RU2845800C1 - Method for differential diagnosis of bladder cancer, kidney cancer and lung cancer - Google Patents

Abstract

FIELD: medical science.

SUBSTANCE: invention refers to medicine, namely to oncology, urology, and can be used for differential diagnostics of bladder cancer, kidney cancer and lung cancer. First morning portion of urine of a patient with malignant neoplasms of unknown localization is centrifuged at 2000 rpm for 20 minutes, then the supernatant is drained, the precipitate is added with 2 ml of phosphate-salt buffer 5 (PBS), re-suspended by pipetting for 1-2 minutes and centrifuged at 2000 rpm for 10 minutes. Described procedure for washing the cell precipitate is carried out twice. Further, the supernatant is drained; the precipitate is added with PBS 0.5 ml and re-suspended by pipetting for 1-2 minutes. Obtained cell suspension is added with monoclonal anti-CD15 antibodies labelled with allophycocyanin, monoclonal anti-CD45 antibodies labelled with fluorescein isothiocyanate, and monoclonal antibodies to CD326, labelled with cyanine fluorescent dye, in amount of 20 mcl. After incubation, fluorescence is measured on a flow cytofluorometer. If CD15+ cell count is 9.7 % or more, the presence of lung cancer is diagnosed, if CD45+ cell count is 9.5 % or more, kidney cancer is diagnosed, if CD326+ cell count is 13.8 % or more, bladder cancer is diagnosed.

EFFECT: method enables reducing an amount of time prior to initiating treatment of the patient with the new growth and thereby improving the favourable prognosis of the disease ensured by flow cytometry of urine sediment with using CD15, CD45 and CD326 monoclonal antibodies according to the preset parameters.

1 cl, 2 tbl, 3 ex

Description

The invention relates to medicine, namely: to oncology, urology, and can be used for differential diagnostics of malignant neoplasms of various localizations.

A method for diagnosing bladder cancer is known based on the analysis of cell cycle indices in urine sediment using flow cytometry. The sediment of morning urine obtained by centrifugation is stained with antibodies to cytokeratin and the material is fixed with propidium iodide and passed through a flow cytometer. An analysis of the quantitative DNA content in the nuclei of epithelial cells is performed and the ratio of the G1 peak of the sample to the reference G1 diploid peak (ploidy index) is calculated. The total number of cells in the S, G2 and M phases of the cell cycle is also calculated, expressed as a percentage (proliferation index). A sample with a proliferation index of >21%, as well as in the presence of aneuploid cells, is considered positive for bladder cancer.

The disadvantages of this method are:

1) It is not intended for differential diagnosis of malignant neoplasms; it only differentiates between the presence/absence of bladder cancer.

2) Lack of specific markers of malignant diseases (eg CD receptors): only the assessment of chromosomal stability and the number of proliferating cells are used. Given the increase in the proliferative index in background processes such as prostatic hyperplasia, cervical epithelial dysplasia, there is a possibility of obtaining false positive results for cancer.

https://searchplatform.rospatent.gov.ru/doc/RU2009122547A_20101220?q=Способ%20

differential diagnostics of bladder diseases

20by%20sediment%20urine&from=search_simple&hash=1707649396

There is a method for diagnosing malignant tumors of the pelvic organs that includes two stages of scanning using positron emission tomography with 18F-fluorodeoxyglucose. This determines the amount of substance accumulating in the tumor. A distinctive feature is that after the first scan, a urinary catheter is inserted into the patient, and the second scan is performed no earlier than 4 hours after the introduction of the substance 18F-fluorodeoxyglucose. If the standardized accumulation rate of 18F-fluorodeoxyglucose in certain foci after the second scan increases by more than 10% compared to the results of the first scan, this indicates the presence of a malignant tumor.

