Method for prediction of clinical outcome of musculo-invasive bladder cancer following combination therapy

FIELD: medicine.

SUBSTANCE: invention refers to medicine, specifically to oncology, and concerns the prediction of the clinical outcome of musculo-invasive bladder cancer. The method involves measuring the activity of 26S proteasomes and NF-kB and p50 amounts in the tumour tissue. If the 26S proteasome activity is more than 18-1,000 units/mg of protein, and if the NF-kB p65/p50 coefficient is more than 1.0, a low probability of the recurrence is predicted; if the 26S proteasome activity is less than 18-1,000 units/mg of protein, and if the NF-kB p65/p50 coefficient is less than 1.0, a high probability of the tumour recurrence is predicted. The invention can be used for detecting the progression of the disease in the form of the tumour recurrence following the transurethral resection of the bladder tumour and M-VAC polychemotherapy.

EFFECT: using the invention provides the higher accuracy and information value for predicting the clinical outcome of the bladder cancer.

2 ex, 2 dwg

 

The invention relates to medicine, specifically to Oncology, relates to methods for predicting outcomes of muscle-invasive bladder cancer and can be used to determine the progression of the disease in the appearance of the tumor relapses after conducting transurethral tumor resection and chemotherapy scheme M-VAC.

Currently used traditional methods of predicting the course of disease, primarily involve the analysis of the parameters characterizing the clinical features of the disease, namely the size of the tumor, the nature and extent of the process. According to the literature there are data on the prognostic significance of parameters such as the presence of microsatellite DNA sequences, mutations of proanagen, oncosuppressor, cell cycle regulation of angiogenic factors [3, 4, 7] and adhesion molecules [5] regarding the risk of recurrence of the disease. However, these data are controversial.

Closest to the claimed is a method of forecasting, which includes the determination of the polymorphism of the GSTP1 gene (glutatine S-transferase), which is associated with the metabolism of xenobiotics (RF Patent No. 2393772, publ 23.03.2009). Polymorphic variants of the gene associated with the change of enzyme activity, which can affect the ri is to the development of recurrence of malignant tumors. From lymphocytes of peripheral venous blood secrete DNA, carried out PCR analysis with subsequent restriction of the amplified fragments polymorphic locus Ile105Val GSTP1 gene. Upon detection of patients with superficial forms RMP homozygous genotype Val/Val predict a high risk of recurrence of bladder cancer (RMP). Homozygous genotype Ile/Ile is associated with minimal risk for the development of recurrence RMP.

The disadvantages of this method are the low sensitivity, accuracy, and comprehension, as well as the limited scope of the method, due to the lack of communication polymorphic variants of genes with enzyme activity, which is associated not only with them, but may be changed due to post-translational modifications of the polypeptide chain, and do not take into account the biological behavior of the tumor itself, which can have a pronounced effect on carcinogenesis.

A new technical challenge - improving the accuracy and usefulness of the prediction of disease progression in the risk of recurrence.

To solve the problem in the method for predicting the outcome of muscle-invasive bladder cancer after combined treatment by studies of the enzymes of metabolism determine the activity of the 26S proteasome and the content of NF-KB p65 and p50 in the tissue of the op is Holi and activity of 26S proteasomes more 18-1000 U /mg protein and when you factor NF-kB p65/p50 more than 1.0 predict a low likelihood of recurrence; when activity of 26S proteasomes less 18-1000 U /mg protein and factor NF-KB p65/p50 less than 1.0 predict a high probability of relapse development.

The method is as follows.

Spend fluorometrically the determination of the activity of the 26S proteasome, as well as enzyme-linked immunosorbent determining the expression of NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells - nuclear transcription factor kB) p65 and p50 in the tumor tissue. To do this, the operating material take samples of tumor tissue within 2-3 hours after the operation, clear of areas of necrosis, hemorrhage and placed in liquid nitrogen. Tissue samples to determine the activity of enzymes stored at -70°C and thawed no more than 1 time for research activity and maintenance of markers. Before determining the activity of the investigated enzymes frozen tumor tissue homogenized to a powder in liquid nitrogen. 26S proteasome allocate method vysalivaniya using ammonium sulfate to 40% saturation [12]. Chymotrypsinogen activity of 26S proteasomes, ongoing chymotrypsinogen centers of proteasomes, judged by the hydrolysis fluorogenic of oligopeptides Suc-LLVY-AMC (Sigma, USA) [1]. The resulting product register on fluorimetry "Hitachi-850 (Japan) at the wavelength of excitation 380 nm and emission 440 nm. To evaluate the activity of impurity PR is teas in the samples used specific inhibitor of proteasome - MG132. Per unit activity of proteasomes take the amount of enzyme, which hydrolyzes 1 nmol Suc-LLVY-AMC for 1 min Specific activity of proteasomes expressed in units of activity per 1 mg of protein. The protein content determined by the method of Lowry.

