Methods for diagnosing uveal melanoma

Abstract

The proposed invention relates to medicine, more specifically to ophthalmology, and relates to a method for diagnosing uveal melanoma. The method comprises first inducing tearing. The tear is then applied as a drop to a microscope slide and dehydrated for 45-180 minutes at room temperature. A microscopic examination of the tear is carried out. Uveal melanoma is diagnosed if 10-50 bush-like structures are detected in the outer zone of the tear sample. The method is highly accurate and does not require the use of expensive equipment and reagents, and is therefore accessible to a large proportion of the general population, and is also non-invasive and easy for physicians to perform.

Description

[Technical field]

[0001] The proposed invention relates to medicine, more specifically to ophthalmology, and relates to a method for diagnosing uveal melanoma, which can be used for the diagnosis of uveal melanoma.

[0002] The incidence of uveal melanoma is currently increasing; in 2002, the incidence was 0.5-0.8 cases per 100,000 adult population in the Russian Federation and 0.5 cases per 100,000 worldwide [Saakyan SV, Zakharova GP, Myakoshina EB, Cellular microenvironment of uveal melanoma: clinical and morphological correlations and predictors of bad prognosis], Molekulyarnaya meditsina 2020; 18 (3):27-33. https: / / doi. org / 10.29296 / 24999490-2020-03-04]. In the CIS countries, the rate of consultation with a physician for uveal melanoma is 0.32-0.35 per 100,000 people [Oftal'moonkologiya: rukovodstvo dlya vrachey [Ophthalmic oncology: A guide for physicians] / ed. AF Brovkina. - M.: Meditsina, 2002. - 424 p.]. According to the latest data, in the Moscow region, the incidence of uveal melanoma reaches 12-13 new cases of the disease per 1 million adult population per year. The disease is registered mainly in patients of working age [Brovkina AF, Panova IE, Saakyan SV, Oftal'moonkologiya: novoye za posledniye dva desyatiletiya [Ophthalmic oncology: achievements over the last two decades], Vestnik oftal'mologii. 2014; 130 (6): 13-19.].

[0003] The problem of early diagnosis remains a challenge. Comprehensive testing using several types of equipment is required, which is often not available at first and second level medical institutions (general clinics and clinical diagnostic centers), and sometimes even at the third level (hospitals). To date, according to the 2020 clinical guidelines, patients suspected of uveal melanoma should undergo the following standard tests: - biomicroscopy of the eye, gonioscopy with maximum pupil dilation; - Ultrasound examination (B-scan) to reveal the presence of a protrusion, its height and to measure the diameter of the tumor; - Color Doppler scan to determine the presence of an endogenous neovascular network; - Ultrasound biomicroscopy if pathology of the anterior segment is suspected; - Fluorescence angiography to detect the endogenous vascular network of the tumor and visualize hidden growth zones; - Optical coherence tomography to detect morphometric signs of tumors; - Computed tomography or magnetic resonance imaging to exclude or detect tumors occurring outside the eye; - computed tomography of the abdomen and chest with intravenous contrast to exclude or detect the presence of metastases [Clinical guidelines ''uveal melanoma'' of the Ministry of Health of the Russian Federation (protocol of April ''10'', 2020, No. 17 / 2-3-4)] is receiving.

[0004] Fluorescence angiography (FAG) is considered the most informative and allows to establish the main signs of uveal melanoma, namely patchy hyperfluorescence, transition to confluence in the venous phase and prolonged residual fluorescence of the tumor. However, a similar pattern can be observed with giant nevus, pseudotumorous disease or choroidal hemangioma. In addition, for many patients, the procedure is contraindicated due to the presence of severe physical conditions and allergies. Another limitation of fluorescence angiography is the need for transparency of the intraocular media, which will not be due to concomitant diseases such as cataracts. The implementation of FAG itself is impossible without a fundus camera, computer analysis equipment, means of providing emergency assistance in case of an allergic reaction to fluorescein, which makes the method itself unavailable in the ophthalmology department of a general clinic.

