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Expression of class III [beta]-tubulin in colorectal carcinomas: an immunohistochemical study using TU-20 & TuJ-1 antibody.

Background & objectives: Expression of class III [beta]-tubulin represents newly discovered marker of resistance to taxol-based chemotherapy in a wide spectra of carcinomas. However, very little is known about its expression in colorectal carcinomas. This study was done to determine class III [beta]-tubulin expression in a large series of colonic carcinomas, covering tumours with different degree of differentiation in order to evaluate its prospective significance in resistance to taxol-based chemotherapeutics and to compare the immunostaining profile of two widely used monoclonal antibodies, TU-20 and TuJ-1.

Methods: Sixty patients with colorectal carcinoma were enrolled; all of them were treated surgically by the resection. Twenty tumours were histologically assessed as G1, 20 as G2 and 20 as G3. Routine immunohistochemical procedure using TU-20 and TuJ-1 mouse monoclonal antibodies was applied to all 60 specimen and slides were evaluated using an optical microscope.

Results: Expression of class III [beta]-tubulin was detected in 14 tumours (23.3%), while remaining tumours were negative. Relatively higher frequency of class III [beta]-tubulin expression was observed in G3 tumours (10 cases) in comparison with G1 (3 cases) and G2 (1 case), respectively. Seven tumours displayed positive immunostaining with both tested antibodies TU-20 and TuJ-1. Six tumours showed expression of class III [beta]tubulin in more than 1 per cent of neoplastic cell population. In remaining 8 tumours only individual scattered neoplastic cells exhibited class III [beta]-tubulin expression either with TU-20, or with TuJ-1 antibody.

Interpretation & conclusion: Higher frequency of immunoreactivity was observed in poorly differentiated tumours. However, more than 90 per cent of neoplastic cell population did not express class III [beta]-tubulin in almost all tumours. These negative cells of colonic cancer could represent the potential target for taxane-based chemotherapy in the future. Our results indicate that TU-20 and TuJ-1 antibodies exhibit very similar immunoreactivity in neoplastic tissue.

Key words Class III [beta]-tubulin--colorectal cancer

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Tubulin superfamily is a large group of molecules that play a complex role during lifespan of the cell, mainly in the formation of cytoskeleton (1). One of these molecules, class III [beta]-tubulin, is a 50 kDa protein (2), encoded by gene located at the long arm of chromosome 16 (3). Together with other tubulin superfamily members it participates in formation of microtubules. The expression of class III [beta]-tubulin correlates with the resistance to microtubule-acting chemotherapeutics in some malignant tumours (4-7), but no information about class III [beta]-tubulin expression in carcinomas of colon is available. Class III [beta]-tubulin expression has been originally used as a marker of early phases of neuronal differentiation in human embryonic development (8-10). However, wide spectra of different human neuronal and non-neuronal tumours also exhibit class III [beta]-tubulin expression (3,11). In neuronal tumours, class III [beta]-tubulin is connected to neuritogenesis and low growth potential. In contrary, some authors associate class III [beta]-tubulin expression in non neuronal tumours with histological signs of higher malignancy (3). Relation between class III [beta]-tubulin expression in tumorous cells and lower degree of differentiation in some non neuronal tumours was proposed (3). In our previous pilot study (12) we tried to verify this presumption in different carcinomas, however, the set of tumours was relatively heterogeneous and the reproducibility of these data was thus limited. Based on the results of this pilot work, we decided to study expression of class III [beta]-tubulin in a group of 60 adenocarcinomas of colon, stratified into three subgroups including proportional number of G1, G2 and G3 tumours. Immunostaining using two commercially available monoclonal antibodies that recognize different epitopes of class III [beta]-tubulin molecule, TU-20 and TuJ-1 was performed. Comparative analysis of differences in staining properties of TU-20 and TuJ-1 was also done.

Material & Methods

Patients: Sixty patients with primary carcinomas of the large bowel were enrolled consecutively during 2006 and 2007 in this retrospective study. The diagnosis of carcinoma was performed by histopathological analysis (13) of the material obtained during surgical operation. All patients signed the informed consent. Paraffin-embedded tissue samples were obtained from the files of the Department of Pathology, 3rd Faculty of Medicine, Charles University and Faculty Hospital King's Vineyards, Prague; clinical data were obtained from the files of Department of Oncology, 3rd Faculty of Medicine, Charles University and Faculty Hospital King's Vineyards, Prague, collected in years 1996-2004. Representative slides with satisfactory amount of neoplastic tissue were selected by two pathologists (VM and TJ). There were no other specific inclusion/exclusion criteria for the selection of carcinomas from files in this study. Human cerebellar tissue obtained by autopsy was used as a positive control; samples incubated without primary antibodies were used as a negative control. This study was done in 2006 and 2007 at the Department of Pathology, 3rd Faculty of Medicine, Charles University and Faculty Hospital King's Vineyards in Prague.

