Correlation of Expression of CHI3L1 and Nogo-A and their Role in Angiogenesis in Invasive Ductal Breast Carcinoma.

BACKGROUND/AIM: Chitinase 3 like 1 (CHI3L1) is a secretion glycoprotein. Elevated levels of this protein are observed in cancer diseases. The biological role of CHI3L1 is not yet fully known, but the connection between CHI3L1 and angiogenesis has been shown. Recent reports also describe the association of Nogo isoforms and Nogo-B receptor (NgBR) with a proliferative potential, cancer cell invasiveness, and angiogenesis. The aim of this study was to evaluate the levels of CHI3L1, Nogo-A, Nogo-A/B, and NgBR and correlate them with clinical-pathological data, to study their role in angiogenesis in invasive ductal breast carcinoma (IDC). MATERIALS AND METHODS: A total of 77 IDC cases were used in the study. Immunohistochemistry was used to determine the level of expression of CHI3L1, Nogo-A, Nogo-A/B, NgBR and vascular endothelial growth factors (VEGFA, VEGFC and VEGFD). The obtained results were subjected to statistical analysis including clinicalpathological data. RESULTS: A statistically significant positive correlation of CHI3L1 and Nogo-A expression (r=0.474, p>0.0001) and a positive correlation of Nogo-A and VEGFC expression (r=0.280, p=0.013) were found. CONCLUSION: CHI3L1 and Nogo-A are important in angiogenesis in IDC.

Sulfatide decreases the resistance to stress-induced apoptosis and increases P-selectin-mediated adhesion: a two-edged sword in breast cancer progression.

BACKGROUND: We have previously shown that galactosylceramide (GalCer) affects the tumourigenic and metastatic properties of breast cancer cells by acting as an anti-apoptotic molecule. Since GalCer is a precursor molecule in the synthesis of sulfatides, the present study was aimed to define the role of sulfatides in apoptosis and breast cancer progression. METHODS: Expression of GAL3ST1 in breast cancer cell lines and breast cancer tissue specimens was analysed using real-time PCR, western blotting and immunohistochemistry analysis. The amount of sulfatide, GalCer and ceramide was analysed by thin-layer chromatography binding assay and by the modified hydrophilic interaction liquid chromatography coupled with electrospray mass spectrometry methodology. The tumourigenicity of cancer cells was analysed by an in-vivo tumour growth assay. Apoptotic cells were detected based on caspase-3 activation and the TUNEL assay. The interaction of breast cancer cells with P-selectin or E-selectin was analysed using the flow adhesion assay. The ability of sulfatide-expressing cells to activate and aggregate platelets was studied using the flow-cytometry-based aggregation assay. RESULTS: Using two models of breast cancer, T47D cells with blocked synthesis of sulfatide and MDA-MB-231 cells with neosynthesis of this glycosphingolipid, we showed that high sulfatide levels resulted in increased sensitivity of cancer cells to apoptosis induced by hypoxia and doxorubicin in vitro, and decreased their tumourigenicity after transplantation into athymic nu/nu mice. Accordingly, a clinical study on GAL3ST1 expression in invasive ductal carcinoma revealed that its elevated level is associated with better prognosis. Using MDA-MB-231 cells with neosynthesis of sulfatide we also showed that sulfatide is responsible for adhesion of breast cancer cells to P-selectin-expressing cells, including platelets. Sulfatide also acted as an activating molecule, increasing the expression of P-selectin. CONCLUSIONS: This study demonstrates that increased synthesis of sulfatide sensitises cancer cells to microenvironmental stress factors such as hypoxia and anticancer drugs such as doxorubicin. However, sulfatide is probably not directly involved in apoptotic cascades, because its increased synthesis by GAL3ST1 decreased the amounts of its precursor, GalCer, a known anti-apoptotic molecule. On the other hand, our data support the view that sulfatides are malignancy-related adhesive molecules involved in activating and binding P-selectin-expressing platelets to breast cancer cells.

