RU2635534C1 - Method for predictiction of early recurrence of superficial bladder cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method including an enzyme immunoassay and immunohistochemical examination of the tumour tissue is described, where the level of matrix metalloproteinase-2 is determined in patients with superficial bladder cancer in the morning urine collected prior to the operation, the ratio of the enzyme to the level of excreted creatinine, as well as in the tumour tissue removed during the operation is calculated, the level of matrix metalloproteinase-2 expression is determined. When the metalloproteinase-2 content is 7.3 pg/mcmol of creatinine and more in urine, and is 52% and more positive cells in the tumour tissue, a high risk of recurrence in a period of 3 to 12 months is predicted.

EFFECT: prediction of the risk of disease recurrence, objective monitoring of malignant process development, a possibility of patients inclusion into risk groups for intensive monitoring, timely measures for appointment of adjuvant treatment.

2 ex

Description

The invention relates to medicine, namely to Oncology, and can be used to predict early recurrence of superficial bladder cancer.

An important task of clinical oncology remains the active search for predictive criteria for the occurrence and development of relapses after surgical interventions, regardless of the location of the tumor. This is especially true in the case of superficial bladder cancer (PRMP), because, despite a favorable prognosis, the percentage of early recurrence is very high. As a consequence of this, the need for careful monitoring of patients and the implementation of repeated cystoscopies, which makes the treatment of this nosology extremely expensive. A non-invasiveness, diagnostic accuracy, ease of use and low cost are considered to be a positive property of relapse prognosis factors. The detection of such markers of cancer progression contributes to the choice of adjuvant treatment tactics and determines the timing and quality of life of patients.

From literature there is a known method for predicting the course of bladder cancer using an assessment of UBC - an antigen of bladder cancer (see Sergeeva N.S. New tumor markers in oncourology // Actual problems of the treatment of oncourological diseases. - Obninsk, 2003. - P. 144- 145). The authors indicate an increase in the UBC level in the morning urine in 70% of patients with relapse, including in the presence of relapse at the preclinical stage, which allows the use of this marker in patient monitoring to identify superficial and invasive relapses after organ-preserving treatment.

However, this forecasting method has certain disadvantages. In most studies, the dynamics of changes in the level of UBC antigen with progression of bladder cancer were not monitored. Levels of prognostic value of UBC were detected for blood serum, and for urine - determined in single studies, which should be confirmed in similar studies. The UBC test can only be considered as an additional diagnostic marker, suggesting the presence of an invasive nature of tumor growth, as well as to evaluate the effectiveness of non-adjuvant chemotherapy.

A method for predicting the course of bladder cancer based on urinary excretion of matrix metalloproteinases is known from the same sources (see Tkachuk V.N., Al-Shukri A.S., Danilchenko D.I. Value of determination of matrix metalloproteinases in urine in patients with urinary cancer bubble // Urological statements. - 2011. - No. 1. - S. 30-32). In this work, the authors showed that the concentration of proteolysis enzymes MMP-2 and MMP-9 in urine, determined by the method of cytography, depends on the depth of invasion and the degree of differentiation of the tumor and indicates a prognosis of the course of the disease. However, this method for predicting the course of bladder cancer reveals a relationship between the level of matrix metalloproteinases-2 and -9 and tumor recurrence only indirectly, indicating that their content is associated with the invasive potential of the tumor in a static state in independent groups, and not in dynamics in the same sample . In addition, the cymography method used by the authors in this study, despite the advantage in diagnostic accuracy, is difficult to use and expensive.

From literature there is a known method for predicting the recurrence and progression of bladder cancer without muscle invasion based on clinical and morphological parameters (see Rolevich A.I., Mishustin S.I. Prognostic classification for bladder cancer without muscle invasion // Oncological Journal. - 2016.- T. 10, No. 1 (37) .- S. 64-70). The authors retrospectively evaluated the clinical and morphological factors for predicting relapse and tumor progression in 905 cases of combined surgical treatment of patients and performed mono- and multivariate analyzes using the Cox proportional risk model. It was found that the main factors in the prognosis of relapse and progression in PRMP were the multifocal bladder lesions and the degree of differentiation, which does not introduce novelty into the prognosis and is widely known.

In addition, clinical and morphological indicators, despite the high diagnostic significance of each individual, are numerous, have an epidemiological, and not pathogenetic, connection with the progression of cancer and, in particular, with recurrence. The most appropriate, from a clinical point of view, may be the simultaneous determination of a small number of complementary indicators that can fully characterize the malignant potential of the tumor.

The technical result of the invention is to develop a method to predict early recurrence of superficial bladder cancer.

The objective of the invention is to identify criteria for predicting early recurrence in patients with superficial bladder cancer.

The technical result is achieved by the fact that in patients with superficial bladder cancer in the morning portion of urine collected before surgery, the level of matrix metalloproteinase-2 is determined, the ratio of the enzyme to the level of excreted creatinine is calculated, and the expression level is determined in the tumor tissue removed during the operation matrix metalloproteinases-2 and when the content of metalloproteinases-2 in urine is 7.3 pg / μmol creatinine or more, and in tumor tissue equal to 52% or more positive cells, a high s the risk of recurrence in a period of 3 to 12 months.

