KR101738766B1 - Method of providing information for diagnosis of malignant mesothelioma - Google Patents

Abstract

The present invention relates to a method for providing information for accurate diagnosis of malignant mesothelioma. More particularly, the present invention relates to a method for diagnosing malignant mesothelioma by observing microvilli located between tumor cells using a paraffin tissue fragment of a tumor and a scanning electron microscope To a method of providing information. The method of the present invention makes it possible to clearly observe characteristic features of long and thin microvilli by using a scanning electron microscope. In addition, the paraffin tissue block used for H & E staining and immunohistochemical staining can be easily used, so that there is no need to use a separate invasive method for preparing the sample. In addition, when scanning electron microscopy is performed using paraffin tissues, the cut-off value for diagnosing malignant mesothelioma when the length and diameter ratio (length / diameter ratio) of the microvilli on the tumor cell surface exceeds 5.0 value. Therefore, the method of the present invention can be used for accurately diagnosing malignant mesothelioma together with H & E staining or immunohistochemical staining.

Description

[0001] The present invention relates to a method of providing information for the diagnosis of malignant mesothelioma,

The present invention relates to a method for providing information for accurate diagnosis of malignant mesothelioma. More particularly, the present invention relates to a method for diagnosing malignant mesothelioma by observing microvilli located between tumor cells using a paraffin tissue fragment of a tumor and a scanning electron microscope To a method of providing information.

Mesothelioma is a type of mesothelioma that occurs mainly in the mesothelium that covers the surface of the lungs surrounding the lungs, the peritoneum that protects the stomach and liver, and the pericardium surrounding the heart. It is divided into benign and malignant . All benign mesothelioma is focal and can be cured almost through surgery, but malignant mesothelioma has poor prognosis. Since malignant mesothelioma is mainly caused by long-term exposure to asbestos, the government has recently compensated for malignant mesothelioma patients by exposure to asbestos. Therefore, accurate diagnosis of malignant mesothelioma is not only for therapeutic purposes It's a problem.

In order to accurately diagnose malignant mesothelioma, it is important to observe the morphologic findings of the specimens taken from tumor lesions accurately. It is difficult to accurately distinguish between different types of cancer with common optical microscopy, Several pathological examinations need to be used. Hematoxylin and eosin (H & E) staining is a common method commonly used to observe morphological changes of specimens taken from tumor lesions. However, the types of tumors are becoming increasingly subdivided, and tumor markers and prognostic factors that can be used to diagnose tumors are newly known, so that conventional hematoxylin- There is a problem that information is difficult to obtain. Immunohistochemical staining is commonly used to confirm the origin of undifferentiated cells, to detect the presence of enzymes, hormones, tumor markers and prognostic factors, to distinguish between sarcoma and sarcoma, It has been used effectively to differentiate tumors from malignant tumors and to estimate the primary lesion of metastatic cancer. Immunohistochemical staining is an extremely sensitive and specific test, and has been used as an indispensable test for diagnosis pathology and morphological studies in the last 20-30 years. Although such immunohistochemical staining is widely performed for the diagnosis of malignant mesothelioma, it is often difficult to confirm the malignant mesothelioma because the result is unclear. Therefore, there is a need for an objective diagnostic method that can supplement this.

Therefore, the present inventors have studied to develop a new diagnostic method for identifying malignant mesothelioma using the paraffin residual tissue piece produced during the pathological examination for suspected malignant mesothelioma, and then, through the scanning electron microscopic observation of the paraffin block specimen, The present inventors have completed the present invention by identifying and characterizing characteristic microvilli findings present on the cell surface.

Korean Patent Laid-Open Publication No. 2013-0136345 International Patent Publication No. WO 13/115476

It is an object of the present invention to provide an information providing method for diagnosing malignant mesothelioma which can distinguish malignant mesothelioma based on the characteristic morphology of microvilli observed between tumor cells in a paraffin tissue section of a specimen collected from a tumor lesion .

