RU2705110C1 - Method for differential diagnosis of thyroid neoplasms - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to endocrine surgery, and can be used for differential diagnosis of thyroid neoplasms. Thyroid ultrasonography is performed, nodal thyroid formations are detected. Puncture biopsy is performed, followed by cystic cytological examination. If the cytological indications are not clear, a molecular-genetic examination is performed, wherein the pre-dried cytological preparation is washed with 200 mcl lysis buffer from the cytological glasses. Further, DNA and microRNA are recovered from cytological preparations of a fine-needle aspiration puncture biopsy of thyroid neoplasms using assay kits. Detection and quantitative assessment of the diagnostically significant microRNAs is carried out based on the polymerase chain reaction method with fluorescent recording of the analysis result in real time using the CFX96(Bio-Rad Laboratories, USA) thermal cycler. If the HMGA2 values are more than 0.09, microRNA 221 is more than 0.0105 and microRNA 375 more than -12.1213, a follicular tumor with signs of malignancy is determined. If the microRNA-146b values are more than 0.1721, the papillary cancer is determined. If the siRNA-375 values are more than 5.2514, the medullary cancer is determined. Ratio of mitochondrial DNA / nuclear DNA ratio more than 5716.3013 defines B-cell cancer. Presence of the V600 mutation in the BRAF gene determines a papillary cancer and is a risk of high biological aggressiveness of the given papillary cancer and a common process.

EFFECT: method provides reducing the number of unreasonable surgical interventions that can lead to a significant decrease in the quality of life by having specific postoperative complications (laryngeal paresis of about 4–6 % in specialized institutions, up to 20 % in general surgical institutions, postoperative hypoparathyroidism 4–30 %, which can lead to persistent irreversible changes of bone tissue) due to higher reliability of thyroid tumor verification by molecular genetic diagnosis.

1 cl, 4 tbl, 4 ex

Description

The present invention relates to medicine, namely to endocrine surgery and can be used in the diagnosis of nodules of the thyroid gland.

The growing interest in the problem of determining the malignancy of thyroid tumors (TGS) is largely associated with an increase in the incidence of thyroid cancer. Thyroid cancer (thyroid) is the most common endocrine system disease and has a tendency to increase in all countries of the world. According to foreign experts, while maintaining the current rate of increase in incidence, thyroid cancer (thyroid cancer) by 2020 will become the most common tumor in women. In Russia, according to the P.A. Moscow Cancer Research Institute Herzen, the increase in the absolute number of cases of thyroid cancer in the period from 2004 to 2014 was 36.5% in women and 15.5% in men, both sexes (rough indicators per 100,000 population) - 18.5%. Of all malignant tumors, thyroid cancer accounts for about 0.5-1% among the male population and 1-4.4% of the female. Thyroid cancer is found 3 times more often in women than in men, and is usually presented in the form of nodular formations, which are detected (according to various sources) in 4 to 40% of the population. According to WHO, over the past 20 years, the incidence of thyroid cancer in Europe and the USA has doubled mainly due to young and middle-aged people.

The main non-invasive method for diagnosing nodules is ultrasound (ultrasound) of the thyroid gland. With the help of an ultrasound, suspicious echographic signs of a tumor lesion of the thyroid gland will be determined. Suspicious signs are (evaluated only in solid nodes or in solid sections of nodes): hypoechoic solid structure, uneven, fuzzy or polycyclic contour, point hyperechoic inclusions (microcalcinates), the predominance of the height of the node over the width ("higher than wider"), enlarged lymph nodes neck. (Russian clinical guidelines for the diagnosis and treatment of highly differentiated thyroid cancer in adults, 2017. // Endocrine surgery. - 2017. - T. 11. - No. 1. - C. 6-27.doi: 10.14341 / serg201716-27) .

