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Association of TMPRSS2-ERG gene fusion with clinical characteristics and outcomes: results from a population-based study of prostate cancer.

FitzGerald LM, Agalliu I, Johnson K, Miller MA, Kwon EM, Hurtado-Coll A, Fazli L, Rajput AB, Gleave ME, Cox ME, Ostrander EA, Stanford JL, Huntsman DG - BMC Cancer (2008)

Bottom Line: Genotyping of four ERG and one TMPRSS2 SNPs using germline DNA was also performed in a sample of the cases (n = 127).However, evidence for reduced prostate cancer-specific survival was apparent in those cases whose tumor had multiple copies of the fusion.If replicated, the results presented here may provide insight into the mechanism by which the TMPRSS2-ERG gene fusion arises and also contribute to diagnostic evaluations for determining the subset of men who will go on to develop metastatic prostate cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., Seattle, WA 98109, USA. lfitzger@fhcrc.org

ABSTRACT

Background: The presence of the TMPRSS2-ERG fusion gene in prostate tumors has recently been associated with an aggressive phenotype, as well as recurrence and death from prostate cancer. These associations suggest the hypothesis that the gene fusion may be used as a prognostic indicator for prostate cancer.

Methods: In this study, fluorescent in situ hybridization (FISH) assays were used to assess TMPRSS2-ERG fusion status in a group of 214 prostate cancer cases from two population-based studies. The FISH assays were designed to detect both fusion type (deletion vs. translocation) and the number of fusion copies (single vs. multiple). Genotyping of four ERG and one TMPRSS2 SNPs using germline DNA was also performed in a sample of the cases (n = 127).

Results: Of the 214 tumors scored for the TMPRSS2-ERG fusion, 64.5% were negative and 35.5% were positive for the fusion. Cases with the TMPRSS2-ERG fusion did not exhibit reduced prostate cancer survival (HR = 0.92, 95% CI = 0.22-3.93), nor was there a significant difference in cause-specific survival when stratifying by translocation or deletion (HR = 0.84, 95% CI = 0.23-3.12) or by the number of retained fusion copies (HR = 1.22, 95% CI = 0.45-3.34). However, evidence for reduced prostate cancer-specific survival was apparent in those cases whose tumor had multiple copies of the fusion. The variant T allele of the TMPRSS2 SNP, rs12329760, was positively associated with TMPRSS2-ERG fusion by translocation (p = 0.05) and with multiple copies of the gene fusion (p = 0.03).

Conclusion: If replicated, the results presented here may provide insight into the mechanism by which the TMPRSS2-ERG gene fusion arises and also contribute to diagnostic evaluations for determining the subset of men who will go on to develop metastatic prostate cancer.

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FISH detection of TMPRSS2 and ERG gene status. A) No TMPRSS2-ERG fusion. B) Positive for the TMPRSS2-ERG fusion with translocation – arrows indicate the separate green 5' ERG signal. C) Positive for the TMPRSS2-ERG fusion with deletion – arrows indicate the combined blue/red TMPRSS2 and 3' ERG signal and the absence of the green 5' ERG signal. FISH signals in these pseudo-colored images are red (telomeric to TMPRSS2; RP11-35C4), green (5' ERG; RP11-95I21) and blue (3' ERG; RP11-476D17). Note that in non-rearranged chromosomes the proximity of the green and blue signals can result in an aqua-colored signal.
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Figure 2: FISH detection of TMPRSS2 and ERG gene status. A) No TMPRSS2-ERG fusion. B) Positive for the TMPRSS2-ERG fusion with translocation – arrows indicate the separate green 5' ERG signal. C) Positive for the TMPRSS2-ERG fusion with deletion – arrows indicate the combined blue/red TMPRSS2 and 3' ERG signal and the absence of the green 5' ERG signal. FISH signals in these pseudo-colored images are red (telomeric to TMPRSS2; RP11-35C4), green (5' ERG; RP11-95I21) and blue (3' ERG; RP11-476D17). Note that in non-rearranged chromosomes the proximity of the green and blue signals can result in an aqua-colored signal.