Preparation of nuclear extracts for determination of NF-kB (p50 and p65 forms) is carried out in accordance with the recommendations of the manufacturer Caymanchem (USA). Frozen tissue (100 mg) were homogenized in liquid nitrogen, then resuspending. The homogenate was centrifuged at 2400g and 4°C to obtain a residue, which resuspended in 50 μl of 50 mM Tris-HCl buffer (pH 7.5) and then centrifuged at 14000g and 4°C. the resulting supernatant used to determine the expression of transcription factors. The expression of activated forms of NF-kB p50, P65 determined in nuclear extracts by ELISA. The results of the determination of the content of transcription factors expressed in arbitrary units per mg of protein in the hole. And activity of 26S proteasomes more 18-1000 U /mg protein and when you factor NF-KB p65/p50 more than 1.0 predict a low likelihood of recurrence; activity of 26S proteasomes less 18-1000 U /mg protein and factor NF-KB p65/p50 less than 1.0 predict a high probability of relapse development.

This approach to predict the risk of recurrence for bladder cancer due to R the house premises.

One of the pathogenetic mechanisms underlying the development and progression of bladder cancer, is the change in the activity of proteasome system and expression regulated by these substrates, including NF-kB. It should be noted that regulatory step in many physiological and pathological processes is an intracellular protein degradation in the proteasome. 26 proteasome is able to activate NF-kB due to the rapid degradation of its own inhibitor IkB, which promote the development p105 - precursor of the p50 subunit of NF-kB [5; 10]. It is known that post-translational modification p105 - predecessor NF-kB p50 is performed using the 20S proteasome without the process of ubiquitination [9]. It is shown that the expression of NF-kB and the products of its regulation high in the case of transitional cell carcinoma of the bladder. The level factor associated with lymphogenous metastasis of tumors [8; 11]. Great importance is given to the ratio of NF-kB p65/p50 [2], which is associated with the presence of the active form of factor.

The information content of the selected criteria is justified by the presence of communication activity of 26S proteasome and the expression of NF-kB p50 and p65 with recurrences of bladder cancer, which was carried out in 54 patients RMP T2-3N0M0(the average age 61,8±1.5 years), treated in the Department of General Oncology fsbi " research Institute on Oncology the GII WITH the RAMS from 2006 to 2012. In all patients the diagnosis was morphologically verified. Treatment consisted of 2 courses of systemic chemotherapy in the neoadjuvant mode according to the standard scheme MVAC (methotrexate 30 mg/m2intravenous in the 1st, 15th and 22nd day, vinblastine 4 mg/m2in the 1st, 15th and 22nd day of intravenous cisplatin 70 mg/m2in the 2nd day intravenous doxorubicin 30 mg/m2intravenously at day 2), a break between courses was 4-6 weeks. Subsequently these patients underwent transurethral resection (TUR) of the bladder and another 2 courses of adjuvant chemotherapy according to the above scheme.

Tumor tissue was taken from the operational material no later than 2-3 hours after surgery, frozen in liquid nitrogen. Then in tumor tissue was determined by the activity of the 26S proteasome and the expression of NF-kB p50 and p65.

The average term monitoring of patients included in the study was 22 months. The progression of the process was noted in 19 patients (35%) due to the development of recurrence of the tumor.

To assess the prognostic significance of the activity of 26S proteasome and the expression of NF-kB P50 and P65 in the tumor tissue for bladder cancer used univariate analysis of the prognostic significance of signs (Survival Analysis, Statistica 6.0). Figures 2-year relapse-free survival was analyzed using build distorting the x survival by the method of Kaplan-Meier. Comparison of survival rates in the groups was carried out according to the criterion of Gehan-Wilcoxon [13].

When evaluating the prognostic significance of enzyme activity systems of intracellular proteolysis in tumor tissue for bladder cancer revealed that a statistically significant factors in relation to disease-free survival, are the activity of the 26S proteasome and the expression of NF-kB p50 and p65. In Fig.1 presents the performance of 2-year disease free survival rates depending on the activity of 26S proteasomes in the tumor, Fig.2 - depending on factor NF-kB p65/P50 in the tumor, which confirms the high likelihood of relapse in the group of patients with the activity of 26S proteasomes more 18-1000 U /mg protein and when you factor NF-kB p65/p50 more than 1.0.