Expression of p16 and SATB1 in Invasive Ductal Breast Cancer - A Preliminary Study.

BACKGROUND/AIM: An impaired cell-cycle control and genetic material organization are crucial elements of carcinogenesis. p16 is a tumor suppressor protein which decelerates promotion of the cells from G1 to S phase, whereas special AT-rich sequence-binding protein 1 (SATB1) is a nuclear matrix protein that binds to specific regions of the DNA and ensures its proper organization and function. Increased levels of both markers are observed in various types of cancers. The aim of this study was to investigate the expression of p16 and SATB1 proteins in regard to expression of the Ki-67 antigen and available clinicopathological data (i.a. receptor status, staging and grading). MATERIALS AND METHODS: The study was performed on 130 samples of archived invasive ductal breast cancers. Immunohistochemical reactions were performed on freshly prepared tissue microarrays and subsequently scanned by a histologic scanner. Reactions were evaluated separately in the cytoplasm (p16c, SATB1c) and nucleus (p16n, SATB1n, Ki-67) with use of a quantification software under researcher supervision. RESULTS: Expression was observed for Ki-67 in 100%, p16c in 90%, p16n in 89.2%, SATB1c in 98.5% and SATB1n in 87.7% of cancer cases. Statistical analysis showed strong positive correlations: p16c vs. p16n and SATB1c vs. SATB1n (p<0.001 for both) and weak positive correlations: p16c vs. SATB1c and p16c vs. SATB1n (p=0.008, p=0.027; respectively). Expression of p16n was stronger in G1 vs. G2 (p=0.034) while Ki-67 expression was stronger in cases with negative progesterone receptor status (p=0.011). All other analyzed associations were statistically insignificant. CONCLUSION: A weak association between immunohistochemical expression of p16 and SATB1 indicated limited possibility of their independent usage. Further studies concerning determination of a wider panel of proteins controlling cell cycle should be considered.

The Role of CHI3L1 Expression in Angiogenesis in Invasive Ductal Breast Carcinoma.

BACKGROUND/AIM: An increased level of chitinase 3 like 1 protein (CHI3L1) expression is observed in patients with cancer and may have potential prognostic value. The aim of this study was to evaluate the role of CHI3L1 in angiogenesis in invasive ductal breast carcinoma (IDC) (n=110). MATERIALS AND METHODS: Immunohistochemistry was used to assess the expression of CHI3L1, CD31, CD34, vascular endothelial growth factor (VEGFA, VEGFC and VEGFD). Real-time polymerase chain reaction and western blot were used to determine the level of CHI3L1 mRNA and protein. RESULTS: Immunohistochemistry demonstrated positive correlation between CHI3L1 expression and angiogenesis markers: CD31 (r=0.34, p=0.0003), CD34 (r=0.24, p=0.012), VEGFD (r=0.24, p=0.013). Higher CHI3L1 expression in estrogen receptor-negative (p=0.041) and progesterone receptor-negative (p=0.014) cancer was observed. Higher CHI3L1 expression was reported in cancer tissues in comparison to non-malignant breast lesions. CONCLUSION: These results suggest a potential role of CHI3L1 in angiogenesis in IDC and may suggest its involvement in cancer progression.

Aberrant Expression of PIWIL1 and PIWIL2 and Their Clinical Significance in Ductal Breast Carcinoma.

