WO2023121515A1

WO2023121515A1 - Method for diagnosing uveal melanoma

Info

Publication number: WO2023121515A1
Application number: PCT/RU2022/050395
Authority: WO
Inventors: Елена Владимировна ЦЫГАНОВА
Original assignee: Елена Владимировна ЦЫГАНОВА; ОЛЕГ АНАТОЛЬЕВИЧ, Шашкин; ВАУЛИНА, Людмила Вячеславовна
Priority date: 2021-12-24
Filing date: 2022-12-14
Publication date: 2023-06-29
Also published as: WO2023121515A8

Abstract

The proposed invention relates to medicine, and more particularly to ophthalmology, and concerns a method for diagnosing uveal melanoma. The present method includes first triggering lacrimation. The lacrimal fluid is then applied as a drop to a microscope slide and dehydrated for 45-180 minutes at room temperature. A microscopic examination of the lacrimal fluid is performed. If 10-15 bush-like structures are detected in the outer region of the lacrimal fluid sample, uveal melanoma is diagnosed. The method is highly accurate, does not require the use of expensive equipment and reagents and is therefore accessible to large sections of the public, and is also non-invasive and easy for doctors to administer.

Description

METHOD FOR DIAGNOSING UVEAL MELANOMA

The present invention relates to medicine, namely to ophthalmology, and concerns a method for diagnosing uveal melanoma, which can be used to diagnose uveal melanoma.

The incidence of uveal melanoma is currently increasing, in 2002 the incidence was 0.5-0.8 cases per 100 thousand adults in the Russian Federation and 0.5 cases per 100 thousand in the world [Saakyan S. V., Zakharova G.P., Myakoshina E.B. Cellular microenvironment of uveal melanoma: clinical and morphological correlations and predictors of poor prognosis. Molecular medicine. 2020; 18(3):27-33. https://doi. org/10.29296/24999490-2020-03-04]. In the CIS countries, the referral rate for UM is 0.32-0.35 per 100,000 population [. Ophthalmooncology: a guide for physicians / Ed. A.F. Brovkina. - M.: Medicine, 2002. - 424 p.]. In the Moscow region, according to the latest data, the incidence of UM reaches annually up to 12-13 new cases of the disease per 1 million adults. The disease is recorded in patients predominantly of working age [Brovkina A. F., Panova I. E., Sahakyan S. V. Ophthalmooncology: over the past two decades. Bulletin of ophthalmology. 2014; 130 (6): 13-19.]. The problem of early diagnosis remains relevant: a comprehensive examination is needed using several types of equipment, which in most cases are not available in medical institutions of the 1st and 2nd levels (polyclinics and clinical diagnostic centers), and sometimes even the 3rd (hospitals). To date, according to the 2020 clinical guidelines, the following standard examinations are performed for patients with suspected UM:

- eye biomicroscopy, gonioscopy with maximal mydriasis;

- Ultrasound (b-scan) to clarify the presence of pominence, its height, measurement of the diameter of the tumor;

- color Doppler scanning to determine the presence of its own neovascular network;

- ultrasonic biomicroscopy in case of suspected damage to the anterior segment of the eye;

- fluorescein angiography to identify the tumor's own vascular network, visualization of the hidden growth zone;

- optical coherence tomography to detect morphometric features of the tumor;

- computed tomography or magnetic resonance imaging to exclude or detect tumor growth beyond the eyeball;

- computed tomography of the abdominal and thoracic regions with intravenous contrast to exclude or detect the presence of metastasis [Clinical recommendations for "uveal melanoma" of the Ministry of Health of the Russian Federation (protocol dated April 10, 2020 No. 17/2-3-4)].

The most informative is fluorescein angiography (FA), which makes it possible to establish the main signs of uveal melanoma, namely: spotty hyperfluorescence, turning into confluent in the venous phase, as well as long-term residual fluorescence of the tumor. However, a similar picture can be observed with giant nevi, pseudotumorous diseases or hemangiomas of the choroid. In addition, this manipulation is contraindicated for a number of patients due to a severe somatic condition and the presence of allergies. Another limitation of fluorescein angiography is the need for transparency of intraocular media, which may not be due to comorbidities such as cataracts. The FAG itself is impossible without a fundus camera, a computer analyzer, emergency aid in the event of an allergic reaction to fluorescein, which makes the method itself inaccessible to ophthalmological rooms in polyclinics.