The invention "A method for predicting early recurrence in patients with superficial bladder cancer" is new, because it is not known from the level of medicine in the search for disease prognosis criteria by studying proteolysis factors in the urine and tumor tissue of patients with PRMP.

The novelty of the invention lies in assessing the individual characteristics of urinary excretion and expression of extracellular proteolysis enzymes in the tumor tissue, which are responsible for the degradation of the basement membrane and contribute to the metastasis and invasive growth of malignant tumors.

The invention "A method for predicting early recurrence in patients with superficial bladder cancer" is industrially applicable, as it can be used in health facilities, cancer research institutes, cancer clinics and other medical institutions involved in the treatment of bladder cancer.

The invention of "A method for predicting early recurrence of superficial bladder cancer" is as follows.

In the morning portion of urine collected before surgery according to the standard method, in patients with superficial bladder cancer TaN0M0 and T1N0M0, the level of MMP-2 is determined by enzyme-linked immunosorbent assay using test kits. To standardize the results and eliminate differences in patient diuresis, the ratio of enzymes to the level of excreted creatinine is calculated. Content determination results are expressed in pg / μmol creatinine. Within 12 months after surgery, patients are monitored and cases of relapse are detected. Samples of tumor tissue taken during surgery were prepared for immunohistochemical studies according to standard methods. Monoclonal antibodies were used to detect MMP-2, and the number of positive cells was counted per 100 cells in at least 10 randomly selected fields of view with an increase in light microscope × 40 to assess the expression level. The differential point of separation of the concentration of MMP-2 in the urine and tissue of patients with PRMP to predict a high risk of disease recurrence was determined by the method of ROC analysis. For this purpose, the condition of 152 patients of the clinical group was ranked as follows: 1 - there is a relapse, 0 - there is no relapse. Next, the level of MMP-2 in urine and expression in tumor tissue were determined, upon reaching which the diagnostic sensitivity and specificity of predicting the recurrence of the disease were the highest in value. The critical value of the level of MMP-2, determined before surgery in the urine of patients with PRMP with a high risk of relapse, was 7.3 pg / µmol creatinine. If the patient has PRMP, the content of MMP-2 in the urine is ≥7.3 pg / μmol creatinine, then the risk of disease recurrence in the next 12 months after surgery is high. The critical value of the expression level of MMP-2 detected in the PRMP tumor tissue obtained during surgery, with a high risk of relapse, was 52% of positive cells. If the patient has PRMP, the expression level of MMP-2 in the tissue is more than 52%, then the risk of disease recurrence in the next 12 months after surgery is high.

To test the developed method for predicting the recurrence of cancer of the oral mucosa, under our dynamic observation for 150 months there were 150 patients of both sexes who were diagnosed with superficial bladder cancer TaN0M0 and T1N0M0 upon admission. Patients underwent surgical and chemotherapeutic treatment with verification of the stage of the process. All patients underwent a study of the content of MMP-2 in the urine before surgery. In 117 urine samples of patients with PRMP, the indicator value was 7.3 pg / μmol creatinine and higher. The detected level of MMP-2 in the urine was accompanied by the development of local relapses in 98 of 117 patients in the period from 3 to 12 months. Among patients with MMP-2 in urine below 7.3 pg / μmol creatinine, a relapse of the disease from 3 to 12 months after surgery was detected in 19 of 33 patients. The diagnostic sensitivity of predicting a high risk of disease recurrence was 75.5%, and the diagnostic specificity was 76.2%.

All patients underwent a study of the expression level of MMP-2 in the tumor tissue obtained during surgery. In 110 samples of tumor tissue, PRMP was 52% and higher. The detected level of MMP-2 in the tissue was accompanied by the development of local relapses in 83 of 110 patients in the period from 3 to 12 months. Among patients with an MMP-2 content below 52% of positive cells, a relapse of the disease from 3 to 12 months after surgery was detected in 15 of 40 patients. The diagnostic sensitivity of predicting a high risk of disease recurrence was 91.1%, and the diagnostic specificity was 72.5%.

We present clinical examples of the use of the method.

Example No. 1.

Patient X., 69 years old. Complaints at admission: pain during urination, periodic admixture of blood in the urine. Ultrasound from 10.23.15 - in the bladder along the left wall, the tumor is 13 × 10 × 11 mm in the projection of the mouth of the left ureter. The prostate gland is 4.4 × 5.2 × 4.1 cm, not infiltrated. Seminal vesicles b / o. The ureters on the right and left are not dilated.

Clinical diagnosis: bladder cancer T1N0M0, cl. column 2.

On October 29, 2015, the patient underwent transurethral resection of tumors with vaporization of the base. During revision of the bladder, the mucous membrane is hyperemic, the mouths of both ureters are located in typical places, on the left side wall 0.3 mm from the mouth of the left ureter, an exophytic tumor is about 2 cm in diameter, and a tumor of 0.3 cm in diameter in the neck of the bladder.

