Clinical and Biological Significance of ESR1 Gene Alteration and Estrogen Receptors Isoforms Expression in Breast Cancer Patients.

The amplification of estrogen receptor alpha (ERα) encoded by the ESR1 gene has been described as having a prognostic role in breast cancer patients. However, increased dosage of the ESR1 gene (tested by real-time PCR) is also observed in ER-negative breast cancers, which might suggest the expression of alternative isoforms of ERα (other than classical ERα of 66 kDa). In the current work, we have investigated the ESR1 gene dosage in 402 primary breast cancer patients as well as the expression of ERα isoforms-ERα66 and ERα36-on mRNA and protein levels. The obtained results were correlated with clinicopathological data of the patients. Results showed that increased ESR1 gene dosage is not related to ESR1 gene amplification measured by fluorescent in situ hybridization (FISH), but it correlates with the decreased expression of ERα66 isoform (p = 0.01). Interestingly, the short ER isoform ERα36 was expressed in samples with increased ESR1 gene dosage, suggesting that genomic aberration might influence the expression of that particular isoform. Similarly to ESR1 increased gene dosage, high ERα36 expression was linked with the decreased disease-free survival of the patients (p = 0.05), which was independent of the status of the classical ERα66 level in breast tumors.

CD99 correlates with low cyclin D1, high topoisomerase 2 status and triple negative molecular phenotype but is prognostically irrelevant in breast carcinoma.

CD99 is a protein initially described in the Ewing sarcoma family of tumors, but growing evidence has shown its expression in other tumors of mesenchymal, hematopoietic and even epithelial origin. Some articles report CD99 in metaplastic carcinoma of the breast, a subtype of breast carcinoma (BC) with pronounced epithelial to mesenchymal (EMT) phenotype. Our aim was to analyse the potential relationship between CD99 and selected EMT (vimentin, E-cadherin, Twist) and proliferation markers (Ki-67, c-myc, cyclin D1, topoisomerase 2), molecular subtypes of BC, as well as overall survival (OS) and progression-free survival (PFS). In a group of 122 cases CD99 membrane expression was seen in 14 (11.5%) cases: strong in 11 (9%) and moderate in 3 (2.5%). Expression of CD99 correlated with low cyclin D1 index, high level of topoisomerase 2 expression and lack of progesterone receptor (PR) but not with EMT characteristics. Additionally, strong expression of CD99 correlated with triple negative molecular BC phenotype. CD99 was prognostically irrelevant for OS and PFS. CD99 correlates with selected proliferative markers and low ER/PR receptor status but not with patients' outcome in BC. Further studies are required to explain precisely its role in molecular pathogenesis of BC.

Prognostic significance of TOP2A gene dosage in HER-2-negative breast cancer.

BACKGROUND: Previous studies showed the prognostic and predictive impact of human epidermal growth factor receptor 2 (HER-2) gene alterations analyzed separately and jointly with topoisomerase II α (TOP2A) gene alterations; however, the role of TOP2A gene abnormalities alone has not been thoroughly investigated. Additionally, TOP2A aberrations were typically studied in HER-2-positive (HER-2(+)) tumors because these genes are frequently coamplified. Therefore, the knowledge concerning the impact of TOP2A abnormalities in HER-2-negative (HER-2(-)) patients is scarce. This study aimed to investigate the clinical significance of TOP2A anomalies in breast cancer patients with HER-2(-) and HER-2(+) tumors. MATERIALS AND METHODS: Snap-frozen tumor samples from 322 consecutive stage I-III breast cancer patients were analyzed for TOP2A gene dosage using quantitative real-time PCR (qPCR). RESULTS: A high TOP2A gene dosage was found in 94 tumors (29%)-32% and 27% of HER-2(+) and HER-2(-) tumors, respectively. The mean TOP2A gene dosages in the HER-2(+) and HER-2(-) groups were 1.49 ± 1.03 and 1.09 ± 0.35, respectively. High TOP2A gene dosage had an inverse prognostic impact in terms of shorter disease-free survival (DFS) and overall survival (OS) times in the entire group and in both the HER-2(-) and HER-2(+) subgroups. The unfavorable prognostic impact of TOP2A gene dosage was maintained in the multivariate Cox regression analysis in the entire group and in HER-2(-) patients. CONCLUSIONS: A high gene dosage of TOP2A determined using qPCR occurs frequently both in HER-2(+) and HER-2(-) tumors and has a strong adverse prognostic impact.