A known method for the differential diagnosis of uveal melanoma from a progressive choroidal nevus (RU 2147129 C1, class G01N 33/53, G01N 33/48, publ. 03/27/2000), including the study of immunocompetent cells in peripheral blood serum, in which the diagnosis is carried out according to the absolute index populations of normal killer cells CD16+ and their specific activity, and with their simultaneous increase beyond the norm, initial uveal melanoma is stated, and in the absence of changes in indicators - progressive choroidal nevus. The method allows differential diagnosis in clinically complex and atypical cases, which makes it possible to timely and more effectively conduct adequate treatment in order to lengthen the life expectancy of ophthalmic oncological patients.

The disadvantage of this method is the invasiveness of the method, the technical complexity of implementation and the need to use expensive reagents.

A known method for the differential diagnosis of uveal melanoma and other intraocular tumors (RU 2218069 C1, class A61B 5/00, publ. 12/10/2003), in which the identified clinical symptoms are evaluated on a point scale, the total number of points is summed up and intraocular metastasis is diagnosed by the final amount , melanocytoma, nevus choroid, hemangioma or uveal melanoma. The method allows to improve the quality of diagnosis.

The disadvantage of this method is the subjectivity of the assessment, as well as the impossibility of visualizing the tumor by ophthalmoscopy in peripheral localizations.

More common is the method of two-dimensional grayscale echography (B - method). It is non-invasive, painless, highly informative, does not require special preparation of patients, allows multiple studies of patients of any age without fear of complications. The method has a high diagnostic value in case of insufficient transparency of the optical media of the eye and is the only objective method for assessing the condition of the fundus when ophthalmoscopic visualization is impossible. However, the method does not allow to judge the state of the tumor tissue. In particular, it does not give a quantitative characteristic of the acoustic density of the tumor tissue (its densitometric characteristic), which, in turn, can significantly affect the determination of the course of the tumor process.

For example, there is a known method for the differential diagnosis of morphological forms of uveal melanoma (RU 2577237 C1, class A61V 8/08, A61V 8/10, publ. 17 MHz. Assess the acoustic density of the tumor tissue. When identifying the average acoustic density of 80 and less conventional. units spindle cell form is diagnosed. At an acoustic density of more than 80 arb. units - epithelioid cell and mixed cell form. The method allows to clarify the nature of the tumor tissue and to predict the course of the tumor process due to densitometric analysis based on tissue histograms of the tumor tissue. The disadvantage of this method is the inefficiency in the early stages of the disease, which is associated with a worse prognosis for patients.

In addition to the frequent lack of the necessary equipment and limited diagnostic capabilities, namely: insufficient information content in the early stages of the disease (stages T1 - base <3-9 mm with prominence <6 mm, T2 - base 10-16 mm, prominence 3-10 mm) , the considered approaches are expensive and not applicable for mass screening of the population. Diagnostic methods such as TIAB (fine needle aspiration biopsy) are categorically contraindicated in order to avoid metastasis, as well as possible complications (retinal detachment, hemophthalmos). Also, for patients, the problem of the cost of performing available necessary examinations in private medical centers becomes relevant.

In view of the problems described above, for most patients with uveal melanoma, only one treatment option becomes possible: enucleation of the eyeball. This operation leads to disability of the able-bodied population. In addition, before and after its implementation, psychological work with people is necessary: there are often cases of refusal of a timely operation due to the rejection of oneself in the future with such a defect as the absence of an eye. According to various authors, enucleation is performed in 26-66% of patients with uveal melanoma, while a high mortality of patients after performing this operation (20.9-40.7%) is reported [Stoyukhina, A. S. Results of enucleation as a method of treating large uveal melanomas / A. S. Stoyukhina, E. E. Grishina, D. V. Davydov // Ophthalmological journals. - 2010. - T. 3. - No. 1. - S. 16-21]. The process of UM metastasis is observed in 12-16% of patients [Saakyan, SV Analysis of metastasis and survival of patients with uveal melanoma / SV Saakyan, TV Width // Tumors of the head and neck. - 2012. - No. 2. - S. 53-57]. In 60-70% of all cases, primary metastases of uveal melanoma are found in the liver, in almost 40% of cases, foci are initially continued growth are detected in the lungs, soft tissues and lymph nodes [Molecular markers of uveal melanoma / M. V. Yakimova, E. N. Zhilyaeva, A. V. Medved [et al.] // News of Surgery. - 2019. - T. 27. - No. 4. - S. 443-452. - DOI 10.18484/2305-0047.2019.4.443].