[0005] A method of differential diagnosis of uveal melanoma from progressive choroidal nevus is known (RU 2147129 C1, cl. G01N 33 / 53, G01N 33 / 48, publ. March 27, 2000), which involves the examination of immunocompetent cells in the surrounding serum, in which the diagnosis is made by the absolute indicators of the population of normal killer cells CD16+ and their specific activity, the simultaneous increase of which above the normal limits leads to the diagnosis of early uveal melanoma, and the absence of any change in the indicators leads to the diagnosis of progressive choroidal nevus. This method makes it possible to make a differential diagnosis of clinically complex and atypical cases, which allows timely and more effectively to perform the appropriate treatment and thus to increase the life expectancy of patients with ophthalmic tumors.

[0006] The disadvantages of this method are the invasiveness of the method, the technical complexity of its implementation and the need to use expensive reagents.

[0007] A method for the differential diagnosis of uveal melanoma and other ocular tumors (RU 2218069 C1, cl. A61B 5 / 00, publ. December 10, 2003) is known, in which detected clinical symptoms are evaluated on a points scale, the total number of points is added up, and based on the final sum, intraocular metastasis, melanocytoma, choroidal nevus, hemangioma or uveal melanoma is diagnosed. This method makes it possible to improve the quality of diagnosis.

[0008] A drawback of the known methods is that the assessment is subjective and that it is not possible to visualize the tumor by means of ophthalmoscopic examination in the peripheral area.

[0009] The method of two-dimensional seroscale echography (method B) is becoming more widespread. It is non-invasive, painless, very informative, does not require special preparation of the patient and allows repeated examinations to be performed on patients of any age without fear of complications. This method has a high diagnostic value in cases of insufficient transparency of the optical medium of the eye and is the only objective way to assess the state of the fundus when ophthalmoscopic visualization is not possible. However, this method does not make it possible to assess the state of tumor tissue. In particular, it does not provide quantitative characteristics of the acoustic density of tumor tissue (its densitometric properties), which can significantly affect the determination of the nature of the course of the tumor process.

[0010] Thus, for example, a method for differential diagnosis of morphological forms of uveal melanoma is known (RU 2577237 C1, cl. A61B 8 / 08, A61B 8 / 10, publ. March 10, 2016), in which ultrasound examination is carried out by high-frequency two-dimensional cell scale scanning at a scanning frequency of 15-17 MHz. The acoustic density of the tumor tissue is evaluated. If an average acoustic density index of 80 or less relative units is detected, a spindle cell type is diagnosed. If the acoustic density is more than 80 relative units, an epithelioid cell type and a mixed cell type are diagnosed. The method, by densitometric analysis based on the tissue histogram of the tumor tissue, makes it possible to identify the characteristics of the tumor tissue and to predict the course of the tumor process.

[0011] A drawback of the known methods is that they are ineffective in the early stages of the disease, which is associated with a poor prognosis for the patient.

[0012] In addition to the frequent lack of necessary equipment and limited diagnostic possibilities, i.e. insufficient information at the early stages of the disease (stages T1-fundus <3-9 mm, protrusion ≤ 6 mm, T2-fundus 10-16 mm, protrusion 3-10 mm), the aforementioned approaches are expensive and not applicable for mass screening of the population. Diagnostic methods such as FNAB (fine needle aspiration biopsy) are strictly contraindicated to avoid metastases and potential complications (retinal detachment, ophthalmopathy). For patients too, the problem of the cost of carrying out the necessary tests available in private medical centers is becoming a challenge.

[0013] Considering the above problems, for most patients with uveal melanoma, only one treatment option is available: enucleation. This operation leads to disability in the healthy population. In addition, psychological care with people is necessary before and after its implementation: it is not uncommon for people to refuse timely surgery because they cannot accept in the future that they have defects such as missing eyes. According to data from various authors, enucleation is performed in 26-66% of patients with uveal melanoma, and a high mortality rate of patients after this operation is reported (20.9-40.7%) [Stoyukhina, AS, Rezul'taty enukleatsiy kak metoda lecheniya bol'shikh uveal'nykh melanoma [Results of enucleations as a method of treatment of large uveal melanomas] / AS Stoyukhina, EE Grishina, DV Davydov / / Oftal'mologicheskiye vedomosti. - 2010. - Vol. 3. - No. 1. - p. 16-21]. The process of uveal melanoma metastasis is observed in 12-16% of patients [Saakyan, SV, Analysis of metastasis and survival rate of patients with uveal melanoma [Analysis of metastasis and survival rate of patients with uveal melanoma] / SV Saakyan, TV Shirina / / Opukholi golovy and shei. - 2012. - No. 2. - p. 53-57].In 60-70% of all cases, the first metastases of uveal melanoma are found in the liver, and in almost 40% of cases, early foci of continued growth are detected in the lungs, soft tissues and lymph nodes [Molekulyarnyye markery uveal'noy melanomy [Molecular markers of uveal melanoma] / MV Yakimova, EN Zhilyayeva, AV Medved' [et al] / / Novosti khirurgii. - 2019. - Vol. 27. - No. 4. - p. 443-452. - DOI 10.18484 / 2305-0047.2019.4.443].