Expression of epithelial to mesenchymal transition-related markers in lymph node metastases as a surrogate for primary tumor metastatic potential in breast cancer.

BACKGROUND: Breast cancers are phenotypically and genotypically heterogeneous tumors containing multiple cancer cell populations with various metastatic potential. Aggressive tumor cell subpopulations might more easily be captured in lymph nodes metastases (LNM) than in primary tumors (PT). We evaluated mRNA and protein levels of master EMT regulators: TWIST1, SNAIL and SLUG, protein levels of EMT-related markers: E-cadherin, vimentin, and expression of classical breast cancer receptors: HER2, ER and PgR in PT and corresponding LNM. The results were correlated with clinicopathological data and patients outcomes. METHODS: Formalin-fixed paraffin-embedded samples from PT and matched LNM from 42 stage II-III breast cancer patients were examined. Expression of TWIST1, SNAIL and SLUG was measured by reverse-transcription quantitative PCR. Protein expression was examined by immunohistochemistry on tissue microarrays. Kaplan-Meier curves for disease-free survival (DFS) and overall survival (OS) were compared using F-Cox test. Hazard ratios (HRs) with 95% confidence intervals (95% CI) were computed using Cox regression analysis. RESULTS: On average, mRNA expression of TWIST1, SNAIL and SLUG was significantly higher in LNM compared to PT (P < 0.00001 for all). Gene and protein levels of TWIST1, SNAIL and SLUG were highly discordant between PT and matched LNM. Increased mRNA expression of TWIST1 and SNAIL in LNM was associated with shorter OS (P = 0.04 and P = 0.02, respectively) and DFS (P = 0.02 and P = 0.01, respectively), whereas their expression in PT had no prognostic impact. Negative-to-positive switch of SNAIL protein correlated with decreased OS and DFS (HR = 4.6; 1.1-18.7; P = 0.03 and HR = 3.8; 1.0-48.7; P = 0.05, respectively). CONCLUSIONS: LNM are enriched in cells with more aggressive phenotype, marked by elevated levels of EMT regulators. High expression of TWIST1 and SNAIL in LNM, as well as negative-to-positive conversion of SNAIL confer worse prognosis, confirming the correlation of EMT with aggressive disease behavior. Thus, molecular profiling of LNM may be used as surrogate marker for aggressiveness and metastatic potential of PT.

Activation of epithelial-mesenchymal transition process during breast cancer progression - the impact of molecular subtype and stromal composition.

Breast cancer (BC) is a heterogeneous disease with different molecular subtypes, which can be defined by oestrogen (ER), progesterone (PR) and human epidermal growth factor (HER2) receptors' status as luminal, HER2+ and triple negative (TNBC). Molecular subtypes also differ in their epithelial-mesenchymal phenotype, which might be related to their aggressiveness, as activation of the epithelial-mesenchymal transition (EMT) is linked with increased ability of cancer cells to survive and metastasize. Nevertheless, the reverse process of mesenchymal-epithelial transition was shown to be required to sustain metastatic colonization. In this study we aimed to analyse activation of the EMT process in primary tumours (PT), which have (N+) or have not (N-) colonized the lymph nodes, as well as the lymph nodes metastases (LNM) themselves in 88 BC patients. We showed that luminal N- PT have the lowest activation of the EMT process (27%), in comparison to N+ PT (48%, p=0.06). On the other hand, TNBC do not show statistically significant EMT activation at the stage before lymph colonization (N-, 83%) and after colonization of the lymph nodes (N+, 63%, p=0.58). TNBC are also the least plastic (unable to change the EMT phenotype) in terms of turning EMT on or off between matched PT and LNM (0% EMT plasticity in TNBC vs 36% plasticity in luminal tumours). Moreover, in TNBC activation of EMT was correlated with increased cell division rate of the PT- in mesenchymal TNBC PT median Ki-67 was 45% in comparison to 10% in epithelial TNBC PT (p=0.002), whereas in PT of luminal subtypes Ki-67 did not differ between epithelial and mesenchymal phenotypes. Profiling of immunotranscriptome of epithelial and mesenchymal luminal BC with Nanostring technology revealed that N- PT with epithelial phenotype were enriched in inflammatory response signatures, whereas N+ mesenchymal cancers showed elevated MHC class II antigen presentation. Overall, activation of EMT changes during cancer progression and metastatic colonization of the lymph nodes depending on the PT molecular subtype and is related to differences in stromal signatures. Activation of EMT is associated with colonizing phenotype in luminal PT and proliferative phenotype of TNBC.

