Jarle B. Arnes

Expression of Enhancer of Zeste Homologue 2 Is Significantly Associated with Increased Tumor Cell Proliferation and Is a Marker of Aggressive Breast Cancer

The polycomb group protein enhancer of zeste homologue 2 (EZH2) has been linked to invasive properties of aggressive breast cancer. In this report, tissue microarray analysis of 190 breast carcinomas from a nested case-control study shows that EZH2 is significantly associated with interval breast cancers. Further, a strong relationship was found with tumor cell proliferation (by Ki-67 expression), locally advanced disease, metastasis at presentation, markers of the basal epithelial phenotype (positivity for cytokeratin 5/6 or P-cadherin), and p53 status. EZH2 expression was also significantly associated with glomeruloid microvascular proliferation, an aggressive angiogenic phenotype. For prediction of aggressive disease (any event of locally advanced disease, lymph node spread, or distant spread), EZH2 was the only variable of significance in multivariate analysis, whereas no additional information was given by Ki-67. Although EZH2 expression was significant in univariate survival analysis, only tumor cell proliferation and lymph node status were significant in the final multivariate model. In conclusion, our findings indicate an important relationship not only between EZH2 and markers of tumor cell proliferation but also with aggressive disease. These findings might be practically important and relevant because the polycomb group proteins have recently been suggested as candidates for targeted therapy.

Placental cadherin and the basal epithelial phenotype of BRCA1-related breast cancer.

Purpose:BRCA1-related breast cancer frequently has a basal epithelial phenotype, and P-cadherin is a basal marker. We undertook a detailed evaluation of the relationship among P-cadherin, prognostic markers in breast cancer, and outcome. Experimental Design: This study was restricted to 292 cases of first primary invasive breast cancer diagnosed in Ashkenazi Jewish women between 1980 and 1995. All available blocks were stained for P-cadherin, and 261 were included in the final statistical analyses, including 27 germ line BRCA1 mutation carriers and 8 BRCA2 mutation carriers. Descriptive analyses were done followed by survival analyses and a Poisson regression analysis. Results: P-cadherin was present in 80 of the 261 breast cancers (31%) and was more frequently present in tumors that have a basal epithelial phenotype [i.e., high-grade, estrogen receptor– and KIP1 (p27Kip1)–negative tumors, with expression of cytokeratin 5/6, cyclin E, TP53, and presence of BRCA1 mutations and vascular nests (all P < 0.001)]. In a univariate survival model, expression of P-cadherin was associated with a relative risk (RR) of death from breast cancer at a 10-year follow-up of 2.9 (95% confidence interval, 1.8-4.7; P < 0.0001) and was a predictor of poor univariate survival in both lymph node–negative and –positive breast cancers. In a multivariate analysis, the effect of P-cadherin levels was not independent of other basal-related markers. Multivariable interaction modeling showed that P-cadherin positivity was highly predictive of a poor prognosis in small, node-negative breast cancers (RR, 7.1; P = 0.006). Conclusions: P-cadherin is a marker for basal-like breast cancers and is strongly associated with the presence of a BRCA1 mutation. It is an adverse prognostic factor, particularly in small, node-negative breast cancers.

Early detection of breast cancer based on gene-expression patterns in peripheral blood cells

IntroductionExisting methods to detect breast cancer in asymptomatic patients have limitations, and there is a need to develop more accurate and convenient methods. In this study, we investigated whether early detection of breast cancer is possible by analyzing gene-expression patterns in peripheral blood cells.MethodsUsing macroarrays and nearest-shrunken-centroid method, we analyzed the expression pattern of 1,368 genes in peripheral blood cells of 24 women with breast cancer and 32 women with no signs of this disease. The results were validated using a standard leave-one-out cross-validation approach.ResultsWe identified a set of 37 genes that correctly predicted the diagnostic class in at least 82% of the samples. The majority of these genes had a decreased expression in samples from breast cancer patients, and predominantly encoded proteins implicated in ribosome production and translation control. In contrast, the expression of some defense-related genes was increased in samples from breast cancer patients.ConclusionThe results show that a blood-based gene-expression test can be developed to detect breast cancer early in asymptomatic patients. Additional studies with a large sample size, from women both with and without the disease, are warranted to confirm or refute this finding.