[0014] The crystallization of the structures is thought to depend 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: this is crucial for the formation of the main structures of the facies, the "ferns" [Structure and microanalysis of tear film ferning of camel tears, human tears, and Refresh. PlusMolecular Vision 2018; 24:305-314].

[0015] The detailed mechanism of crystal distribution and structure formation on the surfaces is described in the monograph by Shabalin VN and Shatokhina SN ''Morfologiya zhidkikh sred glaza (novaya teoriya involyutivnogo kataraktogeneza)'' [''Morphology of liquid media of the eye (new theory of involutional cataractogenesis)''], which describes the composition of tears and aqueous humor, biochemical properties of the liquid medium of the eye in senile cataracts. The authors compared the morphological patterns of serum, tear and aqueous humor surfaces in patients with senile cataracts and also revealed certain features of the morphological pattern of tears in patients with an artificial lens [Morfologiya zhidkikh sred glaza (novaya teoriya involyutivnogo kataraktogeneza) [Morphology of liquid media of the eye (new theory of involutional cataractogenesis)] / VN Shabalin, SN Shatokhina, AA Devyatkin [et al]. - Moskva: Izdatel'stvo ''Meditsina'', 2004. - 244 p. - ISBN 5225046886]. Using this method, it became possible to diagnose senile cataracts at an early stage, when such clinical signs as lens opacity and decreased visual acuity are not present.

[0016] In addition to ophthalmology, methods of crystallography are used in gynecology, in the early diagnosis of malignant neoplasms, in the diagnosis of adhesion processes in children [Structural components of biological fluids in pregnant women with gestosis [Structural components of biological fluids in pregnant women with gestosis] / VD Tadzhiyeva, LI Trubnikova, SN Shatokhina [et al] / / Akusherstvo i ginekologiya. - 2005. - No. 2. - p. 35-39].

[0017] Thus, for example, a method for diagnosing malignant neoplasms is known (RU 2235323 C2, cl. G01N 33 / 48, G01N 21 / 21, G01N 33 / 49, publ. August 27, 2004), in which serum is obtained, dried at room temperature and then examined, in which 0.01-0.02 ml of serum is applied to a microscope slide, drying is carried out at 55-60% relative humidity under a cover glass for 72-96 hours, and examination is carried out by microscope in polarized light. Upon detection of anisotropic spherulites with a single small multicolored inclusion in the center of the spherulite, a preclinical stage of malignant growth in the organism is diagnosed, and upon detection of anisotropic spherulites with developed colored inclusions, an early stage of malignant neoplasms is diagnosed. This method provides an increased accuracy of diagnosis of malignant neoplasms and is simple to carry out.

[0018] A drawback of the known methods is their invasiveness, i.e. the need to draw blood, which implies damage to the body's external barrier.

[0019] A method for predicting adhesive disease in children who have undergone surgical interventions on abdominal organs (RU 2608658 C1, cl. G01N 33 / 48, publ. January 23, 2017) is also known, in which the examination of the peritoneal exudate is carried out. A sample is taken 10-15 hours after the operation, which is thermostatically treated at 37 ° C for 30-40 minutes, after which a drop of the supernatant part of the exudate is taken and a microscopic examination is carried out by the wedge-shaped dehydration method, and if lingual fields are detected in the outer areas of the face, adhesive disease is predicted. This method makes it possible to quickly and effectively identify children at high risk of developing adhesive disease for early and effective prescription of anti-adhesion therapy.

[0020] Disadvantages of the known methods are their invasiveness and the limitations and contraindications to their use, for example the presence of other local concomitant inflammatory processes.