BACKGROUND/AIM: P-Element-induced wimpy testis (PIWI) proteins in complex with PIWI-interacting RNA (piRNA) are involved in epigenetic regulation of gene expression in germline cells. Aberrant expression of piRNA and PIWI proteins have been identified in various types of tumour cells. The aim of this study was to evaluate the expression profiles of PIWI-like protein-1, -2 (PIWIL1 and PIWIL2), their immunohistochemical (IHC) characteristics in ductal breast cancer, and determine their correlation with clinicopathological parameters of this type of cancer. MATERIALS AND METHODS: Material for IHC studies comprised of 101 invasive ductal carcinoma (IDC) cases and 31 mastopathy tissues. Frozen fragments of paired tissue specimens (tumour and adjacent non-malignant breast tissue) taken from 55 patients with IDC and 18 samples of mastopathy were used for molecular studies using real-time polymerase chain reaction (RT-PCR). RESULTS: A statistically significantly higher level of PIWIL1 and PIWIL2 was found in IDC compared to mastopathy samples (p≤0.0001). Increased expression of PIWIL1 was correlated with increased PIWIL2 expression in breast cancer tissue. Surprisingly, PIWIL1 mRNA was detected only in cancer and mastopathy, but was not found in most normal breast tissues, although it is noteworthy that the PIWIL2 mRNA level was statistically significantly lower in mastopathy and IDC samples compared to normal breast tissues. CONCLUSION: Our results affirm the hypothesis that reactivation of PIWI expression in various caner types is crucial for cancer development.

Expression of periostin in breast cancer cells.

Periostin (POSTN) is a protein involved in multiple processes important for cancer development, both at the stage of cancer initiation and progression, as well as metastasis. The aim of this study was to determine the expression of POSTN in the cells of non-invasive ductal breast carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) and to correlate it with clinicopathological data. Immunohistochemical studies (IHC) were conducted on 21 cases of fibrocystic breast change (FC), 44 cases of DCIS and 92 cases of IDC. POSTN expression at mRNA (real-time PCR) and protein level (western blot analysis) was also confirmed in selected breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231 and BO2). Statistically significant higher level of POSTN expression in IDC and DCIS cancer cells compared to FC was noted. Also, the level of POSTN expression in the cytoplasm of IDC cells was shown to increase with the increasing degree of tumour malignancy (G) and significantly higher expression of POSTN was observed in each degree of tumour malignancy (G) relative to FC. Statistically significant higher POSTN expression was observed in tumours with estrogen receptor-negative (ER-) and progesterone receptor-negative (PR-) phenotypes in comparison to estrogen receptor-positive (ER+) and progesterone receptor-positive (PR+) cases, as well as significant negative correlation between POSTN expression in cancer cells and expression of ER and PR (p<0.05). Additionally, statistically significant differences in POSTN expression were shown between particular breast cancer cell lines, both at mRNA and protein level. Observed POSTN expression was the lowest in the case of MCF-7, and the highest in MDA-MB-231 and BO2 of the most aggressive potential clinically corresponding to G3 tumours. POSTN expression in the cytoplasm of IDC cancer cells may play an important role in cancer transformation mechanism.

Expression of metallothionein 3 in ductal breast cancer.

Metallothionein 3 (MT-3) has the ability to regulate the growth of nerve cells, but the significance of MT-3 expression outside the central nervous system and its participation in carcinogenesis have not yet been clarified. The aim of our study was to investigate the expression of MT-3 in ductal breast cancer and to determine its relationship with well-defined clinicopathological factors in this type of tumor. The study was conducted on 134 cases of invasive ductal breast carcinoma (IDC), 42 samples of non-malignant breast tissue (NMBT), and 26 cases of mastopathy. Moreover, selected breast cancer cell lines (MCF-7, SKBR-3, MDA-MB-231, BO2) and normal human breast epithelial cells (hTERT-HME1) were used. The expression of MT-3 was examined on the protein level using immunohistochemistry and on the mRNA level using real-time PCR. It was shown that the MT-3 protein in cells of IDC and mastopathy appeared in the cytoplasm as well as in the cell nuclei. Both the cytoplasmic and nuclear expression of MT-3 was significantly lower in IDC than in the mastopathies (p<0.0001 and p<0.001). However, no significant correlation was demonstrated between the level of MT-3 protein and the studied clinicopathological factors. The mRNA expression of MT-3 in IDC was also lower than in non­malignant breast tissue (p<0.0001). Furthermore, in the cases of IDC with lymph node metastasis, the level of MT-3 mRNA was significantly lower than in the cases without metastasis (p=0.0199). The expression of MT-3 mRNA in breast cancer cell lines was significantly lower than in the normal human breast epithelial cell line (p<0.001). These results suggest that MT-3 may play a role in the malignant transformation of breast epithelial cells and in tumor progression.

