Retinoic Acid-Binding Protein in Human Breast Cancer and Dysplasia

Seventy-five specimens of human breast tissue were checked for the presence of cellular retinoic acid-binding protein (cRABP). Fifty-two percent of the primary carcinomas and 43% of the dysplastic breast lesions (stage MIl) contained detectable amounts of cRABP, whereas no cRABP was found in normal tissue. Sucrose gradient centrifugation and electrophoresis on agarose were used for analysis of the presence of cRABP. The cRABP of human origin (normal uterus and neoplastic mammary tissue) differed in its mobility in agarose electrophoresis from that of rat testis cRA

Long-term outcomes of breast cancer patients after endoscopic axillary lymph node dissection: a prospective analysis of 52 patients

Summary: Background. Reports on long-term outcomes after endoscopic axillary lymph node dissection (ALND) of breast cancer patients are still lacking in the medical literature. The objective of this prospective study was to assess the oncological and functional outcomes in breast cancer patients after endoscopic ALND. Methods. Fifty-five breast cancer patients were prospectively enrolled, of whom 52 were available for follow-up with a median of 71.9 months (range 11-96). The following oncological and functional endpoints were evaluated during follow-up at several time points: occurrence of local, axillary and distant metastases, seroma or infection, shoulder mobility (range of motion), numbness, pain, presence of lymphoedema as well as restriction in activities of daily living. Results. In 52 patients endoscopic ALND of level I and II was successfully performed. Two port-site metastases (2/52, 4%) occurred, one of which in a patient with negative axillary lymph nodes. The same patient suffered from the only axillary recurrence (1/52, 2%). Three patients (3/52, 6%) developed lymphoedema. No other functional adverse events (shoulder mobility, pain, numbness, hypertrophic scar) were noticed at the end of the observation period. Conclusion. The present investigation with long-term follow-up after endoscopic ALND - the first one in the literature - reveals minor morbidity, good functional and cosmetic results. In contrary to conventional surgery, the endoscopic procedure is associated with the occurrence of port-site metastases, not seen in the open approach. Axillary recurrences do not appear more frequently when compared with results after conventional ALND. In the meantime the less invasive sentinel lymph node (SLN) biopsy is the established standard technique in evaluating the axillary lymph node statu

Patterns of HER-2/neu Amplification and Overexpression in Primary and Metastatic Breast Cancer

Background: Only 25% of patients with HER-2/neu-positive metastatic breast tumors respond favorably to trastuzamab (Herceptin) treatment. We hypothesized that a high failure rate of patients on trastuzamab could result if some of the metastases were HER-2 negative and these metastases ultimately determine the course of the disease. Methods: We used tissue microarrays (TMAs) containing four samples each from 196 lymph node-negative primary tumors, 196 lymph node-positive primary tumors, and three different lymph node metastases from each lymph node-positive tumor to estimate HER-2 gene amplification by fluorescence in situ hybridization (FISH) and Her-2 protein overexpression by immunohistochemistry (IHC). Results: FISH and IHC analyses gave the same result with respect to HER-2 status for 93.7% of the tissues contained in the TMAs. Tissue samples were, therefore, considered to be HER-2 positive if they were positive for either HER-2 DNA amplification or Her-2 protein expression and HER-2 negative if both FISH and IHC gave a negative result. The HER-2 status of lymph node-positive primary tumors was maintained in the majority of their metastases. For HER-2-positive primary tumors, 77% (95% confidence interval [CI] = 59% to 90%) had entirely HER-2-positive metastases, 6.5% (95% CI = 8% to 21%) had entirely HER-2-negative metastases, and 16.3% (95% CI = 5% to 34%) had a mixture of HER-2-positive and HER-2-negative metastases. For HER-2-negative primary tumors, 95% (95% CI = 88% to 98%) had metastases that were entirely negative for HER-2. Conclusions: Our data suggest that differences in HER-2 expression between primary tumors and their lymph node metastases cannot explain the high fraction of nonresponders to trastuzamab therap

Detecting Activation of Ribosomal Protein S6 Kinase by Complementary DNA and Tissue Microarray Analysis

