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(mammaglobin and gross cystic disease fluid protein 15) in lung and pleural tumors. Arch Pathol Lab Med. 2008;132:239-243 цитат 38. Trupiano JK, Geisinger KR, Willingham MC, et al. Diffuse malignant mesothelioma of the peritoneum and pleura, analysis of markers. Mod Pathol. 2004;17:476-481
39. Ye J, Findeis-Hosey JJ, Yang Q. et al. Combination of napsin A and TTF-1 immunohistochemistry helps in differentiating primary lung adenocarcinoma from metastatic carcinoma in the lung. Appl Immunohistochem Mol Morphol. 2011;19(4):313-7
DIAGNOSTIC VALUE OF THE IMMUNOHISTOCHEMICAL STUDY FOR MALIGNANT PLEURAL MESOTHELIOMA AND LUNG ADENOCARCINOMAS
Peshev Zhivko Vladimirov Belovezhdov Veselin Todorov
Associate Professor at the Department of General and Clinical Pathology, Medical University, Plovdiv, Bulgaria
Novakov Ivan Petkov
Associate Professor at the Clinic of Special Surgery, St George University Hospital, Plovdiv, Bulgaria
The diagnosis of pleural tumors, despite the progress in clinical and morphological methods, is still difficult. In this anatomic region develop a few primary tumors, and the most common of them is the malignant pleural mesothelioma (MPM). This is also the location of numerous metastases from different malignant tumors of various histogenesis. This diversity of malignant tumors requires first of all to differentiate them from the pleural mesothelioma - a tumor of high malignancy and unfavourable prognosis. Its difficult histological diagnosis is confirmed either rarely in life with reliable electronic microscopic study or after death with an autopsy. The uncertain imaging diagnostics and the difficult interpretation of cytological material from the pleura require a necessary im-munehistochemical study of tissue biopsy material. [1, p. 2427]
Malignant mesothelioma is a rare, asbestos-associated tumor, which originates from the pleural mesothelium, peritoneum, pericardium and tunica vaginalis of testis. Its frequency is less than 1% of all malignancies. It most commonly affects the pleura (80-90%), the peritoneal cavity (10-20%) and there
are only isolated cases of other locations. [8. pp. 1-11], [7 p. 395]
The aim of this study was to adopt into practice an optimized immunohistochemical panel for correct diagnosis of MPM.
Material and Methods
Tumor Samples
We conducted a study on 50 mesotheliomas diagnosed over a 6-year period in the Department of Clinical Pathology at St George University Hospital.
The distribution of malignant mesotheliomas by histo-logical type is shown in Figure 1. According to the WHO his-tological classification for 2004, it includes the following types of malignant mesothelioma:
• epithelioid - 34 (64.0%)
• biphasic - 13 (26.0%)
• sarcomatoid - 3 (8.0%)
• desmoplastic - 1 (2.0%)
biphasic; 13; 25%
sarcomatoidi_desmoplastic 1; 2%
epitheloid biphasic sarcomatoid desmoplastic
epitheloid; 34; 67%
Fig. 1 Histological types of malignant pleural mesothelioma
In the immunohistochemical study of biopsy pleural material was used a panel of 10 antibodies from different companies. (see Table 1)
Immunohistochemical Analysis This panel was applied to all 50 mesotheliomas, which included three main histological types - epithelioid, biphasic and sacromatoid.
The aim of this study was to determine the level of expression for the different histological mesotheliomas and then
compare them to those of metastatic lung adenocarcinomas. For that reason it was necessary to appraise the specificity and sensitivity of each of them and in accordance with the obtained results to select the most suitable antibodies to use in the diagnosis of malignant mesotheliomas and metastatic pleural carcinomas. Therefore was developed a rating scale for the intensity of reaction colour and percentage of cells with positive expression. The intensity of reaction colour adopted the following values: 0 - missing; 1 - weak; 2 - moderate; 3 - strong.
the expected results, specified in the protocols for the respective antibodies of the company producer. All the cases with expression of more than 10%, were accepted as positive. For the statistical analysis and in accordance with the above scales, the tumors can be divided into three groups:
