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Complete absence of M2-pyruvate kinase expression in benign pancreatic ductal epithelium and pancreaticobiliary and duodenal neoplasia.

Aloysius MM, Zaitoun AM, Bates TE, Albasri A, Ilyas M, Rowlands BJ, Lobo DN - BMC Cancer (2009)

Bottom Line: Benign pancreatic ductal epithelium, all primary pancreaticobiliary and duodenal premalignant lesions and cancers (and lymph node metastasis) showed complete lack of expression (IHS = 0).Complete lack of M2-PK expression was observed in benign pancreatic ducts, premalignant lesions and cancer.M2-PK is present only in benign non-ductal epithelium in normal pancreas and peri-tumoural tissue.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gastrointestinal Surgery, Nottingham Digestive Diseases Centre NIHR Biomedical Research Unit, Nottingham University Hospitals, Queen's Medical Centre, Nottingham NG7 2UH, UK. mark.aloysius@nottingham.ac.uk

ABSTRACT

Background: Elevated serum concentrations of M2-pyruvate kinase (M2-PK) correlate with poor prognosis in patients with pancreaticobiliary and duodenal cancer, but the expression of M2-PK in formalin-fixed pancreatic tissue is unknown. We aimed to characterise the immunohistochemical expression of M2-PK in archived specimens of pancreaticobiliary and duodenal cancers, premalignant lesions, chronic pancreatitis, and normal pancreas.

Methods: Immunohistochemical staining was performed with mouse anti-M2-PK monoclonal antibody (clone DF-4) at an optimal dilution of 1:25 on tissue microarrays constructed from formalin-fixed paraffin-embedded pancreatic tissue of 126 consecutive patients undergoing pancreatic resections between June 2001 and June 2006. 104 underwent resection for cancer and 22 for chronic pancreatitis. 78 specimens of chronic pancreatitis tissue were obtained adjacent to areas of cancer. Normal pancreatic tissue was obtained from the resection specimens in a total of 30 patients. Metastatic tumours in 61 regional lymph nodes from 61 patients were also studied. A further 11 premalignant pancreaticobiliary and duodenal lesions were studied. M2-PK expression was quantified with the immunohistochemical score (IHS; Range 0-12).

Results: Benign non-ductal tissue in chronic pancreatitis and normal pancreas showed variable expression of M2-PK (IHS = 1 in 25%, IHS = 2-3 in 40%, IHS>3 in 40%). Benign pancreatic ductal epithelium, all primary pancreaticobiliary and duodenal premalignant lesions and cancers (and lymph node metastasis) showed complete lack of expression (IHS = 0).

Conclusion: Complete lack of M2-PK expression was observed in benign pancreatic ducts, premalignant lesions and cancer. M2-PK is present only in benign non-ductal epithelium in normal pancreas and peri-tumoural tissue.

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Strong expression of M2-pyruvate kinase in a positive control (lung cancer) and benign pancreatic non-ductal epithelium: Immunohistochemical staining for M2-pyruvate kinase on formalin fixed lung adenocarcinoma (× 20), demonstrating a strong uptake of the stain. This was used as a positive control (top panel); Immunohistochemical staining for M2-pyruvate kinase on chronic pancreatitis (× 20), demonstrating moderate staining of benign non-ductal pancreatic epithelium (middle panel); Immunohistochemical staining for M2-pyruvate kinase on normal non-ductal pancreatic epithelium (× 20), demonstrating a strong uptake of stain (bottom panel).
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Figure 1: Strong expression of M2-pyruvate kinase in a positive control (lung cancer) and benign pancreatic non-ductal epithelium: Immunohistochemical staining for M2-pyruvate kinase on formalin fixed lung adenocarcinoma (× 20), demonstrating a strong uptake of the stain. This was used as a positive control (top panel); Immunohistochemical staining for M2-pyruvate kinase on chronic pancreatitis (× 20), demonstrating moderate staining of benign non-ductal pancreatic epithelium (middle panel); Immunohistochemical staining for M2-pyruvate kinase on normal non-ductal pancreatic epithelium (× 20), demonstrating a strong uptake of stain (bottom panel).