Spectrum of Epithelial-Mesenchymal Transition Phenotypes in Circulating Tumour Cells from Early Breast Cancer Patients.

Circulating tumour cells (CTCs) can provide valuable prognostic information in a number of epithelial cancers. However, their detection is hampered due to their molecular heterogeneity, which can be induced by the epithelial-mesenchymal transition (EMT) process. Therefore, current knowledge about CTCs from clinical samples is often limited due to an inability to isolate wide spectrum of CTCs phenotypes. In the current work, we aimed at isolation and molecular characterization of CTCs with different EMT status in order to establish their clinical significance in early breast cancer patients. We have obtained CTCs-enriched blood fraction from 83 breast cancer patients in which we have tested the expression of epithelial, mesenchymal and general breast cancer CTCs markers (MGB1/HER2/CK19/CDH1/CDH2/VIM/PLS3), cancer stem cell markers (CD44, NANOG, ALDH1, OCT-4, CD133) and cluster formation gene (plakoglobin). We have shown that in the CTCs-positive patients, epithelial, epithelial-mesenchymal and mesenchymal CTCs markers were detected at a similar rate (in 28%, 24% and 24%, respectively). Mesenchymal CTCs were characterized by the most aggressive phenotype (significantly higher expression of CXCR4, uPAR, CD44, NANOG, p < 0.05 for all), presence of lymph node metastases (p = 0.043), larger tumour size (p = 0.023) and 7.33 higher risk of death in the multivariate analysis (95% CI 1.06⁻50.41, p = 0.04). Epithelial-mesenchymal subtype, believed to correspond to highly plastic and aggressive state, did not show significant impact on survival. Gene expression profile of samples with epithelial-mesenchymal CTCs group resembled pure epithelial or pure mesenchymal phenotypes, possibly underlining degree of EMT activation in particular patient's sample. Molecular profiling of CTCs EMT phenotype provides more detailed and clinically informative results, proving the role of EMT in malignant cancer progression in early breast cancer.

Aggressive Phenotype of Cells Disseminated via Hematogenous and Lymphatic Route in Breast Cancer Patients.

Intratumoral heterogeneity of breast cancer remains a major challenge in successful treatment. Failure of cancer therapies can also be accredited to inability to systemically eradicate cancer stem cells (CSCs). Recent evidence points to the role of epithelial-mesenchymal transition (EMT) in expanding the pool of tumor cells with CSCs features. Thus, we assessed expression level as well as heterogeneity of CSCs markers in primary tumors (PT), lymph node metastasis (LNM), and circulating tumor cells (CTCs)-enriched blood fractions in order to correlate them with signs of EMT activation as well as clinicopathological data of breast cancer patients. Level of CSCs markers (ALDH1, CD44, CD133, OCT-4, NANOG) and EMT markers was quantified in PT (N=107), LNM (N=56), and CTCs-enriched blood fractions (N=85). Heterogeneity of CSCs markers expression within each PT and LNM was assessed by calculating Gini Index. Percentage of ALDH1-positive cells was elevated in PT in comparison to LNM (P = .005). However, heterogeneity of the four CSCs markers: ALDH1 (P = .019), CD133 (P = .009), OCT-4 (P = .027), and CD44 (P < .001) was decreased in LNM. Samples classified as mesenchymal (post-EMT) showed elevated expression of CSCs markers (OCT-4 and CD44 in PT; OCT-4 in LNM; ALDH1, OCT-4, NANOG, CD44 in CTCs). Patients with mesenchymal-like CTCs had worse prognosis than patients with epithelial-like or no CTCs (P = .0025). CSCs markers are enriched in PT, LNM, and CTCs with mesenchymal features, but their heterogeneity is decreased in metastatic lymph nodes. Mesenchymal CTCs phenotype correlates with poor prognosis of the patients.

