Zorica Milosevic

Lactate dehydrogenase, Catalase, and Superoxide dismutase in Tumor Tissue of Breast Cancer Patients in Respect to Mammographic Findings

Lactate dehydrogenase (LDH), marker of anaerobic metabolism, is associated with highly invasive and metastatic breast cancer. Novel studies show that increased anaerobic metabolism (LDH), as well as activity of antioxidative enzymes (superoxide dismutase (SOD) and catalase (CAT)), is correlated with higher mammographic density, as known predictor of breast cancer risk. In this study, we measured LDH, MDH, and SOD activity in tumor and adjacent tissues of breast cancer patients by spectrophotometric assay. Mammograms were evaluated according to the American College of Radiology Breast Imaging Reporting and Data system. Mammographically dense breast tissue is associated with higher activity of LDH in tumor tissue of breast cancer patients. Moreover, patients with masses have significantly higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Patients with spiculated mass margin had higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Activity of LDH in patients significantly increases, while activity of CAT significantly decreases with the increase of BIRADS category. These results suggest that the association of activity of LDH and CAT in tumor tissue with mammographic characteristics could help in defining aggressive breast cancers.

Breast ductal carcinoma in situ: morphologic and kinetic MRI findings.

Background Adequate diagnosis of ductal carcinoma in situ (DCIS) could lead to efficacious treatment. Due to the fact that DCIS lesions can progress to invasive carcinomas and that the sensitivity of the standard examination – mammography – is between 70 and 80%, use of a more sensitive diagnostic tool was needed. In detection of DCIS, contrast-enhanced magnetic resonance imaging (CE-MRI) has the sensitivity up to 96%. Objectives Morphological features and kinetic parameters were evaluated to define the most regular morphological, kinetic and morpho-kinetic patterns on MRI assessment of breast ductal carcinoma in situ (DCIS). Patients and Methods We retrospectively assessed eighteen patients with 23 histologically confirmed lesions (mean age, 52.4 ± 10.5 years). All patients were clinically and mammographically examined prior to MRI examination. Results DCIS appeared most frequently as non-mass-like lesions (12 lesions, 52.17%). The differences in the frequency of lesion types were statistically significant (P<0.05). The following morphological patterns were detected: A: no specific morphologic features, B: linear/branching enhancement, C: focal mass-like enhancement, D: segmental enhancement, E: segmental enhancement in triangular shape, F: diffuse enhancement, G: regional heterogeneous enhancement in one quadrant not conforming to duct distribution and H: dotted or granular type of enhancement with patchy distribution. The difference in the frequency of the proposed patterns was statistically significant (P<0.05). There were eight lesions with mass enhancement, and six with segmental lesions: regional and triangular. There was no statistically significant difference in the frequency of enhancement curve types (P>0.05). There was no significant difference in the frequency of morpho-kinetic patterns. Conclusion Non-mass-like lesions, lesions with focal or segmental distribution, with a “plateau” enhancement curve type were the most frequent findings of DCIS lesions on MRI.

Purine nucleoside analog--sulfinosine modulates diverse mechanisms of cancer progression in multi-drug resistant cancer cell lines.