In order to accomplish the above object, the present invention provides a method for preparing a sample for SEM observation using a paraffin tissue slice prepared from a tissue of a patient suspected of having malignant mesothelioma; Observing the SEM using a scanning electron microscope to measure and verify the length and diameter of the microvilli located on the surface of the tumor cells; And comparing the length / diameter ratio of the microvilli with the length / diameter ratio of the microvilli in the control metastatic adenocarcinoma.

In an embodiment of the present invention, the step of comparing the length / diameter ratio of the microvilli with the length / diameter ratio of the microvilli of the metastatic adenocarcinoma of the control group includes a step of obtaining the sensitivity and the specificity by a statistical method. In one embodiment of the invention, the statistical method may be, but is not limited to, a Chi-square test.

In one embodiment of the present invention, the step of comparing the length / diameter ratio of the microvilli with the length / diameter ratio of the microvilli in the control metastatic adenocarcinoma can be performed by a cut-off value. In one embodiment of the present invention, the cut-off value of the length / diameter ratio of the malignant mesenchymal microvilli distinguishable from metastatic adenocarcinoma is preferably 5 or more.

In one embodiment of the present invention, the thickness of the paraffin cross-section may be 4 to 5 μm, but is not limited thereto.

In one embodiment of the present invention, the SEM observation sample may be manufactured by a method including the following steps.

(A) fixing the paraffin-embedded tissues to a glass slide, followed by deparaffinization;

(B) allowing the deparaffinized tissue to function using ethanol;

(C) pre-fixing the hydrolyzed tissue using glutaraldehyde;

(D) postfixing the pre-fixed tissue with OsO ₄ ;

(E) dewatering the postfixed tissue;

(F) substituting isoamyl acetate for the dehydrated tissue;

(G) drying the tissue substituted with isoamyl acetate at a critical point and fixing the silver powder; And

(H) depositing the sample surface on which silver is fixed with Pt-Pd.

In one embodiment of the present invention, the method for preparing a sample for SEM observation comprises the steps of immersing in 0.1 M phosphate buffer for 10 minutes after completion of the pre-fixation step of step (C) and the post-fixation step of step (D) And then spout cleaning.

In one embodiment of the present invention, the diagnostic tissue of step (A) may be cut into pieces of 3 to 6 탆 after paraffin embedding. In one embodiment of the present invention, the deparaffinization step of step (A) may be carried out by immersing the glass slide in xylene four times at room temperature for 2 to 10 minutes. In one embodiment of the present invention, the ethanol function of step (B) is carried out by immersing the glass slides in 100%, 95%, 90%, 80%, 70% ethanol and distilled water at room temperature for 2 to 10 minutes . In one embodiment of the present invention, the dehydration process of step (E) may be carried out by dipping at 70%, 80%, 90% and 95% and 100% ethanol (twice) at room temperature for 2 to 10 minutes . In one embodiment of the present invention, the step (F) may be carried out at room temperature for 5 to 15 minutes by using isoamyl acetate. In one embodiment of the present invention, the step of replacing isoamyl acetate in step (F) with t-butyl alcohol, and the step of substituting with t-butyl alcohol may be performed at 30 to 40 ° C for 20 to 40 minutes . In one embodiment of the present invention, the step of depositing Pt-Pd in the step (H) may be performed by using a metal ion evaporator to deposit 5 to 10 nm thick.

In one embodiment of the present invention, the tumor diagnostic tissue staining method is characterized in that after the post-fixation of the step (D), the step (D-1) is performed by using 2% tannic acid at room temperature for 20 to 40 minutes Dyeing step; And (D-2) fixing the conduction-stained sample with 1% OsO ₄ . In one embodiment of the present invention, the method further comprises a step of immersing in 0.1 M phosphate buffer for 10 minutes after completion of the conductive dyeing at step (D-1) and the fixing at step (D-2) .

In the conventional histological examination method using an optical microscope, it is impossible to clearly observe microvilli present in the gaps between the tumor cells due to the limitation of the resolving power. However, the method of the present invention uses a scanning electron microscope to observe long and thin microvilli It is possible to observe distinctive features clearly. In addition, the paraffin tissue block used for H & E staining and immunohistochemical staining can be easily used, so that there is no need to use a separate invasive method for preparing the sample. Therefore, the method of the present invention can be used for accurately diagnosing malignant mesothelioma together with H & E staining or immunohistochemical staining.

