Expression of CD151/Tspan24 and integrin alpha 3 complex in aid of prognostication of HER2-negative high-grade ductal carcinoma in situ.

The pro-tumorigenic and pro-metastatic functions of the tetraspanin protein CD151 (Tspan24) are thought to be dependent on its ability to form complexes with laminin-binding integrin receptors (i.e. alpha6beta1, alpha3beta1, alpha6beta4). We have previously reported that in invasive ductal carcinoma (IDC), CD151/alpha3beta1 complex was of prognostic value in patients with HER2-negative tumors. Extrapolating these findings to the pre-invasive setting, we aimed to make an assessment of a potential relationship between expression of the CD151/alpha3beta1 complex in DCIS and Van Nuys prognostic index (VNPI) in high-grade ductal carcinoma in situ (DCIS) in relation to the HER2 status. Protein distributions were analyzed in 49 samples of pure DCIS using immunohistochemistry. For each case immunoreactivity was assessed in at least 5 ducts (325 ducts in total) and an average score was taken for statistical analyses. When analyzed in the whole cohort, there was no statistical association between the VNPI and any of the proteins scored either separately or in combination. When stratified according to the HER2 status, in the HER2-negative subgroup, CD151 assessed in combination with alpha3beta1 was significantly correlated with VNPI (P = 0.044), while neither protein analyzed individually showed any significant link with the prognostic index. Expression of the CD151/alpha3beta1 complex in HER2-negative DCIS might reflect tumor behavior relevant to the patient outcome and thus might aid prognostication of the disease.

Prognostic value of HER3, PTEN and p-HER2 expression in patients with HER2positive breast cancer.

BACKGROUND: HER2 overexpression is an unfavorable prognostic factor in patients with breast cancer, but it is also a target for the monoclonal antibody trastuzumab, which is effective in adjuvant and palliative settings. HER2 positivity is an inclusion criterion for immunotherapy, but it is not a positive predictive factor, and only half of patients benefit from the treatment. AIM: The aim of this study was to evaluate the prognostic and predictive value of HER3, PTEN and phosphorylated HER2 (p-HER2) expression in primary breast tumors of patients treated with trastuzumab in an adjuvant or palliative regimen. MATERIAL/METHODS: Immunohistochemical (IHC) analysis with 3 antibodies specific to the proteins was performed in tumor specimens obtained from 81 HER2-positive patients treated with trastuzumab. RESULTS: HER3 overexpression was present in 55.6% of the examined tumors, and PTEN or pHER2 positivity was present in 32.0% and 34.6% of them, respectively. HER3 overexpression and PTEN positivity correlated with larger tumor size (p=0.016 and p=0.008, respectively). p-HER2 positivity correlated with more advanced clinical stage of the disease (p=0.032). There was no correlation between the proteins' expression and survival for 31 patients treated with trastuzumab in the palliative regimen. DISCUSSION: HER3 overexpression, PTEN positivity and p-HER2 positivity in tumor cells of HER2-positive patients correlate with more advanced clinical stage of breast cancer. Expression of these proteins does not predict outcome of trastuzumab treatment.

Association of the Arg194Trp and the Arg399Gln polymorphisms of the XRCC1 gene with risk occurrence and the response to adjuvant therapy among Polish women with breast cancer.