Postoperative adjuvant chemotherapy followed by adjuvant tamoxifen versus nil for patients with operable breast cancer: a randomised phase III trial of the European Organisation for Research and Treatment of Cancer Breast Group.

BACKGROUND: The contribution of adjuvant tamoxifen in breast cancer patients after receiving adjuvant chemotherapy is not fully established. We investigated the impact of tamoxifen, given sequentially after completion of adjuvant chemotherapy in patients with operable breast cancer. PATIENTS AND METHODS: Between March 1991 and June 1999, 1863 women with stages I-IIIA operable breast cancer who had undergone surgery and completed six cycles of adjuvant combination chemotherapy with either CMF, CAF, CEF, FAC or FEC were randomised to receive either tamoxifen 20 mg daily for 3 years or no further treatment. Irrespective of menstrual status and hormone receptor content of the primary tumour, patients were stratified by institute, chemotherapy scheme and age (above 50 years or younger). The main end-point was to detect a 5% increase in the 5 year survival (from 80% to 85%) in favour of antioestrogen therapy. Secondary end-points were relapse free survival (RFS), local control, incidence of second primary breast cancer and correlation of results with hormone receptor content. RESULTS: After exclusion of all patients from three sites because of inadequate documentation, a total of 1724 patients (93%) were analysed (Tam 861 and Control 863). At a median follow-up of 6.5 years, 5-year RFS on tamoxifen was 73% versus 67% in controls (p=0.035). No difference was seen in overall survival. The benefit of tamoxifen therapy was mainly seen in the subgroup of patients with histologically documented positive axillary nodes (5-year RFS on tamoxifen 71% versus 64% in the control group, p=0.044) and in patients with tumours expressing the ER and PR positive phenotype (5-year RFS on tamoxifen 77% versus 70% in the control group, p=0.014). CONCLUSIONS: Tamoxifen administered for 3 years after completion of adjuvant chemotherapy in this otherwise unselected group of patients for endocrine sensitivity had a limited impact on relapse and had no detectable effect on overall survival. The beneficial effect of tamoxifen is mainly confined to the subgroup of patients with node-positive disease and to patients with tumours expressing the ER and PR positive phenotype.

Gemcitabine, epirubicin, and paclitaxel versus fluorouracil, epirubicin, and cyclophosphamide as first-line chemotherapy in metastatic breast cancer: a Central European Cooperative Oncology Group International, multicenter, prospective, randomized phase III trial.

BACKGROUND: The objectives of this phase III trial were to compare the time to progressive disease (TtPD), overall response rate (ORR), overall survival, and toxicity of gemcitabine, epirubicin, and paclitaxel (GET) versus fluorouracil (FU), epirubicin, and cyclophosphamide (FEC) as first-line therapy in patients with metastatic breast cancer (MBC). PATIENTS AND METHODS: Female patients aged 18 to 75 years with stage IV and measurable MBC were enrolled and randomly assigned to either gemcitabine (1,000 mg/m(2), days 1 and 4), epirubicin (90 mg/m(2), day 1), and paclitaxel (175 mg/m(2), day 1) or FU (500 mg/m(2), day 1), epirubicin (90 mg/m(2), day 1), and cyclophosphamide (500 mg/m(2), day 1). Both regimens were administered every 21 days for a maximum of eight cycles. RESULTS: Between October 1999 and November 2002, 259 patients (GET, n = 124; FEC, n = 135) were enrolled. Baseline characteristics were well balanced across treatment arms. After a median of 20.4 months of follow-up, median TtPD was 9.1 months and 9.0 months in the GET and FEC arms, respectively (P = .557). The ORR was 62.3% in the GET arm (n = 114) and 51.2% in the FEC arm (n = 129; P = .093). Grade 3 and 4 toxicities, including neutropenia, thrombocytopenia, anemia, stomatitis, neurosensory toxicity, and allergy, occurred significantly more often in the GET arm. CONCLUSION: No significant differences in terms of TtPD and ORR were observed between the two treatment arms. Treatment-related toxicity was higher in the GET arm.