FIG. 1 is a photograph of normal mesothelial cells (A) and malignant mesothelioma cells (B), showing the existence of long and slender microvilli on the surface of tumor cells unlike metastatic cancer cells (C).
FIG. 2 is a graph showing the results of histology (A) using a paraffin block made for pathological diagnosis and a glass slide with a tissue piece for scanning electron microscope observation in order to examine microvilli observed in the space between mesangial cells of malignant mesothelioma 3 is a photograph showing a glass slide (B) trimmed with a knife.
FIG. 3 is a photograph showing a photograph of a gap (A) observed between malignant mesothelioma cells in an optical microscope using the sample according to the present invention, and a number of microvilli (B) present at this site by scanning electron microscopic observation.

The present invention relates to a method of providing information for the diagnosis of malignant mesothelioma. In particular, when the hematoxylin and eosin (H & E) staining or immunohistochemical staining alone is difficult to differentiate from other adenocarcinomas, It is a method that can easily diagnose malignant mesothelioma by observing the microvilli of tumor cells using a scanning electron microscope utilizing the remaining specimen.

The term "tumor" as used herein encompasses any benign or malignant tumor of the human or animal body as a non-therapeutic, non-systemic proliferation of cell growth. Tumors are invasive and are said to be malignant or cancerous when they have metastatic properties. Invasive refers to the tendency of a tumor to penetrate the underlying basal layer that forms the barrier of tissue and penetrate into surrounding tissues and into the circulatory system of the human body. Tumor refers to the tendency of tumors to migrate to other regions and to separate from early developmental sites to form proliferative regions.

As used herein, the term "diagnosis" refers to identifying a molecular or pathological condition, disease or pathology, such as identifying a malignant mesothelioma, or identifying a malignant mesothelioma patient . ≪ / RTI > In one embodiment of the present invention, diagnosis refers to the diagnosis of malignant mesothelioma by identifying the elongated microvilli present on the surface of the tumor cells.

As used herein, the term "room temperature" means the ambient temperature of the room, which means around 15-25 ° C.

The present inventors focused on the fact that the results of the immunohistochemical staining performed widely for the diagnosis of tumors are ambiguous and it is difficult to confirm the diagnosis of the tumor. Therefore, by using a paraffin residual tissue piece produced during the histopathological examination, The present inventors completed the present invention while studying a sample preparation technique that can more accurately confirm microvilli findings characteristic of a specific tumor when observed. Recently, for the purpose of diagnosing malignant mesothelioma or metastatic adenocarcinoma, a small invasive method, which is a less invasive method, is often performed in a clinical setting rather than collecting large tissue from a patient. Therefore, the size of a specimen for pathological diagnosis is small, All of them are made into paraffin tissue blocks, which are cut (4-5 μm in thickness) and thin sections are used for H & E staining and immunohistochemical staining. However, H & E and immunohistochemical staining can not distinguish malignant mesothelioma from adenocarcinoma. In this case, malignant mesothelioma (or adenocarcinoma) can not be treated properly. In these cases, scanning electron microscopy can be performed for the diagnosis of malignant mesothelioma using paraffin tissue pieces used in patient specimens. However, scanning electron microscopy (SEM) of malignant mesothelioma using paraffin sections has not been performed in the pathology department of the hospital because of technical difficulties and there is no research on the diagnosis method of malignant mesothelioma by SEM.

Therefore, the present inventors carried out a study to devise a diagnostic standard by scanning electron microscopy (SEM) using a paraffin tissue piece that has been used for diagnostic purposes and further thinned (thin paraffin tissue slice 4-5 μm thick as in immunohistochemical analysis) Respectively. In this case, the elongated microvilli on the surface of the malignant mesothelioma of the mesothelioma are cut off in the process of fibrillation, and the length of the microvilli is generally shortened in the truncated tissue pieces (thickness 4-5 μm). When the same paraffin three slices are used, The cut-off value, which is statistically meaningful to distinguish between the two, was determined to be 5.0 (Table 1), because the microvilli length / diameter ratio of adenocarcinoma tumor cells was lowered under the same conditions.