[0021] The closest to the proposed method is a method for diagnosing senile cataracts (RU 2273024 C2, cl. G01N 33 / 48, A61F 9 / 00, publ. March 27, 2006), which involves a microscopic examination of dehydrated tears applied in the form of droplets to a degreased microscope slide. Beforehand, a tube with stimulated tears is kept in a thermostat at a temperature of 35-37 ° C for 10-15 minutes. A drop of tear is then dehydrated on a microscope slide for 45-60 minutes and observed microscopically in transmitted light, and if punctate heterogeneous isotropic inclusions are detected in the outer area of ​​the tear sample, senile cataracts are diagnosed. The invention realizes simple execution, is quick to implement, can be successfully used as a screening test, and is adopted as the closest prior art.

[0022] A drawback of the closest prior art method is that it does not make it possible to diagnose uveal melanoma.

[0023] The problem underlying the present invention is the development of a new method for diagnosing uveal melanoma that is highly accurate, does not require the use of expensive equipment and reagents and is therefore accessible to a wider range of the population, is easy for physicians to perform, and is non-invasive.

[0024] This objective is achieved by the fact that the method for diagnosing uveal melanoma involves preliminary induction of lacrimation, then applying the tear fluid in the form of droplets to a microscope slide, dehydrating at room temperature for 45-180 minutes, performing a microscopic examination of the tear fluid, and diagnosing uveal melanoma if bush-like structures in the amount of 10-50 pieces are detected in the outer zone of the tear fluid sample. [Brief description of the drawings]

[0025] [Figure 1] FIG. 2 shows the morphological pattern of the tear surface of a patient according to Example 1. [Diagram 2] FIG. 1 shows the morphological pattern of the tear surface of a patient according to Example 2. [Diagram 3] FIG. 13 shows the morphological pattern of the tear surface of a patient according to Example 3. [Figure 4] FIG. 13 shows the morphological pattern of the tear surface of a patient according to Example 4. [Diagram 5] FIG. 13 shows the morphological pattern of the tear surface of a patient according to Example 5. [Figure 6] FIG. 13 shows the morphological pattern of the tear surface of a patient according to Example 6.

[0026] A proposed method for diagnosing uveal melanoma is as follows.

[0027] Beforehand, lacrimation is induced in the patient. The tear fluid is collected in an Eppendorf with a volume of 0.1 ml or more by the tip of a hypodermic needle. The tear fluid is then applied by a mechanical pipette in the form of droplets with a volume of about 0.05 ml onto a degreased microscope slide. Dehydration is then performed and the microscope slide with the tear fluid is left at room temperature for 45-180 minutes until the formation of a surface. The morphological pattern is examined under an optical microscope connected to a digital camera. Uveal melanoma is diagnosed if bush-like structures, present in an amount of 10-50 pieces, are detected in the outer zone of the tear sample. Disturbances in the patterning of the outer (protein) zone, fading or complete absence of the transition zone, absence or disturbance of the patterning of the "fern" shape, filling of the central zone with bush-like structures are detected only at the later stages of the tumor process. There may also be the presence of punctate heterogeneous isotropic inclusions, indicating concomitant senile cataract.

[0028] The resulting morphological structure of the surface can be recorded in any electronic medium.

[0029] The proposed method was used to diagnose 19 patients with confirmed choroidal / ciliary uveal melanoma. The diagnosis was made clinically by ultrasound examination, ophthalmoscopic data under conditions of maximum pupillary dilation, and confirmed by morphological examination of the enucleated eye specimens. The obtained morphological structure of the surface showed that in 17 of the 19 patients, bushy microstructures of different diameters were present in the amount of more than 10 pieces in the tear surface samples taken from the eye in which the tumor was localized. This allowed a correlation between the amount of microstructures and the size and stage of the neoplasm. In 2 of the 19 patients diagnosed with uveal melanoma, bushy structures were also observed, however, this was only in the tear surface taken from the paired (healthy) eye, and these structures were not present in the surface samples from the affected eye.

[0030] First clinical case Volunteer F, born in 1999 (20 years old at the time of tear collection - 2019), physically healthy. A 0.05 ml sample of the volunteer's tear was applied to a microscope slide, then the sample was dried for 3 hours at room temperature until a surface was obtained, then the morphological pattern of the obtained surface was examined under a microscope in transmitted light and also recorded by a camera (Figure 1). In the surface, a clear division into three zones was observed: peripheral, transitional and central. In all three zones, typical structures that form in the tear surface of healthy people were present: fissures in the radial peripheral zone, "scale" type structures in the transitional zone, and "ferns" in the central zone. This structure formation is characteristic for all conditionally healthy people in the age category of 18-30 years.