Histology: All bioptic specimens were fixed in buffered formalin and embedded in paraffin. Five micrometer thin sections were stained with hematoxylin and eosin. For immunohistochemical purposes, the sections were placed on poly-L-lysine (Sigma Diagnostics, Sant-Louis, Missouri, USA)-coated glass slides.

Antibodies: Two different widely used primary mouse monoclonal antibodies recognizing class III [beta]-tubulin, clones TU-20 and TuJ-1 were kindly gifted by Exbio (Prague, Czech Republic). TU-20 was diluted 1:50; the dilution of TuJ-1 was 1:200.

Immunohistochemistry: Standard immunohistochemical procedure (14) was applied to all specimens: five micron thick representative tissue sections were deparaffinized in xylene and hydrated in graded alcohol. Different antigen retrieval procedures were used to TU-20 and TuJ-1 antibodies. Pre-treatment with 0.1 per cent trypsin solution (DIFCO, Lawrence, USA) in distilled water for 30 min at 37[degrees]C was chosen as optimal for TuJ-1, and boiling in a microwave oven (power 750 W) in the citrate buffer (DakoCytomation, Glostrup, Denmark), pH 6, for 3x5 min was applied as optimal antigen retrieval procedure for TU-20 antibody. Both primary antibodies were applied to slides for 1.2 h at 4[degrees]C. The En-Vision[TM] Kit purchased from DakoCytomation (Glostrup, Denmark) and 3, 3-diaminobenzidine (DAB, Fluka, Buchs, Switzerland) were used to visualize immunohistochemical reactions. The slides were counterstained with Harris's haematoxylin (Bio-Optica, Milano, Italy). Immunostaining results were evaluated semiquantitatively using optical light microscope (Olympus Vanox-S) in the whole tissue sections as follows: 0- no positive staining; 1- up to 1 per cent cells positive; 2- 1-10 per cent cells positive; 3- 10-50 per cent cells positive. In order to compare the immunostaining profile of TU-20 and TuJ-1 antibodies. Chi Square test was applied on the categorical data (Table). Because only one sample had been classified as a member of group 3 (Table, sample No. 50 for TU-20 parameter), the category 3 was not considered during the statistical analysis; therefore, only 3 categories (labelled from 0 to 2) were considered. The exceptional sample (No. 50) was included into the category 2 for the statistical testing.

Results

From 26 women and 34 men age from (41 to 85 yr; mean, 69 [+ or -] 11 yr) tissue samples were obtained by surgical operation. The site of primary tumour was: caecum 14 cases, ascending colon 8 cases, transverse colon 5 cases, lienal flexure 4 cases, descending colon 2 cases, sigmoideum 11 cases and rectum 4 cases. In 12 cases the site of primary tumour was classified as rectosigmoideal. Microscopically, the tumours were classified as tubular adenocarcinomas. Twenty of these tumours were scored grade 1, 20 grade 2 and remaining 20 grade 3.

Expression of class III [beta]-tubulin was detected in 14 tumours (23.3%) using at least one antibody, while 46 tumours (76.7%) were negative. Of these positive tumours, three were scored G1, one G2 and ten G3. All positive neoplastic cells exhibited firmly fibrillary diffuse cytoplasmic staining using both TU-20 and TuJ-1 (Fig.). In cases no. 1 and 16 (both G1 tumours), positive staining was detected only in minor focuses of tumour nests formed by less differentiated neoplastic cells with moderate grade of structural atypia (Fig. E), where the staining was focally pronounced at the periphery of necrotic zone within the tumour mass; the well-differentiated component of both tumours was negative (Fig. F). Ten cases revealed positive immunostaining either with TU-20 or with TuJ-1 in less than 1 per cent tumour cells only. In six cases, up to 10 per cent of neoplastic cells were positive and in one case of G3 tumour (no. 50) up to 50 per cent of tumour cells revealed expression of class III [beta]-tubulin using TU-20 antibody. In seven cases, class III [beta]-tubulin was detected by one antibody only (in six cases by TU-20 and in 1 case by TuJ-1, respectively), while the immunoreactivity with the second antibody was negative. In seven cases both TU-20 and TuJ-1 revealed positive immunostaining within tumour mass, in six of these tumours the expression of class III [beta]-tubulin was detected in more than 1 per cent of neoplastic population (Table). Statistical analysis did not confirm a significant difference between imunostaining profile of Tu20 and TuJ-1 antibodies (P>0.1). In cerebellar tissue which was used as positive control, diffuse cytoplasmic staining within different types of neurons including Purkinje cells (see Fig. E and F) was detected as well as positive staining in intestinal myenteric plexus within investigated samples; negative immunostaining was obtained in all negative controls.