When activity of 26S proteasomes in the tumor over 18*1000 IU/mg protein 3 (13%) patients out of 23 was revealed recurrence of the process. Then, as if enzyme activity of less than 18*1000 IU/mg protein, at 16 (51%) patients out of 31 were diagnosed with recurrence. Disease-free survival in the first case amounted to 86.9 per cent, which was significantly higher compared with the second group of patients, where this indicator was equal to 49% (p<0,05). The sensitivity of the method for determining the activity of the 26S proteasome to assess the risk of recurrence of the tumor was 84%, specificity 57%, predictive value Polo is sustained fashion of the - 51%.

When the ratio of NF-kB p65/p50 above 1.0 in 3 (15%) patients out of 20 were revealed recurrence of bladder cancer, disease-free survival was equal to 84%. In 12 (60%) patients out of 20 when you factor NF-kB p65/p50 less than 1.0 were identified recurrence of the tumor after treatment. Relapse-free survival rate in this case was 40%. When comparing relapse-free survival in groups of patients with different threshold ratio NF-kB p65/p50 was statistically significant difference on this measure (p<0,05). The detection sensitivity of the ratio NF-kB p65/p50 relative risk of recurrence of the tumor was 80%, specificity of 68%, the positive predictive value of the result is 60%.

Clinical examples:

Example 1. Patient G., 60 years old, came in fsbi research Institute of Oncology WITH the RAMS, where the results of a comprehensive survey was diagnosed with bladder cancer T2N0M0. Received 2 courses of systemic chemotherapy in the neoadjuvant mode according to the standard scheme MVAC. In subsequent years this patient was performed TUR of the bladder and another 2 courses of adjuvant chemotherapy according to the above scheme. Histological examination of the surgical material is diagnosed with transitional cell bladder cancer moderate degree of differentiation. The activity of the 26S proteasome in the tumor is fabric made 17-1000 IU/mg protein, factor NF-kB p65/p50 was 0.9 units, which corresponded to an adverse prognosis in relation to risk of tumor recurrence. Dynamic observation of patients in the 14 months after treatment was diagnosed with a relapse, about which has been further treatment. And 20 months after treatment was detected relapse of the disease, and the patient was held cystectomy. The total period of observation was 3 years old.

Example 2.

Patient I., 55 years old, appealed to the state scientific research Institute of Oncology WITH the RAMS, where the results of a comprehensive survey was diagnosed with bladder cancer T3N0M0. Received 2 courses of systemic chemotherapy in the neoadjuvant mode according to the standard scheme MVAC. Further treatment was to conduct a TOUR of the bladder and another 2 courses of adjuvant chemotherapy according to the above scheme. Histological examination of the surgical material is diagnosed with transitional cell bladder cancer moderate degree of differentiation. The activity of 26S proteasomes in tumor tissue was 28-1000 IU/mg protein, factor NF-kB P65/P50 was equal to 1.2 units, which corresponded to a favorable prognosis in relation to risk of tumor recurrence. The period of dynamic observation of patients was 36 months. Data for the program is the financing of the disease was not detected.

Thus, the proposed method for predicting the outcome of muscle-invasive bladder cancer together with the main clinico-morphological parameters allows for a more accurate and informative to predict the occurrence of relapses, which gives the opportunity to adjust our tactics in patients.

Sources of information

1. Ben-Shahar, S., Komlosh A., Nadav E. et al. 26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted an epitope from an inlact protein substrate // The J of Biol. Chem. - 1999. - Vol.274. - No.31. - P. 21963-21972.

2. Conner J. R., Smirnova I., Moseman, A. P., A. Poltorak IRAK1BP1 inhibits inflammation by promoting nuclear translocation of NF-kappaB p50 // Proc. Natl. Acad. Sci. USA. - 2010. - V. 107(25). - P. 11477-11482.

3. Garcia-Closas m, Malats n, Real, F. X. et al. Large-scale evaluation of candidate genes identifies association between VEGF polymorphism and bladder cancer risk // PloS. Genet. 2007. Vol.3. P. 287-293.

4. Goddard.J.C. Sutton C. D., P. N. Furness et al. Microvessel density at presentation predicts subsequent muscle invasion in superficial bladder cancer // Clin Cancer Res. 2003. Vol.9. P. 2583-2586.

5. Goldberg A. L. Functions of the proteasome: from protein degradation and immune surveillance to cancer therapy // Biochemical Society Transactions 2007 Vol.35. - P. 12-17.

6. Habuchi T., Marberger M., Droller, M. J., G. P. Hemstreet et al. Prognostic markers for bladder cancer: International Consensus Panel on bladder tumor markers. // Urology. - 2005. Vol.66(6). P. 64-74.

7. Inoue K., Kamada M., Slaton, J. W. et al. The prognostic value of angiogenesis and metastasis-related genes for progression oftarnsitional cell carcinoma of the renal pelvis and ureter // Clin. Cancer. Res. 2002. Vol.8. P. 1863-1870.

