[Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Contributes to Tamoxifen Resistance in Estrogen-Positive Breast Cancer Patients].

Crosstalk between the estrogen receptors and the receptor tyrosine kinases, including vascular endothelial growth factor receptor type II (VEGFR2), is a key mechanism in breast cancer resistance to antiestrogen therapy with tamoxifen. A high level of VEGFR2 expression in a tumor serves as a marker of tamoxifen resistance. The tamoxifen efficacy prognostic value of functional polymorphisms in the VEGFR2/KDR gene has not been established. Using qRT-PCR, we detected the rs2071559 and the rs2305948 variants and the levels of KDR gene expression in 122 breast tumor tissue samples from cohorts of patients with progression (distant metastases or relapse) and patients with no progression during tamoxifen therapy. The expression levels of VEGFR2 protein were analyzed by immunohistochemistry. The frequency of heterozygous and mutant genotypes of the rs2305948 SNP was significantly higher in patients without progression than in the cohort with progression. KDR rs2305948 was associated with high survival rates in breast cancer patients. A correlation between the mRNA of the ESR1 and KDR genes in patients without progression was detected. The results indicate the prognostic value of rs2305948 and its potential contribution to the tumor phenotype sensitive to tamoxifen.

Breast tumour cell subpopulations with expression of the MYC and OCT4 proteins.

The MYC and OCT4 genes are known factors associated with maintaining pluripotency and are linked with a more aggressive course, progression, and resistance to therapy in cancer. Determining the subpopulations of tumour cells expressing the Myc and Oct4 proteins will provide an opportunity to understand which tumour cell subpopulations expressing MYC and OCT4 are associated with metastasis and resistance and which subpopulations can be targeted by anti-MYC and anti-OCT4 therapy. The study included paraffin-embedded tissue from tumours from 27 patients with luminal B breast cancer obtained after neoadjuvant chemotherapy (NACT). Immunofluorescence staining was used to identify subpopulations of tumour cells expressing Myc, Oct4 and Snai2 (Opal™ 7-Color Kit (PerkinElmer, Hopkinton, MA). The following tumour cell subpopulations were identified with the Myc and Oct4 proteins and the Snai2 EMT marker: stem/progenitor tumour cells with/without Myc, Oct4 or Snai2 expression; differentiated tumour cells with/without Myc, Oct4 or Snai2 expression; and other nontumour cells (CK7-EpCAM-CD44+/-Myc+/-(Oct4, Snai2)+/-) within the inflammatory infiltrate in the tumour parenchyma and stroma. The circulating tumour cell subpopulations with Oct4 protein expression in the bloodstream were studied by flow cytometry. It was found that in patients with partial regression (PR) in response to NACT, the frequency of tumour stem cells was 3.6-fold increased (p = 0.038) in the non-EMT state (CK7+EpCam+CD44+Snai2-). In patients with metastases, there was a statistically significant 2.5-fold increase in the frequency of differentiated tumour cells with Myc expression (CK7+EpCam+CD44-Myc+) and a 2.7-fold increase in the frequency of cells with Oct4 expression (CK7+EpCam+CD44-OCT4+). In the next stage, the frequencies of subpopulations with expression of the Oct4 protein and signs of EMT among circulating tumour cells (CTCs) were determined. In patients with metastases, the frequency of tumour stem cells in the EMT state (CD326+CD44+CD24-CD325+) (p = 0.015) was more than fourfold increased, and the frequency of progenitor tumour cells with expression of the Oct4 stem protein (CD326+CD44+CD24+Oct4+) (p = 0.016) was almost sixfold higher than that in patients without metastases. Nonstem (differentiated) tumour cells with expression of the stemness proteins Myc and Oct4 were present in the breast tumour. Their content was significantly higher in residual tumours after NACT in patients who subsequently developed metastases compared with that in patients without metastases. Such cells are a new in situ marker of metastasis.

VHL Expression in Kidney Cancer: Relation to Metastasis Development, Transcription and Growth Factors and Component of Akt/m-TOR Signaling Pathway.

Von Hippel-Lindau protein (VHL) is associated with the development and progression of kidney cancer. An increase in VHL expression was found in patients with the disseminated form of the disease compared to the localized cancer, which was combined with a uniform distribution of decreased (<1.0) and increased (>1.0) VHL mRNA levels in renal cancer patients depending on the dissemination of the process. The increase in VHL expression was accompanied an increase in the level of mRNA for NF-κB p65 and kinases PDK1 and Akt. The revealed data indicate the importance of molecular biological parameters in oncogenesis.

Integral characteristic of the immune system state predicts breast cancer outcome.

