1 Centre of Gastrointestinal Disease, Ersta Hospital, Stockholm; 2 Department of Oncology and Pathology, Karolinska University Hospital, SE 171 76 Stockholm; 3 Department of Oncology, Radiology and Clinical Immunology, Akademiska Sjukhuset, SE 751 85 Uppsala, Sweden
*Correspondence to: Dr P. J. Nilsson, Centre of Gastrointestinal Disease, Ersta Hospital, PO Box 4622, SE 116 91, Stockholm, Sweden. Tel: +46-8-714-61-00; Fax: +46-8-714-66-80; Email: per.nilsson{at}ersta.se
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Abstract |
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Patients and methods:: From a population-based consecutive series of patients who were prospectively recorded, it was possible to investigate 209 (76%) of the pretreatment biopsies. Immunohistochemistry with a monoclonal antibody for the 2 chain of laminin-5 was used to detect tumour budding (defined as dissociated single cancer cells or clusters of up to five cells).
Results:: Tumour budding was detected in 104 (50%) of the 209 samples. No significant correlation was found between tumour budding and clinicopathological characteristics. Patients with tumour budding had a statistically significantly better 5-year overall survival rate compared with patients lacking tumour budding (74% versus 64%, P <0.05). Albeit not statistically significant, other outcome variables such as tumour-specific survival, recurrence after initial complete response and rate of distant metastases, were all in favour of patients with tumour budding. Multivariate analysis reveals tumour budding as an independent positive prognostic factor.
Conclusions:: Tumour budding detected by laminin-5 immunohistochemistry may be of prognostic value in the treatment of epidermoid anal cancer. However, further studies are needed to clarify the possible clinical implications.
Key words: anus neoplasm, radiotherapy, prognosis, tumour budding, laminin
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Introduction |
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The presence of single cancer cells or clusters of up to five cancer cells just ahead of the invasive front of an invasive carcinoma has been described as tumour budding [4]. In colorectal adenocarcinomas, tumour budding is associated with poor prognosis [5
7
]. Studies of the prognostic importance of tumour budding in squamous cell carcinomas in general, and in epidermoid anal cancer in particular, are infrequent.
Tumour budding is possible to detect in routine staining [6]. However, there are several reports describing immunostaining of laminin-5 (Ln-5) in tumour buds [8
11
], which greatly facilitates the detection of budding cancer cells. The presence of Ln-5 immunostaining in tumour buds has also been shown in squamous cell carcinoma [12
14
]. In non-neoplastic tissue, laminins are a group of extracellular matrix proteins mainly localised in the basement membrane of various epithelial tissues. The isoform laminin-5, and in particular the
2 chain, is preferentially expressed in the cytoplasm of cancer cells along the advancing edge of a tumour and has been associated with cancer growth and invasion [8
, 9
, 12
]. Ln-5
2 chain has been associated with the formation of tumour buds [15
] and has also been proposed as an aid in identifying micrometastasis in tissues surrounding carcinoma [11
].
The aims of this present study were to evaluate the prognostic and predictive potential of tumour budding detected by means of Ln-5 2-chain immunohistochemistry in a large, population-based, consecutive series of epidermoid anal carcinomas.
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Materials and methods |
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Immunohistochemistry
The monoclonal antibody 6C12 (mAb 6C12) was produced by hybridoma techniques. In brief, GST-laminin 2-III fusion protein containing amino acid residues 391567 of human laminin
2 chain was used as an antigen. Balb/c mice were immunised four times after which mouse spleen cells were fused with mouse myeloma cells (P3X63Ag8.653). The production of the anti-laminin
2 antibody by the hybridoma clones was screened by the aid of immunohistology, ELISA and western blotting. The best clone was picked up and cloned again (single-cell cloning) to ensure that the produced hybridoma cell line was monoclonal. For immunohistochemistry, IgG was purified from the hybridoma cell culture media using Protein G Sepharose column (Amersham Pharmacia Biotec). The antibody specificity was checked using purified full-length human
1 and
2 chains that were electrophoresed by SDS-PAGE. The electrophoresed proteins were stained with Coomassie blue to confirm the presence of
1- and
2-chain proteins. The protein preparations were transferred to nylon membranes and immunoreacted with the purified mAb 6C12 generated against the recombinant GST-laminin
2-III fusion protein and specific reaction only with the
2 chain could be observed.
Representative 4 µm paraffin wax-embedded sections were cut and subjected to the standard horseradish peroxidase avidin-biotin complex technique (VECTASTAIN® Elite ABC kit, cat PK-6100; Vector Laboratories Inc., Burlingame, CA). The sections were first deparaffinised in xylene, rehydrated in an ethanol series and rinsed in water. The sections were treated enzymatically with Pronase 0.05% (Sigma P5147) for 15 min at 2022 °C and rinsed. Following the addition of 0.5% H2O2 to the slides, they were incubated at room temperature for 20 min and then rinsed in water. To prevent unspecific staining, 40 µl 1% bovine serum albumin (BSA) in 1x phosphate buffered saline (PBS) was added to each slide, which were then incubated for 30 min at room temperature in a humid chamber. The mAb 6C12 (2.7 mg/ml) was diluted 1:100 in 1% BSA/1 x PBS, and 40 µl was added to each slide. After incubation overnight at 4 °C, biotinylated anti-mouse IgG (Vector), diluted 1:200, was added to the sections for 30 min, followed by incubation in the avidinbiotinperoxidase complex for 30 min. Tris buffered saline (pH 7.6) was used for washing in between steps. Diaminobenzidine tetrahydrochloride was used to develop the peroxidase reaction. The slides were counterstained in Mayer's hematoxylin for 46 min, dehydrated, cleaned in xylene and mounted in a xylene-soluble mounting medium.
