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Breast cancers are a heterogeneous group of tumors classified according to their histological growth patterns and receptor expression characteristics. Intratumor heterogeneity also exists, with subpopulations of cells with different phenotypes found in individual cancers, including cells with stem or progenitor cell properties. At least two types of breast cancer stem cells (CSCs) exist, the epithelial and the basal/mesenchymal subtypes, although how these phenotypes are controlled is unknown. ΔNp63 is a basal cell marker and regulator of stem/progenitor cell activities in the normal mammary gland and is expressed in the basal-like CSC subpopulation in some estrogen receptor-positive (ER+) and/or human epidermal growth factor receptor 2-positive (HER2+) breast adenocarcinomas. Whilst p63 is known to directly impart CSC properties in luminal breast cancer cells, how p63 is regulated and induced in these cells is unknown. We initially confirmed the existence of a small subpopulation of ΔNp63+ cells in lymph node metastases of ER+ human ductal adenocarcinomas, indicating together with previous reports that ΔNp63+ tumor cells are present in approximately 40% of these metastases. Notably, ΔNp63+ cells show a preferential location at the edge of tumor areas, suggesting possible regulation of ΔNp63 by the tumor microenvironment. Subsequently, we showed that the high levels of ΔNp63 in basal non-transformed MCF-10A mammary epithelial cells rely on insulin in their culture medium, whilst ΔNp63 levels are increased in MCF-7 ER+ luminal-type breast cancer cells treated with insulin or insulin-like growth factor 1 (IGF-1). Mechanistically, small molecule inhibitors and siRNA gene knockdown demonstrated that induction of ΔNp63 by IGF-1 requires PI3K, ERK1/2, and p38 MAPK activation, and acts through FOXO transcriptional inactivation. We also show that metformin inhibits ΔNp63 induction. These data reveal an IGF-mediated mechanism to control basal-type breast CSCs, with therapeutic implications to modify intratumor breast cancer cell heterogeneity and plasticity.

A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.

ΔNp63, also known as p40, regulates stemness of normal mammary gland epithelium and provides stem cell characteristics in basal and HER2-driven murine breast cancer models. Whilst ΔNp63/p40 is a characteristic feature of normal basal cells and basal-type triple-negative breast cancer, some receptor-positive breast cancers express ΔNp63/p40 and its overexpression imparts cancer stem cell-like properties in ER + cell lines. However, the incidence of ER + and HER2 + tumours that express ΔNp63/p40 is unclear and the phenotype of ΔNp63/p40 + cells in these tumours remains uncertain. Using immunohistochemistry with p63 isoform-specific antibodies, we identified a ΔNp63/p40 + tumour cell subpopulation in 100 of 173 (58%) non-triple negative breast cancers and the presence of this population associated with improved survival in patients with ER - /HER2 + tumours (p = 0.006). Furthermore, 41% of ER + /PR + and/or HER2 + locally metastatic breast cancers expressed ΔNp63/p40, and these cells commonly accounted for <1% of the metastatic tumour cell population that localised to the tumour/stroma interface, exhibited an undifferentiated phenotype and were CD44 + /ALDH - . In vitro studies revealed that MCF7 and T47D (ER + ) and BT-474 (HER2 + ) breast cancer cell lines similarly contained a small subpopulation of ΔNp63/p40 + cells that increased in mammospheres. In vivo, MCF7 xenografts contained ΔNp63/p40 + cells with a similar phenotype to primary ER + cancers. Consistent with tumour samples, these cells also showed a distinct location at the tumour/stroma interface, suggesting a role for paracrine factors in the induction or maintenance of ΔNp63/p40. Thus, ΔNp63/p40 is commonly present in a small population of tumour cells with a distinct phenotype and location in ER + and/or HER2 + human breast cancers., (© 2019 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Accurate classification of breast tumors is vital for patient management decisions and enables more precise cancer treatment. Here, we present a quantitative proteotyping approach based on sequential windowed acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry and establish key proteins for breast tumor classification. The study is based on 96 tissue samples representing five conventional breast cancer subtypes. SWATH proteotype patterns largely recapitulate these subtypes; however, they also reveal varying heterogeneity within the conventional subtypes, with triple negative tumors being the most heterogeneous. Proteins that contribute most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2 and are associated with estrogen receptor (ER) status, tumor grade status, and HER2 status. Although these three key proteins exhibit high levels of correlation with transcript levels (R > 0.67), general correlation did not exceed R = 0.29, indicating the value of protein-level measurements of disease-regulated genes. Overall, this study highlights how cancer tissue proteotyping can lead to more accurate patient stratification., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Mutations in the BRCA1 and BRCA2 genes are associated with a high risk of developing breast cancer. Tumors arising from this mutation are expected to be more sensitive to platinum-based drugs. The role of platinum-based drugs in systemic neoadjuvant BRCA1/2 breast cancer therapy, and its efficacy in increasing the probability of pathological complete remission (pCR) are discussed repeatedly; however, there are no clear recommendations., Patients and Methods: We retrospectively evaluated the contribution of a platinum-based antineoplastic drug to the achievement of pCR in a set of patients with BRCA1/2 mutant breast cancer treated with neoadjuvant chemotherapy from 2010 to 2017. The response to neoadjuvant chemotherapy was evaluated by a pathologist using definitive surgical specimens. A pCR was defined as a condition in which complete invasive breast cancer, and (eventually) positive lymph nodes, had disappeared., Results: Of 76 patients (median age, 39 years; 62% with triple negative breast cancer (TNBC); 70% with BRCA1 positivity), 37 were treated with platinum-based drugs. More patients treated with platinum derivatives achieved pCR (57% vs. 23%, p = 0.005). Patients treated in a neoadjuvant setting with platinum-based antineoplastic drugs had a 4.4× greater chance of achieving pCR than those not treated with platinum, assuming the same tumor phenotype (TNBC or SR+/HER2)., Conclusion: Neoadjuvant platinum-based chemotherapy for patients with a BRCA1/2 mutation is associated with a higher probability of achieving pCR, which is important for subsequent prognosis. This treatment should be considered particularly for patients with BRCA1 mutation and a TNBC phenotype. This work was supported by grant of the Ministry of Health of the Czech Republic - Conceptual. Development of a Reaserch Organization (MMCI 00209805). This work was supported by grant of the Ministry of Education, Youth and Sport of the Czech Republic (NPU I - LO1413). The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 21. 3. 2019 Accepted: 14. 5. 2019.

