Effect of Preanalytic Variables on an Automated PTEN Immunohistochemistry Assay for Prostate Cancer.
Author(s): Guedes LB, Morais CL, Fedor H, Hicks J, Gurel B, Melamed J, Lee P, Gopalan A, Knudsen BS, True LD, Scher HI, Fine SW, Trock BJ, De Marzo AM, Lotan TL
Publication: Arch Pathol Lab Med, 2019, Vol. 143, Page 338-348
PubMed ID: 30295067 PubMed Review Paper? No
Purpose of Paper
This paper investigated whether PTEN immunostaining in benign and tumor prostate specimens is adversely affected by cold ischemia time (0.5 - 48 h); fixative (10% neutral buffered formalin, NBF; Bouins, Hollande); the duration of formalin fixation (4-48 h), FFPE block storage (2 weeks - 23 y), unstained FFPE slide storage (<1 month, 5 y); institution-specific tissue processing protocols; or automated staining platform choice. Specimens were assembled into a tissue microarray prior to analysis. Immunohistochemical staining was either visually scored by a pathologist blind to the experimental assignments or quantified using open source software.
Conclusion of Paper
PTEN immunostaining results were not significantly affected by cold ischemia time, duration of formalin fixation, or automated staining platform choice. Significant differences were observed among different fixatives as the intensity of PTEN immunopositive staining was significantly stronger among prostate specimens fixed in 10% NBF compared to Bouins (P=0.03) or Hollande's fixative (P=0.004). While a probable effect of prolonged FFPE block storage was observed among one sample set (P=0.02), analysis of a second sample set failed to confirm the finding. Equivalent PTEN immunostaining was observed among prostate biopsy specimens that underwent tissue processing at 11 different institutions according to institution-specific protocols.
Studies
-
Study Purpose
The purpose of this study was to determine the effects of cold ischemia time (the time between specimen excision and fixation) on intensity of PTEN immunohistochemical staining in formalin-fixed, paraffin-embedded benign prostate tissue specimens. Five biopsy cores (7 mm) were collected from 10 different prostates obtained by radical prostatectomy and subjected to 0, 1, 2, 4, or >4 hours of cold ischemia at room temperature in a humidified chamber prior to preservation. Tissue was preserved in 10% neutral buffered formalin (NBF) for 24 h, processed, and paraffin-embedded. A second sample set of biopsies collected from three radical prostatectomies containing benign tissue that were subjected to a cold ischemia time at room temperature of 0.5 to 48 h was also evaluated. Between two and four cores (0.6 mm) were punched from each FFPE block and assembled into a tissue microarray block. Automated immunohistochemical staining of PTEN was detected after antigen retrieval with Epitope Retrieval Solution 2 for 20 min using the Leica Bond platform. Immunostaining was quantified using FrIDA TMAJ software.
Summary of Findings:
Cold ischemia time and the intensity of PTEN immunostaining in benign prostate FFPE specimens were not correlated in the first sample set of specimens subjected to a cold ischemia time at room temperature of 0-4 h. Similarly, the intensity of PTEN immunostaining in the second sample set did not differ significantly among benign prostate tissue subjected to cold ischemia times that ranged between 0.5 - 48 h
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform Protein Tissue microarray Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 0 min
30 min
1 h
2 h
4 h
≤ 48 h
Immunohistochemistry Specific Targeted peptide/protein PTEN
-
Study Purpose
The purpose of this study was to determine if the intensity of PTEN immunohistochemical staining was affected by the duration of formalin fixation in benign prostate specimens. Six cores (7 mm) were collected from 16 radical prostatectomy benign tissue specimens; subjected to a cold ischemia time of less than 1 h; and fixed in 10% NBF for 4, 8, 12, 24, or 48 h; washed and stored in phosphate buffered saline at 4°C; processed; and paraffin-embedded. A control designated as 0 h in 10% NBF was also included although details relating to the exact time and method of fixation were not specified. Between two and four cores (0.6 mm) were punched from each FFPE block and assembled into a tissue microarray block. Automated immunohistochemical staining of PTEN was detected after antigen retrieval with Epitope Retrieval Solution 2 for 20 min using the Leica Bond platform. Immunostaining was quantified using FrIDA TMAJ software.