Background: Studies by comparative genomic hybridization (CGH) have shown that chromosomal region 17q23 is amplified in up to 20% of primary breast cancers. We used microarray analyses to measure the expression levels of genes in this region and to explore their prognostic importance. Methods: A microarray that contained 4209 complementary DNA (cDNA) clones was used to identify genes that are overexpressed in the MCF-7 breast cancer cell line as compared with normal mammary tissue. Fluorescence in situ hybridization was used to analyze the copy number of one overexpressed gene, ribosomal protein S6 kinase (S6K), and to localize it to the 17q23 region. Northern and western blot analyses were used to measure S6K gene and protein expression, and an enzymatic assay was used to measure S6K activity. Tumor tissue microarray analysis was used to study amplification of S6K and the HER-2 oncogene, another 17q-linked gene, and the relationship between amplification and prognosis was analyzed. The Kaplan-Meier method was used for data analysis, and the log-rank test was used for statistical analysis. All P values are two-sided. Results: S6K was amplified and highly overexpressed in MCF-7 cells relative to normal mammary epithelium, and protein expression and enzyme activity were increased. S6K was amplified in 59 (8.8%) of 668 primary breast tumors, and a statistically significant association between amplification and poor prognosis (P = .0021) was observed. Amplification of both S6K and HER-2 implied particularly poor survival (P = .0001). Conclusions: The combination of CGH information with cDNA and tissue microarray analyses can be used to identify amplified and overexpressed genes and to evaluate the clinical implications of such genes and genomic rearrangements. S6K is likely to be one of the genes at 17q23 that is amplified during oncogenesis and may adversely affect the prognosis of patients with this amplificatio

Total loss of MHC class I is an independent indicator of good prognosis in breast cancer

Tumours can be recognised by CTL and NK cells. CTL recognition depends on expression of MHC Class I loaded with peptides from tumour antigens. In contrast, loss of MHC Class I results in NK activation. In our study a large set of samples from patients with primary operable invasive breast cancer was evaluated for the expression of MHC Class I heavy and light by immunohistochemical staining of 439 breast carcinomas in a tissue microarray. Forty-seven percent (206 of 439) of breast carcinomas were considered negative for HLA Class I heavy chain (HC10), whereas lack of anti-β2m-antibody staining was observed in 39% (167 of 424) of tumours, with only 3% of the β2m-negative tumours expressing detectable HLA Class I heavy chain. Correlation with patient outcome showed direct relationship between patient survival and HLA-negative phenotype (log rank = 0.004). A positive relationship was found between the intensity of expression of MHC Class I light and heavy chains expression and histological grade of invasive tumour (p < 0.001) and Nottingham Prognostic Index (p < 0.001). To investigate whether HLA Class I heavy and light chains expression had independent prognostic significance, Cox multivariate regression analysis, including the parameters of tumour size, lymph node stage, grade and intensity of HC10 and anti-β2m staining, was carried out. In our analysis, lymph node stage (p < 0.001), tumour grade (p = 0.005) and intensity of MHC Class I light and heavy chains expression were shown to be independent prognostic factors predictive of overall survival (p-values HC10 = 0.047 and β2m = 0.018)

Gene expression variation between distinct areas of breast cancer measured from paraffin-embedded tissue cores

BACKGROUND: Diagnosis and prognosis in breast cancer are mainly based on histology and immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) material. Recently, gene expression analysis was shown to elucidate the biological variance between tumors and molecular markers were identified that led to new classification systems that provided better prognostic and predictive parameters. Archived FFPE samples represent an ideal source of tissue for translational research, as millions of tissue blocks exist from routine diagnostics and from clinical studies. These should be exploited to provide clinicians with more accurate prognostic and predictive information. Unfortunately, RNA derived from FFPE material is partially degraded and chemically modified and reliable gene expression measurement has only become successful after implementing novel and optimized procedures for RNA isolation, demodification and detection. METHODS: In this study we used tissue cylinders as known from the construction of tissue microarrays. RNA was isolated with a robust protocol recently developed for RNA derived from FFPE material. Gene expression was measured by quantitative reverse transcription PCR. RESULTS: Sixteen tissue blocks from 7 patients diagnosed with multiple histological subtypes of breast cancer were available for this study. After verification of appropriate localization, sufficient RNA yield and quality, 30 tissue cores were available for gene expression measurement on TaqMan(R) Low Density Arrays (16 invasive ductal carcinoma (IDC), 8 ductal carcinoma in situ (DCIS) and 6 normal tissue), and 14 tissue cores were lost. Gene expression values were used to calculate scores representing the proliferation status (PRO), the estrogen receptor status and the HER2 status. The PRO scores measured from entire sections were similar to PRO scores determined from IDC tissue cores. Scores determined from normal tissue cores consistently revealed lower PRO scores than cores derived from IDC or DCIS of the same block or from different blocks of the same patient. CONCLUSION: We have developed optimized protocols for RNA isolation from histologically distinct areas. RNA prepared from FFPE tissue cores is suitable for gene expression measurement by quantitative PCR. Distinct molecular scores could be determined from different cores of the same tumor specimen

Prognostic Significance of High Expression of ER-beta in Surgically Treated ER-Positive Breast Cancer Following Endocrine Therapy