8. Karashima T, Sweeney P, Kamat A, Huang S, Kim SJ, Bar-Eli M, McConkey DJ, Dmney CP. Nuclear factor-kappaB mediates angiogenesis and metastasis of human bladder cancer through the regulation of interleukin-8. // Cln Cancer Res. 2003. Vol.9(7). P. 2786-2797.

9. Moorthy, A. K., Savinova O. V., Ho J. Q., Wang, V. Y., Vu, D., Ghosh G. The 20S proteasome processes NF-kappaB1 p105 into p50 in a translation-independent manner. // EMBOJ. 2006. Vol.25(9). P. 1945-56.

10. Shah, S. A., Potter, M. W., McDade T. P., Ricciardi R, Perugini R. A., Elliot P. J., Adams J., Gallery M. P. 26S proteasome inhibition dosage apoptosis and limits growth of human pancreatic cancer. // J. Cell Biochem. - 2001. - Vol.82(1). P. 110-122.

11. Xie DH. Tang XD. Xia SJ, Tan JM, Wang XH, Cai Y. Expression of NF-kappa In human bladder cancer and its clinical significance // Ai Zheng. - 2002. - Vol.21(6). P. 663-667.

12. Abramova, E. B., Astakhov, T. M., Yerokhov P. A., Sharov, N. P. The multiplicity of forms of the proteasome and some approaches to their division, Izv. RAI Ser. Biol. - 2006. No. 2. S. 150-156.

13. Borovikov, V. P. STATISTICA statistical analysis and data processing in the Windows environment / B. N. Borovikov. P. P. Borovikov. - M.: Eagle, 1998. - 608 S.

A method for predicting the outcome of bladder cancer in the study of enzymes of metabolism in the tumor, characterized in that the research activity of the 26S proteasome and the content of NF-kB p65 and p50; activity of 26S proteasomes more 18-1000 U/mg protein and when you factor NF-kB p65/p50 more than 1.0 predict a low likelihood of recurrence; activity of 26S proteasomes less 18-1000 U /mg protein and when you factor NF-kB p65/p50 less than 1.0 predict a high likelihood of tumor recurrence.



 

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3 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and represents a method for accelerated staining of histological specimen for identifying tuberculosis mycobacteria involving the preparation of the histological specimen according to a common technique, the Ziehl-Nelsen staining differing by the fact that the standard preparation of the specimen for staining is followed by a de-waxing procedure by placing them successfully into xylol and alcohols of the decreasing concentration for 5 minutes in a thermostat at 54°C and then placing into water, then in a 1% periodic acid for 2 minutes and washing in flowing water for 10 seconds; thereafter, Ziehl carbol-fuxine is poured on a section coated with a filter paper and heated on an alcohol burner lamp until evaporating for 2 minutes; the stain is left on the section after the heating procedure is completed, for 3-5 minutes; the filter paper is removed, and the sections are washed in water, differentiated in a 1% acid buffer, washed in main water for 1 minute; thereafter Leffler methylene blue is placed on the section and heated on the alcohol burner lamp until evaporating, washed in water, differentiated in a 1% acid buffer and washed with a plenty of water, de-watered in alcohols of the increasing concentration for 1 minute, placed in xylol and balm.

EFFECT: developing the method for the accelerated stain of the histological specimen.

1 ex

FIELD: medicine, analytical biochemistry.

SUBSTANCE: invention relates to laboratory methods of investigations. Method involves sampling specimen from patient to be inspected, extraction of serotonin and histamine from a specimen, chromatography of extract and determination of concentration of serotonin and histamine by the fluorescence intensity value. Saliva is used as biological fluid. Saliva by volume 1 ml is extracted with 4 ml of 1 N hydrochloric acid solution, 2 g of anhydrous potassium carbonate and 5 ml of mixture of butanol and chloroform in the ratio 3:2 are added, extract is shaken up and centrifuged. Organic phase (4 ml) is sucked off from extract and passed through chromatography column (diameter is 3 mm, height is 16 mm) filled with ion-exchange resin KB-4 or KB-4P-2 or Bio Rex-70 in H+-form, size of granules is 0.1 ± 0.02 mm. Histamine is eluted with 4 ml of 0.1 N hydrochloric acid at the rate of eluting solution 0.4 ml/min. Histamine concentration is determined by reaction with ortho-phthalic aldehyde dissolved in ethanol. Serotonin concentration is determined by reaction with ninhydrin in organic passed through column. Method provides assaying the saliva concentration of serotonin and histamine with high precision.

EFFECT: improved assay method.

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