The immune system dramatically contributes to the pathogenesis of cancer. An integral estimation of immune system state is considered to be perspective as a prognostic criterion for cancer. We hypothesize that the integral characteristic, uniting numerous parameters of immune response to tumor and presenting the state of the immune system of a cancer patient, may be of prognostic significance. The aim of this work was to verify this hypothesis. MATERIALS AND METHODS: We have evaluated 17 parameters cha-racterizing key innate and adaptive arms of immune system in 146 patients with primary diagnosed local breast cancer (BC) before cancer treatment. Using the original integrative approach of NovoSpark Corporation (Canada), we have presented the state of the immune system by a single visual image for each patient. RESULTS: We classified all BC patients in two groups: with favorable and unfavorable immune system states according to our previous data demonstrating dramatic differences of the visual images of immune system states in patients with good or poor disease outcomes. Then, we have examined the relationship between integral characteristic of the immune system state and the clinical outcome. The 3-year disease-free survival (DFS) of BC patients with favorable immune system state was more than 96.0% vs 65.4% in patients with unfavorable status (p = 0.00006). Univariate Cox proportional hazards regression analysis showed that the integral characteristic of immune system state assessed prior to cancer treatment as unfavorable, was predictive of the poorer DFS (HR = 15.70 [2.15-114.90], p = 0.0007). CONCLUSION: The integral characteristic of the immune system state is a significant prognostic factor in BC patients. The BC patients with favorable integral immune system state can be considered as a target group for immunotherapeutic approach.

Changes in the Activity of Proteasomes and Calpains in Metastases of Human Lung Cancer and Breast Cancer.

In patients with breast cancer and lung cancer, chymotrypsin-like and caspase-like activities of proteasomes and total activity of calpains in the primary tumor nodes and lymphogenic metastasis are elevated in comparison with the corresponding normal tissues. The development of lymphogenic metastases of breast cancer and lung cancer was associated with opposite change in caspase-like activity of proteasomes. These results can be useful for the development of methods for evaluation of aggressiveness of breast and lung cancer.

[Transcription factors NF-kB, HIF-1, HIF-2, growth factor VEGF, VEGFR2 and carboanhydrase IX mRNA and protein level in the development of kidney cancer metastasis].

Here, we have investigated the participation of nuclear factors NF-kB, HIF-1 and HIF-2, VEGF, VEGFR2, and carboanhydrase IX in clear-cell renal cancer. We have determined the expression and protein level of transcription factors, VEGF, VEGFR2, and carboanhydrase IX in tumor and normal tissues of 30 patients with kidney cancer. The Real-Time PCR and ELISA were used in the study. The low levels of HIF-1 mRNA expression associated with high levels of HIF-1 protein were also associated with metastasis. The expression levels of VEGF, VEGFR2, and their protein levels are increased in primary tumors of patients with disseminated kidney cancer compared to nonmetastatic cancer. No correlation was revealed between the content of mRNA and encoded proteins in the kidney cancer tissues. The changes in the ratios of mRNA levels and the respective proteins (HIF-1α, HIF-2, NF-kB, VEGF, VEGFR2, and carboanhydrase IX) may contribute to kidney-cancer metastasis.

Genome-wide association study of loss of heterozygosity and metastasis-free survival in breast cancer patients.

One of the factors providing the diversity and heterogeneity of malignant tumors, particularly breast cancer, are genetic variations, due to gene polymorphism, and, especially, the phenomenon of loss of heterozygosity (LOH). It has been shown that LOH in some genes could be a good prognostic marker. AIM: To perform genome-wide study on LOH in association with metastasis-free survival in breast cancer. MATERIALS AND METHODS: The study involved 68 patients with breast cancer. LOH status was detected by microarray analysis, using a high density DNA-chip CytoScanTM HD Array (Affymetrix, USA). The Chromosome Analysis Suite 3.1 (Affymetrix, USA) software was used for result processing. RESULTS: 13,815 genes were examined, in order to detect LOH. The frequency of LOH varied from 0% to 63%. The association analysis identified four genes: EDA2R, PGK1, TAF9B and CYSLTR1 that demonstrated the presence of LOH associated with metastasis-free survival (log-rank test, p < 0.03). CONCLUSIONS: The presence of LOH in EDA2R, TAF9B, and CYSLTR1 genes is associated with metastasis-free survival in breast cancer patients, indicating their potential value as prognostic markers.

Effect of Target Therapy on the Content of Transcription and Growth Factors, Protein Kinase TOR, and Activity of Intracellular Proteases in Patients with Metastatic Renal Cell Carcinoma.

