Bogomir Dimitrijević

Three-dimensional Total Synchronous Luminescence Spectroscopy Criteria for Discrimination Between Normal and Malignant Breast Tissues

Abstract Specimens of malignant and normal female human breast tissues were analyzed after surgery by means of synchronous luminescence spectroscopy. Measurements were performed in the ranges of excitation wavelengths from 330 to 650 nm and synchronous wavelengths from 30 to 120 nm to obtain ordinary and first derivative three-dimensional total synchronous luminescence spectra (3d-TSLS) of each specimen. Arithmetic mean of these spectra has been calculated for normal and malignant specimens and analyzed to establish criteria for tissue differentiation. Spectral domain volumes (volumes below luminescence intensity surface) and mean spectral slopes have been calculated and also analyzed as tissue discrimination criteria. The obtained results are discussed in view of the possible relevance of synchronous luminescence spectroscopy in discrimination between normal and malignant breast tissue.

Application of supervised self-organizing maps in breast cancer diagnosis by total synchronous fluorescence spectroscopy.

Data from total synchronous fluorescence spectroscopy (TSFS) measurements of normal and malignant breast tissue samples are introduced in supervised self-organizing maps, a type of artificial neural network (ANN), to obtain diagnosis. Three spectral regions in both TSFS patterns and first-derivative TSFS patterns exhibited clear differences between normal and malignant tissue groups, and intensities measured from these regions served as inputs to neural networks. Histology findings are used as the gold standard to train self-organizing maps in a supervised way. Diagnostic accuracy of this procedure is evaluated with sample test groups for two cases, when the neural network uses TSFS data and when the neural network uses data from first-derivative TSFS. In the first case diagnostic sensitivity of 87.1% and specificity of 91.7% are found, while in the second case sensitivity of 100% and specificity of 94.4% are achieved.

The impact of PTEN tumor suppressor gene on acquiring resistance to tamoxifen treatment in breast cancer patients

Tamoxifen is a standard therapeutical treatment in patients with estrogen receptor positive breast carcinoma. However, less than 50% of estrogen receptor positive breast cancers do not respond to tamoxifen treatment whereas 40% of tumors that initially respond to treatment develop resistance over time. The underlying mechanisms for tamoxifen resistance are probably multifactorial but remain largely unknown. The primary aim of this study was to investigate the impact of PTEN tumor suppressor gene on acquiring resistance to tamoxifen by analyzing loss of heterozygosity (LOH) and immunohystochemical expression of PTEN in 49 primary breast carcinomas of patients treated with tamoxifen as the only adjuvant therapy. The effect of PTEN inactivation on breast cancer progression and disease outcome was also analyzed. Reduced or completely lost PTEN expression was observed in 55.1% of samples, while 63.3% of samples displayed LOH of PTEN gene. Inactivation of PTEN immunoexpression significantly correlated with the PTEN loss of heterozygosity, suggesting LOH as the most important genetic mechanism for the reduction or complete loss of PTEN expression in primary breast carcinoma. Most importantly, LOH of PTEN and consequential reduction of its immunoexpression showed significant correlation with the recurrence of the disease. Besides, our study revealed that LOH of PTEN tumor suppressor was significantly associated with shorter disease free survival, breast cancer specific survival and overall survival. In summary, our results imply that LOH of PTEN could be used as a good prognostic characteristic for the outcome of breast cancer patients treated with tamoxifen.

