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Kisspeptin and GPR54 immunoreactivity in a cohort of 518 patients defines favourable prognosis and clear cell subtype in ovarian carcinoma.

Prentice LM, Klausen C, Kalloger S, Köbel M, McKinney S, Santos JL, Kenney C, Mehl E, Gilks CB, Leung P, Swenerton K, Huntsman DG, Aparicio SA - BMC Med (2007)

Bottom Line: The latter might suggest that their overexpression would be associated with a better prognosis in cancer.Both kisspeptin positive and GPR54 positive cases are strongly associated with the ovarian carcinoma clear cell subtype (p < 0.0001, p < 0.0001), and GPR54 is significantly associated with favourable prognosis in overall survival within the clear cell subtype (p = 0.0102).Kisspeptin and GPR54 immunoreactivity are significantly associated with favourable prognosis in both disease specific and overall survival, as well as being significantly associated with the clear cell ovarian carcinoma subtype, thereby creating the first independent prognostic biomarkers specific for ovarian clear cell carcinomas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Oncology and Breast Cancer Program, British Columbia Cancer Research Centre and Department of Pathology, University ofBritish Columbia, Vancouver, British Columbia, Canada. Leah.Prentice@vch.ca

ABSTRACT

Background: Kisspeptins and their G-protein coupled receptor, GPR54 are required for GnRH release and have been associated with anti-metastatic tumour cell behaviour in model systems. The latter might suggest that their overexpression would be associated with a better prognosis in cancer. However, kisspeptin/GPR54 interactions (autocrine, paracrine, and/or endocrine) could also impact tumour behaviour in a negative manner. Here, for the first time, we associate the immunoreactivity of the kisspeptin/GPR54 ligand-receptor pair with favourable prognosis in a large cohort of ovarian carcinomas.

Methods: Immunohistochemical analysis for kisspeptin and GPR54 was performed on a tissue microarray (TMA) consisting of 518 early stage ovarian carcinomas, all with linked clinical outcome data. The TMA was scored using a staining intensity scale of 0 (negative), +1 (mild-moderate), and +2 (strong). Strong staining cases were considered either kisspeptin or GPR54 positive and designated as 1, while all other cases were considered negative and designated 0. All statistical analysis was conducted using two-sided tests and a p value equal to or less than 0.05 was considered significant.

Results: Kisspeptin and GPR54 immunoreactive cases show a favourable prognosis in univariable disease specific survival (p = 0.0023, p = 0.0092), as well as in overall survival (p = 0.0006, p = 0.0002). Furthermore, kisspeptin is an independent marker for favourable prognosis as determined by multivariable disease specific (p = 0.0046) and overall survival analysis (p = 0.0170), while GPR54 is an independent marker for overall survival only (p = 0.0303). Both kisspeptin positive and GPR54 positive cases are strongly associated with the ovarian carcinoma clear cell subtype (p < 0.0001, p < 0.0001), and GPR54 is significantly associated with favourable prognosis in overall survival within the clear cell subtype (p = 0.0102).

Conclusion: Kisspeptin and GPR54 immunoreactivity are significantly associated with favourable prognosis in both disease specific and overall survival, as well as being significantly associated with the clear cell ovarian carcinoma subtype, thereby creating the first independent prognostic biomarkers specific for ovarian clear cell carcinomas.

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IHC controls. Less than 10-week-old human placenta used as a positive control. (A) Kisspeptin-IR shows intense cell-type specific staining in the syncytiotrophoblasts (black arrow), while the cytotrophoblast layers remain unaffected (black arrowhead). (B) GPR54-IR shows intense staining in the villous cytotrophoblasts (black arrow), the extravillous cytotrophoblasts (black arrowhead), and moderate staining on the syncytiotrophoblast membrane (grey arrow). (C) Schematic of the 1–145 amino acid (aa) KiSS-1 pro-peptide. Metastin (Kp-54) is encoded within the 68–121 aa sequence, while Kp-10 is encoded within this same region from 112–121 aa. The specific blocking peptide is encoded within the 100–120 aa sequence. (D) Varying kisspeptin-IR was found among the different blocking peptides used. Blocking the primary antibody with full-length metastin (Kp-54) and blocking peptide resulted in complete loss of immunoreactivity, while Kp-10 was unable to block any detectable staining.
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Figure 1: IHC controls. Less than 10-week-old human placenta used as a positive control. (A) Kisspeptin-IR shows intense cell-type specific staining in the syncytiotrophoblasts (black arrow), while the cytotrophoblast layers remain unaffected (black arrowhead). (B) GPR54-IR shows intense staining in the villous cytotrophoblasts (black arrow), the extravillous cytotrophoblasts (black arrowhead), and moderate staining on the syncytiotrophoblast membrane (grey arrow). (C) Schematic of the 1–145 amino acid (aa) KiSS-1 pro-peptide. Metastin (Kp-54) is encoded within the 68–121 aa sequence, while Kp-10 is encoded within this same region from 112–121 aa. The specific blocking peptide is encoded within the 100–120 aa sequence. (D) Varying kisspeptin-IR was found among the different blocking peptides used. Blocking the primary antibody with full-length metastin (Kp-54) and blocking peptide resulted in complete loss of immunoreactivity, while Kp-10 was unable to block any detectable staining.