Independent prognostic value of the basal-like phenotype of breast cancer and associations with EGFR and candidate stem cell marker BMI-1

Aims:  To study the relationship between basal‐like breast cancers, epidermal growth factor receptor (EGFR) and candidate stem cell markers (BMI‐1, EZH2, Oct‐4) in a population‐based setting.

Frequency of the basal-like phenotype in African breast cancer

Basal‐like breast carcinoma has been recognized as a subtype with specific prognostic implications. However, there is a lack of reports about this category of breast tumors in African women. The aim of this study was to explore the basal‐like phenotype in breast cancer patients in an African population, and a registry‐based series was included from the well‐defined Kyadondo County in Uganda (1.7 millions). We studied a total of 65 archival paraffin blocks of invasive breast cancer using antibodies against cytokeratin 5/6 and P‐cadherin, and these markers were expressed in 34% of all cases and in 52% of ER (estrogen receptor)‐negative tumors. All basal‐like tumors were ER negative (p<0.0005) and PR (progesterone receptor) negative (p=0.002). Basal‐like breast carcinomas were of a higher histologic grade (p=0.001), had high mitotic counts (p=0.002), and marked nuclear pleomorphism (p=0.002). P‐cadherin‐positive tumors had a high Ki‐67 proliferative rate (p=0.039). In conclusion, the basal‐like phenotype is frequent in this African series of breast cancer and is strongly associated with poor prognostic factors. Our findings might be significant in relation to clinical management of these patients, including novel targeted therapy.

Evaluation of Ki67 Expression across Distinct Categories of Breast Cancer Specimens: A Population-Based Study of Matched Surgical Specimens, Core Needle Biopsies and Tissue Microarrays

Introduction Tumor cell proliferation in breast cancer is strongly prognostic and may also predict response to chemotherapy. However, there is no consensus on counting areas or cut-off values for patient stratification. Our aim was to assess the matched level of proliferation by Ki67 when using different tissue categories (whole sections, WS; core needle biopsies, CNB; tissue microarrays, TMA), and the corresponding prognostic value. Methods We examined a retrospective, population-based series of breast cancer (n = 534) from the Norwegian Breast Cancer Screening Program. The percentage of Ki67 positive nuclei was evaluated by visual counting on WS (n = 534), CNB (n = 154) and TMA (n = 459). Results The median percentage of Ki67 expression was 18% on WS (hot-spot areas), 13% on CNB, and 7% on TMA, and this difference was statistically significant in paired cases. Increased Ki67 expression by all evaluation methods was associated with aggressive tumor features (large tumor diameter, high histologic grade, ER negativity) and reduced patient survival. Conclusion There is a significant difference in tumor cell proliferation by Ki67 across different sample categories. Ki67 is prognostic over a wide range of cut-off points and for different sample types, although Ki67 results derived from TMA sections are lower compared with those obtained using specimens from a clinical setting. Our findings indicate that specimen specific cut-off values should be applied for practical use.

Expression of EGFR and c-kit is associated with the basal-like phenotype in breast carcinomas of African women

Epidermal growth factor receptor (EGFR) and c‐kit are tyrosine kinase growth factor receptors which are frequently expressed in basal‐like breast carcinomas, and tyrosine kinase inhibition is now a promising strategy in treatment of breast cancer. The aim of this study was to evaluate the expression of EGFR and c‐kit in breast cancer with special focus on the basal‐like phenotype (BLP) and other prognostic factors in an African population. We analyzed 65 archival tissues immunohistologically. EGFR and/or c‐kit were expressed in 55% of basal‐like tumors. Expression of EGFR and/or c‐kit was strongly associated with high histologic grade (P=0.001), high nuclear grade (P=0.017), high mitotic counts (P=0.002), ER negativity (P=0.003), PR negativity (P=0.007), and HER2 negativity (P=0.014). EGFR and/or c‐kit positive tumors were more likely to express the BLP (OR 9.1, CI 2.6–32.0, P<0.0005) than the negative tumors. In conclusion, there is a high expression of EGFR and/or c‐kit in basal‐like breast carcinoma in this series from Uganda and their expression is associated with features of poor prognosis. More studies are required to assess the clinical significance of EGFR and c‐kit in breast cancer patients in Uganda.

Expression of epidermal growth factor receptor in relation to BRCA1 status, basal-like markers and prognosis in breast cancer.