BACKGROUND: The XRCC1 gene encoding the X-ray cross-complementing group 1 protein (XRCC1) is involved in the base excision repair (BER) pathway. METHODS: The aim of this study was to investigate an association of the Arg194Trp and Arg399Gln polymorphisms of the XRCC1 gene with a risk of breast cancer occurrence and the response to adjuvant treatment among Polish women. Overall survival (OS) and disease-free survival (DFS) were investigated in groups of patients with breast cancer treated with (1) all types of adjuvant therapy, (2) concomitant radiotherapy and chemotherapy, (3) chemotherapy alone, or (4) radiotherapy alone. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was used to evaluate the genotype distribution of the XRCC1 gene among 185 patients with breast cancer and 205 female controls. RESULTS: We showed a higher risk of breast cancer occurrence for the Trp allele and the Arg194Trp genotype of the XRCC1 gene. However there was no significant difference in distribution of the Arg399Gln genotype of XRCC1 between patients and the control group. In the patient subgroup treated with adjuvant therapy, Kaplan-Meier survival analysis showed a significantly higher OS as well as DFS for carriers of the Gln399Gln genotype when compared with carriers of the Arg399Gln and Arg399Arg genotypes. The Gln399Gln genotype was associated with a significantly higher DFS in the subgroup of patients treated with chemotherapy alone or with concomitant radiotherapy and chemotherapy. CONCLUSION: We suggest that the polymorphism of the XRCC1 gene may be considered a predictive factor associated with the risk of occurrence and the survival outcome in breast cancer among Polish women.

Influence of genomic variation in FTO at 16q12.2, MC4R at 18q22 and NRXN3 at 14q31 genes on breast cancer risk.

Breast cancer is a major cause of cancer-related deaths in women. It is known that obesity is one of the risk factors of breast cancer. The subject of our interest was genes: FTO, MC4R and NRXN3-associated with obesity. In this study we have analyzed frequencies of genomic variants in FTO, MC4R and NRXN3 in the group of 134 breast cancer patients. We genotyped two polymorphic sites located in FTO gene (rs993909 and rs9930506), one polymorphic site of MC4R gene (rs17782313) and one polymorphic site of NRXN3 gene (rs10146997). Our hypothesis was that above mentioned SNPs could participate in carcinogenesis. Our research has showed that only rs10146997 was significantly (P = 0.0445) associated with higher risk of breast cancer development (OR = 0.66 (95% CI 0.44-0.99)). Moreover, G allele carriers in rs10146997 of the NRXN3 gene were the youngest patients at onset of breast cancer. On the basis of our research we suggest that further functional may elucidate the role of genomic variation in breast cancer development.

Analysis of cancer tissues by means of spectroscopic methods.

The personalized approach in cancer treatment stimulates the search for new analytical techniques, including spectroscopic methods such as Raman spectroscopy, mass spectrometry MALDI (matrix-assisted laser desorption/ionization) imaging and high-resolution magic angle spinning nuclear magnetic resonance (HR MAS NMR). The purpose of these studies is determination of metabolic profiles of cancer tissues, and their application in diagnostics and therapy of cancers. The review is mainly focused on application of HR MAS NMR technique. Qualitative and quantitative analysis of metabolites by means of this method is described for breast cancer tissues. In the near future HR MAS NMR in vitro studies of metabolic profiles combined with in vivo studies using MRI scanners may be applied as a new diagnostic tool.

Basal keratin expression in breast cancer by quantification of mRNA and by immunohistochemistry.

Definitions of basal-like breast cancer phenotype vary, and microarray-based expression profiling analysis remains the gold standard for the identification of these tumors. Immunohistochemical identification of basal-like carcinomas is hindered with a fact, that on microarray level not all of them express basal-type cytokeratin 5/6, 14 and 17. We compared expression of cytokeratin 5, 14 and 17 in 115 patients with operable breast cancer estimated by real-time RT-PCR and immunohistochemistry. Despite the method of dichotomization and statistical analysis, there were cases with discordant results comparing immunohistochemistry and RT-PCR analysis. For dichotomisation based on quartiles and ROC, 14% of cases were negative on immunohistochemical examination for CK5/6, but presented high CK5 mRNA levels. There were also 48-55% cases, which were CK5/6-immunopositive, but were negative by mRNA examination. Similar discordances were observed for CK14 and CK17. Basal keratin mRNAs did not correlate with ER mRNA levels, while immunohistochemistry produced significant relationship with ER status. Our observation suggest that both method may produce different results in a small proportion of cases. Discordance between immunohistochemistry and RT-PCR may confound attempts to establish a simple methods for identification of basal-like tumors.

Breast cancer relapse prediction based on multi-gene RT-PCR algorithm.