1. Negative (-) • all tumors with no colour reaction or which is up to 10% of the tumor cells.
2. Weak positive (+) • with sum of the two indexes from 2 to 4
3. Strong positive (++) • from 5 to 6. See Table 2
Table 1
Antibodies used in immunohistochemical study
Antibody Source Clone Dilution Pretreatment Expression
Mesothelin Leica Biosystems Novocastra Ltd. Newcastle UK 5B2 ММА* RTU** High temperature with 0.01M CB*** (pH 6.0). membranous
WT1 Leica Biosystems WT49 ММА 1:100 High temperature pH 9.0 for 15 min. nuclear
Calretinin Leica Biosystems 5A5 ММА RTU High temperature with 0.01M CB (pH 6.0) for 20 min. nuclear and cytoplasmic
Anti-D240 antibody Abcam Cambridge. UK D240 ММА 1:40 High temperature 95-100oC CB (pH 6.0), for 20 min. membranous
TTF-1 thyroid transcription factor Leica Biosystems SPT24 ММА 1:200 Water bath 980C with pH 6.0 for 20 min. nuclear
Cytokeratin 7 Leica Biosystems OV-TL 12/30 RTU High temperature with 0.01 M citrate buffer pH 6.0. cytoplasmic
Cytokeratin 20 Leica Biosystems KS20.8 ММА RTU High temperature with 0.01 M pH 6.0. cytoplasmic
EMA Epithelial Membrane Antigen Leica Biosystems GP1,4 ММА RTU Protease cytoplasmic and membranous
CEA carcinoembyonic antigen Leica Biosystems 12-140-10 ММА 1:200 Protease cytoplasmic
Cytokeratin 5/6 DAKO. Agilent Technologies comp. Denmark D5/16 B4 ММА 1:100 100° C for 20 min in 10 mmol/L Tris buffer pH 9.0. cytoplasmic
*MMA - mouse monoclonal antibody, **RTU- ready to use, ***CB - citrate buffer
Interpretation of Results
Table 2
__Immunohistochemical study of different histologic variants of MPM _
Antibody Epithelioid Biphasic Sarcomatoid Mesotheliomas Mesotheliomas total
mesothelioma mesothelioma mesothelioma total n=50 n=50
n=32 n=13 n=5
group group Group group group group
1* 2* 3* 1 2 3 1 2 3 1 2 3 (+/-) %р
(-) (+) (++) (-) (+) (++) (-) (+) (++) (-) (+) (++)
CK 5/6 2 4 26 0 4 9 3 1 1 5 9 36 45/5 90%
D2-40 0 5 27 1 5 7 1 3 1 2 13 35 48/2 96%
Calretinin 2 4 26 1 3 9 3 1 1 6 8 36 44/6 88%
Mesothelin 6 5 21 3 4 6 5 0 14 9 27 36/14 72%
WT1 4 8 20 3 3 7 3 1 1 10 12 28 40/10 80%
ЕМА 11 8 13 4 5 4 3 1 1 18 14 18 32/18 64%
CK 7 14 4 14 2 4 7 3 2 0 19 10 21 31/19 62%
CK 20 28 3 1 13 0 0 5 0 0 46 3 1 4/46 8%
CEA 32 0 0 11 2 0 5 0 0 48 2 0 2/48 4%
TTF1 28 1 0 15 0 0 5 0 0 49 4 0 1/49 2%
1*— negative; 2 *— weak positive; 3* - strong positive
Strong intensity is considered the reaction colour of the positive control. The percentage of positively stained tumor cells was determined as follows: 0 - 10%=0; 10 - 39%=1; 40 -69%=2; 70 - 100%=3. The sum of the numerical values of the two indexes ranged from 0 to a maximum value of 6. This allowed us to appraise in more detail the weak positive and focal staining reactions, which expressed the presence of the respective tissue antigen. Reading the respective expression in the nucleus, cytoplasm or cell membrane took into consideration
With the different histological types of MPM, the percentage distribution of the positive results for the used antibodies was highest for D2-40 (96%), CK 5/6 (90%) and Calretinin (88%). The next in order were WT1 (80%), Mesothelin (72%), reaching the lowest level of expression with TTF1 - 2%.
Table 3
Percentage of positive expression (%p), sensitivity and specificity for mesotheliomas and metastatic lung adenocarcinomas
Table 3 shows the summarized results, obtained from the expression of the same antibodies, which only accounted for the presence or absence of expression, and as positive also accepted all the cases with intensity of colour higher than 10%.
Antibody Epithelioid and biphasic mesothelioma n=45 Metastatic tumors Sensitivity and specificity
Lung adenocarcinoma n=41 Sensitivity Specificity
+/- %p +/- %p % %
Antibodies positive for mesothelioma
CK 5/6 43/2 95.55 9/32 21.95 95.55 78.04
D2-40 44/1 97.77 2/39 4.87 97.77 95.12
Calretinin 42/3 93.33 4/37 9.75 93.33 90.24
Mesothelin 36/9 80 4/37 9.75 80 90.24
WT1 38/7 84.44 8/33 19.51 84.44 80.48
ЕМА 30/15 66.66 32/9 78.04 66.66 21.95
Antibodies positive for adenocarcinomas
CK 7 25/20 55.55 35/6 85.36 85.36 44.44
СК 20 4/41 8.88 6/35 14.63 14.63 91.11
CEA 2/43 4.44 33/7 24.39 80.48 95.55
TTF1 1/44 6.66 40/1 97.56 97.56 97.77
Note. Due to their specific histological picture, the sacromatoid mesotheliomas were taken out of the study in order to distinguish MPM from the metastatic lung adenocarci-nomas.