Evaluation
All slides were evaluated by a senior pathologist (CR) who was blinded to the clinical data and therapeutic outcome. Tumour budding was defined as dissociated single cancer cells or clusters of up to five cancer cells with cytoplasmic Ln-5 immunostaining, ahead of the invasive front. Fifty randomly selected slides were re-evaluated by another investigator (PJN) with a 96% concurrence regarding the presence of tumour budding.
Statistical methods
To assess the correlation between proportions, the chi-square test and the MannWhitney U-test were used where appropriate. Survival curves were plotted using the method of Kaplan and Meier. Comparison between survivals in the different groups was by the log rank test. Cox proportional hazards model was used to perform uni- and multivariate analyses on parameters related to survival. T-stage, N-stage, histology and tumour budding were included in the Cox analyses. P values <0.05 were considered statistically significant.
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Results |
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Discussion |
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In contrast to these results, tumour budding is generally regarded as an indicator of poor prognosis [57
]. However, previous investigations on budding have, for the most part, concerned adenocarcinomas and not squamous cell cancers. The treatment of colorectal adenocarcinomas is generally surgical, whereas our cohort of patients with epidermoid anal cancers has been treated mainly with radiotherapy. This difference in therapeutic approach may have an influence on the prognostic impact of tumour budding.
Tumour budding is a representation of the dissociation of the invasive front of a tumour and is related to the invasive and metastatic capability of a tumour [5]. Epidermoid anal carcinomas generally have a low capability of distant metastasis, although locally aggressive and with frequent nodal spread [17
]. In the investigated cohort of patients, 50% showed tumour budding but the rate of distant metastasis was only 7%. In addition, no correlation between lymph node engagement and presence of tumour budding was found in the present study. It is conceivable that tumour budding also represents other intrinsic tumour characteristics that are related to long-term outcome, i.e. survival, but not the immediate response to therapy.
Earlier reports on Ln-5 expression in various squamous cell carcinomas have generally described Ln-5 positivity as an indicator of poor prognosis [13, 14
, 18
]. However, in those studies, immunoreactivity for the
2 chain of Ln-5 per se, and not the presence or absence of tumour budding was evaluated. The
2 chain of Ln-5 has been associated with the formation of tumour buds [15
]. Giannelli et al. [11
] suggested that Ln-5 can be used to detect micrometastasis in peritumoural tissue and in the present study, immunohistochemistry was used for the detection of tumour budding. Thus, the results from previous investigations on Ln-5 in different squamous cell carcinomas are not directly comparable to those obtained in the present study. However, the full meaning of Ln-5 expression in various tumour types remains to be elucidated.
In the treatment of epidermoid anal cancer the aims are cure and, whenever possible, sphincter preservation. When non-surgical therapy fails and sphincter preservation is impossible, an abdominoperineal resection is generally undertaken. In patients where this is the case, the previous radiation therapy renders patients susceptible to post-operative complications. Predictors of radiation therapy response could facilitate planning of a therapeutic approach [19] and curative radiotherapy at a high dose, which has a risk of increasing surgical complications, could be avoided if the chances of tumour control are low. When exploring the possibilities of evaluating the predictive potential of tumour budding, either in the entire material or in a subset of patients with locally advanced tumours, no correlation could be obtained. Bearing in mind the relatively small number of patients investigated with respect to this, the present study does not suggest a predictive potential of tumour budding regarding tumour response to (chemo)radiotherapy.
Previously, different prognostic markers in epidermoid anal cancer have been investigated. For example, Holm et al. [20] found shorter overall survival for p21-negative tumours in a series of 94 epidermoid anal cancer patients and Wong et al. [21
] reported an independent prognostic value for p53 in 49 patients treated with radiotherapy, 5-fluorouracil and mitomycin C. However, in a recent review of earlier studies on prognostic factors in epidermoid anal cancer, it was concluded that results so far were unable to provide reliable guidance in treatment decisions [22
]. In the present study, the possible role of tumour budding as a prognostic factor has been investigated in a comparatively large number of patients with epidermoid anal cancer and it can be concluded that tumour budding detected by Ln-5 immunohistochemistry can be of prognostic value. However, further studies are needed to clarify the possible clinical implications.
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Acknowledgements |
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This work was supported by the Public Health and Medical Service Committee of Stockholm County Council, the Swedish Cancer Society and the Cancer Society in Stockholm. The authors are grateful to Inga Maurin and Ulla Aspenblad for excellent technical assistance.
Received for publication September 28, 2004. Accepted for publication January 7, 2005.
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