Prognostic Significance of NOGO-A/B and NOGO-B Receptor Expression in Malignant Melanoma - A Preliminary Study.

BACKGROUND: Neurite outgrowth inhibitor type B (NOGO-B) and its receptor (NGBR) were shown to regulate various crucial cellular processes and may be therefore potential factors influencing carcinogenesis. MATERIALS AND METHODS: Expression of NOGO-A, NOGO-A/B and NGBR was studied in benign melanocytic lesions and primary tumors and metastases of malignant melanoma (MM). RESULTS: Cytoplasmic expression of the studied antigens was detected in melanocytes and MM cells. NOGO-A/B expression was significantly lower in metastatatic MM cases compared to primary MM tumors (p<0.01) and bening melanocytic lesions (p<0.001). In primary MM tumors, NOGO-A expression intensity positively correlated with NOGO-A/B (r=0.32, p<0.05) and NGBR expression (r=0.53, p<0.0001). NOGO-B and NGBR immunoreactivity correlated negatively with depth of primary MM infiltration (both p<0.01). Moreover, low NOGO-A/B expression was a factor of poor prognosis of primary MM. CONCLUSION: NOGO-A/B may be a negative prognostic factor of MM.

Impact of SOX18 expression in cancer cells and vessels on the outcome of invasive ductal breast carcinoma.

PURPOSE: SOX18 is a transcription factor known to be involved in hair follicle, blood and lymphatic vessel development, as well as wound healing processes (together with SOX7 and SOX17). In addition, it has been reported that SOX18 may affect the growth of cancer cells in vitro. Until now, the exact role of SOX18 expression in invasive ductal breast carcinoma (IDC) has remained unknown. METHODS: In this study, we have investigated SOX18 expression in cancer cells and endothelial cells in 122 IDC samples using immunohistochemistry (IHC). SOX18 expression was also determined using real-time PCR and Western blotting in a series of breast cancer-derived cell lines (i.e., MCF-7, BT-474, SK-BR-3, MDA-MB-231, BO2). RESULTS: Using IHC, we observed SOX18 nuclear expression in cancer cells, as well as in blood and lymphatic vessels of the IDC samples tested. SOX18 expression in the IDC samples correlated with a higher malignancy grade (Grade 2 and Grade 3 versus Grade 1; p = 0.02 and p = 0.009, respectively) and VEGF-D expression (r = 0.27, p = 0.007). SOX18 expression was also associated with HER2 positivity (p = 0.02). A significantly higher SOX18 expression was found in the HER2-positive cell line BT-474, and a significantly lower expression in the triple negative cell lines MDA-MB-231 and BO2. Laser capture microdissection of IDC samples revealed significantly higher mRNA SOX7, SOX17 and SOX18 expression levels in the vessels as compared to the cancer cells (p = 0.02 and p = 0.0002, p < 0.0001, respectively). SOX18 positive intratumoral and peritumoral microvessel counts (MVC) were associated with higher malignancy grades (p = 0.04 and p = 0.02, respectively). Moreover, peritumoral SOX18 positive MVC were found to act as an independent marker for a poor prognosis (p = 0.04). CONCLUSION: SOX18 expression may serve as a marker for a poor prognosis in IDC.

Impact of different tumour stroma assessment methods regarding podoplanin expression on clinical outcome in patients with invasive ductal breast carcinoma.