Mentions: Sections from formalin-fixed and paraffin-embedded tissues were deparaffinized with xylene and rehydrated with a graded series of alcohols. Wet heat-induced antigen retrieval was performed in a steamer for 20 min with a modified citrate buffer (pH 6.1, Dako, Mississauga, Ontario, Canada). Following antigen retrieval, sections were treated with 3% hydrogen peroxide (H2O2) in phosphate buffered saline (PBS) for 30 min to quench endogenous peroxidase activity. All of the aforementioned steps were followed by three washes with PBS for 5 min each. Slides were subsequently blocked for 30 min with serum-free protein block (Dako) and incubated overnight at 4°C with a polyclonal goat anti-(KiSS-1) antibody (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:400 in serum-free protein block. Kisspeptin immunoreactivity (IR) was detected with the CSA II biotin-free tyramide signal amplification system and 3,3'-diaminobenzidine chromogen solution (Dako). Specifically, rabbit anti-goat horseradish peroxidase conjugate (HRP) was applied for 15 min followed by fluorescyl-tyramide amplification reagent for 15 min and anti-fluorescein HRP for 15 min. All of the steps subsequent to the incubation with primary antibody were followed by three washes with Tris-buffered saline containing 1% Tween (TBST) for 5 min each. Slides were counterstained with Harris hematoxylin (Sigma-Aldrich, Oakville, Ontario, Canada) and mounted in a xylene-based mounting medium. Based on previously published data showing cell-type restriction of GPR54 and kisspeptins in different trophoblast layers of human placenta [3], less than 10-week old human placenta was used as a specificity control (courtesy of Vancouver Coastal Health archives), in conjunction with two blocking peptides (21 residues and 54 residues; Figure 1). Omission of the primary antibody was used as a negative control.


Kisspeptin and GPR54 immunoreactivity in a cohort of 518 patients defines favourable prognosis and clear cell subtype in ovarian carcinoma.

Prentice LM, Klausen C, Kalloger S, Köbel M, McKinney S, Santos JL, Kenney C, Mehl E, Gilks CB, Leung P, Swenerton K, Huntsman DG, Aparicio SA - BMC Med (2007)

IHC controls. Less than 10-week-old human placenta used as a positive control. (A) Kisspeptin-IR shows intense cell-type specific staining in the syncytiotrophoblasts (black arrow), while the cytotrophoblast layers remain unaffected (black arrowhead). (B) GPR54-IR shows intense staining in the villous cytotrophoblasts (black arrow), the extravillous cytotrophoblasts (black arrowhead), and moderate staining on the syncytiotrophoblast membrane (grey arrow). (C) Schematic of the 1–145 amino acid (aa) KiSS-1 pro-peptide. Metastin (Kp-54) is encoded within the 68–121 aa sequence, while Kp-10 is encoded within this same region from 112–121 aa. The specific blocking peptide is encoded within the 100–120 aa sequence. (D) Varying kisspeptin-IR was found among the different blocking peptides used. Blocking the primary antibody with full-length metastin (Kp-54) and blocking peptide resulted in complete loss of immunoreactivity, while Kp-10 was unable to block any detectable staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2200658&req=5