Depending on the number of suspicious echographic signs suggesting malignancy or benignness of the thyroid gland node, the TI-RADS system (from the English Thyroid Imaging Reporting and Data System) proposed in 2009 by E. Horvath et al. and modified in 2011 by J.Y. Kwak, which determines the risk of thyroid cancer. (Horvath E., Majlis S., Rossi R. et al. An ultrasonogram reporting system for thyroid nodules stratifying cancer risk for clinical management. J. Clin. Endocrinol. Metab. 2009; 94 (5): 1748-1751. DOI: 10.1210 / jc. 2008-1724).

Patients with unchanged thyroid parenchyma were assigned to TIRADS1. In the TIRADS 2 category, there are no signs of malignancy (these are colloid thyroid cysts, spongy cysts, cysts with a parietal component) - a thyroid cancer risk of 0-4%; with TIRADS 3 - no signs of malignancy, but with the presence of iso- or hyperechoic nodes with eccentrically located anechogenic inclusions) - the risk of thyroid cancer is less than 5%; TIRADS 4a - the presence of one sign of malignancy - the risk of thyroid cancer is 5-10%; TIRADS 4b - two signs or more, but in the absence of regional lymphadenopathy - the risk of thyroid cancer is 10-80%; TIRADS 5 - by the presence of three to five signs or detected metastatic lesions of the regions of regional lymphatic drainage - the risk of thyroid cancer is more than 80%. However, this technique is subjective in nature and very much depends on the qualifications of the doctor of ultrasound diagnostics and the resolution of the U3 apparatus.

The main method of preoperative diagnosis of thyroid nodes, taken as the closest analogue that determines the need for surgical treatment, is a fine-needle aspiration puncture biopsy (TAPB) followed by cytological examination of the preparations obtained. Specialists in cytological diagnostics around the world currently use the classification of thyroid lesions according to the Bethesda system (Cibas and Ali, 2009).

In this case, experts assign each category the so-called risk of malignancy, i.e. in what percentage of cases when a cytological diagnosis is made, a malignant tumor is histologically detected.

Bethesda I - Non-diagnostic material (blood, artifacts, poor or absent cellular composition, cyst contents). In this category, repeated puncture is indicated.

Bethesda II - Benign changes (colloid node, autoimmune thyroiditis, toxic goiter) The risk of malignancy is 0-3%. There are no indications for surgical treatment in this category. The observation in dynamics is shown.

Bethesda III - The Atypia of Unclear Meaning. The risk of a malignant process is 5-15%. Repeated TAPB after 1-3 months is indicated.

Bethesda IV - Follicular neoplasia or suspected follicular neoplasia (including from Guertl cells) The risk of malignancy is 15-30%. Surgical treatment is indicated.

Bethesda V - Suspicion of cancer (papillary, medullary, low-grade) The risk of malignancy is 60-75%. Surgical treatment is indicated.

Bethesda VI - Cancer (papillary, medullary, low-grade, lymphoma, etc.). The risk of a malignant process is 97-99%. Surgical treatment is indicated.

TAPB followed by a cytological conclusion requires a lot of experience from the performer and relatively often leads to errors (Stevens et al., 2009). According to various estimates, TAPB in 15-30% of cases cannot differentiate benign and malignant thyroid tumors. In these cases, the conclusions of the cytological examination are formulated as “follicular neoplasia”, “follicular tumor” or “thyroid tumor”, which are a heterogeneous group of so-called “uncertain” changes - both malignant and benign diseases, which have a similar cytomorphological picture. Only 10-15% of them during histological examination really turn out to be malignant (I.I., Kuznetsov N.S., Melnichenko G.A. Guide for doctors: Endocrine surgery. - M.: GEOTAR-Media, 2014) The only evidence of a highly differentiated follicular cancer is the detection of invasion of the tumor into the vessels and capsule, which cannot be detected by cytological examination. The belonging of the sample to the first category generally does not allow diagnosis in cytological examination. Thyroid epithelial cells are closely interconnected, cellular connections are broken with difficulty, therefore, obtaining informative material during TAPB is a difficult task. The proportion of non-informative punctures in various medical institutions is 5-12%. Meta-analysis by M. Bongiovanni et al. (2012), showed that the frequency of an "indefinite" cytological conclusion can reach 20-30%.