Summary of Findings:
No significant differences in the intensity of PTEN epithelial immunostaining were observed among benign prostate tissue specimens preserved in formalin for different durations (4, 8, 12, 24, 48 h) and analyzed via tissue microarray. The authors state that similar results were obtained with a second sample set fixed in formalin for 8, 16, 24, 48, 64, 72, 96, 120, or 128 h that were collected and preserved at a different institution and assembled into a tissue microarray of 1.2 mm cores, although data was not shown.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform Protein Tissue microarray Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 0 h
4 h
8 h
12 h
24 h
48 h
Immunohistochemistry Specific Targeted peptide/protein PTEN
-
Study Purpose
The purpose of this study was to determine if the intensity of PTEN immunohistochemical staining was affected by the type of fixative used to preserve benign prostate specimens. Three cores (7 mm) were collected from five radical prostatectomy benign tissue specimens, subjected to a cold ischemia time of less than 1 h, and fixed in either 10% NBF, Bouin's, or Hollande's fixative for 12 h. Between two and four cores (0.6 mm) were punched from each FFPE block and assembled into a tissue microarray block. Automated immunohistochemical staining of PTEN was detected after antigen retrieval with Epitope Retrieval Solution 2 for 20 min using the Leica Bond platform. Immunostaining was quantified using FrIDA TMAJ software.
Summary of Findings:
The intensity of epithelial PTEN immunostaining was significantly stronger in benign prostate specimens preserved in 10% NBF compared to either Bouin's (P=0.03) or Hollande's fixative (P=0.004).
Biospecimens
Preservative Types
- Formalin
- Other Preservative
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Immunohistochemistry Specific Targeted peptide/protein PTEN
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Bouin's fixative
Hollande's fixative
-
Study Purpose
The purpose of this study was to assess potential differences in PTEN immunostaining intensity among FFPE prostate biopsy specimens that underwent tissue processing at 11 different institutions according to institution-specific protocols. Eleven cores (7 mm) were collected from each of two radical prostatectomy tumor specimens, subjected to a cold ischemia time of less than 1 h, placed in 10% NBF, and distributed to 11 different institutions for processing using their own protocols as part of the Inter-Prostate SPORE Biomarker study. Details regarding the extent that protocols differed from one another are provided in supplemental material and include differences in the duration and temperature of formalin fixation, dehydration, clearing, paraffin infiltration, and total processing time. The reagents used by individual institutions were not provided. FFPE blocks were then sent to a central location for sectioning, slide preparation, and immunohistochemical analysis. Automated immunohistochemical staining of PTEN was detected after antigen retrieval with Epitope Retrieval Solution 2 for 20 min using the Leica Bond platform. Nuclear and cytoplasmic PTEN immunostaining was quantified visually by a pathologist blinded to the details of specimen processing for consistency and weak or absent staining. Effects attributable to the storage of unstained slide-mounted FFPE sections were also investigated by comparing PTEN immunostaining of freshly cut slides (<1 month) and those stored for 5 y prior to immunohistochemical analysis.
