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Identification of prefoldin amplification (1q23.3-q24.1) in bladder cancer using comparative genomic hybridization (CGH) arrays of urinary DNA.

López V, González-Peramato P, Suela J, Serrano A, Algaba F, Cigudosa JC, Vidal A, Bellmunt J, Heredero O, Sánchez-Carbayo M - J Transl Med (2013)

Bottom Line: Supervised hierarchical clustering identified the gain at 1q23.3-q24.1 significantly correlated to stage (p = 0.011), and grade (p = 0.002).Moreover, PFND2 overexpression was significantly associated with poor disease-specific survival (p ≤0.0005).Genomic profiles of urinary DNA mirrowed bladder tumors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Center, Melchor Fernandez Almagro 3, Madrid E-28029, Spain.

ABSTRACT

Background: Array-CGH represents a comprehensive tool to discover genomic disease alterations that could potentially be applied to body fluids. In this report, we aimed at applying array-CGH to urinary samples to characterize bladder cancer.

Methods: Urinary DNA from bladder cancer patients and controls were hybridized on 44K oligonucleotide arrays. Validation analyses of identified regions and candidates included fluorescent in situ hybridization (FISH) and immunohistochemistry in an independent set of bladder tumors spotted on custom-made tissue arrays (n = 181).

Results: Quality control of array-CGH provided high reproducibility in dilution experiments and when comparing reference pools. The most frequent genomic alterations (minimal recurrent regions) among bladder cancer urinary specimens included gains at 1q and 5p, and losses at 10p and 11p. Supervised hierarchical clustering identified the gain at 1q23.3-q24.1 significantly correlated to stage (p = 0.011), and grade (p = 0.002). The amplification and overexpression of Prefoldin (PFND2), a selected candidate mapping to 1q23.3-q24.1, correlated to increasing stage and tumor grade by means of custom-designed and optimized FISH (p = 0.013 and p = 0.023, respectively), and immunohistochemistry (p ≤0.0005 and p = 0.011, respectively), in an independent set of bladder tumors included in tissue arrays. Moreover, PFND2 overexpression was significantly associated with poor disease-specific survival (p ≤0.0005). PFND2 was amplified and overexpressed in bladder tumors belonging to patients providing urinary specimens where 1q23.3q24.1 amplification was detected by array-CGH.

Conclusions: Genomic profiles of urinary DNA mirrowed bladder tumors. Molecular profiling of urinary DNA using array-CGH contributed to further characterize genomic alterations involved in bladder cancer progression. PFND2 was identified as a tumor stratification and clinical outcome prognostic biomarker for bladder cancer patients.

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PFND2 expression patterns are associated with tumor progression and clinical outcome. A, B, C. Representative FISH images of PFND2 on tissue arrays containing bladder tumors (n = 181) showing cases with normal (A), gained (B), and amplified (C) hybridization patterns. D.E. Representative immunostainings by immunohistochemistry showing cases with low (+) (D) and high (+++) (E) intensity of PFND2 cytoplasmic expression on tissue arrays. F. Kaplan-Mayer curve survival analysis indicating that a cytoplasmic protein expression of PFND2 with medium (++) or high (+++) intensity measured by immunohistochemistry on tissue arrays was associated with shorter disease-specific overall survival (log rank, p ≤0.0005).
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Figure 4: PFND2 expression patterns are associated with tumor progression and clinical outcome. A, B, C. Representative FISH images of PFND2 on tissue arrays containing bladder tumors (n = 181) showing cases with normal (A), gained (B), and amplified (C) hybridization patterns. D.E. Representative immunostainings by immunohistochemistry showing cases with low (+) (D) and high (+++) (E) intensity of PFND2 cytoplasmic expression on tissue arrays. F. Kaplan-Mayer curve survival analysis indicating that a cytoplasmic protein expression of PFND2 with medium (++) or high (+++) intensity measured by immunohistochemistry on tissue arrays was associated with shorter disease-specific overall survival (log rank, p ≤0.0005).