We analyzed the dynamics of the expression of transcription factors, VEGF and its receptor VEGFR2, serine-threonine protein kinase mTOR and activity of proteasome and calpain in patients with metastatic renal cancer during therapy with tyrosine kinase inhibitor Votrient and mTOR blocker Afinitor. The expression of hypoxic nuclear factor HIF-1α in the tumor tissue decreased during therapy with the target preparations. The decrease of VEGF and its receptor VEGFR2 was observed only in patients treated with mTOR inhibitor. The increase in calpain activity in the tumor tissue was observed in both groups. These findings extend our understanding of the mechanism of action of target anticancer preparations as allow considering the studied markers as predictors in choosing optimal therapy.

Activation of AKT of signaling pathway and the level of mTOR substrates in tumor of patients with kidney cancer, connection with prevalence of malignancy.

Activation of AKT signaling pathway and mTOR substrates of kidney tumor tissue occurs by improving AKT, its phosphorylated form, the serine / threonine proteinkinase m-TOR, the exchange regulator glycogen GSK-3-beta and also the inhibitor of 4E-BP1transcription. Increasing the size of primary tumor is followed by increasing the content of therein c-Raf and decreasing the content of phospho-m-TOR. The development of disseminated forms of the disease was associated with a reduction PTEN and phospho-AKT in tumor.

Invasive and drug resistant expression profile of different morphological structures of breast tumors.

In order to understand invasive/adhesive and drug resistant properties of intratumor morphological heterogeneity of breast cancer, we compared the expression of genes responsible for the cell adhesion and for the drug resistance between distinct morphological structures of breast tumors. Tubular (hollow-like), alveolar (morula-like), trabecular, solid structures/patterns, and discrete (small) groups of tumor cells were isolated from invasive carcinoma of no special type (n=3) and invasive micropapillary carcinoma (n=1) of the breast using laser microdissection. The gene expression of cadherins, catenins, integrins, ABC transporters, GSTP1, and drug targets was analyzed using qRT-PCR. Expression of catenin genes was identified in almost all structures. In contrast, the expression of cadherin and integrin genes significantly varied depending on the morphological variant. Cadherin expression declined in the row: solid - alveolar and trabecular structures - discrete groups of tumor cells. Expression of integrins declined in the row: solid and alveolar - trabecular structures - discrete groups of tumor cells. For drug resistance genes, trabecular structures more often demonstrated activity of genes coding for ABC transporters compared to other morphological variants. These results indicate that intratumoral morphological heterogeneity in breast cancer correlates with expression profile of adhesion and drug resistance genes reflecting different patterns of invasive growth and responsiveness to chemotherapy.

[Ethnic aspects of hereditary breast cancer in the region of Siberia].

BACKGROUND: Ethnic diversity of the population in the region of Siberia suggests the existence of different germline mutations in the BRCA1/2 genes associated with breast and ovarian cancer in different ethnic populations, but spectrum of these mutations has not been studied. OBJECTIVE: Our aim was to evaluate the frequency of the most common mutations BRCA1/2 (BRCA1 5382insC, BRCA1 185delAG, BRCA1 4153delAG, BRCAI T300G, BRCA2 6174delT) in women diagnosed with breast cancer among indigenous people and newcomers living in Siberia. METHODS: We tested 1281 genomic DNA samples for the presence of BRCA1 5382insC mutation in patients diagnosed with breast cancer considering no family history. 72 patients having hereditary cancer signs were tested for the mutations BRCA1 185delAG, BRCA1 4153delAG, BRCA1 T300G, BRCA2 6174delT. RESULTS: Out of 765 patients of Slavic ethnic group, 27 women (3.5%) were carriers of allele BRCA1 5382insC. The frequencies of mutations in patients with signs of hereditary cancer were: 8.3% in group of young patients (under 40 years), 20.0% in patients with bilateral cancer and 5.7% in patients with family history of breast or ovarian cancers. We tested 516 BC patients residing on the territory of the Buryat-Aginsky district, Republics of Tyva and Altai. Out of them, there were 197 patients among the indigenous population (buryats, tuvinians, altaians), and 319 patients among newcomers (Slavic ethnics). Mutations BRCA1 5382insC were detected only in women from Slavic ethnic groups. The frequency of BRCA1 5382insC mutation was 6% in the group where family history was excluded and 14% in the group of patients with characteristics of family cancer. Allele BRCA1 5382insC was not found in indigenous breast cancer patients, although 59 patients had signs of hereditary cancer. In women from Slavic ethnic group, the BRCA1 185delAG, BRCA1 4153delAG and BRCA1 T300G mutations were detected in 9.1% of cases and were not found in patients among the indigenous population. CONCLUSION: studies of mutations in the BRCA1 gene in breast cancer patients from Siberia confirmed data on the high frequency of "founder mutation" BRCA1 5382insC in Slavic population and indicate the advisability of further studies to identify the genes responsible for the occurrence of hereditary breast cancer in the indigenous population.

[Chymotripsin-like activity and subunit composition of proteasomes in human cancers].