BACKGROUND: Podoplanin, a small mucin-type transmembrane protein has been shown in several studies to be expressed in cancer-associated fibroblasts (CAFs) and affect patient outcome. MATERIALS AND METHODS: We evaluated podoplanin expression in CAFs in a cohort of 257 patients with invasive ductal breast carcinomas (IDCs) using three different assessment scales based on the number of positive cells alone or in combination with the reaction intensity. RESULTS: Two of the utilized scales yielded prognostic information concerning patients' overall survival (OS), but scores were not independent prognostic factors in the multivariate analysis. On the contrary, two scales based on the combination of cell positivity and reaction intensity had no significant impact on patients' OS, but they were significantly correlated with a greater number of analysed clinicopathological parameters. CONCLUSION: In summary, podoplanin expression in CAFs may be considered a possible marker of poor prognosis in IDC, however, caution should be taken as the results varied regarding the utilized scales.

Discrepancies between HER2 assessment from core needle biopsies and surgical specimens of invasive ductal breast carcinoma.

BACKGROUND: The assessment of HER2 status is particularly important for qualifying patients for trastuzumab treatment of invasive ductal breast carcinoma (IDC). HER2 assessment in core needle biopsies (CNBs) of IDC could contribute to a better therapy schedule. OBJECTIVES: The study aimed at examining the relationship between HER2 immunohistochemistry assessment scores in paired CNBs and whole tissue sections of IDC. MATERIAL AND METHODS: The study was performed on paired samples of CNBs and whole tissue sections from 49 IDC patients operated on at the Lower Silesian Oncology Center in Wroclaw, Poland. RESULTS: Discrepancies in HER2 scores were noted in eleven (22.45%) of the paired samples analyzed. Three cases (6.12%) were underscored in the CNB specimens as compared to the surgical HER2 specimens, whereas eight cases (16.33%) were overscored in the CNB specimens. CONCLUSIONS: Based on the high level of discrepancy between the tested pairs of IDC tissues, the authors recommend caution in assessing HER2 in CNB tissue specimens as a standard procedure. Wherever possible whole tissue sections should be utilized for HER2 assessment.

Expression of melatonin receptor MT1 in cells of human invasive ductal breast carcinoma.

In humans, two main types of membrane melatonin receptors have been identified, MT1 and MT2. Expression of MT1 in neoplastic cells seems to increase the efficacy of melatonin's oncostatic activity. The purpose of this study was to determine the distribution and the intensity of MT1 expression in breast cancer cells and to correlate it with clinicopathological factors. Immunohistochemical studies (IHC) were conducted on 190 cases of invasive ductal breast carcinomas (IDC) and molecular studies were performed on 29 cases of frozen tumor fragments and selected breast cancer cell lines. Most of the studied tumors manifested a membranous/cytoplasmic IHC expression of MT1. In IDC, the MT1 expression was higher than in fibrocystic breast disease. MT1 expression was higher in estrogen receptor positive (ER+) and HER2 positive (HER2+) tumors. Triple negative tumors (TN) manifested the lowest MT1 expression level. The lowest MT1 protein expression level was noted in the TN breast cancer cell line MDA-MB-231 compared with ER+ cell lines MCF-7 and SK-BR-3. MT1 mRNA expression was negatively correlated with the malignancy grade of the studied IDC cases. Moreover, higher MT1 expression was associated with patients' longer overall survival (OS) in the group of ER+ breast cancers and treated with tamoxifen. Multivariate analysis indicated that MT1 was an independent prognostic factor in the ER+ tumors for OS and event-free survival in the ER+ tumors. The results of this study may point to a potential prognostic and therapeutic significance of MT1 in IDC.

Expression of SATB1 protein in the ductal breast carcinoma tissue microarrays - preliminary study.