Unilateral follicular variant of papillary thyroid carcinoma with unique KRAS mutation in struma ovarii in bilateral ovarian teratoma: a rare case report

BackgroundStruma ovarii (SO) is a rare form of ovarian mature teratoma in which thyroid tissue is the predominant element. Because of its rarity, the differential diagnosis between benign and malignant SO has not been clearly defined. It is believed that malignant transformation of SO has similar molecular features with and its prognosis corresponds to that of malignant tumors originating in the thyroid.Case presentationWe report 35-year-old woman with bilateral ovarian cysts incidentally detected by ultrasound during the first trimester of pregnancy. Four months after delivery of a healthy child without complication she was admitted to the hospital for acute abdominal pain. Laparoscopic left adnexectomy was performed initially in a regional hospital; right cystectomy was done later in a specialized clinic. Intraoperative frozen section and a final pathology revealed that the cyst from the left ovary was composed of mature teratomatous elements, normal thyroid tissue (>50%) and a non-encapsulated focus of follicular variant of papillary thyroid carcinoma (PTC).Normal and cancerous thyroid tissues were tested for BRAF and RAS mutations by direct sequencing, and for RET/PTC rearrangements by RT-PCR/Southern blotting. A KRAS codon 12 mutation, the GGT → GTT transversion, corresponding to the Gly → Val amino acid change was identified in the absence of other genetic alterations commonly found in PTC.ConclusionTo the best of our knowledge, this is the first time this mutation is described in a papillary thyroid carcinoma arising in struma in the ovarii. This finding provides further evidence that even rare mutations specific for PTC may occur in such tumors. Molecular testing may be a useful adjunct to common differential diagnostic methods of thyroid malignancy in SO.

Genomic instability and tumor-specific DNA alterations in oral leukoplakias

Leukoplakias, clinically identifiable premalignant lesions, often precede oral squamous cell carcinoma (OSCC). Identification of leukoplakias that have the potential for transformation to malignancy is a key clinical problem. The aim of this study was to assess genomic instability, and to detect tumor-specific genomic alterations, in leukoplakias. Genomic instability was analyzed by comparing the DNA fingerprints of 32 leukoplakias with those of paired normal tissue. In addition, the mutational status of the p53 gene was analyzed using polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) and polymerase chain reaction-heteroduplex DNA (PCR-HET), and the mutations were subsequently confirmed by DNA sequencing. Moderate-to-significant genomic instability was detected in all leukoplakias analysed. Nine unique amplicons, present in leukoplakias but not in normal tissue, were retrieved and successfully characterized. The p53 gene was mutated in 40.6% of patients. Four patients with moderate instability and mutated p53 developed OSCC. The data obtained in this study support and concretize the thesis that premalignant lesions possess many of the alterations found in cancer before the development of a malignant phenotype. Inactivation or mutation of the p53 tumor-suppressor might be an early event contributing to genomic instability and increasing the risk of malignant transformation.

Different associations of estrogen receptor β isoforms, ERβ1 and ERβ2, expression levels with tumor size and survival in early- and late-onset breast cancer

BACKGROUND In breast cancer, little is known about the consequences of co-expression of ERα with the second estrogen receptor, ERβ, and its isoforms in light of their joint prognostic value. Previously reported correlations have been based mostly on independent ERα and ERβ expression levels in breast tumors. PURPOSE To address whether the expression ratio of ERα and ERβ and its isoforms may be a more important parameter than their absolute levels, we analyzed relative mRNA expression ratios of ERβ1 to ERβ2 and ERα in 74 clinical samples of invasive breast cancer including 39 early-onset and 35 late-onset breast cancers. Expression levels were correlated with clinical and histopathological parameters and disease-free interval. RESULTS A specific correlation of ERβ1 expression levels with tumor size was detected in early-onset breast cancer patients and of ERβ2 levels with tumor size in late-onset patients. Expression of both ERβ isoforms inversely correlated with expression of the two estrogen regulated genes, progesterone receptor and pS2 in both groups. Higher levels of ERβ2 than ERβ1 isoform were associated with a better outcome in late-onset patients. CONCLUSIONS Our results suggest that different isoforms of ERβ may be involved in suppression of tumor growth in young and elder patients and may have different prognostic values.

Analysis of 5' non-coding region in hepatitis C virus by single-strand conformation polymorphism and low-stringency single specific primer PCR.