To appraise the sensitivity and specificity of the markers used in the differential diagnosis of MPM and pleural metastases from lung adenocarcinomas, the standard 2x2 table was used. The markers used were divided into two groups -positive for mesothelioma and positive for metastatic adeno-carcinomas.
With mesotheliomas, from the positive markers the highest sensitivity and specificity were displayed by D2-40 and Calretinin, followed by Mesothelin and WT1. CK 5/6 had a very high sensitivity, but lower specificity, whereas Meso-thelin had low sensitivity and high specificity, which makes it a suitable ancillary marker in confirming the mesothelial origin of the tumor.
With the metastatic lung adenocarcinomas TTF1 had very high sensitivity and specificity, followed CEA. CK7 had good sensitivity but low specificity. Figure 2.
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- : V
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D
CVn^Vii• '¡S* ■
._-
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Fig. 2 IHC study of epithelioid mesothelioma A - НЕ x100.
Positive markers; B - D2-40. x100; C - CK 5/6 x100; D -mesothelin x100; E - calretinin x200; F - WT1 x100;
Negative markers: H - TTF1. x100; G - CEA x100
Discussion:
With its wide differential diagnosis, difficult therapy and unfavourable prognosis, the accurate diagnosis of the malignant mesothelioma nowadays is crucial for the treatment of the patient. [3, p. 1-11]
The histological picture of the malignant mesothelioma and the metastatic tumors in most part are very similar and are often indistinguishable in routine colouring with hematoxylin and eosin. Is particularly refers to metastases of lung adeno-carcinomas. The only way to a correct diagnosis of pleural tumor remains the immunohistochemical study of biopsy material. There are great difficulties in defining a unique reproductive immunophonotype of MPM, which we can use in differential diagnosis with metastatic pleural tumors. [9, p. 1316]
The immunohistochemical study (ICH) has existed for more than 25 years. In the diagnostic practice for that time were offered various antibodies and combinations of them, but still the "gold standard" for this study has not been found yet. Today it plays an important part in diagnosing MPM. Without it the differential diagnosis of this kind of tumor is most of the times impossible. The literature has described dozens of bigger and smaller antibody panels, but none of them has proven entirely accurate and efficient for a correct morphological diagnosis. That's why most of the authors today suggest big panels of 10-12 antibodies, which makes the immunohistochemical study significantly more expensive. [11 pp. 1-19]
There are many reasons for these, sometimes significant differences in the obtained results from the immunohisto-chemical study, but the main one is the phenotypical flexibility of the mesothelium, typical for the embryonic differentiation of the mesoderm. Second are the objectively limited possibilities of the immunohistochemical method itself. Today there
are dozens of companies offering different antibodies with different sensitivity and specificity. Despite the existing automated systems for IHC study, the records for processing the material are still too cumbersome and bulky. We shouldn't also forget the very important subjective factor in assessing the results from this study. [12, p. 253]
The lack of absolutely specific and sensitive immunohistochemical markers for mesothelioma makes the differential diagnosis of this tumor very difficult. It depends entirely on using the immunohistochemical panel comprised of markers positive for mesothelioma (i.e. markers which are most often expressed in mesothelioma, but not in carcinomas) and epithelial markers negative for mesothelioma (i.e. markers which are most often expressed in carcinomas, but not in mesotheli-omas). The recommended panels constantly change as a result of identifying new antibodies, which can be used in the differential diagnosis of these tumors and of the new information about the diagnostic value of the individual markers [2, p. 397], [12, p. 253]
The obtained results showed that highest sensitivity and specificity as positive markers for MPM were manifested by D2-40 and Calretinin, followed by Mesothelin and WT1. СК 5/6 had a very high sensitivity, but lower specificity, whereas Mesothelin had low sensitivity and high specificity, which makes it a suitable ancillary marker in confirming the meso-thelial origin of the tumor.
ЕМА and СК7 had low specificity, which showed that they are expressed with very close results, in both МРМ and the metastatic lung adenocarcinomas. That's why they are not suitable for differential-diagnostic search in primary and met-astatic pleural tumors. These results are close to those reported by Husain AN et al. 2013 and Ordonez NG 2013, but different
from the data of King JE et al. in 2006, which tells of an evolution in specifying the antibody panel to determine the im-munophenotype of MPM. [4, p. 647], [11, pp. 1-19], [5, p. 223] In the differential diagnosis we considered: 1. Epithelioid and biphasic mesothelioma and lung adenocarcinoma
The number of antibodies used for this purpose today is too large. [6, p. 190] Considering the sensitivity and specificity of the antibodies that we investigated in our study, the recommended positive markers for epithelioid mesothelioma with the highest sensitivity and specificity were D2-40 and Calretinin. For the metastases of lung adenocarcinomas the best sensitivity / specificity ratio was for TTF-1 and СЕА. Conclusion
IHC testing is essential for the diagnosis and differential diagnosis of MPM with metastatic pleural adenocarcino-mas. It must necessarily include a panel of positive and negative (carcinoma-related) markers.