Mentions: A total of 372 paraffin embedded tumor blocks was used to build the TMAs. Of these specimens, eight were selected as blind duplicates for quality control purposes resulting in 380 cores being represented on the TMAs. H&E stained slides were reviewed for each case and areas containing tumor were marked on both the slides and corresponding paraffin blocks for TMA construction. Three cores per case, taken from a single tumor focus, were represented on the TMAs. The TMAs were constructed using a manual arrayer (Beecher Instruments) with tissue core diameters of 0.6 mm. Each section was baked overnight at 60°C, then deparaffinized in xylene and rinsed with 100% ethanol. Sections were pretreated as described previously [27]. FISH analysis was performed using the following BACs (BACPAC Resources Centre, Children's Hospital Oakland Research Institute, Oakland, CA): RP11-95I21 (5' ERG), RP11-476D17 (3' ERG) and RP11-35C4 (telomeric to TMPRSS2; Figure 1). BACs RP11-95I21 and RP11-35C4 were directly labeled by nick translation with Spectrum Green and Spectrum Orange respectively (Vysis, Downer's Grove, IL). BAC RP11-476D17 was indirectly labeled using a modified protocol with Streptavidin-Cy5 (MetaSystems, Belmont, MA) using the BioPrime DNA labeling system (Invitrogen). Probe labeling and FISH were performed using Vysis or MetaSystems reagents according to manufacturers' protocols. FISH signals were visualized on a Zeiss Axioplan epifluorescent microscope and captured using MetaSystems ISIS FISH imaging software (MetaSystems, Belmont, MA). Evaluation of the FISH results from each case was independently performed by 2 operators (KJ, MM). A total of 50 epithelial nuclei per case was evaluated across the three cores and to be classed positive for the TMPRSS2-ERG fusion, evidence needed to be present in at least 20% of the cells. Each individual was scored as follows (Figure 1 and 2): normal (three combined signals indicating no rearranged TMPRSS2 or ERG loci); positive for the fusion with translocation (combined red/blue signals with separate green 5' ERG signal); positive for the fusion with deletion (combined red/blue signals with the absence of the green 5' ERG signal); or not scored (absence of a FISH signal, low cellularity or core(s) dislodged from array). It was also noted whether multiple copies of the particular TMPRSS2-ERG fusion were present in a single nucleus.


Association of TMPRSS2-ERG gene fusion with clinical characteristics and outcomes: results from a population-based study of prostate cancer.

FitzGerald LM, Agalliu I, Johnson K, Miller MA, Kwon EM, Hurtado-Coll A, Fazli L, Rajput AB, Gleave ME, Cox ME, Ostrander EA, Stanford JL, Huntsman DG - BMC Cancer (2008)