It is believed that the crystallization of structures depends on the concentration and interaction of electrolytes, especially sodium and chloride, with mucin and protein macromolecules. The ratio of monovalent Na+ and K+ to divalent Ca2+ and Mn2+ is also important: it is key in the formation of the main structures of the facies - “ferns” [Structure and microanalysis of tear film feming of camel tears, human tears, and Refresh. PlusMolecular Vision 2018; 24:305-314].

Detailed mechanisms of crystal distribution and structure formation in the facies are described in the monograph by V.N. Shabalin. and Shatokhina S.N. "Morphology of the liquid media of the eye (a new theory of involutive cataractogenesis)". It describes the organization of the lacrimal fluid and aqueous humor, the biochemical parameters of the liquid media of the eye in senile cataract. The authors compared the morphological picture of the facies of blood serum, lacrimal fluid and aqueous humor in patients with senile cataracts, and also revealed the features of the morphological picture of the lacrimal fluid in patients with an artificial eye lens [Morphology of the liquid media of the eye (a new theory of involutive cataractogenesis) / V. N. Shabalin , S. N. Shatokhina, A. A. Devyatkin [and others]. - Moscow: Publishing house "Medicine", 2004. - 244 p. - ISBN 5225046886]. Using this method, it became possible to diagnose senile cataracts in the initial stages, when clinical signs, such as clouding of the lens and decreased vision, are absent.

In addition to ophthalmology, the method of crystallography is used in gynecology [Structural components of biological fluids in pregnant women with gestosis / V. D. Tadzhieva, L. I. Trubnikova, S. N. Shatokhina [and others] // Obstetrics and Gynecology. - 2005. - No. 2. - S. 35-39], early diagnosis of malignant neoplasms, in the diagnosis of adhesive processes in children.

For example, there is a method for diagnosing malignant neoplasms (RU 2235323 C2, class G01N 33/48, G01N 21/21, G01N 33/49, publ. 27.08.2004), in which blood serum is obtained, dried at room temperature and then it is examined, while 0.01-0.02 ml of blood serum is applied to a glass slide, and drying is carried out under a coverslip at a relative humidity of 55-60% for 72-96 hours, and the study is carried out under polarized light microscopy. If anisotropic spherulites with single small multi-colored inclusions in the central part of the spherulite are detected, the preclinical stage of malignant growth in the body is diagnosed, if anisotropic spherulites with pronounced color inclusions are detected, the early stage of malignant neoplasm is diagnosed. The method provides an increase in the accuracy of diagnosis of malignant neoplasms, and is also easy to implement.

The disadvantage of the known method is the invasiveness of the method, namely the need to take blood, which implies damage to the external barriers of the body.

Also known is a method for predicting adhesive disease in children who have undergone surgery on the abdominal organs (RU 2608658 C1, class G01N 33/48, publ. 01/23/2017), in which a study of peritoneal exudate is carried out. Sampling is carried out 10-15 hours after the operation, it is thermostated for 30-40 minutes at 37 ° C, after which a drop of the supernatant part of the exudate is taken, a microscopic examination is carried out by the method of wedge-shaped dehydration, and if tongue fields are detected in the marginal zone of the facies, adhesions are predicted. disease. EFFECT: method allows to quickly and effectively identify children at high risk of developing adhesive disease for early and effective prescription of anti-adhesion therapy. The disadvantages of the known method is the invasiveness of the method, as well as the presence of limitations and contraindications for use, such as the presence of concomitant inflammatory processes of other localization.

Closest to the proposed method is a method for diagnosing the development of senile cataracts (RU 2273024 C2, class G01N 33/48, A61F 9/00, publ. subsequent dehydration. The test tube with stimulated lacrimal fluid is preliminarily kept for 10-15 minutes in a thermostat at a temperature of 35-37 °C. Then, a drop of tear fluid is dehydrated for 45-60 minutes on a glass slide, microscoped in transmitted light, and if pinpoint foreign isotropic inclusions are detected in the edge zone of the tear fluid sample, the development of senile cataract is diagnosed. The invention provides ease of execution, quickly feasible, can be successfully used as a screening study, adopted for the closest analogue (prototype).