Meanwhile, in Russia, tens of thousands of operations are performed annually in patients with thyroid diseases, and the number of operations continues to grow steadily. The problem is that the operative path is most often chosen for the treatment of thyroid diseases that are not life threatening. Appointed in this way, the operation does not improve the condition and does not increase the quality of life. According to some estimates, up to 70% of thyroid surgery is performed without indications for surgical treatment. Excessive operative activity, on the one hand, is associated with the use by doctors of outdated principles for determining patient treatment tactics, and on the other, with the lack of reliable methods that rule out thyroid cancer.

Tasks: increasing the reliability of verification of tumor lesions of the thyroid gland, determining an adequate volume of surgery.

Technical result: reduction in the number of unreasonable surgical interventions that can lead to a significant decrease in the quality of life due to the presence of specific postoperative complications (paresis of the larynx about 4-6% in specialized institutions, up to 20% in general surgical institutions, postoperative hypoparathyroidism 4-30% , which can lead to permanent irreversible changes in bone tissue).

The essence of the invention is the molecular genetic method: in case of uncertain cytological conclusions, molecular genetic diagnostics is performed, in which previously dried cytological preparation from cytological glasses is washed off with 200 μl of lysis buffer, then DNA and microRNA are extracted from cytological preparations of fine needle aspiration biopsy (TAPB) of neoplasms Using the kits for isolation, detect and quantify the diagnostically significant microR K, based on the PCR method (polymerase chain reaction) with fluorescence recording of the results of analyzes in real time using a CFX96 thermal cycler (Bio-Rad Laboratories, USA) and provided that HMGA2 values are more than 0.09, miRNA 221 more than 0.0105 and miRNA 375 more - 12,1213 determine a follicular tumor with signs of malignancy, with a miRNA-146b of more than 0.1721, papillary cancer is determined, with an miRNA-375 of more than 5.2514, medullary cancer is determined, and the ratio of mitochondrial DNA / nuclear DNA is equal to or more than 5716,3013 - B-cell cancer, the presence of the V600 mutation in the BRAF-papillary cancer gene is a risk of the presence of high biological aggressiveness of this papillary cancer and the presence of a common process in this patient.

The molecular genetic analysis involved several diagnostically significant types of tumor markers that are most common in thyroid cancer: a change in the level of HMGA2 gene expression, somatic point substitutions in the BRAF genes, a change in the expression level of a number of miRNAs, and also the ratio of mitochondrial and nuclear DNA.

Since molecular genetic diagnosis allows not only to detect the malignant process, but also to determine the type of tumor, the samples were divided into the following groups: benign tumors (BS), follicular tumors without markers of malignancy (FOBMZ), follicular tumors with markers of malignancy (FOMZ), B- cell follicular tumors with markers of malignancy (B-FOMZ), papillary and medullary cancers. If we draw parallels with the histological classification, the FOBMZ group consists mainly of follicular adenomas with some follicular cancers for which no molecular markers of malignancy were found. The FOMZ group consists of follicular cancers, part of the follicular variants of papillary cancer and a small number of follicular adenomas for which molecular markers of malignancy have been identified. The group includes patients with HMGA2 greater than 0.0918, miRNA-375 greater than -12.1213, miRNA-221 greater than 0.0105. And with miRNA-375 values greater than 5.2514, medullary cancer is determined. The B-FOMZ group corresponds to B-cell follicular cancers. The method thus allows not only to increase the reliability of verification of thyroid tumors, but also to reduce the number of unreasonable surgical interventions and thus reduce the number of postoperative complications, improve the quality of life. The average diagnostic accuracy of a molecular genetic study is 90.1%, and that of a cytological study is 58.2%.