Achieving an effective treatment of cancer is difficult, particularly when resistance to conventional chemotherapy is developed. P-glycoprotein (P-gp) activity governs multi-drug resistance (MDR) development in different cancer cell types. Identification of anti-cancer agents with the potential to kill cancer cells and at the same time inhibit MDR is important to intensify the search for novel therapeutic approaches. We examined the effects of sulfinosine (SF), a quite unexplored purine nucleoside analog, in MDR (P-gp over-expressing) non-small cell lung carcinoma (NSCLC) and glioblastoma cell lines (NCI-H460/R and U87-TxR, respectively). SF showed the same efficacy against MDR cancer cell lines and their sensitive counterparts. However, it was non-toxic for normal human keratinocytes (HaCaT). SF induced caspase-dependent apoptotic cell death and autophagy in MDR cancer cells. After SF application, reactive oxygen species (ROS) were generated and glutathione (GSH) concentration was decreased. The expression of key enzyme for GSH synthesis, gamma Glutamyl-cysteine-synthetase (γGCS) was decreased as well as the expression of gst-π mRNA. Consequently, SF significantly decreased the expression of hif-1α, mdr1 and vegf mRNAs even in hypoxic conditions. SF caused the inhibition of P-gp (coded by mdr1) expression and activity. The accumulation of standard chemotherapeutic agent – doxorubicin (DOX) was induced by SF in concentration- and time-dependent manner. The best effect of SF was obtained after 72 h when it attained the effect of known P-gp inhibitors (Dex-verapamil and tariquidar). Accordingly, SF sensitized the resistant cancer cells to DOX in subsequent treatment. Furthermore, SF decreased the experssion of vascular endothelial growth factor (VEGF) on mRNA and protein level and modulated its secretion. In conclusion, the effects on P-gp (implicated in pharmacokinetics and MDR), GSH (implicated in detoxification) and VEGF (implicated in tumor-angiogenesis and progression) qualify SF as multi-potent anti-cancer agent, which use must be considered, in particular for resistant malignancies.

Targeting CXCR4 and FAK reverses doxorubicin resistance and suppresses invasion in non-small cell lung carcinoma

BackgroundCurrent high lung cancer mortality rates are mainly due to the occurrence of metastases and therapeutic resistance. Therefore, simultaneous targeting of these processes may be a valid approach for the treatment of this type of cancer. Here, we assessed relationships between CXC chemokine receptor type 4 (CXCR4) and focal adhesion kinase (FAK) gene expression levels and expression levels of the drug resistance-related genes ABCB1 and ABCC1, and tested the potential of CXCR4 and FAK inhibitors to reverse doxorubicin (DOX) resistance and to decrease the invasive capacity of non-small cell lung carcinoma (NSCLC) cells.MethodsqRT-PCR was used for gene expression analyses in primary lung tissue samples obtained from 30 NSCLC patients and the human NSCLC-derived cell lines NCI-H460, NCI-H460/R and COR-L23. MTT, flow cytometry, cell death and β-galactosidase activity assays were used to assess the in vitro impact of CXCR4 and FAK inhibitors on DOX sensitivity. In addition, invasion and gelatin degradation assays were used to assess the in vitro impact of the respective inhibitors on metastasis-related processes in combination with DOX treatment.ResultsWe found that ABCB1 over-expression was significantly associated with CXCR4 and FAK over-expression, whereas ABCC1 over-expression was associated with increased FAK expression. We also found that CXCR4 and FAK inhibitors strongly synergized with DOX in reducing cell viability, arresting the cell cycle in the S or G2/M phases and inducing senescence. Additionally, we found that DOX enhanced the anti-invasive potential of CXCR4 and FAK inhibitors by reducing gelatin degradation and invasion.ConclusionsFrom our data we conclude that targeting of CXCR4 and FAK may overcome ABCB1 and ABCC1-dependent DOX resistance in NSCLC cells and that simultaneous treatment of these cells with DOX may potentiate the anti-invasive effects of CXCR4 and FAK inhibitors.

Positive enhancement integral values in dynamic contrast enhanced magnetic resonance imaging of breast carcinoma: Ductal carcinoma in situ vs. invasive ductal carcinoma