BACKGROUND: Breast cancer is very heterogeneous disease at both the clinical and molecular levels. Most research is based on analysis of a single gene, but only complex investigation of genes involved in different cell processes such as apoptosis or signal transduction can help to better understand the biology of this type of tumour. Novel techniques such as microarrays and real-time RT-PCR allow performance of such complex research. Only this kind of approach can improve cancer treatment through individualisation of disease cases with different molecular backgrounds. MATERIAL/METHODS: We performed quantitative RT-PCR to analyze levels of expression of 10 genes in 119 patient samples: 4 with known good prognosis signature (WWOX, ESR1, CDH, BAX) and 6 previously reported as bad prognosis markers of breast cancer (KRT5, KRT14, KRT17, CCNE1, BCL2, BIRC5). RESULTS: The algorithm composed of 10 genes distinguishes 2 statistically significant groups of patients with different rates of disease-free survival. However, when patients were divided into 2 groups according to estrogen receptor status, this algorithm could be applied only for a group with estrogen receptor negative breast cancer. High algorithm value is a good prognostic factor of disease-free survival for patients with estrogen negative breast cancers (HR=0.26; p=0.0039), but not for patients with ER positive tumors (p>0.05). CONCLUSIONS: The presented multigene algorithm may be used for outcome evaluation for estrogen receptor-negative breast cancer patients.

Detection of P53 mutations in different cancer types is improved by cDNA sequencing.

Recently published data show discrepancies between P53 cDNA and DNA sequencing results in glioblastoma, colorectal cancer and pleomorphic xanthoastrocytoma. We hypothesized that similar discrepancies are observed in other types of human cancers. Using DNA and cDNA direct sequencing, we analyzed 40 cases of invasive breast duct carcinoma, 23 cases of acute myeloblastic leukaemia, 12 cases of astrocytoma and 40 cases of soft tissue sarcoma for P53 mutations. Additionally, we used real-time quantitative PCR to estimate the normalized relative P53 expression. In the comparative study, the P53 mutation was detected more frequently when using cDNA sequencing than DNA sequencing in all of the cancer types. Furthermore, several samples presented missense P53 mutations, with visible wild-type nucleotide on the DNA sequence. In contrast, elimination of the wild-type allele or selective overproduction of the mutated allele was observed on the cDNA sequence. P53 expression was not significantly different between the cases with or without P53 mutations. These results indicate that cDNA sequencing improves the detection of P53 mutations in these cancers. We suggest that the true incidence of P53 mutations in these cancers is underestimated at the DNA level, and evaluation of the alteration should be carried out using cDNA analysis.

Does vimentin help to delineate the so-called 'basal type breast cancer'?

BACKGROUND: Vimentin is one of the cytoplasmic intermediate filament proteins which are the major component of the cytoskeleton. In our study we checked the usefulness of vimentin expression in identifying cases of breast cancer with poorer prognosis, by adding vimentin to the immunopanel consisting of basal type cytokeratins, estrogen, progesterone, and HER2 receptors. METHODS: 179 tissue specimens of invasive operable ductal breast cancer were assessed by the use of immunohistochemistry. The median follow-up period for censored cases was 90 months. RESULTS: 38 cases (21.2%) were identified as being vimentin-positive. Vimentin-positive tumours affected younger women (p = 0.024), usually lacked estrogen and progesterone receptor (p < 0.001), more often expressed basal cytokeratins (<0.001), and were high-grade cancers (p < 0.001). Survival analysis showed that vimentin did not help to delineate basal type phenotype in a triple negative (ER, PgR, HER2-negative) group. For patients with 'vimentin or CK5/6, 14, 17-positive' tumours, 5-year estimated survival rate was 78.6%, whereas for patients with 'vimentin, or CK5/6, 14, 17-negative' tumours it was 58.3% (log-rank p = 0.227). CONCLUSION: We were not able to better delineate an immunohistochemical definition of basal type of breast cancer by adding vimentin to the immunopanel consisted of ER, PgR, HER2, CK5/6, 14 and 17 markers, when overall survival was a primary end-point.

