posttransplant skin cancer. p53 Gene mutation, rather than HPV-induced p53 degradation, may be more significant in the development of these tumors.