[FIGURE OMITTED]

Discussion

Class III [beta]-tubulin is a well-established marker of early phases of neuronal differentiation in developmental studies (1) detected in a wide spectrum of neuronal tumours (3,15). More recently, expression of class III [beta]-tubulin was observed in different epithelial tumours, denoting potential neuronal differentiation of these tumours (3). So neuroendocrine tumours (NET) of the lung and the gastrointestinal tract (carcinoids) display (16,17) relatively high immunoreactivity for class III [beta]-tubulin in underlining previously known feature of NET to express markers of neuronal differentiation, like neuron specific enolase (NSE) and synaptophysin (18). More surprising is the distinctive populations of "pure" epithelial tumours such as renal cell carcinoma, ovarian carcinoma, non-small cell lung carcinoma or breast carcinoma express class III [beta]-tubulin. Class III [beta]-tubulin expression was in some instances demonstrated to correlate with the resistance of tumour cells to administration of chemotherapeutic drugs, namely taxols (19). Some authors suggested the relation of class III [beta]-tubulin expression to higher degree of differentiation in neuroectodermal tumours (3), which was not confirmed in a study encompassing a wide spectrum of malignant epithelial tumours (12).

Immunostaining profile of TU-20 and TuJ-1 antibodies in colorectal carcinomas was also compared. Both antibodies recognize class III [beta]-tubulin molecule, however, TU-20 is raised against ESESQGPK amino acid sequence which corresponds to C-terminal part of human class III [beta]-tubulin, while TuJ-1 recognize terminal 14 aminoacid sequence of chicken class III [beta]-tubulin molecule (2). Differences in staining profile of both these antibodies were previously described (2). Our data showed that in all cases where higher number (more than 1%) of neoplastic cells exhibited class III [beta]-tubulin expression, this was detected by both TU-20 and TuJ-1.

The incidence of colon cancer has an increasing tendency and mortality is high. Surgery followed by chemotherapy plays an important role in the treatment of colon cancer. Newly applied cytostatics in the colon cancer are effective; however, the therapeutic results remain still unsatisfactory. Taxol belongs to very effective cytostatic agents in the treatment of malignant tumours of different types and locations; however, we have not found any studies dealing with the effect of Taxol on colon cancer. Taxanes cause derangement of microtubule dynamics by stabilizing the microtubule against depolymerization. In fact, these enhance microtubule polymerization, promoting the nucleation and elongation phases of the polymerization reaction and reducing the critical tubulin subunit concentration required for microtubule assembly (20). The role of taxanes in treatment of colorectal carcinomas shall be proven in further clinical studies.

In the present study, we demonstrated that expression of class III [beta]-tubulin was a relatively rare event in colorectal carcinomas. Scattered cells displaying immunoreactivity for this marker may represent limited population of tumour cells revealing signs of differentiation towards neuroendocrine phenotype. This finding correlates with already described presence of focal neuroendocrine differentiation in colorectal carcinomas (21).

In conclusion, our data show that class III [beta]-tubulin expression in colorectal carcinomas seems to be exceptional; however, its expression appears to be increased in less differentiated tumours. Also, class III [beta]-tubulin expression should be taken into account in pertinent taxol chemotherapy in patients with G3 colorectal carcinomas.

Received August 16, 2007

Acknowledgment

Authors thank Dr Karel Roubik from the Faculty of Biomedical Engineering, Czech Technical University, Prague for his assistance with statistical tests. This work was supported by the Project "Oncology" No. MSM 0021620808 from the Ministry of Education, Youth and Sports of the Czech Republic.

References

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(4.) Dumontet C, Isaac S, Souquet P J, Bejui-Thivolet F, Pacheco Y, Peloux N, et al. Expression of class III beta tubulin in non-small cell lung cancer is correlated with resistance to taxane chemotherapy. Bull Cancer 2005; 92 : E25-30.

(5.) Ferlini C, Raspaglio G, Mozzetti S, Cicchillitti L, Filippetti F, Gallo D, et al. The seco-taxane IDN5390 is able to target class III beta-tubulin and to overcome paclitaxel resistance. Cancer Res 2005; 65 : 2397-405.

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(7.) Shalli K, Brown I, Heys SD, Schofield AC. Alterations of beta-tubulin isotypes in breast cancer cells resistant to docetaxel. FASEB J 2005; 19 : 1299-301.