Background: Breast cancer can be diagnosed easily in most cases. However, occasionally, we are faced with some conditions that can mimic it. These may include inflammations, benign tumors, cysts, hematomas, or, more rarely, focal necrosis., Case Presentation: This report presents a case of focal breast necrosis following myocardial revascularization with the left internal mammary artery, which is a very rare condition, with only few cases described in the literature. The necrosis becomes usually apparent a few days or weeks after the surgery and is often coincidental with the dehiscence of sternotomy with necrosis of wound edges. As it mostly affects the skin, it can be easily recognized. Also, our patient developed a dehisced sternotomy shortly after the surgery but there were no obvious objective changes on the breast. The condition was first dominated only by non-specific subjective symptom-pain. Later, a lump in the breast occurred, when the sternotomy had already healed. Moreover, an enlarged lymph node was palpable in the axilla. Because of non-typical symptoms, the condition was suggestive of breast cancer for a relatively long time. The patient had suffered from a very strong pain until she was treated by mastectomy with a good clinical result., Conclusions: Mammary necrosis following the coronary artery bypass is rare. In most cases, it manifests on the skin shortly after the surgery concurrently with dehisced sternotomy, so it can be easily diagnosed. However, in sporadic cases, the symptoms may occur later and may mimic breast cancer. Our objective is to raise awareness of this rare condition.