The disadvantage of the prototype method is the impossibility of diagnosing uveal melanoma with it.

The objective of the invention is to develop a new method for diagnosing uveal melanoma, which has high accuracy, does not require the use of expensive equipment and reagents, is accessible due to this for the general population and, at the same time, is easy to perform and non-invasive for the doctor.

This task is achieved by the fact that the method for diagnosing uveal melanoma includes a preliminary call for lacrimation, then the lacrimal fluid is applied to a glass slide in the form of a drop and dehydrated for 45-180 minutes at room temperature, a microscopic examination of the lacrimal fluid is performed and, if a tear fluid is detected in the marginal zone of the sample, fluids of bush-like structures in the amount of 10-50 diagnose the development of uveal melanoma. In FIG. 1 shows the morphological picture of the facies of the lacrimal fluid of the patient according to example 1.

In FIG. 2 shows the morphological picture of the facies of the lacrimal fluid of the patient according to example 2.

In FIG. 3 shows the morphological picture of the facies of the lacrimal fluid of the patient according to example 3

In FIG. 4 shows the morphological picture of the facies of the lacrimal fluid of the patient according to example 4.

In FIG. 5 shows the morphological picture of the facies of the lacrimal fluid of the patient according to example 5.

In FIG. 6 shows the morphological picture of the facies of the lacrimal fluid of the patient according to example 6.

The proposed method for the diagnosis of uveal melanoma is as follows.

Previously, the patient is lacrimated. Collect lacrimal fluid with a needle cone syringe in Eppendorf in a volume of at least 0.1 ml. Next, tear fluid is applied to a defatted glass slide using a mechanical dispenser in the form of a drop in a volume of ~ 0.05 ml. Then, dehydration is carried out, leaving the glass slide with the applied lacrimal fluid for 45-180 minutes at room temperature until the facies is formed. The morphological picture is studied under a light microscope connected to a digital camera. If bush-like structures present in the amount of 10-50 pieces are detected in the marginal zone of the lacrimal fluid sample, the development of uveal melanoma is diagnosed. Violation of the structure of the marginal (protein) zone, shading or complete absence of the transition zone and the absence or violation of the structure of the figures of "ferns", filling the central zone with bush-like structures are found only in the late stages of the tumor process. It is also possible to have dot foreign isotropic inclusions, indicating concomitant senile cataract.

The resulting morphological structure of the facies can be recorded on any electronic media.

The proposed method was diagnosed 19 patients with a confirmed diagnosis of uveal melanoma of the choroid/ciliary body. The diagnosis was made clinically according to ultrasound, ophthalmoscopy in conditions of maximum mydriasis and was confirmed by a morphological study of preparations of enucleated eyes. In the obtained morphological structures of the facies in 17 out of 19 patients, in the samples of the lacrimal fluid facies taken from the eye where the tumor was localized, cluster microstructures were presented in the amount of 10 or more of different diameters. Moreover, a correlation was noted between the number of microstructures and the size and stage of neoplasm. In 2 out of 19 patients diagnosed with uveal melanoma, bushy structures were also noted, but in tear fluid facies taken from the fellow (healthy) eye, while these structures were absent in facies samples from the affected eye.

Clinical case #1.

Volunteer F., born in 1999 (20 years old at the time of tear collection - 2019), somatically healthy. A 0.05 ml sample of the lacrimal fluid of a volunteer was applied to a glass slide, then the sample was dried for 3 hours at room temperature until a facies was obtained, then the morphological picture of the resulting facies was examined under a microscope in transmitted light, and it was also fixed with a camera (figure 1). In the facies, a clear division into three zones was observed: peripheral, transitional and central. In all three zones, typical structures were present that are formed in the facies of the lacrimal fluid of healthy people: cracks along the peripheral zone of a radial orientation, structures like "scales" in the transitional and "ferns" in the central. This structure formation is typical for all conditionally healthy people in the age category from 18 to 30 years.

Clinical case #2.