(CA)n microsatellite polymorphism of ERBB-1 in breast cancer.

The aim of this study was to determine polymorphism of repeated sequences (CA)(n) in the ERBB-1 gene. The study group included 197 breast cancer patients and 180 healthy women. DNA was isolated from fresh-frozen tumour tissue and from peripheral blood. ERBB-1 (CA)(n) microsatellite polymorphism was examined by polymerase chain reaction (PCR). A polymorphic simple sequence repeat region of 9-23 CA repeats was detected in both groups. Homozygotes comprised 22% and 34% of breast cancer patients and controls, respectively (P=0.009). An allelic imbalance (AI), mostly in the shorter allele, was found in 27% of breast cancer patients. AI occurrence was associated with the lack of oestrogen receptors in tumour cells (P=0.05); otherwise, there were no correlations between histoclinical features and (CA)(n) microsatellite polymorphism of ERBB-1. It was concluded that an allelic imbalance is a common feature in breast cancer patients and may coincide with the lack of oestrogen receptors in tumour cells. The clinical relevance of ERBB-1 microsatellite polymorphism in breast cancer remains to be established.

Health-related quality of life parameters as prognostic factors in a nonmetastatic breast cancer population: an international multicenter study.

PURPOSE: The purpose of this research was to evaluate whether baseline health-related quality of life (HRQOL) parameters are prognostic factors for survival in locally advanced breast cancer patients. Although the literature highlights the important role of HRQOL parameters in predicting survival in advanced metastatic disease, little evidence exists for earlier stages. PATIENTS AND METHODS: The overall sample consisted of 448 patients randomly assigned to receive cyclophosphamide, epirubicin, and fluorouracil versus epirubicin, cyclophosphamide, and granulocyte colony-stimulating factor. Patients were enrolled in 12 countries. HRQOL baseline scores were assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30. The Cox proportional hazards regression model was used for both univariate and multivariate analyses of survival. In addition, a bootstrap resampling technique was used to assess the stability of the outcomes. Bootstrap results were then applied for model averaging purposes as a means to account for the observed model selection uncertainty. RESULTS: The final multivariate model retained inflammatory breast cancer (T4d) as the only factor predicting overall survival (OS) with a hazard ratio of 1.375 (95% CI, 1.027 to 1.840; P =.03). The presence of inflammatory breast cancer lowers the median survival time from 6.6 to 4.2 years (36% reduction). None of the preselected HRQOL variables were prognostic for OS or disease-free survival, in either the univariate or multivariate analysis. CONCLUSION: Our findings suggest that baseline HRQOL parameters have no prognostic value in a nonmetastatic breast cancer population.

Modified direct-double-differential PCR for gene dosage quantification of HER2.

HER2 amplification and/or overexpression in breast cancer are adverse prognostic factors and can predict the response to trastuzumab therapy. As assessment of HER2 status in breast cancer is of great importance for clinical decision-making, it is crucial to have reliable methods to quantify HER2. In the present project, we have developed a modification of the direct-double-differential PCR method (dddPCR) for gene dosage quantification of HER2 in breast cancer samples. The study was also undertaken to establish the potential application of the newly developed dddPCR method for HER2 testing in a diagnostic laboratory. The dddPCR validity for determining HER2 amplification in comparison to IHC-based HercepTest for the detection of protein levels was performed. Modified dddPCR was proven to be highly reproducible and reliable. A statistically significant correlation between protein expression and gene dosage of HER2 was found in the examined material. The obtained results indicate that dddPCR provides a fast and reliable diagnostic tool for HER2 quantification and may be considered as a supplementary method to verify equivocal results obtained with IHC-based HercepTest. Notably, dddPCR can be performed in every basic molecular biology laboratory, and is inexpensive and time-saving.