Mentions: The gain of PFND2 detected by array-CGH, and its protein overexpression were initially evaluated by FISH and IHC analysis, respectively, on the paired bladder tumors belonging to the bladder cancer patients providing urinary specimens. The cases showing PFND2 amplification in the urinary specimen displayed amplification in the paired bladder tumor by FISH (Additional file 1: Figure S1B) and also protein overexpression by IHC (Additional file 1: Figure S1C). Paired normal urothelium specimens showed a normal pattern of hybridization (data not shown, similar to Figure 4A). The association between FISH and IHC observations with clinicopathologic variables was then evaluated on tissue arrays containing independent sets of bladder tumors (n = 181). For FISH analyses, three main hybridization patterns were observed: normal (Figure 4A), gains (Figure 4B), and amplifications (Figure 4C). For IHC analyses, the intensity of PFND2 immunostaining was categorized from 1 (Figure 4D), to 3 (Figure 4E). Interestingly, tumor stage was significantly associated with the gene amplification observed by FISH (p = 0.013), and the protein overexpression of PFND2 observed by IHC (p ≤ 0.0005). Tumor grade was also associated with PFND2 amplification (p = 0.023), and its overexpression (p = 0.011). PFND2 amplification and protein overexpression were significantly associated between them (Kendall’s tau = 0.125, p = 0.034), and with increased proliferation as measured by Ki67 staining (Kendall’s tau τ = 0.223, p ≤ 0.0005, and τ = 0.433, p ≤ 0.0005, respectively). Furthermore, patients with high cytoplasmic PFND2 overexpression had shorter disease-specific survival than those with low expression (log rank, p < 0.0005, Figure 4F). Overall, FISH and IHC validation analyses on tissue arrays containing an independent large set of bladder tumors served to associate PFND2 amplification and overexpression with histopathologic variables of tumor progression and clinical outcome of bladder cancer patients.


Identification of prefoldin amplification (1q23.3-q24.1) in bladder cancer using comparative genomic hybridization (CGH) arrays of urinary DNA.

López V, González-Peramato P, Suela J, Serrano A, Algaba F, Cigudosa JC, Vidal A, Bellmunt J, Heredero O, Sánchez-Carbayo M - J Transl Med (2013)