Therefore, the new reference value devised in the present invention can be used as information for the diagnosis of malignant mesothelioma that can be applied to the paraffin tissue using tumor cells in the scanning electron microscopic analysis.

In particular, it can be concluded that malignant mesothelioma can be judged to be a malignant mesothelioma when the microvilli length / diameter ratio of the tumor cell is 5.0 or more. If it is less than 5.0, it can be judged to be metastatic adenocarcinoma.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

Paraffin organization using scanning electron microscope In the sample  Microvilli observation of tumor cells

Preparation of Samples for Scanning Electron Microscopy Using Paraffin Sections and Scanning Electron Microscopy (SEM) were performed as follows.

Twenty cases of malignant mesothelioma and 10 cases of metastatic adenocarcinoma were selected and paraffin sections (thickness of 4-5 μm) were obtained. After washing with 0.1% PB, 2.5% groutaldehyde pretreatment, ₄ post-fixation, washing with 0.1M PB, series alcohol dehydration, isoamyl acetate infiltration, critical point drying, and deposition with Pt-Pd followed by observation with a Hitachi S4200 scanning electron microscope. Specific experimental examples of each step are as follows.

< Experimental Example  1>

Step 1: Create slides

For the diagnosis of malignant mesothelioma, the tissue of the cases to be scanned by scanning electron microscopy was fixed with formalin, fixed with formalin, embedded in paraffin embedded in 4 ~ 5 μm sections, and placed on a glass slide to prepare a single tissue slide for dyeing.

Step 2: Paraffin (20 minutes)

Thereafter, the glass slide is placed in a coplin jar containing Xylene (Junsei, Japan), and the de-paraffin process is performed in which the glass slide is reacted four times at room temperature for 5 minutes.

Step 3: Ethanol function (30 minutes)

Ethanol (Merck, Germany) is used and the glass slides are dipped in 100%, 95%, 90%, 80%, 70% ethanol and distilled water at room temperature for 5 minutes each.

Step 4: 2.5% Groutaldehyde Fixed (30 minutes)

Add 10 mL of 25% Glutaraldehyde (Merck, Germany) to 90 mL of 0.1M phosphate buffer and dilute (2.5% groutaldehyde solution) to fix the sample for 30 min. In this case, 15 ml of 0.1 M phosphate buffer (pH 7.4) was dissolved in 15 ml of sodium phosphate, monobasic (Junsei, Japan) in 1 L of distilled water, and 190 ml of solution A and 14.2 g of sodium phosphate, dibasic (Junsei, Japan) And 810 mL of solution B dissolved in 1 L of distilled water.

Step 5: Wash (10 min)

Thereafter, the pre-fixed sample is immersed in 0.1M phosphate buffer solution for 10 minutes and washed.

Step 6: 1% OsO ₄ Postfix (30 minutes)

After diluting 1 g of Osmium tetroxide (Merck, Germany) with 50 ml of distilled water and 0.1 M phosphate buffer, the washed sample is immersed in the solution for 30 minutes and fixed.

Step 7: Wash (10 minutes)

Thereafter, the postfixed sample is immersed in 0.1 M phosphate buffer for 10 minutes and washed.

Step 8: Dehydration of alcohol (30 minutes)

The washed samples were dipped in ethanol (Merck, Germany) at 70%, 80%, 90% and 95% and 100% ethanol (x 2 times) for 5 minutes each at room temperature.

Step 9: Isoamyl acetate  Substitution (10 min)

The dehydrated sample is reacted with isoamyl acetate (Junsei, Japan) at room temperature for 10 minutes. In this case, t-butyl alcohol (Junsei, Japan) can be used instead of isoamyl acetate, but it is preferable to carry out the reaction at 37 占 폚 for 30 minutes.