Special AT-rich sequence-binding protein 1 (SATB1) is a nuclear matrix protein which interacts with specific regions of DNA, ensuring its proper organization and function in the cell. The expression of SATB1 was primarily found in thymocytes, but its increased levels were observed in various types of cancers. However, the knowledge of the function and application possibilities of this protein is still limited. The aim of this study was to investigate the expression of SATB1 protein using immunohistochemistry and tissue microarray (TMA) technique and determine its possible relationship with the proliferative marker Ki-67, estrogen a (ER) and progesterone (PR) receptors as well as grade of histological malignancy (G). The study was performed on material of 48 archival invasive ductal breast cancers (IDC). The TMAs were prepared with the use of 0.6 mm diameter punches. Immunohistochemical reactions were carried out using antibodies against Ki-67, ER, PR and SATB1 proteins. The intensity of the nuclear reaction was evaluated using a light microscope and computer-assisted image analysis. Expression of Ki-67 and SATB1 protein was observed in 89.58% and 31.25% of cancer cases, respectively. 62.5% of tumors were classified as ER-positive, and 47.92% as PR-positive. Statistical analysis showed a moderate positive correlation between Ki-67 and SATB1 expression (r = 0.291, p = 0.045 independently on the receptor status, and r = 0.392, p = 0.032 in ER-negative tumors). The expression of the Ki-67 antigen increased with higher grade of histological malignancy (G). The results suggest that SATB1 protein may play an indirect role in the cell proliferation and should be evaluated in relation to the other markers. Further studies concerning determination of its role in cancer progression and metastasis, in terms of application as therapeutic target and prognostic marker, are recommended.

Therapeutic radiation induces different changes in expression profiles of metallothionein (MT) mRNA, MT protein, Ki 67 and minichromosome maintenance protein 3 in human rectal adenocarcinoma.

AIM: The study aimed at the evaluation of the effects of radiotherapy on expression of metallothionein (MT) isoforms, both in the form of quantitative alterations in mRNA, and differences in expression of MTI/II proteins in rectal tumours. MATERIALS AND METHODS: Material for the study originated from 21 patients with rectal cancer at stage II or III. Material for immunohistochemical studies [MTI/II, Minichromosome Maintenance Protein 3 (MCM3), Ki-67] and real-time polymerase chain reaction (PCR) (mRNA of MT1F, MT1X and MT2A) was sampled twice: during rectoscopic examination before the start of the preoperative radiotherapy (samples A) and from the post operative specimen, following radiotherapy (samples B). RESULTS: The level of mRNA expression for each of the studied MT isoforms was higher in cancer cells subjected to irradiation. The most extensive differences were observed for the MT2A isoforms (p=0.09). No differences were disclosed between samples A and B in expression of MT I/II protein. The material sampled after radiotherapy manifested a tendency for reduced proliferative activity of the tumour cells: the decrease of MCM3 expression was significant (p=0.022), while in the case of Ki-67, the difference approached statistical significance (p=0.096). CONCLUSION: Application of radiotherapy to rectal adenocarcinoma cells is followed by an increase in MT mRNA expression level, affecting first of all the MT2A isoform. However, we failed to note an increased expression of MTI/II protein coded by the gene. Moreover, application of radiotherapy was followed by a decrease in expression of MCM3 protein. Our results cannot clearly confirm induction of MT after irradiation of human adenocarcinoma cells. The role of MT in radioprotection remains ambiguous.

Hsp-27 expression in invasive ductal breast carcinoma.

The aim of this study was to determine the intensity of Hsp27 protein expression in fibrocystic breast changes (FC) and invasive ductal breast carcinoma (IDC) and to examine its impact on patients' clinico-pathological characteristics and overall survival. Immunohistochemical reactions were conducted on archival samples of 20 cases of FC and 101 cases of IDC treated in 1999-2002. Nuclear-cytoplasmic Hsp27 expression was observed in 92 (92.1%) of the examined cases of IDC, and all the cases of FC. Significantly higher Hsp27 expression was observed in G2 (p < 0.01) and G3 cases (p < 0.0001) compared to FC. HER-2 positive cases had higher Hsp27 expression (p = 0.0153), than HER-2 negative cases. Our research showed that Hsp27 could have an impact on tumor malignancy. Moreover, a positive correlation between the expression of Hsp27 and HER-2 positive cases was demonstrated.