Breast cancer is the most common and molecularly relatively well characterized malignant disease in women, however, its progression to metastatic cancer remains lethal for 78% of patients 5years after diagnosis. Novel markers could identify the high risk patients and their verification using quantitative methods is essential to overcome genetic, inter-tumor and intra-tumor variability and translate novel findings into cancer diagnosis and treatment. We recently identified 13 proteins associated with estrogen receptor, tumor grade and lymph node status, the key factors of breast cancer aggressiveness, using untargeted proteomics. Here we verified these findings in the same set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM), transcriptomics and immunohistochemistry and validated in 5 independent sets of 715 patients using transcriptomics. We confirmed: (i) positive association of anterior gradient protein 2 homolog (AGR2) and periostin (POSTN) and negative association of annexin A1 (ANXA1) with estrogen receptor status; (ii) positive association of stathmin (STMN1), cofilin-1 (COF1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1) and negative associations of thrombospondin-2 (TSP2) and POSTN levels with tumor grade; and (iii) positive association of POSTN, alpha-actinin-4 (ACTN4) and STMN1 with lymph node status. This study highlights a panel of gene products that can contribute to breast cancer aggressiveness and metastasis, the understanding of which is important for development of more precise breast cancer treatment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Expression of the AGR2 oncogene was shown to be associated with estrogen receptor positive tumors. This gene contributes to enhanced cellular proliferation, drug resistance, metastasis development and may also serve as a predictor of poor prognosis. However, our analysis of AGR2 expression in a subset of estrogen-receptor negative tumors revealed that AGR2 could also be upregulated in hormone-independent manner. AGR2 expression was shown to be significantly increased in HER2 positive breast tumors on both the mRNA and the protein level. Moreover, in a subset of estrogen- and progesterone-receptor negative and simultaneously HER2-positive cases, increased AGR2 expression significantly correlated with worse patient prognosis. Subsequent analysis of independent data sets either collected in our institute or obtained from Oncomine cancer microarray database confirmed all these findings., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Inherited mutations in BRCA1 and BRCA2 genes represent the most important cause of hereditary breast cancer. This highly penetrating familial cancer syndrome, including also the onset of ovarian cancer and other malignancies at relatively low age, represents a substantial medical problem. The affected families should be managed actively. When compared to spontaneous tumors, the breast carcinomas in BRCA1 mutation carriers exhibit a relatively different, despite non-specific, phenotype (often triple negative, medullary features) arousing suspicion of hereditary background. In the contrary, the distribution of phenotypes of breast carcinomas in BRCA2 carriers is similar to distribution in the non-affected population. The lymphocytic lobulitis is observed significantly more often in non-cancerous breast tissue of BRCA1/2 mutation carriers, but again, this feature is not specific.

Transgelin and transgelin-2 have been discussed as potential markers of various cancers. Here we identified increased transgelin level in lymph node positive vs. negative, low grade primary breast cancer tissues using 2-DE in the cohort of 12 patients. We further clinically validated 2-DE results in an independent cohort of 48 low grade breast cancer patients through untargeted and targeted proteomics analysis (iTRAQ-2D-LC-MS/MS, mTRAQ-SRM), at transcript level and using immunohistochemistry. Another group of 48 high grade tumors of different breast cancer subtypes was analyzed together with the low grade samples to test transgelin specificity for low grade tumors and to study transgelin relation to known molecular markers and histological features. The results confirmed transgelin connection with the lymph node metastasis. As a marker of a reactive tumor stroma, transgelin can be connected with the higher risk of metastasis development. Moreover, we observed significant down-regulation of transgelin in high vs. low grade tumors caused by decreased content of stromal cells (mainly expressing transgelin) in high grade tumor tissue. We also analyzed expression of transgelin-2 in the second cohort using proteomics and immunohistochemistry. Transgelin-2 was mainly expressed by epithelial cancer cells and its levels were increased in metastatic and poorly differentiated tumors., Biological Significance: Both transgelin and transgelin-2 have been previously described as potential markers of many types of cancer. We are specifying this connection to metastatic affection of lymph nodes and cell differentiation in breast cancer. In the wider context, the results of our study highlight tumor stroma as a source of cancer biomarkers and point out how measured levels of tissue markers can actually reflect cellular feature of cancer mass., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Current prognostic factors are insufficient for precise risk-discrimination in breast cancer patients with low grade breast tumors, which, in disagreement with theoretical prognosis, occasionally form early lymph node metastasis. To identify markers for this group of patients, we employed iTRAQ-2DLC-MS/MS proteomics to 24 lymph node positive and 24 lymph node negative grade 1 luminal A primary breast tumors. Another group of 48 high-grade tumors (luminal B, triple negative, Her-2 subtypes) was also analyzed to investigate marker specificity for grade 1 luminal A tumors. From the total of 4405 proteins identified (FDR < 5%), the top 65 differentially expressed together with 30 previously identified and control markers were analyzed also at transcript level. Increased levels of carboxypeptidase B1 (CPB1), PDZ and LIM domain protein 2 (PDLIM2), and ring finger protein 25 (RNF25) were associated specifically with lymph node positive grade 1 tumors, whereas stathmin 1 (STMN1) and thymosin beta 10 (TMSB10) associated with aggressive tumor phenotype also in high grade tumors at both protein and transcript level. For CPB1, these differences were also observed by immunohistochemical analysis on tissue microarrays. Up-regulation of putative biomarkers in lymph node positive (versus negative) luminal A tumors was validated by gene expression analysis of an independent published data set (n = 343) for CPB1 (p = 0.00155), PDLIM2 (p = 0.02027) and RELA (p = 0.00015). Moreover, statistically significant connections with patient survival were identified in another public data set (n = 1678). Our findings indicate unique pro-metastatic mechanisms in grade 1 tumors that can include up-regulation of CPB1, activation of NF-κB pathway and changes in cell survival and cytoskeleton. These putative biomarkers have potential to identify the specific minor subpopulation of breast cancer patients with low grade tumors who are at higher than expected risk of recurrence and who would benefit from more intensive follow-up and may require more personalized therapy., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In 2012, the new classification of the fourth series WHO blue books of breast tumors was released. The current version represents a fluent evolution, compared to the third edition. Some limited changes regarding terminology, definitions and the inclusion of some diagnostic units were adopted. The information about the molecular biology and genetic background of breast carcinoma has been enriched substantially.