Stromal expression of ALDH1 in human breast carcinomas indicates reduced tumor progression.

Interactions between cancer cells and microenvironment are emerging issue in tumor progression. Aldehyde dehydrogenase 1 (ALDH1) is a recognized cancer stem cell marker but little is known about its role in intratumoral stroma. Therefore, we focused on ALDH1 expression in tumor-associated stroma of breast carcinomas (BrCa). Stromal and tumoral ALDH1 expression was evaluated immunohistochemically in BrCa and their lymph node metastases (LNMs), and related to clinico-pathological characteristics, patients' outcome, presence of CD68, HLADR, retinoic acid (RA) in stroma, and selected proteins in tumor cells. ALDH1(+) stromal cells were detected in 53% of 374 BrCa and 61% of 102 LNMs. ALDH1(+) stroma in primary tumor correlated to longer disease-free (p = 0.030), metastasis-free (p = 0.024), and overall survival (p = 0.043) having an independent prognostic impact on DFS (multivariate analysis, p = 0.047). It was associated with concomitant presence of HLA-DR(+) stromal cells and RA in tumor cells (both p < 0.001), and inversely associated with vimentin expression in tumor cells (p = 0.036). ALDH1(+) stroma in LNMs correlated inversely to presence of disseminated tumor cells in patients' bone marrow (p = 0.014) and was independent prognosticator of shorter DFS and MFS (multivariate analysis, p = 0.004 and p = 0.002, respectively). In conclusion, ALDH1 expression in tumor-associated stromal cells indicates reduced BrCa progression, possibly via RA secretion.

The risks and benefits of hormonal replacement therapy in healthy women and in breast cancer survivors.

For many years hormonal replacement therapy (HRT) has been considered to offer not only effective relief of climacteric symptoms but also a reduction in the risk of osteoporosis and cardiovascular disease, plus a possible prevention of cognitive decline. Randomised trials of HRT in women with preexisting coronary heart disease have not confirmed, however, cardiovascular benefits of HRT and have even suggested increased cardiac risk associated with this management. Numerous retrospective studies demonstrated that the risk of breast cancer is higher in HRT-users and is related to therapy duration. More recent studies suggested that breast cancer risk increases further if oestrogen is coupled with progestogen. On the other hand, some data show that breast cancers diagnosed in women during HRT administration may be less aggressive. Typically, tumours in these patients are smaller and better differentiated. HRT also increases the relative risk of endometrial cancer, particularly if oestrogen alone is administered. Currently, the indications for HRT in healthy subjects should include only reduction of menopausal symptoms and prevention of osteoporosis. The use of HRT in breast cancer survivors is controversial, and until the results from prospective randomised trials are available, cannot be recommended in this group as a standard care.

Radiotherapy for breast cancer in patients undergoing breast reconstruction or augmentation.

Due to increasing indications for postmastectomy radiotherapy and a growing demand for breast reconstruction or augmentation, increasing numbers of patients are currently being exposed to both these treatments. In view of the wide range of available techniques for breast reconstruction, either prosthetic or autologous, and their various sequencing in relation to radiotherapy, physicians can be faced with numerous clinical situations requiring comprehensive knowledge of the topic. This review discusses physical, radiobiological and clinical aspects of combining breast reconstruction and radiotherapy. The available data indicate the feasibility of such combinations, although at the expense of increased risk of complications and less satisfactory cosmesis. Of the two methods of breast reconstruction: using autologous tissue or prosthesis, the former seems to provide better cosmesis and a lower risk of complications in conjunction with radiotherapy. To minimize the risk of unfavourable outcome, the techniques and timing of both breast reconstruction and radiotherapy should be given meticulous attention.

Mesenchymal phenotype of CTC-enriched blood fraction and lymph node metastasis formation potential.