The described method is not without its drawbacks: due to the use of positron emission tomography with 18F-fluorodeoxyglucose, this method carries a large financial burden when used in routine practice. There are also a number of limitations: the impossibility of performing the study in the presence of pregnancy, hypersensitivity to 18F-fluorodeoxyglucose.

https://searchplatform.rospatent.gov.ru/doc/RU2246259C1_20050220?q=Способ%20

differential%20diagnosis%20of%20various%20malignant%20

neoplasms%20prostate%20uterus%20bladder&from=search_simple&hash=779723828

There is information about a method of diagnosing pelvic tumors that involves placing the patient in a certain position and identifying the area that requires biopsy through the skin. A distinctive feature of this method is the use of MRI to calculate the parameters of needle insertion to exclude the possibility of damage to important organs, vessels and nerve structures. The puncture point is determined on a transverse line drawn on the patient's skin that corresponds to the area of interest, taking into account the fat capsule in this area. The angle of the needle puncture is set as the angle between the vertical line passing through the center of the area of interest and the line connecting the puncture point and the center of the area of interest. Then the required depth of immersion and the inclination of the needle are controlled.

The described method has a disadvantage associated with its invasiveness: there is a risk of infectious complications, perforation of hollow organs, and bleeding.

https://searchplatform.rospatent.gov.ru/doc/RU2547686C2_20150410?q=diagnostics%20of%20cancer%20of%20the%20pelvis&from=search_simple&hash=1843518938

The objective of the proposed method is to conduct differential diagnostics of malignant neoplasms of various localizations (bladder cancer, kidney cancer and lung cancer) using the method of flow cytometry of cellular urine sediment using monoclonal antibodies to CD 15, CD 45 and CD 326.

The problem is solved in the following way. The first morning portion of urine of patients with superficial bladder cancer (T1N0M0, TaN0M0 or TisN0M0), kidney cancer or lung cancer was centrifuged at 2000 rpm for 20 min to sediment cellular elements. After that, the supernatant was poured off, 2 ml of phosphate-saline buffer PBS (PBS) were added to the sediment, resuspension was carried out by pipetting for 1-2 min and centrifugation at 2000 rpm for 10 min. The described procedure of washing the cell sediment was carried out twice. Then, the supernatant was poured off, 0.5 ml of PBS was added to the sediment, resuspension was carried out by pipetting for 1-2 min. Monoclonal antibodies to CD15 labeled with allophycocyanin (APC - absorption maximum at 652 nm, fluorescence maximum at 657.5 nm, manufacturer BD Biosciences, USA) in a volume of 20 μl were added to the resulting cell suspension. The mixture was mixed on a personal vortex for 20 sec. Monoclonal antibodies to CD45 labeled with fluorescein isothiocyanate (FITC - fluorescence maximum at 518 nm, manufacturer BD Biosciences, USA) were added in a volume of 20 μl. Monoclonal antibodies to CD326 labeled with cyanine fluorescent dye (Cy5.5 - fluorescence maximum at 694 nm, manufacturer BD Biosciences, USA) were added in a volume of 20 μl. Mixed on a personal vortex for 20 seconds and put in a dark place. Incubation of the cell suspension was carried out at room temperature for 15 minutes. After incubation, fluorescence was measured on a BD FACSCanto II flow cytofluorometer (BectonDickinson, USA).

According to the literature, the CD15 antigen is a carbohydrate adhesion molecule in the composition of proteoglycans, glycolipids and glycoproteins of the cell membrane, expressed on a wide range of cells, including cells of the immune system (phagocytosis, chemotaxis, recognition) and cancer cells, the CD45 antigen is the main leukocyte antigen and marks lymphoid tissue cells, CD326/EpCAM serves as a specific biological indicator of the presence of a tumor. The reliability of differences between the indicators of patient groups was assessed using the nonparametric Mann-Whitney criterion. The expression levels of the listed antigens in patients with malignant neoplasms of various localizations are presented in Table 1.