Many human cancers contain missense TP53 mutations that result in p53 protein accumulation. Although generally considered as a single class of mutations that abrogate wild-type function, individual TP53 mutations may have specific properties and prognostic effects. Tumours that contain missense TP53 mutations show variable p53 stabilization patterns, which may reflect the specific mutation and/or aspects of tumour biology. We used immunohistochemistry on cell lines and human breast cancers with known TP53 missense mutations and assessed the effects of each mutation with four structure-function prediction methods. Cell lines with missense TP53 mutations show variable percentages of cells with p53 stabilization under normal growth conditions, ranging from approximately 50% to almost 100%. Stabilization is not related to structural or functional disruption, but agents that stabilize wild-type p53 increase the percentages of cells showing missense mutant p53 accumulation in cell lines with heterogeneous stabilization. The same heterogeneity of p53 stabilization occurs in primary breast cancers, independent of the effect of the mutation on structural properties or functional disruption. Heterogeneous accumulation is more common in steroid receptor-positive or HER2-positive breast cancers and cell lines than in triple-negative samples. Immunohistochemcal staining patterns associate with Mdm2 levels, proliferation, grade and overall survival, whilst the type of mutation reflects downstream target activity. Inhibiting Mdm2 activity increases the extent of p53 stabilization in some, but not all, breast cancer cell lines. The data indicate that missense mutant p53 stabilization is a complex and variable process in human breast cancers that associates with disease characteristics but is unrelated to structural or functional properties. That agents which stabilize wild-type p53 also stabilize mutant p53 has implications for patients with heterogeneous mutant p53 accumulation, where therapy may activate mutant p53 oncogenic function., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: Various markers are used to identify the unique sub-population of breast cancer cells with stem cell properties. Whether these markers are expressed in all breast cancers, identify the same population of cells, or equate to therapeutic response is controversial., Methods: We investigated the expression of multiple cancer stem cell markers in human breast cancer samples and cell lines in vitro and in vivo, comparing across and within samples and relating expression with growth and therapeutic response to doxorubicin, docetaxol and radiotherapy., Results: CD24, CD44, ALDH and SOX2 expression, the ability to form mammospheres and side-population cells are variably present in human cancers and cell lines. Each marker identifies a unique rather than common population of cancer cells. In vivo, cells expressing these markers are not specifically localized to the presumptive stem cell niche at the tumour/stroma interface. Repeated therapy does not consistently enrich cells expressing these markers, although ER-negative cells accumulate., Conclusions: Commonly employed methods identify different cancer cell sub-populations with no consistent therapeutic implications, rather than a single population of cells. The relationships of breast cancer stem cells to clinical parameters will require identification of specific markers or panels for the individual cancer.