Mentions: Slides were deparaffinised in xylene, then hydrated via graded dilutions of alcohol followed by running tap water. Following a rinse in deionized water, the sections were incubated with Proteinase-K (S3020; Dako UK Ltd., Ely, UK) for 15 min at room temperature to unmask antigenicity, and subsequently a Labelled Streptavidin Biotin (LSAB) immunoperoxidase procedure was performed using 3,3'-diaminobenzidine (DAB) as the chromogen on a TechMate 500+ automated stainer (Dako). The staining protocol consisted of incubating the sections with 0.3% hydrogen peroxide for 10 min to block endogenous peroxidases, washing in phosphate buffered saline (PBS) buffer, followed by incubation in normal goat serum for 20 minutes. The slides were washed then incubated in mouse anti-M2-PK monoclonal antibody, (clone DF-4) from ScheBo® Biotech (Giessen, Germany) at an optimal dilution of 1:25 (optimisation was achieved using a range of dilutions from 1:10 to 1:50) in a standard antibody diluent (S2022; Dako, UK) for 1 hr at room temperature, followed by further washing in Phosphate Buffer Saline (PBS). Sections were then incubated with a biotin linked secondary antibody (30 min) followed by washing in PBS and streptavidin-horseradish peroxidase (30 min) using reagents in a kit from Dako (K5001). Immunostaining was visualized using DAB for 10 minutes, also present in this kit followed by light counterstaining with haematoxylin. Two types of negative control experiments were performed on the same TMAs omitting the incubation with the primary (anti-M2-PK) antibody and the Proteinase K antigen extraction steps. The TMAs did not stain positively in either negative control experiment, confirming the positive staining observed using this protocol was that of M2-PK. Adenocarcinoma of the lung was used as a positive control (Figure 1, top panel) and pancreatic islets as negative controls. Whole tissue sections of pancreatic tumours (n = 5) were also stained by this protocol.


Complete absence of M2-pyruvate kinase expression in benign pancreatic ductal epithelium and pancreaticobiliary and duodenal neoplasia.

Aloysius MM, Zaitoun AM, Bates TE, Albasri A, Ilyas M, Rowlands BJ, Lobo DN - BMC Cancer (2009)

Strong expression of M2-pyruvate kinase in a positive control (lung cancer) and benign pancreatic non-ductal epithelium: Immunohistochemical staining for M2-pyruvate kinase on formalin fixed lung adenocarcinoma (× 20), demonstrating a strong uptake of the stain. This was used as a positive control (top panel); Immunohistochemical staining for M2-pyruvate kinase on chronic pancreatitis (× 20), demonstrating moderate staining of benign non-ductal pancreatic epithelium (middle panel); Immunohistochemical staining for M2-pyruvate kinase on normal non-ductal pancreatic epithelium (× 20), demonstrating a strong uptake of stain (bottom panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749871&req=5