INTRODUCTION: Circulating tumor cells (CTCs) that present mesenchymal phenotypes can escape standard methods of isolation, thus limiting possibilities for their characterization. Whereas mesenchymal CTCs are considered to be more malignant than epithelial CTCs, factors responsible for this aggressiveness have not been thoroughly defined. This study analyzed the molecular profile related to metastasis formation potential of CTC-enriched blood fractions obtained by marker unbiased isolation from breast cancer patients without (N-) and with lymph nodes metastases (N+). MATERIALS AND METHODS: Blood samples drawn from 117 patients with early-stage breast cancer were enriched for CTCs using density gradient centrifugation and negative selection with anti-CD45 covered magnetic particles. In the resulting CTC-enriched blood fractions, expression of CK19, MGB1, VIM, TWIST1, SNAIL, SLUG, HER2, CXCR4 and uPAR was analyzed with qPCR. Results were correlated with patients' clinicopathological data. RESULTS: CTCs (defined as expression of either CK19, MGB1 or HER2) were detected in 41% (20/49) of N- and 69% (34/49) of N+ patients (P = 0.004). CTC-enriched blood fractions of N+ patients were more frequently VIM (P = 0.02), SNAIL (P = 0.059) and uPAR-positive (P = 0.03). Positive VIM, CXCR4 and uPAR status correlated with >3 lymph nodes involved (P = 0.003, P = 0.01 and P = 0.045, respectively). In the multivariate logistic regression MGB1 and VIM-positivity were independently related to lymph node involvement with corresponding overall risk of 3.2 and 4.2. Moreover, mesenchymal CTC-enriched blood fractions (CK19-/VIM+ and MGB1+ or HER2+) had 4.88 and 7.85-times elevated expression of CXCR4 and uPAR, respectively, compared with epithelial CTC-enriched blood fractions (CK19+/VIM- and MGB1+ or HER2+). CONCLUSIONS: Tumors of N+ patients have superior CTC-seeding and metastatic potential compared with N- patients. These differences can be attributed to VIM, uPAR and CXCR4 expression, which endow tumor cells with particularly malignant phenotypes.

Epithelial-mesenchymal transition markers in lymph node metastases and primary breast tumors - relation to dissemination and proliferation.

Epithelial-mesenchymal transition (EMT) was shown to enhance metastatic abilities of cancer cells, but it remains elusive in clinical samples. Moreover, EMT is rarely studied in lymph node metastases (LNM), thus limiting our understanding of its role outside of the primary tumors (PT). We collected a set of samples including triplets - PT, circulating tumor cells (CTCs)-enriched blood samples and LNM from 108 early breast cancer patients. With immunohistochemistry we analyzed levels of EMT effectors - E-cadherin, vimentin and N-cadherin in LNM, central areas and margins of PT. Additionally, expression of EMT core regulators TWIST1, SNAI1, SNAI2 was measured with RT-qPCR. Patients with E-cadherin loss had CTCs in 45% of the cases in comparison to 23% with normal E-cadherin level (P = 0.05). Mesenchymal phenotype of CTCs-enriched blood fractions was five-times more frequent in patients with E-cadherin loss in PT compared to PT with normal E-cadherin levels (P = 0.01). Epithelial/mesenchymal status of matched samples at different stages of dissemination was frequently discordant, especially for pairs involving CTCs, indicating high plasticity of tumor cells. LNM showed increased expression of TWIST1, SNAI1, SNAI2 accompanied by decreased Ki67 labeling index, with median Ki67 of 15% in PT and 10% in LNM (P = 0.0002). Our findings demonstrate that E-cadherin loss, not only in PT margin, might lead to seeding of especially malignant CTCs with mesenchymal phenotype. In comparison to PT, cells in LNM re-express E-cadherin, upregulate EMT transcription factors and reduce cell division rate, which could be viewed as their long-term survival strategy.

Heterogeneity of mesenchymal markers expression-molecular profiles of cancer cells disseminated by lymphatic and hematogenous routes in breast cancer.