Volunteer Sh., born in 1999 (20 years old at the time of tear collection - 2019), somatically healthy. She had a history of rhinoplasty, high myopia, which was considered as one of the factors influencing a possible change in the structure formation and composition of the lacrimal fluid.

A 0.05 ml sample of the lacrimal fluid of a volunteer was applied to a glass slide, then the sample was dried for 3 hours at room temperature until a facies was obtained, then the morphological picture of the resulting facies was examined under a microscope in transmitted light, and it was also fixed with a camera (Fig. 2). In the facies, a clear division into three zones was observed: peripheral, transitional and central. In the peripheral zone, there was a violation of cracking up to complete disappearance. In the transition zone, structural disturbances were observed with inclusions of bush-like structures in the amount of 3 pieces.

Clinical case #3.

Patient G., born in 1949 (70 years at the time of hospitalization - 2019), diagnosis based on a morphological study of the eyeball preparation: spindle cell melanoma of the choroid of the left eye.

A sample of the lacrimal fluid of the patient with a volume of 0.05 ml was applied to a glass slide, then the sample was dried for 3 hours at room temperature until a facies was obtained, then the morphological picture of the resulting facies was examined under a microscope in transmitted light, and it was also fixed with a camera . In sample facies, bush-like structures with radial branching in the peripheral section were observed in the amount of 14 throughout the entire circumference of the facies (Fig. 3). For the most part, the structures are closely adjacent to each other without intermediate intervals. There are single large structures that are 2 times larger in diameter than the rest. In FIG. Figure 3 shows the morphological picture of the patient's facies, with circles marking areas of the facies containing cluster microstructures.

Clinical example No. 4.

Patient K., born in 1941 (78 years at the time of hospitalization - 2019), diagnosis based on morphological examination of the eyeball preparation: melanoma of the ciliary body of the left eye.

A sample of the lacrimal fluid of the patient with a volume of 0.05 ml was applied to a glass slide, then the sample was dried for 3 hours at room temperature until a facies was obtained, then the morphological picture of the resulting facies was examined under a microscope in transmitted light, and it was also fixed with a camera . In the facies, the presence of bush-like microstructures, less branched and smaller than in example No. 1, was observed along the periphery throughout the entire circumference of the facies in the amount of 32 with different intervals not exceeding the diameter of 1 of the bush-like microstructure. In FIG. Figure 4 shows the morphological picture of the patient's facies, with circles marked with areas of the facies containing cluster microstructures.

Clinical example No. 5.

Patient V., born in 1937 (82 years at the time of hospitalization - 2019), diagnosis based on a morphological study of the eyeball preparation: spindle cell melanoma of the choroid of the left eye. A sample of the lacrimal fluid of the patient with a volume of 0.05 ml was applied to a glass slide, then the sample was dried for 3 hours at room temperature until a facies was obtained, then the morphological picture of the resulting facies was examined under a microscope in transmitted light, and it was also fixed with a camera . In the facies, the presence of bush-like microstructures with radial branching was observed, along the periphery along the entire circumference of the facies in the amount of 23 with different intervals not exceeding the diameter of 1 of the bush-like microstructure. In FIG. Figure 5 shows the morphological picture of the patient's facies, with circles marking areas of the facies containing cluster microstructures.

Clinical example No. 6.

Patient A., born in 1955 (65 years at the time of hospitalization - 2020), diagnosis based on a morphological study of the eyeball preparation: mixed cell melanoma of the choroid of the left eye with invasion into the orbit and paranasal sinuses and metastases to the lungs.

A sample of the lacrimal fluid of the patient with a volume of 0.05 ml was applied to a glass slide, then the sample was dried for 3 hours at room temperature until a facies was obtained, then the morphological picture of the resulting facies was examined under a microscope in transmitted light, and it was also fixed with a camera . In FIG. Figure 6 shows the morphological picture of the patient's facies, with 42 areas of the facies containing bush microstructures in the circles marked.

Claims

CLAIM

1 . A method for diagnosing uveal melanoma, includes a preliminary call for lacrimation, then the lacrimal fluid is applied to a glass slide in the form of a drop and dehydrated for 45-180 minutes at room temperature, a microscopic examination of the lacrimal fluid is carried out, and when bush-like structures are detected in the marginal zone of the tear fluid sample in the amount 10-50 diagnose the development of uveal melanoma.