(8.) Easter SS, Jr., Ross LS, Frankfurter A. Initial tract formation in the mouse brain. J Neurosci 1993; 13 : 285-99.

(9.) Katsetos CD, Frankfurter A, Christakos S, Mancall EL, Vlachos IN, Urich H. Differential localization of class III, beta-tubulin isotype and calbindin-D28k defines distinct neuronal types in the developing human cerebellar cortex. J Neuropathol Exp Neurol 1993; 52: 655-66.

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(11.) Katsetos CD, Legido A, Perentes E, Mork SJ. Class III beta-tubulin isotype: a key cytoskeletal protein at the crossroads of developmental neurobiology and tumor neuropathology. J Child Neurol 2003; 18 : 851-66; discussion 67.

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(13.) Hamilton SR, Vogelstein B, Kudo S, Riboli E, Nakamura S, Hainaut P, et al. Tumours of the Colon and Rectum. In: Hamilton SR, Aaltonen LA, editors. World Health Organization classification of tumours pathology and genetics of tumours of the digestive system. Lyon: IARC Press; 2000. p. 104-43.

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Reprint requests: Dr Tomas Jirasek, Department of Pathology, 3rd Faculty of Medicine, Srobarova 50, 10034 Prague, Czech Republic e-mail: tjirasek@fnkv.cz

Tomas Jirasek, Simon Cipro, A. Musilova, M. Kubecova * & V. Mandys

Departments of Pathology & * Radiotherapy & Oncology, 3rd Faculty of Medicine, Charles University Prague, Czech Republic
Table. Immunostaining results of TU-20 and TuJ-1 antibody in group of
colorectal adenocarcinomas

No. G. Age (yr) Sex Loc. TU-20 TuJ-1

l. * 1 82 M AC 1 0
2. I 73 M C 0 0
3. 1 75 M LF 0 0
4. 1 84 M RS 0 0
5. 1 80 F RS 0 0
6. 1 83 M C 2 2
7. 1 54 F RS 0 0
8. 1 78 M C 0 0
9. 1 57 M R 0 0
10. 1 71 F LF 0 0
ll. 1 68 M RS 0 0
12. 1 66 F RS 0 0
13. 1 49 F S 0 0
14. 1 72 M RS 0 0
15. 1 65 F S 0 0
16. * 1 72 M AC 1 2
17. 1 79 M RS 0 0
18. 1 56 M C 0 0
19. 1 60 M S 0 0
20. 1 73 M AC 0 0
21. 2 84 F DC 0 0
22. 2 57 F LF 0 0
23. 2 68 M C 0 0
24. 2 76 F S 0 0
25. 2 65 F C 0 0
26. 2 80 M C 0 0
27. 2 52 M S 2 1
28. 2 72 F C 0 0
29. 2 84 F DC 0 0
30. 2 72 F AC 0 0
31. 2 41 M RS 0 0
32. 2 68 M RS 0 0
33. 2 73 M S 0 0
34. 2 60 F TC 0 0
35. 2 79 M LF 0 0
36. 2 76 F AC 0 0
37. 2 75 M C 0 0
38. 2 72 M TC 0 0
39. 2 85 F TC 0 0
40. 2 79 M AC 0 0
41. 3 70 F S 1 0
42. 3 60 M RS 0 0
43. 3 60 M TC 0 0
44. 3 49 F S 1 0
45. 3 64 M C 1 1
46. 3 75 M S 1 0
47. 3 63 F RS 0 0
48. 3 73 M AC 0 0
49. 3 68 F C 0 0
50. 3 59 M C 3 2
51. 3 83 F C 0 0
52. 3 56 M S 0 0
53. 3 56 M R 0 1
54. 3 72 F TC 1 0
~5. 3 74 F RS 0 0
56. 3 78 F R 0 0
57. 3 67 M AC 0 0
58. 3 61 M S 2 2
59. 3 73 F R 2 2
60. 3 47 F C 1 0

No., number of case; G, tumour grade; Loc., localization of tumour in
large intestine; AC, ascending colon; C, cecum; LF, lienal flexure;
RS, rectosigmoideum; R, rectum; S, sigmoideum; DC, descending colon;
TC, transverse colon; 0, absence of labelling; 1, up to 1 per cent of
tumour cells positive; 2, 1-10 per cent of tumour cells positive; 3,
10-50 per cent of tumour cells positive;
* , cases, where both G 1 and G2 components were present
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Author:Jirasek, Tomas; Cipro, Simon; Musilova, A.; Kubecova, M.; Mandys, V.
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Jan 1, 2009
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