OBJECTIVES The aim of this study was to contribute to the standardization of the numeric positive enhancement integral (PEI) values in breast parenchyma, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) and to evaluate the significance of the difference in PEI values between IDC and parenchyma, DCIS and parenchyma and IDC and DCIS. MATERIALS AND METHODS In the prospective trial, we analyzed the dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) of 60 consecutive patients with histologically confirmed unilateral DCIS (n=30) and IDC (n=30) and defined the PEI values (range; mean ± SD) for the lesions and the breast parenchyma. Tumor-to-non-tumor (T/NT) ratios were calculated for DCIS and IDC and compared. PEI color maps (PEICM) were created. The differences in PEI values between IDC and parenchyma and between DCIS and parenchyma were tested according to t-test. Analysis of variance (ANOVA) was used to test the differences between the mean PEI values of parenchyma, DCIS and IDC. RESULTS IDC showed highly statistically different PEI numeric values compared to breast parenchyma (748.7 ± 32.2 vs. 74.6 ± 17.0; p<0.0001). The same applied to the differences in the group of patients with DCIS (428.0 ± 25.0 vs. 66.0 ± 10.6; p<0.0001). The difference between IDC, DCIS and parenchyma were also considered highly statistically significant (p<0.0001) and so were the T/NT ratios for IDC and DCIS (10.1 ± 2.4 vs. 6.6 ± 1.4; p<0.0001). CONCLUSIONS PEI numeric values may contribute to differentiation between invasive and in situ breast carcinoma.

Concurrent alteration of p16 and PTEN tumor suppressor genes could be considered as potential molecular marker for specific subgroups of NSCLC patients

p16 and PTEN are tumor suppressors that are commonly inactivated in human cancers. Loss of each of these molecules is widely studied in lung cancer, including non-small cell lung carcinoma (NSCLC), its most common clinical form. However, the importance of their mutual alterations for NSCLC pathogenesis has been barely examined so far. In this study we tested hypothesis that aberrant p16 might cooperate with inactive PTEN during pathogenesis of NSCLC, particularly in promoting tumor aggressiveness and invasiveness. Initially, we screened NSCLC tumor samples from patients for the presence of the most common genetic and epigenetic alterations of p16 and further correlated them with previously detected aberrations in PTEN gene. Statistical analyses showed that aberrant p16 directly correlated with altered PTEN. Such significant correlation was also observed in groups of patients with high genomic instability, with squamocellular histological subtype, with disease grade 2 and with lymph node invasion. Finally, survival analyses revealed dramatic decrease in survival rate of patients with mutual alterations of p16 and PTEN, but without prognostic significance. Overall results implicate cooperation between aberrant p16 and PTEN in pathogenesis of NSCLC and suggest that their combination might be considered as potential molecular marker for specific subgroups of NSCLC patients.

The role of breast magnetic resonance imaging in the diagnosis of ductal carcinoma in situ

Ductal carcinoma in situ (DCIS), the noninvasive breast malignant tumor originates from the terminal ductal-lobular units (TDLU). The typical feature of DCSI is the formation of calcifications. Up to 90% of DCIS are diagnosed on mammographic examinations, as clinically asymptomatic. Between 10% and 20% of DCIS remain mammographically occult due to the lack of calcifications and/ or small tumor dimensions. Contrast-enhanced breast magnetic resonance imaging (MRI) detects mammographically occult breast lesions, thus defining morphologic features of the lesion and the dynamics of signal intensity changes due to contrast enhancement. Distribution of contrast enhancement - signal intensity increase in DCIS most frequently includes segmental, ductal and linear patterns, followed by regional enhancement pattern, while the intralesional contrast uptake most frequently includes the nodular pattern with the areas of confluence. Postcontrast signal intensity increase in DCIS is most frequently fast in the initial phase (wash-in), while the whole dynamic of contrast-enhancement includes either of the three possible time-intensity curve (TIC) types (persistent, plateau or washout), although the plateau TIC is considered to be more frequent. Breast MRI has high sensitivity in the diagnosis of invasive breast cancer, varying from 90% to 100%; the sensitivity in the diagnosis of DCIS is lower (77-96%). For the time being, the primary role of MRI in DCIS is planning of breast-conserving surgery (BCS) for the evaluation of lesion extension. Further development of MRI in the diagnosis of DCIS includes the implementation of the principles of functional and molecular imaging.