The TP63 gene gives rise to protein isoforms with different properties and functions due to the presence (TAp63) or absence (ΔNp63) of an N-terminal p53-like transactivation domain. Immunohistochemistry for p63 has clinical value for certain tumour types, but investigations have been hampered by a lack of well characterized antibodies and the inability to discriminate between these N-terminal isoforms with opposite functional properties. We have extensively characterized a series of monoclonal antibodies to recombinant human TAp63 and two commercial p63 monoclonals by Western blot, immunostaining and phage display epitope mapping. Twenty-eight of 29 (96.6 %) novel monoclonals that recognized all p63 isoforms showed substantial cross-reactivity with p73, as did the commercial antibody, 4A4. One novel clone, PANp63-6.1, showed slight cross-reaction with p73 by Western blotting but not immunohistochemistry and the SFI-6 monoclonal did not cross-react with p73 or p53. Phage display revealed that the PANp63-6.1 epitope has one amino acid difference between p63 and p73, the 4A4 epitope is identical in both, whereas the SFI-6 epitope is unique to p63, accounting for these findings. We also produced and characterized a TAp63-specific clone that does not recognize p53 or p73, and we prepared polyclonal sera specific for ΔNp63 isoforms. Immunohistochemistry demonstrated that TAp63 is expressed in a variety of epithelial and other cell types during development, often in a converse pattern to ΔNp63, but has a very limited expression in normal adult tissues and is independent of ΔNp63. TAp63 was expressed in 17.6 % of squamous cancers of cervix that expressed p63, unlike normal cervix where TAp63 was not expressed. TAp63 did not associate with proliferative index, but cervical carcinomas with TAp63 expression showed improved survival. These data highlight the need for rigorous antibody characterization and indicate that p63-isoform identification may improve the clinical value of p63 expression analyses.

Proteomics profiling of intact proteins based on MALDI-TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high-throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC-MS/MS with top-down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top-down based protein profiling at high resolution is discussed., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Triple-negative breast cancer (TNBC) represents a heterogeneous group of breast cancers that do not express ER-α, PgR and Her-2 receptors. Generally, these tumors are aggressive and more common in younger women, in which an association of TNBC with mutations in the BRCA1 gene was documented. The aim of our study was to create a representative group of patients with TNBC, which could be analyzed and the data gathered to build basic epidemiological, molecular and clinical characteristics of Czech patients with TNBC., Patients and Methods: We performed basic clinical-pathologic correlations in a group of 335 patients diagnosed and/or treated for TNBC at the Masaryk Memorial Cancer Institute between 2004 and 2009. We also performed immunohistochemical examination of expression of cytokeratin 5/6, cytokeratin 14 and EGFR to identify the basal-like subset of TNBC., Results: The median age of patients with TNBC was 56 years, range 25-88 years. A total of 9.25% of TNBC cases were diagnosed in patients under the age of 34, and another 15.22% of cases were in the age group of 35 to 44 years. 'Basal-like' carcinomas accounted for 75% of TNBC. We confirmed the aggressive nature of this disease: in the follow-up period we observed a relapse in 25% of patients: 55% of deaths due to disease progression occured within 2 years after diagnosis of the disease. Treatment strategies include chemotherapy, in most cases (88.4%). Chemotherapy was mostly based on regimens with anthracyclines or in combination with taxanes. The most important negative prognostic factors in relation to OS (disease specific OS) were: higher clinical stage (p < 0.0001), pN - positive status (p < 0.0001), high proliferative activity (as measured by Ki-67, cut-off 50%, HR = 0.4740, p = 0.0411) and positive expression of CK5/6 (HR = 0.4274, p = 0.0338). In relation to DFS, the negative prognostic significance was found for these factors: higher clinical stage (p < 0.0001), pN positive status (p < 0.0001), high proliferative activity (Ki-67, cut-off 50%, HR = 0.04993, p = 0.0240). DFS was longer in patients with a higher number of applied cycles of anthracycline-based chemotherapy (> 4 cycles, HR = 1.7273, p = 0.0467)., Conclusion: TNBC is an aggressive form of breast cancer, which may occur in patients of all ages, but more frequently in younger patients. Only early detection of disease and intensive treatment gives a high chance of cure. Unfortunately, no reliable predictive factors have been identified so far. Better therapeutic results can be expected from targeted therapy.

Transcriptomic screens in breast cancer cell lines have identified a protein named anterior gradient-2 (AGR2) as a potentially novel oncogene overexpressed in estrogen receptor (ER) positive tumours. As targeting the ER is responsible for major improvements in cure rates and prevention of breast cancers, we have evaluated the pro-oncogenic function of AGR2 in anti-hormone therapeutic responses. We show that AGR2 expression promotes cancer cell survival in clonogenic assays and increases cell proliferation and viability in a range of cancer cell lines. Chromatin immunoprecipitation and reporter assays indicate that AGR2 is transcriptionally activated by estrogen through ERalpha. However, we also found that AGR2 expression is elevated rather than inhibited in response to tamoxifen, thus identifying a novel mechanism to account for an agonistic effect of the drug on a specific pro-oncogenic pathway. Consistent with these data, clinical analysis indicates that AGR2 expression is related to treatment failure in ERalpha-positive breast cancers treated with tamoxifen. In contrast, AGR2 is one of the most highly suppressed genes in cancers of responding patients treated with the anti-hormonal drug letrozole. These data indicate that the AGR2 pathway represents a novel pro-oncogenic pathway for evaluation as anti-cancer drug developments, especially therapies that by-pass the agonist effects of tamoxifen.