Summary of Findings:
Tumor prostate specimens that underwent tissue processing and paraffin embedding at different institutions using institution-specific protocols yielded comparable PTEN immunostaining results despite differences in the durations and temperatures of fixation, dehydration, clearing, and paraffin infiltration. While representative micrographs from specimens processed at three institutions were provided, additional micrographs and tallies of specimens with weak or absent PTEN immunostaining were not. Storage of unstained slide-mounted FFPE sections for 5 years did not significantly affect PTEN immunostaining of prostate tumor biopsy specimens, as PTEN scoring was identical among freshly sectioned and stored slides.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Tissue microarray Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative ≤ 9 h
11 h
12 h
18 h
24 h
27 h
45 h
75 h
Biospecimen Preservation Dehydration duration/condition Room temperature
25°C
35°C
37°C
40°C
42°C
57°C
5 min/stage
7 min/stage
15 min/stage
20 min/stage
30 min/stage
40 min/stage
45 min/stage
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Pen-fix
Storage Storage duration <1 month
5 y
Biospecimen Preservation Duration of tissue processing 70 min
75 min
94 min
185 min
205 min
260 min
510 min
570 min
Biospecimen Preservation Embedding duration/condition Room temperature
57°C
58°C
60°C
5-7 min/stage
15-25 min/stage
30-45 min/stage
Immunohistochemistry Specific Targeted peptide/protein PTEN
Biospecimen Preservation Clearing duration/condition Room temperature
25°C
35°C
37°C
40°C
42°C
57°C
10 min/stage
14 min/stage
30 min/stage
45 min/stage
50 min/stage
75 min/stage
90 min/stage
120 min/stage
-
Study Purpose
The purpose of this study was to determine the effects of FFPE block storage duration on the intensity of PTEN immunohistochemical staining in prostate tumor specimens. The percentage of specimens that exhibited weak or absent PTEN immunostaining was determined in 277 needle biopsies collected, fixed, and processed at a single institution within 2 weeks of specimen embedding as well as meta-analysis of previously published data from 278 biopsies that had been stored as FFPE blocks for 2 to 15 y. A third sample set consisting of a tissue microarray representing 508 prostate tumors that had been stored for 9-23 y prior to sectioning and immunohistochemical staining was also compared. All samples had a Gleason score of 6 or 7 prior to storage. Automated immunohistochemical staining of PTEN was detected after antigen retrieval with Epitope Retrieval Solution 2 for 20 min using the Leica Bond platform. Nuclear and cytoplasmic PTEN immunostaining was quantified visually by a pathologist blinded to the details of specimen processing for consistency and weak or absent staining.
Summary of Findings:
The percentage of prostate tumor specimens that displayed weak or absent PTEN increased with progressive FFPE block storage reflecting 2% of specimens stored for less than 5 y (6/399), 4% (4/101) stored for 5-10 y, and 14% (8/55) stored for 11-15 y; indicating a probable effect with the χ2 test (P=0.02). Conversely, examination of the tumor prostate sample set with the largest range of FFPE storage durations exhibited a less marked effect of block storage on PTEN immunostaining with weak or absent PTEN immunostaining observed in 2% of specimens stored for 9-13 y (2/85) and 5% of specimens stored for 19-23 y (12/249) but 16% of specimens stored for 15-17 y (13/82). Importantly, the authors attribute the rise in ambiguous PTEN staining among specimens stored for 15-17 y to a brief institutional change in fixation methods (microwave formalin fixation without formalin injection).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Tissue microarray Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Immunohistochemistry Specific Targeted peptide/protein PTEN
Storage Storage duration <2 weeks
2-15 y
9-23 y
-
Study Purpose
The purpose of this study was to assess the variability introduced by using different autostainer platforms and machines on the results of PTEN immunohistochemistry of prostate tumor specimens. A tissue microarray representing 77 prostate tumor specimens and 253 individual cores (0.6 mm) were analyzed for PTEN immunostaining in a blind fashion. Three different Ventana platforms were compared to one another (Benchmark Ultra, Discovery Ultra, Discovery XT), and the Ventana Benchmark platform was compared to the Leica Bond automated staining platform.
Summary of Findings:
There was a 98% (247/253 cores) agreement between the Ventana Benchmark and Leica Bond automated autostaining platforms for PTEN scores. The three Ventana machines (Benchmark Ultra, Discovery Ultra, Discovery XT) produced identical PTEN results.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Tissue microarray Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Immunohistochemistry Specific Targeted peptide/protein PTEN
Immunohistochemistry Specific Detection method Ventana Discovery Ultra
Ventana Benchmark Ultra
Ventana Discovery XT
Leica Bond