Table 1

Expression levels of CD15 CD45 and CD326 antigens in patients with superficial bladder cancer

Indicator Patients with RMP
N=58
Me
(C25-C75)
1 Patients with RP
N=42
Me
(C25-C75)
2 Patients with RL
N=46
Me
(C25-C75)
3 Reliability Number of CD15 ⁺ cells, % 6.20
(2.80-9.10) 7.55
(2.50-9.70) 9.70
(5.90-13.20) p _1-3 >0.01
p _2-3 >0.01 CD45 ⁺ cell count, % 4.20
(1.80-8.00) 9.50
(6.25-19.55) 2.80
(2.30-4.80) p _1-2 >0.01
p _2-3 >0.01 Number of CD326 ⁺ cells, % 13.80
(9.30-21.00) 8.95
(4.60-13.60) 3.90
(2.40-5.00) p _1-2 >0.05
p _1-3 >0.0001

Thus, the maximum level of CD15 expression is observed in patients with LC, while patients with RP demonstrate the maximum level of CD45 expression, and patients with BC show the maximum expression of the CD326 antigen.

Thus, by determining the expression levels of CD15, CD45 and CD326 antigens in the cellular sediment of urine in patients with an unknown localization of a malignant neoplasm, it is possible to conduct differential diagnostics to establish the localization of malignant neoplasms (bladder cancer, kidney cancer and lung cancer).

The method is carried out as follows:

When diagnosing a patient with complaints that the physician may interpret as a suspicion of a malignant neoplasm of unknown localization, the first morning portion of urine is collected from such patients. The above-described method is used to measure the content of surface antigen expression in the urine sediment. The probability of bladder cancer, kidney cancer, or lung cancer in such patients is calculated according to Table 2.

Table 2

The patient's risk of having bladder cancer, kidney cancer, or lung cancer

Indicator CD15 ⁺ cell count CD45 ⁺ cell count CD326 ⁺ cell count High risk of having RMP ≥13.8% High risk of having RP ≥9.5% High risk of having RL ≥9.7%

The sensitivity of the proposed method of differential diagnosis of malignant neoplasms of various localizations (bladder cancer, kidney cancer and lung cancer) is 68.8%, specificity is 84.0%, diagnostic accuracy is 92.0%.

The obtained results allow us to determine the localization of a malignant neoplasm in the patient’s body, which reduces the time before the start of treatment and increases the favorable prognosis.

Example #1

Patient A, born in 1965, came to the Krasnoyarsk Regional State Healthcare Institution “Krasnoyarsk Regional Children's Hospital named after A.I. Kryzhanovsky” (Krasnoyarsk) on 12/18/2023 on the referral of a urologist from the local clinic with complaints of pain when urinating and blood in the urine.

History: has been observed by an oncologist at his place of residence after surgical treatment - left nephrectomy for kidney cancer since October 2022. In December 2023, the patient began to note the complaints described above. In the general urine analysis, signs of microhematoria (20-25 red blood cells in the field of vision). During ultrasound examination of the pelvic organs, uniform thickening of the bladder wall in the fundus area measuring 2.7×3.0 cm was detected. According to magnetic resonance imaging of the pelvic organs with contrast enhancement, the bladder wall in the fundus area is unevenly thickened, lumpy over 3.0 cm with signs of active accumulation of contrast agent 2.5×3.0 cm with the greatest thickness of 2.0 cm. Also, during MRI of the abdominal organs and retroperitoneal space with contrast enhancement, a volumetric formation of irregular shape was detected in the area of the left kidney bed measuring 15×19 mm, non-uniformly accumulating contrast agent.

As a result of using the claimed method, expression of the CD 326 antigen was determined to be 15.1%, CD 45 9.67%, which indicates that patient A has bladder cancer. The percentage result of the CD 45 marker may be alarming in relation to recurrence of kidney cancer.

On 21.02.2024, a simultaneous operation was performed: transurethral resection of the posterior wall of the bladder. Biopsy of the tumor of the retroperitoneal space on the left. GI No. 1256-26 high-grade muscle-invasive urothelial cancer. GI No. B124-4432 - highly differentiated clear cell renal cell carcinoma G1. Postoperative diagnosis: bladder cancer IIIA st. pT4aN0M0. Recurrence of kidney cancer on the left.