Recent studies have suggested that genetic polymorphisms in the TP53 pathway influence tumour formation, progression and response to therapy. We analysed the three most common TP53 gene polymorphisms as potential genetic markers to predict the development and prognosis of breast cancer. The incidence of R72P, PIN3 Ins 16bp and PIN6 G13494A polymorphisms was determined in a cohort of 117 breast cancer tissues and 108 control specimens by PCR-RFLP. No significant difference was observed in the polymorphism variants in breast cancer specimens compared to controls. Furthermore, no statistically significant association of these polymorphisms with the outcome of the patients was observed. On the other hand we found positive correlation of lymph node metastases with both PIN3 Ins 16bp and PIN6 G13494A polymorphisms. The association of intronic TP53 variants with an aggressive breast cancer phenotype may represent a useful predictive biomarker, particularly in patients of clinical stage I with low or intermediate risk.

The present pilot study constitutes a proof-of-principle in the use of a quantitative LC-MS/MS based proteomic method for the comparative analysis of representative low-grade breast primary tumor tissues with and without metastases and metastasis in lymph node relative to the nonmetastatic tumor type. The study method incorporated iTRAQ stable isotope labeling, two-dimensional liquid chromatography, nanoelectrospray ionization and high resolution tandem mass spectrometry using the hybrid QqTOF platform (iTRAQ-2DLC-MS/MS). The principal aims of this study were (1) to define the protein spectrum obtainable using this approach, and (2) to highlight potential candidates for verification and validation studies focused on biomarkers involved in metastatic processes in breast cancer. The study resulted in the reproducible identification of 605 nonredundant proteins (p < or = 0.05). A quantitative comparison revealed 3/3 proteins with significantly increased/decreased level in metastatic primary tumor and 13/6 proteins with increased/decreased level in lymph node metastasis compared to nonmetastatic primary tumor (p < 0.01). Changes in selected differentially expressed proteins were verified with qRT-PCR. Although our pilot scale study does not warrant general biological conclusions, the synergic regulation of some proteins with related function (e.g., heme binding proteins, proteins of energetic metabolism, interferon induced proteins, proteins with adhesive function) determined in our sample set reflects the ability of our method in providing biologically meaningful data. The main conclusion from this pilot study was that our quantitative proteomic method constitutes a novel way of analyzing cancerous breast tissue biopsy samples that can be extended as part of a larger scale biomarker discovery program.

Objectives: SNP309 polymorphism (T-G) at the promoter region of MDM2 has been reported to cause increased binding affinity of transcriptional activator Sp1 followed by increased MDM2 both in mRNA and protein level. This model was proposed in vitro in the small panel of cell lines that indicated an on average 8-fold higher level of MDM2 mRNA in cells bearing the GG genotype., Methods: The incidence of SNP309 was determined in a cohort of 158 breast, 17 endometrium, 13 cervix and 45 ovarian cancer tissues by PCR-RFLP. The expression of p53 and MDM2 protein levels in the cohort was immunohistochemically investigated and statistically correlated with SNP309 polymorphism using Pearson chi(2) and t test., Results: No significant difference was observed in the G allele incidence in breast cancer specimens compared to 149 noncancer controls. Furthermore, no statistically significant association of the G allele frequencies and p53 and MDM2 protein expression levels was observed., Conclusions: Our data clearly show neither association between SNP309 and cancer risk, nor the responsibility of G allele for increased MDM2 or decreased of p53 protein levels in human primary breast tumors., (Copyright 2008 S. Karger AG, Basel.)