A Foley catheter 3-way No. 20 is installed in the bladder, the balloon is inflated by 20 ml, there is no hematuria. In histological analysis No. 89248-249 / 15 - G2, urothelial cancer with invasion of the subepithelial layer. The patient was determined to use the therapeutic tactics of intravesical chemotherapy (50 mg of doxorubicin, a full exposure time of 60 minutes), 6 weekly injections.

In the patient’s urine taken before surgery in the TUR volume by ELISA, the level of MMP-2 was determined, which amounted to 5.3 pg / μmol creatinine. The immunohistochemical method was used to determine the expression level of MMP-2 in tumor tissue taken during surgery, which amounted to 34% of positive cells.

Currently, the follow-up period is 14 months. According to a comprehensive examination, including ultrasound and urinalysis, no signs of relapse and progression were found.

Example No. 2.

Patient M., 67 years old. Complaints at admission: on a periodic admixture of blood in the urine.

Ultrasound from 10.19.15: a hyperechoic formation is determined in the bladder in the base projection, with uneven contours of 1.4 cm. Diffuse changes in the liver parenchyma, cysts of the right lobe of the liver. Calicoectasis of the left kidney.

Clinical diagnosis: bladder cancer T1N0M0, cl. column 2.

11/26/2015 performed transurethral resection of tumors with vaporization of the bases. During the revision of the bladder, the mucous membrane is pale pink, the mouths of both ureters are located in typical places, a creeping tumor around the mouth of the right ureter, an exophytic tumor on the right side wall is about 0.4 cm in diameter, at the base of the bladder about 1.5 cm in diameter.

The mouth of the right ureter is preventively catheterized by the ureteral catheter Ch No. 6. A 3-way No. 22 Foley catheter is installed in the bladder, the balloon is inflated by 25 ml, there is no hematuria. (Macro drug - fragments of a tumor of the bladder). Histological examination: G.A. No. 98028/15 - G1 urothelial cancer with invasion of the subepithelial layer.

The patient determined therapeutic tactics: adjuvant intravesical chemotherapy with doxorubicin (50 mg, full exposure time 60 minutes), 6 weekly injections.

In the patient’s urine taken before surgery in the volume of TUR by ELISA, the level of MMP-2 was determined, which amounted to 7.3 pg / μmol creatinine. The immunohistochemical method was used to determine the expression level of MMP-2 in tumor tissue taken during surgery, which amounted to 53% of positive cells.

After 8 months (in July 2016), a routine examination revealed a relapse of the disease. Complaints at admission: weakness. An ultrasound scan dated 06/14/16: in the bladder along the left side wall, hyperechoic formation of 1-2 S / S with uneven contours of 1.0 × 1.6 cm. Diffuse changes in the liver parenchyma, cysts of the right lobe of the liver. Calicoectasis of the left kidney.

Clinical diagnosis: T1N0M0 bladder cancer, relapse, cl. column 2.

On June 24, 2016, the patient underwent repeated transurethral resection of the tumor with vaporization of the base.

When the bladder is inspected, the mucous membrane is pale pink, the ureteral mouths are located in typical places, scars are visualized after previous surgical treatment, an exophytic tumor about 1.5 cm in diameter is revealed on the left side wall with the transition to the back wall. A 3-way No. 22 Foley catheter is installed in the bladder, the balloon is inflated by 15 ml, there is no hematuria. (Macrodrug - fragments of a bladder tumor.) During histological analysis - G.A. No. 52126-27 / 16 - fragments of a highly differentiated papillary urothelial carcinoma. The patient was discharged 05.07.16 with a diagnosis of Bladder cancer T1N0M0, relapse, class. column 2, the state after complex treatment.

The technical and economic effectiveness of the method consists in the possibility of an objective assessment of the state of the orientation of the development of the pathological process; timely prediction of the development of relapse; taking adequate therapeutic measures.

The technical and economic effectiveness of the claimed invention “A method for predicting early recurrence of superficial bladder cancer” allows us to predict the risk of recurrence of the disease, to objectively monitor the development of the malignant process (superficial bladder cancer), to suggest the development of relapses before their clinical manifestation and to include patients at risk for intensive observation, timely take measures to prescribe adjuvant treatment.

The availability, relative simplicity and practicality of the assessment of proteolysis factors allows their use in everyday clinical practice in order to individualize adjuvant treatment. Individual planning of specific therapy is the key to its effectiveness and minimal risk of treatment complications, which underlies the improvement of patients' quality of life.

Claims (1)

A method for predicting early recurrence of superficial bladder cancer, including enzyme-linked immunosorbent assay and immunohistochemical study of tumor tissue, characterized in that in patients with superficial bladder cancer in the morning portion of urine collected before surgery, the level of matrix metalloproteinase-2 is determined, the ratio of enzyme to level is calculated creatinine excreted, as well as in the tumor tissue removed during surgery, the expression level of matrix metalloproteinase-2 and when the urinary metalloproteinase-2 content is 7.3 pg / μmol creatinine or more, and in tumor tissue equal to 52% or more positive cells, a high risk of relapse in the period from 3 to 12 months is predicted.