The method is as follows: the primary diagnostic link is an ultrasound of the thyroid gland. In the presence of a nodular formation of more than 1 cm or the presence of suspicious signs of thyroid cancer in nodular formations of less than 1 cm, a fine-needle puncture aspiration biopsy (TAPB) is performed followed by cytological examination. If thyroid cancer is suspected, surgical treatment is indicated. Upon receipt of the uncertain results of a cytological examination: Bethesda class III and IV, conflicting results of cytological, ultrasound and histological studies, with multiple uninformative findings of a puncture biopsy, a molecular genetic study is carried out: 200 μl of lyse buffer is washed off the cytological preparation previously dried from cytological glasses, then produce Isolation of DNA and miRNA from cytological preparations of fine-needle aspiration puncture biopsy (TA B) thyroid neoplasms using isolation kits, detect and quantify diagnostically significant miRNAs based on the PCR method (polymerase chain reaction) with fluorescence recording of the results of analyzes in real time using a CFX96 thermal cycler (Bio-Rad Laboratories, USA) and Under conditions of miRNA-146b indicators greater than 0.1721, papillary cancer is determined, with miRNA-375 indicators greater than 5.2514, medullary cancer is determined, and a mitochondrial DNA / nuclear DNA ratio greater than 5716.3013 determines B-cell cancer, the presence of muta ii V600 gene BRAF-papillary cancer, is the risk of having high biological aggressiveness of papillary carcinoma and the presence of the patient's common process. Based on the conclusion, the final tactics and the need for surgical treatment are determined.

To prove the achievement of the tasks at hand, the results of examination and treatment of 61 patients of the endocrine surgery department of GBUZ KKB No. 2, operated on for mononodous non-toxic goiter in 2016 - 2017, were subjected to analysis. Cytological preparations obtained during a standard fine-needle aspiration biopsy were subjected to molecular genetic analysis, at the Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk.

Among the patients, there were 50 (82%) women and 11 (18%) men aged 19 to 64 years. Only patients with mononodous thyroid lesions were analyzed, which excluded errors in comparing the results of ultrasound, histological and molecular genetic research methods.

1. Evaluation of U3 diagnostics:

In this examination, 3 patients were included in the TIRADS 2 group (benign changes in the thyroid gland.), 2 cases were diagnosed with colloid goiter, in one patient - follicular adenoma. In the TIRADS 3 group (probably benign changes in the thyroid gland), there were 33 patients: in 2 cases (6.1%), histological examination revealed benign lesions, in 17 cases (51.5%) revealed follicular adenoma, and in 14 cases (42, 4%) revealed a malignant tumor, which illustrates the unreliability of ultrasound diagnosis of tumors in this group.

In the TIRADS IVa group (the risk of malignancy is 5-10%), papillary cancer was detected in all 2 patients (100%). TIRADS IVb group (risk of malignancy 10-80%) included 14 patients: 2 cases (14.3%) were diagnosed with follicular adenoma, and 12 (85.7%) showed malignant tumors. With TIRADS V, a malignant process was detected in all 9 patients. All data are summarized in table 1.

2. Assessment of cytological diagnosis

In the Bethesda II group (18 patients) in 12 (61.1%) patients with PID, a benign process was detected, but in 6 (38.9%) - a malignant process.

In the Bethesda IV group (27 patients), 15 (55.6%) showed a malignant tumor. In 12 (44.4%), follicular adenoma was revealed. In this group, two patients had signs of a malignant process (the presence of preoperative paresis of the larynx confirmed by examination by an ENT doctor), however, a follicular tumor was diagnosed by TAPB.

In the Bethesda VI group, all 15 patients confirmed malignant tumors. All data are summarized in table 2.