The suitable optimal combination to distinguish the malignant mesothelioma from metastatic lung adenocarcino-mas should be a panel of antibodies with the highest possible sensitivity and specificity. This gives us reason to recommend an economically optimized immunohistochemical panel of two antibodies positive for mesothelioma: D2-40 and Calretinin^ and two negative, but positive for lung adenocarcinomas: TTF1 and СЕА.
References
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2. Alberto MM. Application of Immunohistochemistry to the Diagnosis of Malignant Mesothelioma. Arch Pathol Lab Med 2008;132:397-401
3. Galateau-Salle F. Pathology of Malignant Mesothelioma Springer-Verlag London Limited 2006:1-11
4. Husain AN., ThomasVC., Nelson GO., et al. Guidelines for Pathologic Diagnosis of Malignant Mesothelioma 2012 Update of the Consensus Statement from the International Mesothelioma Interest Group. Arch Pathol Lab Med 2013;137:647-667
5. King JE, Thatcher N, Pickering CA at al. Sensitivity and specificity of immunohistochemical markers used in the diagnosis of epithelioid mesothelioma: a detailed systematic analysis using published data. Histopathol-ogy 2006;48: 223-232
6. Kushitani K, Takeshima Y, Amatya VJ, Furonaka O, Sakatani A, Inai K. Immunohistochemical marker panels for distinguishing between epithelioid mesotheli-oma and lung adenocarcinoma. Pathol Int. 2007;57:190-199
7. Loggie BW. Malignant peritoneal mesothelioma. CurrTreat Options Oncol. 2001;2:395-399.
8. Moore AJ., Robert JP., John W. Malignant mesothelioma. Orphanet Journal of Rare Diseases 2008;(3)34:1-11
9. Mani H. Dani SZ. Immunohistochemistry. Applications to the Evaluation of Lung and Pleural Neoplasms: Part 1. Chest 2012; 142:(5):1316-1323
10. 10 Ordonez NG., Immunohistochemical Diagnosis of Epithelioid Mesothelioma An Update. Arch Pathol Lab Med. 2013;129:1407-1414
11. Ordonez NG. Application of immunohistochemistry in the diagnosis of epithelioid mesothelioma: a review and update. Human Pdthology 2013;44(1):1-19
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ПРИМЕНЕНИЕ ВЫСОКОЙ СПИНАЛЬНОЙ АНЕСТЕЗИИ В АБДОМИНАЛЬНОЙ
ХИРУРГИИ
Плеханов Александр Николаевич
докт. мед. наук, профессор, Бурятский государственный университет, заведующий кафедрой факультетской
хирургии, г. Улан-Удэ Якушевский Андрей Борисович
Бурятский государственный университет, кафедра факультетской хирургии, аспирант, г. Улан-Удэ
Введение. Ещё в прошлом веке Б.А. Петров дал такую высокую оценку спинальной анестезии: «Нет ни одного вида местного или проводникового обезболивания, который давал бы столь полную анестезию, как спинномозговое обезболивание. Нет ни одного способа более простого по технике, который в такой степени обеспечивал хирургу наилучшие условия производства самых сложных операций в брюшной полости» (1954) [1, с.34]. К положительным сторонам спинальной анестезии относят: вялый паралич мыщц - миоплегия и миорелаксация. А использование современных местных анестетиков, применение одноразовых малого диаметра игл (соответственно малотравматичных - значительное уменьшение травмати-зации паравертебральных тканей), простой интраопераци-онный мониторинг позволяют применять спинальную анестезию при различных заболеваниях органов брюшной полости, требующий операционного вмешательства: как в плановом, так и в экстренном порядке [3, с.145; 4, с.62].
Но необходимо помнить, что у спинальной анестезии имеются и отрицательные моменты - снижение общего периферического сопротивления сосудов с последующим падением артериального давления и перераспределением внутрисосудистого объёма жидкости с резким снижением тонуса ёмкостных и резистивных сосудов [2, с.132]. А при высокой спинальной анестезии может развиться и парез дыхательной мускулатуры. Поэтому актуальным является поиск новых методов регионального обезболивания при операциях на верхнем этаже брюшной полости, обладающих высокой эффективностью и не приносящих побочных и нежелательных эффектов для больного.
Целью работы разработка новой методики высокой спинальной анестезии с оценкой её эффективности и безопасности при выполнении операций на верхнем этаже органов брюшной полости.