CD151 regulates tumorigenesis by modulating the communication between tumor cells and endothelium.

The tetraspanin CD151 forms stoichiometric complexes with laminin-binding integrins (e.g., alpha3beta1, alpha6beta1, and alpha6beta4) and regulates their ligand-binding and signaling functions. We have found that high expression of CD151 in breast cancers is associated with decreased overall survival (3.44-fold higher risk of death). Five-year estimated survival rates were 45.8% (95% confidence interval, 16.4-71.4%) for CD151-positive patients and 79.9% (95% confidence interval, 62.2-90.0%) for CD151-negative patients. Furthermore, CD151 was positively associated with axillary lymph node involvement. To study the biological significance of this observation, we investigated the contribution of CD151 in breast cancer tumorigenesis using MDA-MB-231 cells as a model system. Stable down-regulation of this tetraspanin by short-hairpin RNA decreased the tumorigenicity of these cells in mice. Detailed immunohistologic analysis of CD151(+) and CD151(-) xenografts showed differences in tumor vascular pattern. Vascularization observed at the subcutaneous border of the CD151(+) tumors was less pronounced or absent in the CD151(-) xenografts. In vitro experiments have established that depletion of CD151 did not affect the inherent proliferative capacity of breast cancer cells in three-dimensional extracellular matrices, but modified their responses to endothelial cells in coculture experiments. The modulatory activity of CD151 was dependent on its association with both alpha3beta1 and alpha6beta4 integrins. These data point to a new role of CD151 in tumorigenesis, whereby it functions as an important regulator of communication between tumor cells and endothelial cells. These results also identify CD151 as a potentially novel prognostic marker and target for therapy in breast cancer.

Prognostic relevance of cyclin E expression in operable breast cancer.

BACKGROUND: Cyclin E is an important regulator of cell-cycle progression. High levels of cyclin E protein in breast cancer have been reported in association with higher disease stage, poor histological differentiation of tumor, and lack of steroid receptors. Data concerning the prognostic relevance of cyclin E expression in breast cancer are conflicting. The aim of this retrospective study was to evaluate the prognostic relevance of cyclin E expression assessed by immunohistochemistry in patients with operable invasive ductal breast cancer. MATERIAL/METHODS: The expression of cyclin E was analyzed by immunostaining in 174 women with breast cancer after radical mastectomy with a median follow-up period of 58 months. Tumor samples were judged to be negative (<2%) or positive (> or =2%) according to the percentage of cells showing the nuclear staining pattern. Ninety-nine (56.9%) tumor samples were regarded as cyclin E-positive. RESULTS: Positive staining for cyclin E determined poor prognosis compared with cyclin E-negative patients in all cases (five-year cancer-specific survival rate of 64.5 vs. 84.5%, p=0.005), in the node-positive group (50.9 vs. 82.1%, p=0.008), and in patients treated with adjuvant chemotherapy (71.0 vs. 96.6%, p=0.008). In a multivariate analysis, high expression of cyclin E was associated with a higher risk of death in the node-positive group (hazard ratio: 3.2, 95%CI: 1.3-8.2, p=0.015). CONCLUSIONS: It was demonstrated that a high expression of cyclin E measured by immunohistochemistry was a significant factor of poor prognosis, especially in the node-positive group.

Recurrence of cholangiogenous carcinoma in port-sites two years after laparoscopic removal of noncancerous gallbladder.

We present a unique case of carcinoma diagnosed in port-site, two years after uncomplicated laparoscopic cholecystectomy for benign cholecystitis. Analysis of morphology and cytokeratin profile (CK19+ and CK20+/-) of resected port-site tumor allows us to establish the diagnosis of tubular carcinoma with probable cholangiogenic origin. The primary carcinoma was not diagnosed in archival gallbladder tissue, despite repeated histological examination. No other primary tumor was identified during follow-up. Patient history and histological/immunohistochemical picture of the recurrent tumor suggested that primary carcinoma was probably located in the gallbladder, but was not detected during initial and repeated histological examinations of postoperative specimen. The patient is still alive, 12 months after the first port-site recurrence and 36 months after initial laparoscopy.