Figure 1: Strong expression of M2-pyruvate kinase in a positive control (lung cancer) and benign pancreatic non-ductal epithelium: Immunohistochemical staining for M2-pyruvate kinase on formalin fixed lung adenocarcinoma (× 20), demonstrating a strong uptake of the stain. This was used as a positive control (top panel); Immunohistochemical staining for M2-pyruvate kinase on chronic pancreatitis (× 20), demonstrating moderate staining of benign non-ductal pancreatic epithelium (middle panel); Immunohistochemical staining for M2-pyruvate kinase on normal non-ductal pancreatic epithelium (× 20), demonstrating a strong uptake of stain (bottom panel).
Mentions: Slides were deparaffinised in xylene, then hydrated via graded dilutions of alcohol followed by running tap water. Following a rinse in deionized water, the sections were incubated with Proteinase-K (S3020; Dako UK Ltd., Ely, UK) for 15 min at room temperature to unmask antigenicity, and subsequently a Labelled Streptavidin Biotin (LSAB) immunoperoxidase procedure was performed using 3,3'-diaminobenzidine (DAB) as the chromogen on a TechMate 500+ automated stainer (Dako). The staining protocol consisted of incubating the sections with 0.3% hydrogen peroxide for 10 min to block endogenous peroxidases, washing in phosphate buffered saline (PBS) buffer, followed by incubation in normal goat serum for 20 minutes. The slides were washed then incubated in mouse anti-M2-PK monoclonal antibody, (clone DF-4) from ScheBo® Biotech (Giessen, Germany) at an optimal dilution of 1:25 (optimisation was achieved using a range of dilutions from 1:10 to 1:50) in a standard antibody diluent (S2022; Dako, UK) for 1 hr at room temperature, followed by further washing in Phosphate Buffer Saline (PBS). Sections were then incubated with a biotin linked secondary antibody (30 min) followed by washing in PBS and streptavidin-horseradish peroxidase (30 min) using reagents in a kit from Dako (K5001). Immunostaining was visualized using DAB for 10 minutes, also present in this kit followed by light counterstaining with haematoxylin. Two types of negative control experiments were performed on the same TMAs omitting the incubation with the primary (anti-M2-PK) antibody and the Proteinase K antigen extraction steps. The TMAs did not stain positively in either negative control experiment, confirming the positive staining observed using this protocol was that of M2-PK. Adenocarcinoma of the lung was used as a positive control (Figure 1, top panel) and pancreatic islets as negative controls. Whole tissue sections of pancreatic tumours (n = 5) were also stained by this protocol.

Bottom Line: Benign pancreatic ductal epithelium, all primary pancreaticobiliary and duodenal premalignant lesions and cancers (and lymph node metastasis) showed complete lack of expression (IHS = 0).Complete lack of M2-PK expression was observed in benign pancreatic ducts, premalignant lesions and cancer.M2-PK is present only in benign non-ductal epithelium in normal pancreas and peri-tumoural tissue.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gastrointestinal Surgery, Nottingham Digestive Diseases Centre NIHR Biomedical Research Unit, Nottingham University Hospitals, Queen's Medical Centre, Nottingham NG7 2UH, UK. mark.aloysius@nottingham.ac.uk

ABSTRACT

Background: Elevated serum concentrations of M2-pyruvate kinase (M2-PK) correlate with poor prognosis in patients with pancreaticobiliary and duodenal cancer, but the expression of M2-PK in formalin-fixed pancreatic tissue is unknown. We aimed to characterise the immunohistochemical expression of M2-PK in archived specimens of pancreaticobiliary and duodenal cancers, premalignant lesions, chronic pancreatitis, and normal pancreas.

Methods: Immunohistochemical staining was performed with mouse anti-M2-PK monoclonal antibody (clone DF-4) at an optimal dilution of 1:25 on tissue microarrays constructed from formalin-fixed paraffin-embedded pancreatic tissue of 126 consecutive patients undergoing pancreatic resections between June 2001 and June 2006. 104 underwent resection for cancer and 22 for chronic pancreatitis. 78 specimens of chronic pancreatitis tissue were obtained adjacent to areas of cancer. Normal pancreatic tissue was obtained from the resection specimens in a total of 30 patients. Metastatic tumours in 61 regional lymph nodes from 61 patients were also studied. A further 11 premalignant pancreaticobiliary and duodenal lesions were studied. M2-PK expression was quantified with the immunohistochemical score (IHS; Range 0-12).

Results: Benign non-ductal tissue in chronic pancreatitis and normal pancreas showed variable expression of M2-PK (IHS = 1 in 25%, IHS = 2-3 in 40%, IHS>3 in 40%). Benign pancreatic ductal epithelium, all primary pancreaticobiliary and duodenal premalignant lesions and cancers (and lymph node metastasis) showed complete lack of expression (IHS = 0).

Conclusion: Complete lack of M2-PK expression was observed in benign pancreatic ducts, premalignant lesions and cancer. M2-PK is present only in benign non-ductal epithelium in normal pancreas and peri-tumoural tissue.

Show MeSH
Related in: MedlinePlus