3. Assessment of the molecular genetic method of diagnosis

In all studies from the Beshesda II group (6 people), for which a malignant process was diagnosed by PIP (pathological examination), a malignant process was also diagnosed by molecular genetic research.

For 6 cases of discrepancy between the results of molecular analysis and PIP, an expert histological study was carried out, as a result of which in 3 cases the histological diagnosis of follicular adenoma was changed to malignant (2 FVPR and 1 FR), and 3 cases the histopathological diagnosis did not change.

The results of the comparison of molecular analysis and PIP are shown in table 3.

Table 3. Comparison of molecular analysis and histopathological studies.

DO - benign formations

FOBMZ - follicular tumor without markers of malignancy

FOMZ is a follicular tumor with markers of malignancy.

PR - papillary cancer

B-FOMZ - B-cell tumor with markers of malignancy.

MP is medullary cancer.

Diagnostic characteristics

The data obtained during the study allowed us to determine the main diagnostic characteristics of different methods: sensitivity, specificity, predictive value of a positive result (PCR), predictive value of a negative result (PCR), diagnostic accuracy. These data for cytological and molecular analysis (taking into account the revision of 3 PIP results) are shown in table 4.

A molecular genetic analysis of material from cytological glasses of 16 patients with neck lymphadenopathy with cytologically detected thyroid cancer was also performed. The diagnostic criterion for confirming metastasis to the lymph nodes of the neck is the level of HMGA2 expression. With HMGA2 values in the lymph node of more than 0.0255, then we are talking about metastasis of papillary cancer in the lymph nodes of the neck, if the indicator is less than 0.0255, there is no pathology.

In 6 patients who underwent simultaneous thyroidectomy with fascial-sheath lymphodissection, molecular genetic analysis confirmed metastasis to the paravasal lymph nodes of the neck, diagnosed by cytological examination. In 1 patient, a RET-PTC mutation was detected. In 5 patients, a BRAF V600E mutation was detected, which confirms a more aggressive form of the course of papillary cancer disease with a mutation in the BRAF gene. 5 patients confirmed the absence of damage to the lymph nodes.

In 1 patient with ultrasound of the lymph nodes of the neck for thyroid cancer, suspicious lymph nodes were detected. With TAB of suspicious lymph nodes of the neck, lesions of paravasal lymph nodes are excluded. Intraoperative thyroidectomy was expanded to remove the lymph nodes of the neck of group III. A histopathological examination confirmed a metastatic lesion of the removed lymph nodes. Molecular genetic analysis confirmed metastasis in the lymph nodes with a BRAF mutation.

In 5 patients who excluded damage to the lymph nodes of the neck according to the TAB data, molecular metastases revealed lymph node metastases. All patients were reevaluated, 3 patients underwent repeated puncture of the lymph nodes, 2 revealed no lymphadenopathy. In 1 patient metastasis to the lymph node of the neck was confirmed. The patient is registered for repeated surgical treatment. And after surgical treatment, metastasis to the paravasal lymph nodes was confirmed. In the rest, the damage to the lymph nodes of the neck was not confirmed, since they were all after radioiodine therapy and most likely the lymph nodes were ablated.

Patient M., 79 years old, was admitted to the Department of Surgery with complaints of hoarseness. Earlier, a patient in a regional oncologic dispensary underwent a fine-needle aspiration puncture biopsy (TAPB), according to the results of which there is a follicular tumor. Bethesda IV. Surgical treatment in the general medical network is recommended. In the clinic, during an additional examination (consultation with an ENT doctor), the patient has laryngeal paresis (which is a sign of thyroid cancer with invasion of the recurrent laryngeal nerve). A repeat TAPB was also performed at the clinic. Result: follicular tumor. When performing molecular genetic analysis, signs of papillary cancer were confirmed (HMGA 2.7. MiRNA 146b - 9.18). Thyroidectomy performed. Histological examination revealed papillary cancer.