Relationship of P-cadherin expression to basal phenotype of breast carcinoma.

BACKGROUND: P-cadherin (P-CD) is a molecule expressed mainly by basal cells involved in cell adhesion. We evaluated expression of P-CD in operable breast carcinomas and its relationship with immunohistochemical markers of the basal-like phenotype and with clinical outcome. MATERIAL AND METHODS: Expression of P-CD was analyzed by immunohistochemistry in 194 tissue specimens of invasive operable ductal breast cancer. RESULTS: 112 cases (57.7%) were identified as being P-CD-positive. P-CD-positive tumors usually lacked steroid receptors (p = 0.042), expressed basal type cytokeratins (p = 0.001), and were positive for cyclin E (p = 0.039). In a univariate analysis of cancer-specific survival with a median follow-up period of 58 months, P-CD expression was not associated with prognosis (5-year survival rate for positive vs. negative patients 67.0 vs. 77.0%, log rank p = 0.121). CONCLUSION: P-CD may be regarded as an additional immunohistochemical marker of basal-like breast carcinomas. However, P-CD expression is not an adverse prognostic factor.

A comparative assessment of HER2 status in operable breast cancer by real-time RT-PCR and by immunohistochemistry.

BACKGROUND: The most commonly used methods for detecting HER2 gene amplification in breast cancer are immunohistochemistry and fluorescence in-situ hybridization. The aim of this retrospective study was to assess HER2 expression by real-time RT-PCR. MATERIAL/METHODS: Expression of HER2 was analyzed by real-time RT-PCR and immunohistochemistry in specimens of invasive ductal breast cancer tissue obtained from 131 women during radical mastectomy. RESULTS: There was a highly significant difference in mean relative gene expression between HER2-positive and HER2-negative patients as assessed by immunostaining (22.10+/-41.89 vs. 3.17+/-8.76; p<0.001). With a median follow-up of 56 months, the cancer-specific survival of HER2-positive patients assessed by RT-PCR was worse in all cases and in the node-positive group. In multivariate analysis, HER2 status was an independent prognostic factor in all patients. CONCLUSIONS: Real-time RT-PCR is a valuable method for determining HER2 status, but the selection of the cut-off point of the relative gene expression differentiating between HER2-negative and -positive tumors is essential.

Ki-67 expression in operable breast cancer: a comparative study of immunostaining and a real-time RT-PCR assay.

The main purpose of this retrospective study was to compare Ki-67 expression in operable breast cancer examined by immunostaining and real-time reverse transcriptase polymerase chain reaction (RT-PCR). Relations between Ki-67 and classic prognostic factors were also investigated, and the prognostic relevance of Ki-67 expression was examined. Expression of Ki-67 was analyzed in specimens of invasive ductal breast cancer tissue obtained from 131 women during radical mastectomy. There was a significant, but weakly positive, correlation between Ki-67 expression assessed by immunostaining and real-time RT-PCR (tau=0.154, p=0.005). Higher Ki-67 expression in immunostaining and RT-PCR was more often seen in grade 3 tumors (p<0.001 and p=0.026, respectively). No significant relationship with age, disease stage, nodal involvement, estrogen receptor or HER2 status was found. In a univariate and multivariate analysis of cancer-specific survival with a median follow-up of 56 months, Ki-67 expression determined by immunostaining or real-time RT-PCR was no prognostic factor. We demonstrated that Ki-67 expression levels measured by immunostaining and real-time RT-PCR were weakly concordant, and both were related to higher tumor grade. Ki-67 expression did not influence survival.

Cyclin E expression in operable breast cancer quantified using real-time RT-PCR: a comparative study with immunostaining.