Aims: BRCA1-related breast cancer is associated with a basal-like phenotype, and is frequently oestrogen receptor (ER) and HER2 negative. The expression of epidermal growth factor receptor (EGFR) has been considered to be one component of the basal-like phenotype, but no standard criteria exist. This study investigates the relationship between EGFR expression, BRCA1 status and basal markers with respect to clinicopathological associations and prognosis, in addition to evaluating different criteria for EGFR assessment by immunohistochemistry. Methods: A tissue microarray comprising 230 available cases, from a series of primary invasive breast cancer diagnosed in Ashkenazi Jewish women during 1980–1995, was stained for EGFR using the Dako PharmDX kit, and evaluated by Webslide virtual microscopy. Results: EGFR was positive in 9–19% according to different criteria. Expression was associated with BRCA1 carrier status and basal-like markers as negative ER, positive cytokeratin 5/6 and positive P-cadherin staining. EGFR was prognostically significant by univariate and multivariate analysis within the group carrying germ-line BRCA1 mutations. Histological grade, axillary lymph node status and P-cadherin status had significant independent value in the final multivariate model including all cases, whereas EGFR was not significant in this model. All five scoring systems gave comparable results concerning clinicopathological associations and patient outcome, although the most restrictive criteria (EGFR-HI) tended to be most sensitive in predicting BRCA1 status, a basal phenotype, and patient prognosis. Conclusions: EGFR expression, being present in 9–19% of the cases, was prognostically significant among BRCA1 mutated cases only. In multivariate survival analysis of all cases, no independent effect was seen. However, EGFR immunostaining might be relevant to predict the response to targeted therapy, and this should be studied further.

Vascular proliferation is a prognostic factor in breast cancer

Angiogenesis is important for the growth and spread of malignant tumors, and anti-angiogenesis treatment is currently being evaluated for breast cancer and other tumors. Although microvessel density is the most commonly used tissue-based marker of tumor associated angiogenesis, it has significant limitations and has not proven effective as a predictive factor in selecting patients for treatment. We here wanted to explore the significance of vascular endothelial cell proliferation in breast carcinoma. We examined microvessel proliferation in breast cancer by dual immunohistochemical staining, using the pan-endothelial marker Factor-VIII combined with proliferation of endothelial cells by Ki-67 expression, in three independent series of breast cancer, including a total of 499 patients and 141 events during follow-up. Common statistical tests of associations as well as univariate and multivariate regression analysis of patient survival were used. By counting vessels with actively proliferating endothelium, we show that microvascular proliferation is a significant predictor of disease progression in breast cancer, especially among high-grade and ER-negative tumors. Our findings indicate that this novel marker of active tumor angiogenesis might be of value in patient management and should be further studied in the context of patient selection for anti-angiogenesis treatment.

Strong Expression of Hypoxia-Inducible Factor-1α (HIF-1α) Is Associated with Axl Expression and Features of Aggressive Tumors in African Breast Cancer.

Purpose Inhibition of hypoxia-inducible factor (HIF) and Axl receptor tyrosine kinase is being evaluated for targeted therapy in solid tumors. Both HIF-1α and Axl influence tumor growth and metastatic potential, and they have been linked to treatment failure in many cancers. However, there is a lack of reports on HIF-1α expression in African breast cancer, which has a poor prognosis, and novel treatment targets must therefore be established. Here, we aimed to evaluate HIF-1α in relation to Axl expression, angiogenesis markers, and other tumor characteristics in a series of African breast cancer. Methods Using immunohistochemistry, we examined 261 invasive breast cancers on tissue microarrays for HIF-1α and Axl as well as several other markers, and a subset of 185 cases had information on VEGF (vascular endothelial growth factor) expression, microvessel density (MVD), proliferating microvessel density (pMVD) and vascular proliferation index (VPI) for important comparisons. Results Strong HIF-1α expression was associated with increased Axl (p = 0.007), VEGF (p<0.0005), and p53 (p = 0.032) expression, as well as high tumor cell proliferation by Ki-67 (p = 0.006), and high tumor grade (p = 0.003). Tumors with strong HIF-1α expression had significantly higher MVD (p = 0.019) and higher pMVD (p = 0.027) than tumors with weak expression. Conclusions High HIF-1α expression is significantly associated with Axl and VEGF expression, and with markers of poor prognosis in this series of breast cancer, suggesting HIF-1α and Axl as potential therapeutic targets in African breast cancer.