Step 10: Trimming

After the infiltration step is completed, the tumor portion attached to the glass slide is trimmed with a glass knife.

Step 11: Dry the critical point (60 minutes)

Dry the critical point using a critical point dryer (Hitachi, HCP-2, Japan) and fix the sample with silver paste (Nissin EM Co, Japan) on the sample stand. In this case, a freeze dryer (Hitachi, E-2030, Japan) can be used instead of the critical point dryer, and in this case, drying is preferably carried out for 120 minutes. Here, the critical point means that the pressure gauge of the HCP-2 device indicates 110 kgf / cm ² at 38 to 40 ° C.

Step 12: Pt - Pd (30 minutes)

Using a metal ion evaporator (Ion sputter, Hitachi, E-1030, Japan), the silver ion-deposited sample surface is ion-deposited to a thickness of 5-10 nm using Pt-Pd in a metal ion evaporator.

Step 13: Scanning electron microscope observation

The ion-deposited sample was observed at an acceleration voltage of 10 KV using a scanning electron microscope (Hitachi, S-4200, Japan).

< Experimental Example  2>

A sample can be prepared by conducting conduction staining additionally to the method described in Experimental Example 1 above. In order to do this, after the post-fixing step of step 6 and the cleaning step of step 7, the following process may be added.

Step 7-1: Conductive dyeing process (30 minutes)

After the post-fixation, the washed sample was reacted at room temperature for 30 minutes using 2% Tannic acid (Kanto, Japan).

Step 7-2: Wash (10 min)

Then, the conduction-stained sample is washed with 0.1 M phosphate buffer for 10 minutes.

Step 7-3: 1% OsO ₄  to  Fixed (30 minutes)

1 g of Osmium tetroxide (Merck Co., Germany) was dissolved in 50 ml of distilled water and diluted with the same amount of 0.1 M phosphate buffer, and the washed sample was immersed and immersed in the solution for 30 minutes.

Step 7-4: Wash (10 minutes)

Thereafter, the postfixed sample is immersed in 0.1 M phosphate buffer for 10 minutes and washed.

< Example  2>

Establish criteria for diagnosis of malignant mesothelioma

When the sample prepared by the method of Example 1 is observed with a scanning electron microscope, microscopic villi that appear to be faint in the gap between tumor cells can be observed clearly by optical microscope observation of the residual tissue of paraffin of the tumor (Fig. 3) . In order to distinguish malignant mesothelioma from adenocarcinoma using the morphologic features of the microvilli of tumor cells that can be observed by scanning electron microscopy, the "length-diameter ratio" criterion for the diagnosis of malignant mesothelioma value) was developed in the following way.

The length and diameter of the microvilli were measured in at least five tumor lesions per each case used in Example 1, and then the "L / D ratio (LDR)" was calculated to prepare experimental data for each case Respectively. Measurements of length and diameter for at least 20 microvilli in each case were obtained and Mann

Whitney U test was used to verify the length / diameter ratio of microvilli, and sensitivity and specificity were determined by Chi-square test. The results were as follows.

Experiment result

The ratio of the length (L) to diameter (D) of the microvilli in malignant mesothelium tumor cells was measured and the length / diameter ratio (LDR) of the microvilli located on the tumor cell surface of the metastatic adenocarcinoma, The diagnosis of malignant mesothelioma was based on the diagnosis of LDR with a diagnostic value of 5.0. The sensitivity and specificity of the diagnostic criteria were high and the diagnostic accuracy was very high. The cut-off value of the new malignant mesothelioma, which can be diagnosed as malignant mesothelioma, was determined (Table 1).