PFND2 expression patterns are associated with tumor progression and clinical outcome. A, B, C. Representative FISH images of PFND2 on tissue arrays containing bladder tumors (n = 181) showing cases with normal (A), gained (B), and amplified (C) hybridization patterns. D.E. Representative immunostainings by immunohistochemistry showing cases with low (+) (D) and high (+++) (E) intensity of PFND2 cytoplasmic expression on tissue arrays. F. Kaplan-Mayer curve survival analysis indicating that a cytoplasmic protein expression of PFND2 with medium (++) or high (+++) intensity measured by immunohistochemistry on tissue arrays was associated with shorter disease-specific overall survival (log rank, p ≤0.0005).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: PFND2 expression patterns are associated with tumor progression and clinical outcome. A, B, C. Representative FISH images of PFND2 on tissue arrays containing bladder tumors (n = 181) showing cases with normal (A), gained (B), and amplified (C) hybridization patterns. D.E. Representative immunostainings by immunohistochemistry showing cases with low (+) (D) and high (+++) (E) intensity of PFND2 cytoplasmic expression on tissue arrays. F. Kaplan-Mayer curve survival analysis indicating that a cytoplasmic protein expression of PFND2 with medium (++) or high (+++) intensity measured by immunohistochemistry on tissue arrays was associated with shorter disease-specific overall survival (log rank, p ≤0.0005).
Mentions: The gain of PFND2 detected by array-CGH, and its protein overexpression were initially evaluated by FISH and IHC analysis, respectively, on the paired bladder tumors belonging to the bladder cancer patients providing urinary specimens. The cases showing PFND2 amplification in the urinary specimen displayed amplification in the paired bladder tumor by FISH (Additional file 1: Figure S1B) and also protein overexpression by IHC (Additional file 1: Figure S1C). Paired normal urothelium specimens showed a normal pattern of hybridization (data not shown, similar to Figure 4A). The association between FISH and IHC observations with clinicopathologic variables was then evaluated on tissue arrays containing independent sets of bladder tumors (n = 181). For FISH analyses, three main hybridization patterns were observed: normal (Figure 4A), gains (Figure 4B), and amplifications (Figure 4C). For IHC analyses, the intensity of PFND2 immunostaining was categorized from 1 (Figure 4D), to 3 (Figure 4E). Interestingly, tumor stage was significantly associated with the gene amplification observed by FISH (p = 0.013), and the protein overexpression of PFND2 observed by IHC (p ≤ 0.0005). Tumor grade was also associated with PFND2 amplification (p = 0.023), and its overexpression (p = 0.011). PFND2 amplification and protein overexpression were significantly associated between them (Kendall’s tau = 0.125, p = 0.034), and with increased proliferation as measured by Ki67 staining (Kendall’s tau τ = 0.223, p ≤ 0.0005, and τ = 0.433, p ≤ 0.0005, respectively). Furthermore, patients with high cytoplasmic PFND2 overexpression had shorter disease-specific survival than those with low expression (log rank, p < 0.0005, Figure 4F). Overall, FISH and IHC validation analyses on tissue arrays containing an independent large set of bladder tumors served to associate PFND2 amplification and overexpression with histopathologic variables of tumor progression and clinical outcome of bladder cancer patients.

Bottom Line: Supervised hierarchical clustering identified the gain at 1q23.3-q24.1 significantly correlated to stage (p = 0.011), and grade (p = 0.002).Moreover, PFND2 overexpression was significantly associated with poor disease-specific survival (p ≤0.0005).Genomic profiles of urinary DNA mirrowed bladder tumors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Center, Melchor Fernandez Almagro 3, Madrid E-28029, Spain.

ABSTRACT

Background: Array-CGH represents a comprehensive tool to discover genomic disease alterations that could potentially be applied to body fluids. In this report, we aimed at applying array-CGH to urinary samples to characterize bladder cancer.

Methods: Urinary DNA from bladder cancer patients and controls were hybridized on 44K oligonucleotide arrays. Validation analyses of identified regions and candidates included fluorescent in situ hybridization (FISH) and immunohistochemistry in an independent set of bladder tumors spotted on custom-made tissue arrays (n = 181).

Results: Quality control of array-CGH provided high reproducibility in dilution experiments and when comparing reference pools. The most frequent genomic alterations (minimal recurrent regions) among bladder cancer urinary specimens included gains at 1q and 5p, and losses at 10p and 11p. Supervised hierarchical clustering identified the gain at 1q23.3-q24.1 significantly correlated to stage (p = 0.011), and grade (p = 0.002). The amplification and overexpression of Prefoldin (PFND2), a selected candidate mapping to 1q23.3-q24.1, correlated to increasing stage and tumor grade by means of custom-designed and optimized FISH (p = 0.013 and p = 0.023, respectively), and immunohistochemistry (p ≤0.0005 and p = 0.011, respectively), in an independent set of bladder tumors included in tissue arrays. Moreover, PFND2 overexpression was significantly associated with poor disease-specific survival (p ≤0.0005). PFND2 was amplified and overexpressed in bladder tumors belonging to patients providing urinary specimens where 1q23.3q24.1 amplification was detected by array-CGH.

Conclusions: Genomic profiles of urinary DNA mirrowed bladder tumors. Molecular profiling of urinary DNA using array-CGH contributed to further characterize genomic alterations involved in bladder cancer progression. PFND2 was identified as a tumor stratification and clinical outcome prognostic biomarker for bladder cancer patients.

Show MeSH
Related in: MedlinePlus