Figure 1: IHC controls. Less than 10-week-old human placenta used as a positive control. (A) Kisspeptin-IR shows intense cell-type specific staining in the syncytiotrophoblasts (black arrow), while the cytotrophoblast layers remain unaffected (black arrowhead). (B) GPR54-IR shows intense staining in the villous cytotrophoblasts (black arrow), the extravillous cytotrophoblasts (black arrowhead), and moderate staining on the syncytiotrophoblast membrane (grey arrow). (C) Schematic of the 1–145 amino acid (aa) KiSS-1 pro-peptide. Metastin (Kp-54) is encoded within the 68–121 aa sequence, while Kp-10 is encoded within this same region from 112–121 aa. The specific blocking peptide is encoded within the 100–120 aa sequence. (D) Varying kisspeptin-IR was found among the different blocking peptides used. Blocking the primary antibody with full-length metastin (Kp-54) and blocking peptide resulted in complete loss of immunoreactivity, while Kp-10 was unable to block any detectable staining.
Mentions: Sections from formalin-fixed and paraffin-embedded tissues were deparaffinized with xylene and rehydrated with a graded series of alcohols. Wet heat-induced antigen retrieval was performed in a steamer for 20 min with a modified citrate buffer (pH 6.1, Dako, Mississauga, Ontario, Canada). Following antigen retrieval, sections were treated with 3% hydrogen peroxide (H2O2) in phosphate buffered saline (PBS) for 30 min to quench endogenous peroxidase activity. All of the aforementioned steps were followed by three washes with PBS for 5 min each. Slides were subsequently blocked for 30 min with serum-free protein block (Dako) and incubated overnight at 4°C with a polyclonal goat anti-(KiSS-1) antibody (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:400 in serum-free protein block. Kisspeptin immunoreactivity (IR) was detected with the CSA II biotin-free tyramide signal amplification system and 3,3'-diaminobenzidine chromogen solution (Dako). Specifically, rabbit anti-goat horseradish peroxidase conjugate (HRP) was applied for 15 min followed by fluorescyl-tyramide amplification reagent for 15 min and anti-fluorescein HRP for 15 min. All of the steps subsequent to the incubation with primary antibody were followed by three washes with Tris-buffered saline containing 1% Tween (TBST) for 5 min each. Slides were counterstained with Harris hematoxylin (Sigma-Aldrich, Oakville, Ontario, Canada) and mounted in a xylene-based mounting medium. Based on previously published data showing cell-type restriction of GPR54 and kisspeptins in different trophoblast layers of human placenta [3], less than 10-week old human placenta was used as a specificity control (courtesy of Vancouver Coastal Health archives), in conjunction with two blocking peptides (21 residues and 54 residues; Figure 1). Omission of the primary antibody was used as a negative control.

Bottom Line: The latter might suggest that their overexpression would be associated with a better prognosis in cancer.Both kisspeptin positive and GPR54 positive cases are strongly associated with the ovarian carcinoma clear cell subtype (p < 0.0001, p < 0.0001), and GPR54 is significantly associated with favourable prognosis in overall survival within the clear cell subtype (p = 0.0102).Kisspeptin and GPR54 immunoreactivity are significantly associated with favourable prognosis in both disease specific and overall survival, as well as being significantly associated with the clear cell ovarian carcinoma subtype, thereby creating the first independent prognostic biomarkers specific for ovarian clear cell carcinomas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Oncology and Breast Cancer Program, British Columbia Cancer Research Centre and Department of Pathology, University ofBritish Columbia, Vancouver, British Columbia, Canada. Leah.Prentice@vch.ca

ABSTRACT

Background: Kisspeptins and their G-protein coupled receptor, GPR54 are required for GnRH release and have been associated with anti-metastatic tumour cell behaviour in model systems. The latter might suggest that their overexpression would be associated with a better prognosis in cancer. However, kisspeptin/GPR54 interactions (autocrine, paracrine, and/or endocrine) could also impact tumour behaviour in a negative manner. Here, for the first time, we associate the immunoreactivity of the kisspeptin/GPR54 ligand-receptor pair with favourable prognosis in a large cohort of ovarian carcinomas.

Methods: Immunohistochemical analysis for kisspeptin and GPR54 was performed on a tissue microarray (TMA) consisting of 518 early stage ovarian carcinomas, all with linked clinical outcome data. The TMA was scored using a staining intensity scale of 0 (negative), +1 (mild-moderate), and +2 (strong). Strong staining cases were considered either kisspeptin or GPR54 positive and designated as 1, while all other cases were considered negative and designated 0. All statistical analysis was conducted using two-sided tests and a p value equal to or less than 0.05 was considered significant.

Results: Kisspeptin and GPR54 immunoreactive cases show a favourable prognosis in univariable disease specific survival (p = 0.0023, p = 0.0092), as well as in overall survival (p = 0.0006, p = 0.0002). Furthermore, kisspeptin is an independent marker for favourable prognosis as determined by multivariable disease specific (p = 0.0046) and overall survival analysis (p = 0.0170), while GPR54 is an independent marker for overall survival only (p = 0.0303). Both kisspeptin positive and GPR54 positive cases are strongly associated with the ovarian carcinoma clear cell subtype (p < 0.0001, p < 0.0001), and GPR54 is significantly associated with favourable prognosis in overall survival within the clear cell subtype (p = 0.0102).

Conclusion: Kisspeptin and GPR54 immunoreactivity are significantly associated with favourable prognosis in both disease specific and overall survival, as well as being significantly associated with the clear cell ovarian carcinoma subtype, thereby creating the first independent prognostic biomarkers specific for ovarian clear cell carcinomas.

Show MeSH
Related in: MedlinePlus