Introduction: Microarray-based gene expression profiling represents a major breakthrough for understanding the molecular complexity of breast cancer. cDNA expression profiles cannot detect changes in activities that arise from post-translational modifications, however, and therefore do not provide a complete picture of all biologically important changes that occur in tumors. Additional opportunities to identify and/or validate molecular signatures of breast carcinomas are provided by proteomic approaches. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) offers high-throughput protein profiling, leading to extraction of protein array data, calling for effective and appropriate use of bioinformatics and statistical tools., Methods: Whole tissue lysates of 105 breast carcinomas were analyzed on IMAC 30 ProteinChip Arrays (Bio-Rad, Hercules, CA, USA) using the ProteinChip Reader Model PBS IIc (Bio-Rad) and Ciphergen ProteinChip software (Bio-Rad, Hercules, CA, USA). Cluster analysis of protein spectra was performed to identify protein patterns potentially related to established clinicopathological variables and/or tumor markers., Results: Unsupervised hierarchical clustering of 130 peaks detected in spectra from breast cancer tissue lysates provided six clusters of peaks and five groups of patients differing significantly in tumor type, nuclear grade, presence of hormonal receptors, mucin 1 and cytokeratin 5/6 or cytokeratin 14. These tumor groups resembled closely luminal types A and B, basal and HER2-like carcinomas., Conclusion: Our results show similar clustering of tumors to those provided by cDNA expression profiles of breast carcinomas. This fact testifies the validity of the SELDI-TOF MS proteomic approach in such a type of study. As SELDI-TOF MS provides different information from cDNA expression profiles, the results suggest the technique's potential to supplement and expand our knowledge of breast cancer, to identify novel biomarkers and to produce clinically useful classifications of breast carcinomas.

Introduction: Trastuzumab is a humanized monoclonal antibody directed against the HER-2 receptor. Trastuzumab-based therapy significantly improves response rate (RR), time to progression (TTP) and overall survival (OS) for women with HER-2 positive metastatic breast cancer. Despite its initial efficacy, acquired resistance to trastuzumab develops in a majority of patients with MBC, and a large subset never responds, demonstrating primary resistance. The purpose of this retrospective study was to determine prognostic factors applicable to clinical practice., Methods: We enrolled 112 women with metastatic breast cancer, who started the trastuzumab-based therapy at Masaryk Memorial Cancer Institute until January 2007. Clinical and laboratory factors, such as: patients conditions, character ofmetastatic spread, histology, estrogen, progesterone and Her-2 receptor status, Her-2/neu gene amplification, and serum tumor markers CEA, CA 15-3 and extracellular domain of Her-2 receptor (S-HER-2 ECD) were monitored. The association of all factors to response to therapy, time to progression (TTP) and overall survival (OS) was assessed., Results: In 95% patients, the trastuzumab was combined with cytostatics (83% taxanes), 88,4% of patients started the trastuzumab as the first or second-line anticancer treatment. The median TTP was 284 days (9,3 months) and the median OS was 612 days (20,1 months) for all patients, RR was 54,5%. The highest RR was associated with the first-line treatment (p<0.0001) and with HER-2 gene/Chromosome 17 ratio > 2,2 (p=0,0092). Eleven patients (9,8%) discontinued the treatment because of toxicity, 7 patients did it as a result of cardiotoxicity (6,2%). CNS metastases occurred in 31 patients (27,7%). The S-HER-2 ECD was the most frequently elevated serum marker at the time of the treatment initialization (72,5%) and at the time of the progression (55,9%). Cox regression analysis identified S-HER-2 ECD levels at the beginning and between day 90 and 130 of the trastuzumab therapy as the best predictors of TTP. On the other hand the best predictor of OS was level of CEA before the treatment started and level of S-HER-2 ECD between day 90 and 130 of the trastuzumab therapy., Conclusions: We confirmed that the only one predictive marker for response to trastuzumab therapy is a proof of HER-2 tumor positivity.The highest prevalence of S-HER-2 ECD positivity among serum tumor markers and the strong association between initial and subsequent S-HER-2 ECD serum concentrations and time to progression and overall survival make the S-HER-2 ECD the most significant prognostic marker.