Abstract Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR in hepatitis C virus (HCV) genotyping were examined for informativeness and reliability. The analysis of HCV isolates included seven type 1 isolates, two type 2 isolates, and two type 3 isolates. We also analyzed five isolates that presented as mixed infections determined by type-specific PCR. Among mixed isolates, one isolate was 1a/1b and four isolates were 1b/3a. SSCP and LSSP-PCR were applied to the analysis of 5′ non-coding region of HCV (−289 to −5) that contains genotype-specific sequences. Direct cycle sequencing of this region determined sequence divergences that define genotype and sequence alterations within the same genotype. Optimized conditions for the SSCP analysis clearly distinguished between genotypes 1, 2 and 3. In addition, the SSCP analysis detected sequence variants within the same genotype. However, the SSCP analysis and DNA sequencing did not confirm the presence of mixed infections. LSSP analysis, not previously employed in HCV genotyping, enabled clear distinction between genotypes 1, 2 and 3, however, this method did not differentiate between sequence variants within a genotype. Importantly, the LSSP profile demonstrated distinction between mixed infection isolates.

Concurrent quantitation of the A and D genotypes of hepatitis B virus

Hepatitis B virus (HBV) infection is a global health problem associated with severe liver disorders. Viral load and HBV genotype affect the clinical outcome, guide antiviral therapy and provide long term prognosis for HBV infected patients. Various types of detection and quantitation assays are currently in use with a different effectiveness. The aim of this study was to develop a method that would provide simultaneous identification and quantitation of genotypes A and D in a single-tube reaction. Sera from infected patients were analyzed by a TaqMan based real time PCR. Optimized reagents were used for HBV DNA quantitation while the genotypes A and D were quantified separately by our design of the assay. Multiplex real time PCR was achieved and was specific for HBV genotypes A and D within a single-tube reaction. Simulation of mixed virus populations was identified reproducibly in vitro. Quantitation of these individual genotypes was exceptionally reliable, so much so that the sum of individual genotypes was equal to the total viral load determined in a separate reaction. Therefore, a straightforward, conceptually simple and reliable approach to issues involving HBV genotypes A and D is submitted. Identity and exact titer of these genotypes in the Caucasian population can now be determined easily.

Hematological toxicity associated with tiazofurin-influence on erythropoiesis.

In this study hematological toxicity was analyzed after the single and repeated applications of tiazofurin (TZF). Cellularity of bone marrow, spleen and peripheral blood was examined, spanning the period of fifty days after the initial application. Analysis of hematological parameters was performed by slightly modified conventional techniques. The fraction of erythroid series was monitored during the experiment. Presented data describe kinetics of damage and recovery of hemopoietic tissue. Our results indicate that the effect of tiazofurin on cellularity of bone marrow and spleen and on erythropoiesis is reversible and dose dependent within tested dose range and therapeutic regimes. Twenty days after the application normal function of hemopoietic tissues was restored. This approach and results can be useful in defining the timing for sequencing and combination therapy with tiazofurin.

Effects of a Modified CMF Treatment (Cyclophosphamide, Methotrexate and 5-Fluorouracil) on Hematopoietic Tissues and Yoshida Sarcoma in Rats

Effects of a modified CMF treatment on hematopoietic tissue and an implanted tumor were studied in rats. The modification of the treatment refers to the application of cyclophosphamide 24 h after methotrexate and 5-fluorouracil. The study was done on Wistar rats bearing Yoshida sarcoma in the ascites form. The controls were a) untreated animals bearing the tumor or b) treated conventionally with the 3 cytostatics and c) tumor-free animals under either conventional treatment or d) modified treatment. We examined survival, the appearance of metastases, and the regeneration of hematopoietic tissues. Improved survival, the absence of metastases, and improved regeneration of hematopoietic tissues was observed when modified CMF treatment was applied. These results support the importance of sequencing cytostatic protocols for basic hematological determinants and anti-tumor activity.