[0031] Second clinical case Volunteer Sh, born in 1999 (age 20 at time of tear collection - 2019), was physically healthy, had a history of rhinoplasty and high myopia, which were thought to be one of the factors influencing potential changes in tear structure formation and composition.

[0032] A 0.05 ml sample of the volunteer's tear was applied to a microscope slide, then the sample was dried for 3 hours at room temperature until a surface was obtained, then the morphological pattern of the obtained surface was examined under a microscope in transmitted light and also recorded by a camera (Figure 2). A clear division of the surface into three zones was observed: peripheral, transitional and central. In the peripheral zone, a disruption of the structure with fissures to complete disappearance was observed. In the transitional zone, a disruption of the structure with three amounts of bush-like structured inclusions was observed.

[0033] Third clinical case Patient G, born 1949 (age 70 at admission - 2019), diagnosis based on morphological examination of the ocular specimen: spindle cell choroidal melanoma of the left eye.

[0034] A 0.05 ml sample of the patient's tear fluid was applied to a microscope slide, then the sample was dried for 3 hours at room temperature until a surface was obtained, then the morphological pattern of the obtained surface was examined under a microscope in transmitted light and also recorded by a camera. In the surface sample, a quantity of 14 bush-like structures with radial branching at the periphery were observed over the entire circumference of the surface (Figure 3). In most cases, the structures are closely adjacent to each other without intermediate intervals. A single large structure is seen that exceeds the others by two times in diameter. Figure 3 shows the morphological pattern of the patient's surface, where the area of ​​the surface containing the bush microstructure is marked with a circle.

[0035] Fourth Clinical Case Patient K, born 1941 (78 years old at admission - 2019), diagnosis based on morphological examination of the ocular specimen: ciliary body melanoma of the left eye.

[0036] A sample of 0.05 ml of the patient's tear fluid was applied to a microscope slide, then the sample was dried at room temperature for 3 hours until a surface was obtained, then the morphological pattern of the obtained surface was examined under a microscope in transmitted light and also recorded by a camera. It was observed that in the surface, there were fewer branched and smaller bush-like microstructures than in Example 1, in an amount of 32, with different intervals not exceeding the diameter of one bush-like microstructure, along the periphery over the entire circumference of the surface. Figure 4 shows the morphological pattern of the patient's surface, where the area of ​​the surface containing the bush-like microstructures is marked with a circle.

[0037] Fifth Clinical Case Patient V, born 1937 (age 82 at admission - 2019), diagnosis based on morphological examination of ocular specimen: spindle cell choroidal melanoma of the left eye.

[0038] A 0.05 ml sample of the patient's tear fluid was applied to a microscope slide, then the sample was dried at room temperature for 3 hours until a surface was obtained, then the morphological pattern of the obtained surface was examined under a microscope in transmitted light and also recorded by a camera. It was observed that in the surface, bush-like microstructures with radial branches were present along the periphery over the entire circumference of the surface, in an amount of 23, with different intervals not exceeding the diameter of one bush-like microstructure. Figure 5 shows the morphological pattern of the patient's surface, where the area of ​​the surface containing bush-like microstructures is marked with a circle.

[0039] Sixth Clinical Case Patient A, born 1955 (age 65 at admission - 2020), diagnosis based on morphological examination of ocular specimen: mixed cell choroidal melanoma of the left eye with budding in the orbit and nasal sinuses and metastasis to the lungs.

[0040] A 0.05 ml sample of the patient's tear fluid was applied to a microscope slide, then the sample was dried for 3 hours at room temperature until a surface was obtained, then the morphological pattern of the obtained surface was examined under a microscope in transmitted light and also recorded by a camera. Figure 6 shows the morphological pattern of the patient's surface, where the areas of the surface containing 42 amounts of bush microstructures are marked with circles. In the center, the absence of the "fern"-type structures characteristic of tear surface samples from healthy people and the appearance of round structures resembling bush-like structures are observed.

Claims

[Claim 1] 1. A method for diagnosing uveal melanoma, comprising: preliminarily inducing tearing, then applying the tear fluid in the form of droplets to a microscope slide, dehydrating at room temperature for 45-180 minutes, performing a microscopic examination of the tear fluid, and diagnosing the uveal melanoma if an amount of 10-50 bush-like structures is detected in the outer zone of the tear fluid sample.

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