Matrix metalloproteinase (MMP)-2 and MMP-9 expression in tumor infiltrating CD3 lymphocytes from women with endometrial cancer.

OBJECTIVE: In this study, we hypothesized that not only endothelial malignant cells but also lymphocytes infiltrating tumor epithelium, in patients with endometrial cancer, could be an important source of the gelatinases (matrix metalloproteinase [MMP]-2 and MMP-9) extensive production, which in turn, may facilitate tumor cells infiltration and progression due to the extracellular matrix degradation. MATERIALS AND METHODS: First, we isolated lymphocytes from the endometrial carcinoma samples taken from 41 patients who were operated on and from healthy endometrial tissue taken of the same patients after histological verification. Then, we detected the level of CD3-positive cells in endometrial tissues by flow cytometry. Simultaneously, we studied the messenger RNA expression of MMP-2 and MMP-9 in the isolated cells from malignant and unchanged endometrial tissues. Using immunohistochemistry, we compared the protein expression of MMP-2, MMP-9, and CD3 in the studied samples. RESULTS: We showed the enhanced abundance of CD3 lymphocytes both by flow cytometry and immunohistochemistry in the samples from malignant tissues. The expression of MMP-9 in the endometrial carcinoma was increased significantly at the protein level but not at the messenger RNA level. We could not observe any differences concerning MMP-2 expression in both methods of detection. CONCLUSIONS: CD-3 lymphocytes significantly infiltrate endometrial cancer tissue, but they do not seem to be the source of enhanced metalloproteinases 2 and 9 expression in the tumor environment. Still, owing to the immunohistochemistry staining, we could show the significant increase of MMP-9 protein in the very close vicinity of tumor-infiltrating CD3 lymphocytes. Could it be the result of CD3 lymphocyte action, or is it just the imperfection of the detecting method we used? This remains unclear. Further studies explaining the role of tumor infiltrating lymphocytes in mediating the endometrial cancer milieu are needed.

Angiotensin II type 1 receptor (AT-1R) expression correlates with VEGF-A and VEGF-D expression in invasive ductal breast cancer.

Recent studies point to the involvement of angiotensin II (Ang II) receptor type 1 (AT-1R) on processes of metastasing, stimulation of invasiveness and angiogenesis in tumours. In this study, the correlation between intensity of AT-1R expression and expression of lymph- and angiogenesis markers in invasive ductal breast cancers (IDC) was examined. Immunohistochemical studies (IHC) were performed on archival material of 102 IDC cases. Only 28 (27.5%) cases manifested low AT-1R expression while 74 (72.5%) cases demonstrated a moderate or pronounced AT-1R expression. Expression intensity of AT-1R was found to correlate with expressions of VEGF-A (r = 0.26; p = 0.008) and VEGF-D (r = 0.24; p = 0.015). Out of the examined markers of angiogenesis and lymphangiogenesis only the pronounced expression of VEGF-C was found to correlate with patient poor clinical outcome (p = 0.009). The positive correlation between AT-1R and VEGF-A and VEGF-D could point to stimulatory action of Ang II on their expression what might result in augmented lymph- and angiogenesis in IDC.

Tissue microarray technique in evaluation of proliferative activity in invasive ductal breast cancer.