Compare Malignant mesothelioma Metastatic adenocarcinoma p value Average length (L) (um) 2.55 (+ - 076) 0.97 (+/- 0.34) p < 0.001 Average Diameter (D) (um) 0.34 (+/- 0.28) 0.35 (0.08) p = 0.552 L / D ratio 7.73 (+ - 2.21) 2.77 (+/- 0.78) p < 0.001 L / D ratio cutoff standard > = 5.0 <5.0 p < 0.001

The microvilli length on the surface of the mesothelial cells of the actual mesothelioma is known to be very long. However, when using the paraffin-section slice as a specimen for the scanning electron microscope, the length of the microvilli is cut at the end of the sifting process by using thin slices of 4-5 μm. Therefore, when using residual samples of formalin fixed or pleural cytology, Otherwise, the microvilli length of malignant mesothelioma cells may be generally shortened. Thus, the length / diameter ratio (LDR) of microvilli can be relatively lower than in the case of using residual samples of stomach formalin fixed or pleural fluid cytology.

The present invention is a new method that can be further applied in cases where the results of H & E or immunohistochemistry are ambiguous and difficult to diagnose, and can provide crucial information for the diagnosis of malignant mesothelioma using the remaining paraffin tissue used for H & E diagnosis This can be a useful technique. Further, when the method of the present invention is applied, the diagnostic accuracy can be further increased.

Surgical excision of freshly isolated tumor tissue or formalin fixed tumor tissue may be performed by electron microscopy, but the tumor is not surgically removed for the diagnosis of malignant mesothelioma. Recently, a noninvasive method for diagnosis is preferred. A small biopsy, a biopsy or a cytology test is taken from a possible patient and the tumor tissue is minimally taken to the pathology department. All the tumor tissues collected for diagnosis are made into paraffin blocks have. Therefore, there is no other specimen for electron microscopy for the diagnosis of malignant mesothelioma.

However, there is no standard for diagnosing malignant mesothelioma by electron microscopy.

In this regard, this technology has developed a diagnostic criteria for diagnosing accurate malignant mesothelioma by scanning electron microscopy using paraffin block tissue which can be easily obtained in hospitals. As a criterion, the length of microvilli on the surface of tumor cells The cut-off value of diameter ratio (length / diameter ratio) was statistically significant when diagnosed as malignant mesothelioma of 5.0 or more, and it could be diagnosed as metastatic adenocarcinoma when the cut-off value was less than 5.0 &Lt; 0.001). And high specificity and sensitivity when the cutoff value of the length and diameter ratio of microvilli is 5.0 or more.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the presented embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (9)

Sectioning a paraffin-embedded biopsy tissue of a patient suspected of having malignant mesothelioma into a thickness of 4 to 5 占 퐉; preparing a sample for scanning electron microscopic observation; Observing the SEM using a scanning electron microscope to determine the length / diameter ratio by measuring the length and diameter of the microvilli located on the surface of the tumor cells; And
Wherein the malignant mesothelioma is judged to be malignant mesothelioma when the length / diameter ratio of the microvilli is 5 or more, and if it is less than 5, it is judged as metastatic adenocarcinoma. delete delete delete delete The method according to claim 1,
Wherein the sample for SEM observation includes the following step after the step of segmenting:
(A) immobilizing paraffin-embedded biopsy tissue on a glass slide and deparaffinizing it;
(B) allowing the deparaffinized tissue to function using ethanol;
(C) pre-fixing the hydrolyzed tissue using glutaraldehyde;
(D) postfixing the pre-fixed tissue with OsO ₄ ;
(E) dewatering the postfixed tissue;
(F) substituting isoamyl acetate for the dehydrated tissue;
(G) drying the tissue substituted with isoamyl acetate at a critical point and fixing the silver powder; And
(H) depositing the sample surface on which silver is fixed with Pt-Pd. The method according to claim 6,
Further comprising, after completion of the pre-fixing step of step (C) and the post-fixing step of step (D), dipping in 0.1 M phosphate buffer for 10 minutes each. The method according to claim 6,
After the post-fixing of step (D)
(D-1) tannic acid at room temperature for 20 to 40 minutes; And
(D-2) characterized in that further comprising the step of securing the samples stained with the conductive OsO _4. 9. The method of claim 8,
Further comprising the step of immersing in 0.1 M phosphate buffer for 10 minutes after completion of the conductive dyeing at step (D-1) and fixing at step (D-2).

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