FISH detection of TMPRSS2 and ERG gene status. A) No TMPRSS2-ERG fusion. B) Positive for the TMPRSS2-ERG fusion with translocation – arrows indicate the separate green 5' ERG signal. C) Positive for the TMPRSS2-ERG fusion with deletion – arrows indicate the combined blue/red TMPRSS2 and 3' ERG signal and the absence of the green 5' ERG signal. FISH signals in these pseudo-colored images are red (telomeric to TMPRSS2; RP11-35C4), green (5' ERG; RP11-95I21) and blue (3' ERG; RP11-476D17). Note that in non-rearranged chromosomes the proximity of the green and blue signals can result in an aqua-colored signal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: FISH detection of TMPRSS2 and ERG gene status. A) No TMPRSS2-ERG fusion. B) Positive for the TMPRSS2-ERG fusion with translocation – arrows indicate the separate green 5' ERG signal. C) Positive for the TMPRSS2-ERG fusion with deletion – arrows indicate the combined blue/red TMPRSS2 and 3' ERG signal and the absence of the green 5' ERG signal. FISH signals in these pseudo-colored images are red (telomeric to TMPRSS2; RP11-35C4), green (5' ERG; RP11-95I21) and blue (3' ERG; RP11-476D17). Note that in non-rearranged chromosomes the proximity of the green and blue signals can result in an aqua-colored signal.
Mentions: A total of 372 paraffin embedded tumor blocks was used to build the TMAs. Of these specimens, eight were selected as blind duplicates for quality control purposes resulting in 380 cores being represented on the TMAs. H&E stained slides were reviewed for each case and areas containing tumor were marked on both the slides and corresponding paraffin blocks for TMA construction. Three cores per case, taken from a single tumor focus, were represented on the TMAs. The TMAs were constructed using a manual arrayer (Beecher Instruments) with tissue core diameters of 0.6 mm. Each section was baked overnight at 60°C, then deparaffinized in xylene and rinsed with 100% ethanol. Sections were pretreated as described previously [27]. FISH analysis was performed using the following BACs (BACPAC Resources Centre, Children's Hospital Oakland Research Institute, Oakland, CA): RP11-95I21 (5' ERG), RP11-476D17 (3' ERG) and RP11-35C4 (telomeric to TMPRSS2; Figure 1). BACs RP11-95I21 and RP11-35C4 were directly labeled by nick translation with Spectrum Green and Spectrum Orange respectively (Vysis, Downer's Grove, IL). BAC RP11-476D17 was indirectly labeled using a modified protocol with Streptavidin-Cy5 (MetaSystems, Belmont, MA) using the BioPrime DNA labeling system (Invitrogen). Probe labeling and FISH were performed using Vysis or MetaSystems reagents according to manufacturers' protocols. FISH signals were visualized on a Zeiss Axioplan epifluorescent microscope and captured using MetaSystems ISIS FISH imaging software (MetaSystems, Belmont, MA). Evaluation of the FISH results from each case was independently performed by 2 operators (KJ, MM). A total of 50 epithelial nuclei per case was evaluated across the three cores and to be classed positive for the TMPRSS2-ERG fusion, evidence needed to be present in at least 20% of the cells. Each individual was scored as follows (Figure 1 and 2): normal (three combined signals indicating no rearranged TMPRSS2 or ERG loci); positive for the fusion with translocation (combined red/blue signals with separate green 5' ERG signal); positive for the fusion with deletion (combined red/blue signals with the absence of the green 5' ERG signal); or not scored (absence of a FISH signal, low cellularity or core(s) dislodged from array). It was also noted whether multiple copies of the particular TMPRSS2-ERG fusion were present in a single nucleus.

Bottom Line: Genotyping of four ERG and one TMPRSS2 SNPs using germline DNA was also performed in a sample of the cases (n = 127).However, evidence for reduced prostate cancer-specific survival was apparent in those cases whose tumor had multiple copies of the fusion.If replicated, the results presented here may provide insight into the mechanism by which the TMPRSS2-ERG gene fusion arises and also contribute to diagnostic evaluations for determining the subset of men who will go on to develop metastatic prostate cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., Seattle, WA 98109, USA. lfitzger@fhcrc.org

ABSTRACT

Background: The presence of the TMPRSS2-ERG fusion gene in prostate tumors has recently been associated with an aggressive phenotype, as well as recurrence and death from prostate cancer. These associations suggest the hypothesis that the gene fusion may be used as a prognostic indicator for prostate cancer.

Methods: In this study, fluorescent in situ hybridization (FISH) assays were used to assess TMPRSS2-ERG fusion status in a group of 214 prostate cancer cases from two population-based studies. The FISH assays were designed to detect both fusion type (deletion vs. translocation) and the number of fusion copies (single vs. multiple). Genotyping of four ERG and one TMPRSS2 SNPs using germline DNA was also performed in a sample of the cases (n = 127).

Results: Of the 214 tumors scored for the TMPRSS2-ERG fusion, 64.5% were negative and 35.5% were positive for the fusion. Cases with the TMPRSS2-ERG fusion did not exhibit reduced prostate cancer survival (HR = 0.92, 95% CI = 0.22-3.93), nor was there a significant difference in cause-specific survival when stratifying by translocation or deletion (HR = 0.84, 95% CI = 0.23-3.12) or by the number of retained fusion copies (HR = 1.22, 95% CI = 0.45-3.34). However, evidence for reduced prostate cancer-specific survival was apparent in those cases whose tumor had multiple copies of the fusion. The variant T allele of the TMPRSS2 SNP, rs12329760, was positively associated with TMPRSS2-ERG fusion by translocation (p = 0.05) and with multiple copies of the gene fusion (p = 0.03).

Conclusion: If replicated, the results presented here may provide insight into the mechanism by which the TMPRSS2-ERG gene fusion arises and also contribute to diagnostic evaluations for determining the subset of men who will go on to develop metastatic prostate cancer.

Show MeSH
Related in: MedlinePlus