Patient A, 24 years old, was admitted to the Department of Surgery with a diagnosis of multinodular goiter 2 tbsp. Cervical lymphadenopathy. At the place of residence, a puncture biopsy of the nodular formation of the right lobe of the thyroid gland and paravasal lymph node on the right was performed. Result: in the thyroid gland node: goiter colloid on the background of thyroiditis. Bethesda II. TAB of the lymph nodes of the neck: signs of hyperplasia. The clinic performed repeated puncture biopsy. Result: nodular thyroid gland formation: cell-colloidal goiter with lymphoid infiltration. Besthesda II. TAB L / C: lymph node elements. When performing molecular genetic research: in the material from the thyroid gland node: papillary carcinoma (HMGA 1.61, miRNA 146b - 15.00). In the lymph node: metastasis of papillary carcinoma (HMGA-1.5052, miRNA 146b - 7.34). The patient underwent surgical treatment: thyroidectomy, central lymphadenectomy, fascial-sheath lymphadenectomy. According to a histopathological study, papillary cancer in the right lobe of the thyroid gland with metastasis to the central and paravasal lymph nodes of the neck on the right was confirmed.

Patient K, 24 years old, was admitted to the Department of Surgery with a diagnosis of nodular goiter 1 tbsp. The node of the right lobe. Performed a puncture biopsy of the nodular formation of the right lobe of the thyroid gland. Result: cell-colloidal goiter. Bethesda II. According to the ultrasound of the thyroid gland, a nodular formation of 9 mm, suspicious signs of tumor damage. When performing molecular genetic research: in material from the thyroid gland node: papillary carcinoma, follicular variant (HMGA 0.62, miRNA 146b - 3.42). Given the absence of BRAF mutation, tumor size, follicular variant, the degree of biological aggressiveness of the tumor is minimal, hemithyroidectomy was performed. According to a histopathological study, the follicular variant of papillary cancer is confirmed.

Patient D, 47 years old, was admitted to the Department of Surgery with a diagnosis of nodular goiter 1 tbsp. The node of the right lobe. Performed a puncture biopsy of the nodular formation of the right lobe of the thyroid gland. Result: colloid goiter with lymphoid infiltration. Bethesda II. According to the ultrasound of the thyroid gland, a nodular formation of 6 * 5 * 5 mm, suspicious signs of tumor damage. When performing molecular genetic research: a follicular tumor with signs of malignancy: (miRNA221: 1.92, miRNA375: -4.59). Given the absence of the BRAF mutation, the size of the tumor, hemithyroidectomy was performed. According to a histopathological study: follicular variant of papillary cancer.

Claims (1)

A method for the differential diagnosis of thyroid neoplasms (thyroid gland), including ultrasound of the thyroid gland, detection of nodular thyroid formations, puncture biopsy, subsequent cytological examination of punctate, characterized in that, with uncertain cytological findings, a molecular genetic study is performed in which a previously dried cytological preparation 200 μl of lysis buffer is washed off from cytological glasses, then DNA and microRNA are extracted from cytological Using fine-needle aspiration puncture biopsy (TAPB) of thyroid neoplasms with the help of isolation kits, diagnostic-significant microRNAs are detected and quantified based on the polymerase chain reaction (PCR) method with real-time fluorescence recording of the results of analyzes using a CFX96 thermal cycler (Bio- Rad Laboratories, USA) and with HMGA2 values greater than 0.09, miRNAs 221 greater than 0.0105 and miRNAs 375 greater than -12.1213, determine a follicular tumor with signs of malignancy, subject to showing microRNA-146b antibodies is more than 0.1721, papillary cancer is determined, with miRNA-375 more than 5.2514, medullary cancer is determined, and the mitochondrial DNA / nuclear DNA ratio is more than 5716.3013 - B-cell cancer, the presence of V600 mutation in the BRAF gene, papillary cancer and is a risk of the presence of high biological aggressiveness of this papillary cancer and a common process.