OBJECTIVE: The main purpose of this retrospective study was to compare cyclin E expression levels in operable breast cancer patients determined using real-time RT-PCR and immunostaining. The prognostic relevance of cyclin E was also investigated. METHODS: Specimens of invasive ductal breast cancer tissues obtained from 124 women during radical mastectomy were analyzed. RESULTS: Of the tumor samples, 40.3 and 59.7% showed high expression of cyclin E in RT-PCR and immunostaining, respectively. The overall agreement probability was 0.032 according to Scott's pi statistic. With a median follow-up of 55.5 months, cyclin E expression assessed using immunostaining was an independent negative prognostic factor in the node positive group (hazard ratio 3.1; 95% CI 1.0-9.2; P = 0.045). Cyclin E expression correlated with absence of steroid receptors and younger age. RT-PCR results did not predict survival in any group of patients. CONCLUSIONS: Disagreement between real-time RT-PCR and immunostaining was demonstrated. Immunostaining seems to be the more reliable method for assessing cyclin E in breast cancer cells.

Immunohistochemical identification of basal-type cytokeratins in invasive ductal breast carcinoma--relation with grade, stage, estrogen receptor and HER2.

Gene expression analyses with cDNA microarray technology identified distinct groups of breast cancers. Tumors with no ER expression could be divided into three subgroups: "basal-like" subtype, HER2-positive subtype, and "normal breast-like". "Basal-like" subtype was characterized by high expression of keratins 5 and 17, laminin and fatty acid binding protein 7. In the present study, we analyzed the usefulness of immunohistochemistry for separation of the distinct subtypes of the breast ductal carcinomas and provided further characterization of "basal-like subtype". A consecutive series of 195 primary operable invasive breast carcinomas was immunostained for HER2, ER, PGR, CK5/6 and CK17. CK5/6 or CK17 were expressed in 72 cases (36.9%), and 41 cases (21%) presented expression of CK5/6 or CK17 without ER/PGR or HER2. ER/PGR was present in 109 cases (55.9%), but in this group there were 8 cases with HER2 overexpression and 17 cases with basal-cytokeratin positivity. Similarly, in 17 out of 72 "basal-like" tumors there was ER/PGR positivity, and also in 17 of them there was HER2 overexpression. Three of these cases belonged to all three groups, representing expression of all markers. Tumor grade differed significantly (p < 0.001) between luminal and basal cytokeratin- or HER2-positive tumors. Differences for tumor size and lymph node status were not statistically significant. Our study showed that immunohistochemistry is useful for dividing breast cancers into separate subgroups, but further analyses for better characterization of cases presenting two or three markers should be performed.

Prognostic relevance of basal cytokeratin expression in operable breast cancer.

OBJECTIVE: We investigated whether basal cytokeratin (CK5/6 or CK17) expression had an impact on survival in patients with operable breast cancer. METHODS: Expression of CK5/6 or CK17 was analyzed by immunohistochemistry in 195 women with breast cancer. RESULTS: In total, 72 (37%) tumor samples were regarded as being positive for CK5/6 or CK17. The basal-like phenotype as defined by basal cytokeratin expression, lack of estrogen receptor (ER) and absence of HER2 overexpression was found in 48 (25%) cases. Positive staining for CK5/6 or CK17 was associated with worse prognosis when compared with patients negative for basal cytokeratins in all cases (5-year cancer-specific survival rate 59.4 vs. 77.5%, p = 0.0273) and in the node-negative group (70.5 vs. 90.8%, p = 0.0208) but not in the node-positive group (43.9 vs. 65.4%, p = 0.1182). To determine the real prognostic value of basal cytokeratins, survival in a group of ER-negative patients was analyzed depending on CK5/6 or CK 17 expression. No influence on survival was observed. The outcome of patients whose cancers were positive for cyclin E regardless of ER status was not changed by CK5/6 or CK17 expression. In multivariate analysis, independent prognostic factors affecting survival in the whole group included: nodal involvement, HER2 status and cyclin E expression. Neither ER status nor basal cytokeratin expression retained statistical significance. CONCLUSION: We demonstrated that the poor prognosis associated with the basal-like phenotype of breast cancer was determined by ER absence and cyclin E expression and not by CK5/6 or CK17 expression.