AIM: Investigation of the relationship between expression of Ki-67 antigen and minichromosome maintenance 2 (MCM-2) protein by using an immunohistochemical study on whole sections (WS) and sections of invasive ductal breast cancer (IDC) obtained in the tissue microarray (TMA) technique. MATERIALS AND METHODS: Material consisted of 51 archival paraffin blocks of IDC. Tissue microarrays were composed of 0.6 mm core punches. Reactions were performed using specific antibodies (anti-Ki-67 and anti-MCM-2). Intensity of the marker expression was evaluated using computer-assisted image analysis. For statistical purposes, three different tests were used. RESULTS: Spearman rank correlation revealed a strong positive correlation between expression of tested markers: Ki-67 (TMA) vs. Ki-67 (WS) (r=0.91, p<0.05) and MCM-2 (TMA) vs. MCM-2 (WS) (r=0.87, p<0.05). Mann Whitney U-test showed no significant differences between the two markers in both analysed techniques. Moreover, the Bland-Altman plot demonstrated a low level of bias between the analysed methods. CONCLUSION: This study shows that TMA could have a great potential in evaluation of proliferative activity in IDC and that MCM-2 protein might be a specific and sensitive marker of cell proliferation.

Podoplanin expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma.

AIMS: It has recently been shown that podoplanin, a mucin-type glycoprotein, is expressed by cancer cells and cancer-associated fibroblasts (CAFs), and promotes cancer cell migration and invasiveness. The biological role of podoplanin expression in tumour stroma of invasive ductal carcinoma of the breast (IDC) has not been determined. METHODS AND RESULTS: Podoplanin expression was analysed in 117 cases of IDC and 27 cases of fibrocystic change, as well as in breast cancer cell lines, with the use of immunohistochemistry and real-time polymerase chain reaction. In 82.1% of analysed tumours, podoplanin was found only in CAFs. Only two of 117 IDC cases (1.7%) were characterized by expression of this glycoprotein in cancer cells. None of the fibrocystic changes or stroma surrounding normal ducts showed podoplanin expression. Podoplanin-positive CAFs correlated with tumour size (P = 0.0125), grade of malignancy (P = 0.0058), lymph node metastasis (P = 0.0149), lymphovascular invasion (LVI) (P = 0.0486) and Ki67 expression in cancer cells (P = 0.0128). High-level podoplanin expression (>50% of positive stroma) in the tumour stroma was significantly associated with a negative oestrogen status (P = 0.0201). Univariate, but not multivariate, analysis showed that podoplanin expression by CAFs was associated with poor patient outcome (P = 0.0202). CONCLUSIONS: Our results suggest that podoplanin expression by CAFs could be an unfavourable prognostic marker for IDC.

Correlation of intensity of MT-I/II expression with Ki-67 and MCM-2 proteins in invasive ductal breast carcinoma.

The purpose of the study was to evaluate the clinical significance of the intensity of metallothionein (MT-I/II) expression and its relationship to two different proliferation markers, Ki-67 antigen and minichromosome maintaince 2 protein (MCM-2) in 117 patients with invasive ductal breast carcinoma (IDC). A significantly higher MT-I/II expression was noted in the grade 3 (G3) carcinomas as compared to those of G1 and G2. A positive correlation was observed between the MT-I/II expression and both proliferation markers, Ki-67 (r=0.20, p=0.0343) and MCM-2 (r=0.25, p=0.0065). Also a strong positive correlation was noted between Ki-67 and MCM-2 expression (r=0.52, p<0.0001). No significant correlations were found between the analyzed markers and tumour size, lymph node metastasis, oestrogen expression (ER), progesterone receptor (PR) or human epidermal growth-factor receptor (HER-2) expression. Out of the three studied markers only the high expression of Ki-67 exhibited a negative impact on patient overall and event free survival and was an independent prognostic factor. MT-I/II and MCM-2 protein expression was not correlated with poor patient outcome, although MCM-2 expression correlated (Fisher's exact test) positively with grade of malignancy (p=0.0018) and negatively with ER (p=0.0002) and PR (p=0.0056) expression. To conclude, MT-I/II may play a key role in IDC proliferation, but is not a useful prognostic factor of this disease.