Purpose: This study evaluated estrogen receptor (ER)-beta mRNA and ER-beta protein expression and its prognostic implications in hormone receptor-positive breast cancer. Methods: Paraffin sections from 139 hormone receptor-positive breast cancer cases were prepared. The expression of ER-beta mRNA and protein were analyzed by branched-chain assay and immunohistochemistry (IHC), respectively. Results: The Allred score of ER-beta IHC was correlated with smaller tumor size (p=0.043), the Allred score of ER-alpha IHC (p&lt;0.001), and the Allred score of progesterone receptor (PR) IHC (p=0.022) but not with the HER2 IHC score. ER-beta mRNA level was correlated with PR mRNA levels (p&lt;0.001) but not with the Allred score of ER-beta IHC, ER-alpha IHC, and PR IHC, nor with the HER2 IHC score and ER-alpha mRNA level. In survival analysis, high expression of ER-beta mRNA was associated with worse disease-free survival along with poor differentiation, lymph node metastasis and absence of PR protein expression in univariate analysis (p = 0.040, p = 0.002, p = 0.018, and p = 0.007, respectively) and multivariate analysis (p = 0.044, p = 0.002, p = 0.035, and p = 0.007, respectively). Conclusion: High expression of ER-beta mRNA is an independent predictor of disease recurrence in hormone-receptorpositive breast cancer

Reduced expression of the polymeric immunoglobulin receptor in pancreatic and periampullary adenocarcinoma signifies tumour progression and poor prognosis

The polymeric immunoglobulin receptor (pIgR) is a key component of the mucosal immune system that mediates epithelial transcytosis of immunoglobulins. High pIgR expression has been reported to correlate with a less aggressive tumour phenotype and an improved prognosis in several human cancer types. Here, we examined the expression and prognostic significance of pIgR in pancreatic and periampullary adenocarcinoma. The study cohort encompasses a consecutive series of 175 patients surgically treated with pancreaticoduodenectomy for pancreatic and periampullary adenocarcinoma in Malmö and Lund University Hospitals, Sweden, between 2001-2011. Tissue microarrays were constructed from primary tumours (n = 175) and paired lymph node metastases (n = 105). A multiplied score was calculated from the fraction and intensity of pIgR staining. Classification and regression tree analysis was used to select the prognostic cut-off. Unadjusted and adjusted hazard ratios (HR) for death and recurrence within 5 years were calculated. pIgR expression could be evaluated in 172/175 (98.3%) primary tumours and in 96/105 (91.4%) lymph node metastases. pIgR expression was significantly down-regulated in lymph node metastases as compared with primary tumours (p = 0.018). Low pIgR expression was significantly associated with poor differentiation grade (p < 0.001), perineural growth (p = 0.027), lymphatic invasion (p = 0.016), vascular invasion (p = 0.033) and infiltration of the peripancreatic fat (p = 0.039). In the entire cohort, low pIgR expression was significantly associated with an impaired 5-year survival (HR = 2.99, 95% confidence interval (CI) 1.71-5.25) and early recurrence (HR = 2.89, 95% CI 1.67-4.98). This association remained significant for survival after adjustment for conventional clinicopathological factors, tumour origin and adjuvant treatment (HR = 1.98, 95% CI 1.10-3.57). These results demonstrate, for the first time, that high tumour-specific pIgR expression signifies a more favourable tumour phenotype and that low expression independently predicts a shorter survival in patients with pancreatic and periampullary cancer. The mechanistic basis for the putative tumour suppressing properties of pIgR in these cancers merits further study

Automated detection of regions of interest for tissue microarray experiments: an image texture analysis

BACKGROUND: Recent research with tissue microarrays led to a rapid progress toward quantifying the expressions of large sets of biomarkers in normal and diseased tissue. However, standard procedures for sampling tissue for molecular profiling have not yet been established. METHODS: This study presents a high throughput analysis of texture heterogeneity on breast tissue images for the purpose of identifying regions of interest in the tissue for molecular profiling via tissue microarray technology. Image texture of breast histology slides was described in terms of three parameters: the percentage of area occupied in an image block by chromatin (B), percentage occupied by stroma-like regions (P), and a statistical heterogeneity index H commonly used in image analysis. Texture parameters were defined and computed for each of the thousands of image blocks in our dataset using both the gray scale and color segmentation. The image blocks were then classified into three categories using the texture feature parameters in a novel statistical learning algorithm. These categories are as follows: image blocks specific to normal breast tissue, blocks specific to cancerous tissue, and those image blocks that are non-specific to normal and disease states. RESULTS: Gray scale and color segmentation techniques led to identification of same regions in histology slides as cancer-specific. Moreover the image blocks identified as cancer-specific belonged to those cell crowded regions in whole section image slides that were marked by two pathologists as regions of interest for further histological studies. CONCLUSION: These results indicate the high efficiency of our automated method for identifying pathologic regions of interest on histology slides. Automation of critical region identification will help minimize the inter-rater variability among different raters (pathologists) as hundreds of tumors that are used to develop an array have typically been evaluated (graded) by different pathologists. The region of interest information gathered from the whole section images will guide the excision of tissue for constructing tissue microarrays and for high throughput profiling of global gene expression