Association of CCND1 overexpression with KRAS and PTEN alterations in specific subtypes of non-small cell lung carcinoma and its influence on patients' outcome

Cyclin D1 is one of the major cellular oncogenes, overexpressed in number of human cancers, including non-small cell lung carcinoma (NSCLC). However, it does not exert tumorigenic activity by itself, but rather cooperates with other altered oncogenes and tumor suppressors. Therefore, in the present study, we have examined mutual role of cyclin D1, KRAS, and PTEN alterations in the pathogenesis of NSCLC and their potential to serve as multiple molecular markers for this disease. CCND1 gene amplification and gene expression were analyzed in relation to mutational status of KRAS gene as well as to PTEN alterations (loss of heterozygosity and promoter hypermethylation) in NSCLC patient samples. Moreover, the effect of these co-alterations on patient survival was examined. Amplified CCND1 gene was exclusively associated with increased gene expression. Statistical analyses also revealed significant association between CCND1 overexpression and KRAS mutations in the whole group and in the groups of patients with adenocarcinoma, grade 1/2, and stage I/II. In addition, CCND1 overexpression was significantly related to PTEN promoter hypermethylation in the whole group and in the group of patients with squamous cell carcinoma and lymph node invasion. These joint alterations also significantly shortened patients’ survival and were shown to be an independent factor for adverse prognosis. Overall results point that cyclin D1 expression cooperates with KRAS and PTEN alterations in pathogenesis of NSCLC, and they could serve as potential multiple molecular markers for specific subgroups of NSCLC patients as well as prognostic markers for this type of cancer.

Prolonged survival after neoadjuvant chemotherapy related with specific molecular alterations in the patients with nonsmall-cell lung carcinoma.

Lung cancer is the most common cause of neoplasia-related death worldwide. Accounting for approximately 80% of all lung carcinomas, the non-small cell lung carcinoma (NSCLC) is the most common clinical form with its two predominant histological types, adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Although surgical resection is the most favorable treatment for patients with NSCLC, relapse is still high, so neoadjuvant chemotherapy (NAC) is an accepted treatment modality. In this study we examined whether some of the key molecules associated with the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have predictive and prognostic value for the NAC application. To that end we examined the expression status of PTEN, pAKT, pERK and loss of heterozygosity (LOH) of PTEN in two groups of NSCLC patients, those who received and those who did not receive NAC. LOH PTEN and low pERK expression is shown to be correlated with the longest survival of patients with SCC and ADC, respectively, who received NAC. These results point that the application of NAC is beneficial in the NSCLC patients with specific molecular alterations which could further help to improve constant search for the druggable molecular targets used in personalized therapy.

Breast Dynamic Contrast Enhanced MRI: Fibrocystic Changes Presenting as a Non-Mass Enhancement Mimicking Malignancy

Abstract Background We aimed to analyse the morphokinetic features of breast fibrocystic changes (nonproliferative lesions, proliferative lesions without atypia and proliferative lesions with atypia) presenting as a non-mass enhancement (NME)in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) examination. Patients and methods Forty-six patients with histologically proven fibrocystic changes (FCCs) were retrospectively reviewed, according to Breast Imaging Reporting and Data System (BI-RADS) lexicon. Prior to DCE-MRI examination, a unilateral breast lesion suspicious of malignancy was detected clinically, on mammography or breast ultrasonography. Results The predominant features of FCCs presenting as NME in DCE-MRI examination were: unilateral regional or diffuse distribution (in 35 patients or 76.1%), heterogeneous or clumped internal pattern of enhancement (in 36 patients or 78.3%), plateau time-intensity curve (in 25 patients or 54.3%), moderate or fast wash-in (in 31 patients or 67.4%).Nonproliferative lesions were found in 11 patients (24%), proliferative lesions without atypia in 29 patients (63%) and lesions with atypia in six patients (13%), without statistically significant difference of morphokinetic features, except of the association of clustered microcysts with proliferative dysplasia without atypia. Conclusions FCCs presenting as NME in DCE-MRI examination have several morphokinetic features suspicious of malignancy, therefore requiring biopsy (BI-RADS 4). Nonproliferative lesions, proliferative lesions without atypia and proliferative lesions with atypia predominantly share the same predefined DCE-MRI morphokinetic features.