Activity of the proteasome, polyfunctional enzymatic complex, is known to undergo changes during cancer development. This phenomenon is, probably, caused by the changes in subunit composition of proteasomes. In present work, we studied chymotrypsin-like activity of proteasomes, subunit composition and their association in breast cancer, head and neck squamous cell carcinoma, endometrial cancer, renal cancer, bladder cancer, stomach cancer and colorectal cancer. The increase of proteasome activity was revealed in most cancer tissues compared with adjacent tissues except for the renal cell carcinoma. Changes in proteasome activity in cancer tissues compared with correspondent normal tissues were accompanied by modification of its subunit composition. High proteasome activity was observed in combination with an increased expression of immune subunits and/or proteasome activator PA28, associated with activity of 20S proteasome. In breast cancer, head and neck squamous cell carcinoma, bladder cancer, stomach cancer and colorectal cancer we additionally found higher expression of Rpt6 subunit of 26S proteasome. Correlations between chymotrypsin like proteasome activity and subunit expressions were found in human cancer tissues. In summary, we suggest that proteasome ac- tivation and changes in its subunit composition plays an important role in cancer pathogenesis.

Changes in proteasome chymotrypsin-like activity during the development of human mammary and thyroid carcinomas.

Changes in the proteasome chymotrypsin-like activity in mammary and thyroid carcinomas in comparison with the adjacent tissue were studied at stages T(1-4)N(0-3)M(0) and T(2-3)N(0-1)M(0), respectively. The activities changed in a wave-like manner over the course of mammary carcinoma growth in cases with and without metastases. The minimum increment of the activity in the tumor was recorded during the T(2)N(0) stage in the absence of local metastases. The increment of the activity reached the peak in N(1) tumors of the same size with metastases. The activities in the tumor and adjacent tissues virtually did not differ during the T(3-4)N(1-3) stages. The time course of proteasome activity changes in thyroid tumors of the studied stages was similar to that in mammary carcinoma. The results can be used for development of methods for evaluating the aggressiveness of mammary and thyroid tumors.

Quantification of breast cancer blood flow in absolute units using Gjedde-Rutland-Patlak analysis of 99mTc-MIBI uptake.

BACKGROUND: Scintimammography with 99mTc-MIBI has been proven as efficient technique of diagnosis of breast cancer. Nevertheless, quantification of breast carcinoma blood flow (BCBF) in absolute units is not yet developed. To compensate this, we analysed kinetics of 99mTc-MIBI uptake in breast cancer using Gjedde-Rutland-Patlak (GRP) approach. METHODS: If BC is radioactivity in breast cancer quantified by dynamic scintigraphy, C(h)-blood concentration of 99mTc-MIBI and K-transport constant, then, assuming 99mTc-MIBI uptake to breast carcinoma unidirectional for early minutes after injection and subjected to equation d(BC)/dt = K; C(h), classic GRP plot can be obtained from this by integration as BC/C(h)=K x (integral of C(h)(dt))/C(h) + V0 and placing [(integral of C(h)(dt))/C(h)] as X, and (A/Ch) as Y. The K can be then obtained as slope. K is breast cancer clearance equal to product (retention fraction); (blood flow) : K = E; BCBF. K can be calculated from A(t) and Ch(t) as asymptote of 99mTc-MIBI retention function h(t) = F-1[F[A(t)]/F[Ch(t)]], where F depicts Fourier transforms. The BCBF can be then obtained as ratio K/E. We employed the technique in 33 patients with breast carcinoma of stages T(1-3)N(0-3)M(0-1) injecting 99mTc-MIBI (370-510 MBq) as i.v. bolus. In 12 scintigraphy with 99mTc-MAA (370 MBq) injected via catheter intraaortically was performed as validation study. RESULTS: E values were essentially uniform over the population with overall mean 0.58 sd 0.06. Blood clearance curves did not differ between various stages also and were subjected to biexponential approximation. K was in all cases obtained from the slope of initial 3 min part of GRP plot, strongly linear (r > 0.95, p < 0.001) in all cases. 99mTc-MAA validation study revealed significant correlation with 99mTc-MIBI blood flow values (r = 0.94, p < 0.01). The BCBF(as ml/min/100 cm3) was in T1 12.85 sd. 4.76, in T2 15.87 sd. 1.78, in T3 17.35 sd. 2.45, and in T4 23.07 sd. 2.21, expressing tendency to increase with stage. Higher BCBF was significantly associated with metastatic spread and in patients with BCBF over 17 ml/min/100 cm3 distant mets were revealed in all cases. CONCLUSION: Hence, analysis of early kinetics of 99mTc-MIBI in breast carcinoma provides correct estimates of blood flow in the neoplasm and can be applied in clinical studies and for calculation of cytostatic delivery to BC.