Breast cancers can metastasize via hematogenous and lymphatic routes, however in some patients only one type of metastases are detected, suggesting a certain proclivity in metastatic patterns. Since epithelial-mesenchymal transition (EMT) plays an important role in cancer dissemination it would be worthwhile to find if a specific profile of EMT gene expression exists that is related to either lymphatic or hematogenous dissemination. Our study aimed at evaluating gene expression profile of EMT-related markers in primary tumors (PT) and correlated them with the pattern of metastatic spread. From 99 early breast cancer patients peripheral blood samples (N = 99), matched PT (N = 47) and lymph node metastases (LNM; N = 22) were collected. Expression of TWIST1, SNAI1, SNAI2 and VIM was analyzed in those samples. Additionally expression of CK19, MGB1 and HER2 was measured in CTCs-enriched blood fractions (CTCs-EBF). Results were correlated with each other and with clinico-pathological data of the patients. Results show that the mesenchymal phenotype of CTCs-EBF correlated with poor clinico-pathological characteristics of the patients. Additionally, PT shared more similarities with LNM than with CTCs-EBF. Nevertheless, LNM showed increased expression of EMT-related markers than PT; and EMT itself in PT did not seem to be necessary for lymphatic dissemination.

Are bilateral breast cancers different from breast cancers coexisting with ovarian cancer? An immunohistochemical analysis aimed at intrinsic tumor phenotype.

RATIONALE: Bilateral breast cancers (BBC) and breast cancers coexisting with ovarian cancer (BOCS) are associated with genetic predisposition more frequently than sporadic cases. We compared the phenotypes of these tumors to better understand their pathomechanisms and aid the guiding of their clinical management. MATERIALS AND METHODS: Tumor morphology and expression of ER, PgR, HER2, Ki67, CK5/6, E-cadherin, vimentin and EGFR were assessed in a tissue microarray containing cores from 174 BBC, 23 BOCS and 2 BBC + BOCS. RESULTS: BOCS tumors were characterized by higher incidence of EGFR expression, HER2 negativity and lower incidence of intraductal component. HER2-positive phenotypes were marginally more frequent in the BBC group and triple negative tumors - in BOCS. CONCLUSION: Breast cancers from BOCS patients are characterized by more aggressive phenotype, most probably related to their more frequent association with BRCA1 mutation.

Prognostic significance of ESR1 amplification and ESR1 PvuII, CYP2C19*2, UGT2B15*2 polymorphisms in breast cancer patients.

INTRODUCTION: Amplification of the ESR1 gene, coding for estrogen receptor alpha, was shown to predict responsiveness to tamoxifen, however its prognostic impact in breast cancer patients has not been thoroughly investigated. Other factors that could contribute to responsiveness to tamoxifen treatment are polymorphisms in ESR1 gene and genes involved in tamoxifen metabolism. The aim of this study was to assess the prognostic role of ESR1 gene dosage in a consecutive group of breast cancer patients and to correlate this feature with clinico-pathological factors. Additionally, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphisms were analyzed in the tamoxifen-treated subgroup of patients. MATERIALS AND METHODS: Primary tumor samples from 281 stage I-III consecutive breast cancer patients were analyzed for ESR1 gene dosage using real-time PCR with locked nucleic acids hydrolysis probes. In the tamoxifen-treated subgroup of patients, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphism in leukocytes genomic DNA were analyzed. Results were correlated with clinico-pathological factors and with disease-free survival (DFS) and overall survival (OS). RESULTS: ESR1 amplification (with a cut-off level of 2.0) was found in 12% of the entire group of breast cancer patients, and in 18% of the ER-negative subgroup. This feature was associated with decreased DFS both in the entire group (P=0.007) and in the ER-negative subgroup (P=0.03), but not in the tamoxifen-treated patients. Patients with ESR1 PvuII wt/wt genotype and at least one UGT2B15 wt allele had a worse DFS (P=0.03) and showed a trend towards decreased Os (P=0.08) in comparison to patients with ESR1 PvuII wt/vt or vt/vt genotype and UGT2B15 *2/*2 genotype. CONCLUSIONS: ESR1 amplification can occur in ER-negative tumors and may carry poor prognosis. In the tamoxifen-treated subgroup, poor prognosis was related to the combined presence of ESR1 PvuII wt/wt and UGT2B15wt/wt or wt/*2 genotype.