Example #2

Patient G, born in 1971, was referred to the Krasnoyarsk Regional State Healthcare Institution “Krasnoyarsk Regional Children’s Hospital named after A.I. Kryzhanovsky” (Krasnoyarsk) with suspected metastatic lesions in both lungs: according to contrast-enhanced MSCT of the chest organs, multiple focal lesions are detected in all lobes of both lungs with a maximum size of 35 mm in S6 of the left lung.

During diagnostic procedures at the regional oncology dispensary, according to the MSCT data of the retroperitoneal organs, there are CT signs of a volumetric formation of the upper pole of the left kidney with intimate adjacency to the lumbar muscle, measuring 67×72×69 mm with an unclear, bumpy contour, and intense accumulation of contrast agent along the periphery of the formation.

As a result of using the claimed method, the expression of the CD45 antigen was determined to be 24.4%, which indicates the presence of kidney cancer in patient G.

The patient underwent a trephine biopsy of the tumor of the right kidney under ultrasound control; according to the histological report, low-differentiated clear cell renal cell carcinoma G3 was verified.

Example #3

Patient B, born in 1956, has been under dynamic observation at the Krasnoyarsk Regional State Healthcare Institution "Krasnoyarsk Regional Clinical Oncology Dispensary named after A.I. Kryzhanovsky" (Krasnoyarsk) since 2012 with the diagnosis: Peripheral cancer of the right lung, stage IIA (pT2bN0M0). Condition after extended upper lobectomy on 11/12/2023. GI No. 2343-55, the growth of adenocarcinoma G1 is determined in the presented tissues. No tumor growth was detected in the removed lymph nodes.

03/12/2024 The patient began to complain of a dry, hacking cough with hard-to-separate sputum streaked with blood. According to the data of the MSCT of the chest organs with contrast enhancement - in the area of the metal postoperative suture, after the upper lobectomy, a volumetric formation with uneven radiant contours, approximately 45 × 40 × 40 mm in size, is determined.

As a result of using the claimed method, expression of the CD 15 antigen was determined to be 16.1%, which indicates the presence of lung cancer in patient A.

The patient underwent transthoracic biopsy of the left lung tumor. Relapse of lung cancer was verified - GI No. B224-4567 - adenocarcinoma G2.

Claims (1)

A method for the differential diagnosis of bladder cancer, kidney cancer and lung cancer, including collecting morning urine and determining antigen expression levels by flow cytofluorometry, characterized in that the first morning portion of urine of a patient with malignant neoplasms of unknown localization is centrifuged at 2000 rpm for 20 min to precipitate cellular elements, then the supernatant is drained, 2 ml of phosphate buffered saline 5 (PBS) are added to the sediment, resuspension is carried out by pipetting for 1-2 min and centrifugation at 2000 rpm for 10 min, the described procedure for washing the cell sediment is carried out twice, then the supernatant is drained, 0.5 ml of PBS is added to the sediment, resuspension is carried out by pipetting for 1-2 min, monoclonal antibodies to CD15 labeled with allophycocyanin are added to the resulting cell suspension, in in a volume of 20 μl, mixed on a personal vortex for 20 s, add monoclonal antibodies to CD45 labeled with fluorescein isothiocyanate in a volume of 20 μl, add monoclonal antibodies to CD326 labeled with a cyanine fluorescent dye in a volume of 20 μl, mix on a personal vortex for 20 s and put away in a dark place; after incubation, fluorescence is measured on a BD FACSCanto II flow cytofluorometer; if the number of CD15+ cells is 9.7% or more, lung cancer is diagnosed, if the number of CD45+ cells is 9.5% or more, kidney cancer is diagnosed, if the number of CD326+ cells is 13.8% or more, bladder cancer is diagnosed.