Background: The latest clinical trials indicate better performance of aromatase inhibitors, compared to tamoxifen, in adjuvant hormonotherapy of breast carcinoma. The identification of molecular markers, predicting resistance to tamoxifen, could help to identify patients, which are most likely to benefit from aromatase inhibitors in up-front adjuvant hormonotherapy., Material and Methods: Tissue microarrays were constructed from archival paraffin blocks of primary tumors of 179 patients with estrogen receptor positive operable breast carcinoma in stage I-III, subsequently treated with tamoxifen for five years or until relapse, with at least 7 years follow up available. The amplifications of Her-2 and cyclin D1 genes were evaluated by fluorescence in-situ hybridization. The level of progesterone receptor (PR) and Ki67 were estimated by immunohistochemistry., Results: 54 of above patients recurred during follow up. In univariate analysis of disease free survival, the presence of more than three nodal metastases (RR=4,5 p<0,001), grade 3 (RR=2,3 p=0,035), cyclin D1 (RR=3,06 p<0,001) and Her-2 (RR=3,06 p<0,001) amplifications were identified as significant risk factors, together with the negativity of PR (RR=2,1 p=0,013). In multivariate analysis, only clinical stage III (RR=2,6 p=0,003), cyclin D1 (RR=2,7 p=0,001) and Her-2 (RR=2,1 p=0,014) amplifications proved significant. In 77 patients who received adjuvant chemotherapy no statistically significant risk factor was identified. In multivariate analysis of 102 patients without adjuvant chemotherapy only stage III (RR=6,9 p=0,001) and Her-2 amplification (RR=4,5 p=0,001) were confirmed., Conclusion: The advanced clinical stage, cyclin D1 and Her-2 gene amplifications represent factors, predicting the failure of adjuvant tamoxifen treatment, but their predictive value is much lower in patients receiving adjuvant chemotherapy. This fact indicates, they can reflect the common biological aggressiveness of tumor and need not to be tamoxifen specific.

The expression of cytokeratins 7, 8, 14, 18, 19 and vimentin was examined in 100 cases of ductal invasive breast carcinomas. While the predominantly diffuse immunohistological positivity of simple epithelia cytokeratins 7 (in 93), 8 (in 100), 18 (in 100) and 19 (in 97) cases represents a constant feature of these tumors, cytokeratin 14 was detected in only 36 cases which were mostly of low grade and in a focal pattern. Vimentin positivity was found in 53 intermediate and high grade tumors and, again the pattern was also rarely diffuse. The ductal carcinomas can be grouped into four classes according to vimentin and cytokeratin 14 immunoreactivity. This grouping correlates well with tumor grade and with simple histological classification of ductal breast carcinoma, consisting of the low, intermediate and high malignancy categories, as proposed here. The types ofductal carcinomas can be sorted into prognostically different subgroups, according to ICD-O morphologic terminology and commonly adopted results of morphologic and prognostic studies.

A simple classification of ductal breast carcinoma, consisting of the low, intermediate and high malignancy categories is proposed. The tumour subtypes, categorized according to WHO classification, were sorted into prognostically different subgroups, taking the modified Bloom's grading score and previously published histological studies into account. The low grade category includes papillary, cribriform, tubular, colloid and typical medullary (with the lymphoid stroma) carcinomas. The intermediate group consists of ductal not otherwise specified and mixed ductal/lobular tumours. The high grade category includes atypical medullary, comedo and mucin producing carcinomas. This classification is fully compatible with the commonly adopted ICD-O (1990) morphologic terminology. It should simplify the histological reporting of breast cancer so that the results were as reproducible as from the working formula of malignant lymphomas.

We analysed antibodies specific for human p53 in sera from primary breast cancer patients using three different immunoassays and we related these results to the p53 level in tumour tissue detected by immunohistochemistry. Only 44% (11/25) of apparently enzyme-linked immunosorbent assay (ELISA)-positive sera were from patients with a high level of p53 protein in more than 50% of their tumour cells. Surprisingly, 36% (9/25) of the sera originated from patients with no detectable p53 protein at all. Immunoprecipitation data suggested that the reason for this discrepancy is that at least some of the antibodies detected as positive in the ELISA in these sera from patients with clinical stage I and stage II breast cancers may be induced by immunogens other than p53 protein. Many of these reactions give apparently positive signals in a variety of p53 assays, and very stringent analysis is required to avoid possible misinterpretation of these responses as a p53-specific B-cell response in human cancer patients.

We have analyzed p53 protein expression in 121 primary breast cancer biopsies by immunohistochemistry using the monoclonal antibody DO-1 and polyclonal serum CM-1. p53 protein overexpression has correlated in our study with mitotic activity (p=0.001), nuclear atypia (p=0.002), less favorable histological type of tumor and in a lesser extent with tumor size. The inverse, but highly significant, correlation (p=0.007) has been observed with lymph node involvement. There was also a trend for higher p53 positivity among DNA aneuploid tumors as compared with DNA diploid cases, but this was not significant. Our study suggests that p53, at least in some patients, may not be directly involved in the process of metastatic progression in breast cancer. Preliminary data would suggest that the detection of p53 protein overexpression could be a useful additional prognostic parameter in breast cancer.