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Keywords:
Benign prostatic hyperplasia, In situ hybridization, miRNA, Prostate cancer, Xmatrx®
Application Highlights:
• Both benign prostatic hyperplasia (BPH) and prostate cancer remain the most prevalent urologic
health concerns affecting elderly men in their lifetime, frequently coexist and therefore share
common symptoms.
• Distinguishing nonaggressive BPH from aggressive prostate cancer is crucial for effective
treatment and better clinical outcome.
• Several techniques for diagnosing prostate cancer have evolved over the past decades; however
there are concerns on spatial resolution of these diagnostic tools.
• BioGenex Xmatrx®
automated systems and BioGenex miRNA ISH Prostate panel probes were used
to successfully differentiate prostate cancer and BPH.
• Downregulation of miRNAs involved in cellular regulation and upregulation of miRNA in disease
progression were observed.
• The in situ experimental conditions for hybridization were optimized for both BioGenex manual and
automated systems.
BioGenex Products Used:
• #HM125B-100: miR-125b
• #HM017-100: miR-17
• #DF400-YADE: XISH™ One-Step Polymer-HRP ISH Detection Kit (Automation)
• #DF400-50KE: Super Sensitive One-Step Polymer-HRP ISH Detection Kit (Manual)
Accelerating the pace of precision medicine
Introduction:
Prostate cancer and benign prostatic hyperplasia (BPH) are the most frequent pathologies of the
prostate gland that are responsible for morbidity in men. Unlike prostate cancer, BPH is nonmalignant
and nonfatal. BPH and prostate cancer frequently coexist and therefore share common symptoms; over
20% of men with prostate cancer have BPH. There remains an important clinical challenge in
prostate oncology to distinguish the aggressive from the nonaggressive form, without requiring all
patients to undergo a painful tissue biopsy. In the United States, prostate cancer is the second leading
cause of cancer death affecting one in nine men. This year, an estimated 164,690 men in the United
States will be diagnosed with prostate cancer and 29,430 will die from it. Therefore, early diagnosis and
timely detection of disease progression is a very important step for effective treatment and a beneficial
clinical outcome. Although several techniques for diagnosing prostate cancer have evolved over the past
decades, lack of specificity and sensitivity of these diagnostic tools hinder their application in clinical
practice. Additionally, the existing methods to utilize serum prostate-specific antigen (PSA) does not
provide enhanced survival rates nor do these techniques offer absolute results for the differentiation of
prostate cancer and BPH. Elevated levels of PSA have also been reported in non-malignant conditions of
the prostate, including BPH, which is commonly misdiagnosed as prostate cancer, leading to
unnecessary biopsies. Furthermore, metastatic and advanced prostate tumors types
respond very poorly to chemotherapy. Accumulating data suggest that small noncoding
RNAs such as microRNAs (miRNAs) can be utilized as potential biomarkers for
differentiation of prostate cancer and BPH.
Application Note
Ready-to-Use fully optimized SSNA miRNA in situ hybridization (ISH) Kit
Prostate Cancer (PC) and Benign Prostatic Hyperplasia (BPH)
Differentiation by New miRNA Biomarker Panel
miRNAs are small, evolutionarily conserved, noncoding RNAs that negatively regulate gene expression
in divergent cellular processes including development, differentiation, proliferation, cell cycle control,
and apoptosis. Owing to their stability in biological fluids and resistance to various storage conditions,
miRNAs are considered as useful biomarkers for cancer diagnosis, prognosis, and prediction of
treatment efficacy. Previous reports have shown that miRNAs have the potential to improve current
clinical practice to distinguish aggressive from the nonaggressive prostate cancer. Adopting BioGenex
Super Sensitive Nucleic Acid ISH-based microRNA (SSNA miRNA ISH) detection, the
expression pattern of miRNAs in prostate cancer and BPH can be successfully differentiated.
Super Sensitive Nucleic Acid (SSNA) miRNA probes:
BioGenex has developed proprietary SSNA miRNA probes that are specially designed to enhance signals
from the intrinsically low populated miRNAs. These probes have high melting temperatures enabling
stringent washes at elevated temperatures to remove non-specific binding. BioGenex miRNA probes are
dual-end labeled with a fluorophore that amplifies the signal, giving intense stains. Overall, SSNA
miRNA probes aid in studying the lowly expressed miRNA populations to assess the physiological
function of miRNA.
This Application Note addresses the feasibility of using BioGenex SSNA miRNA ISH probes for
differentiation of prostate cancer and BPH. In situ visualization of miRNA provides a benefit of
localization of miRNA inside a tumor cell, which is essentially lost during miRNA isolation used in the
high-throughput screening methods. The original study and the results were presented as posters in
USCAP (1).
Study samples and detection methods:
miRNA expression profiles were evaluated in 166 formalin-fixed paraffin-embedded (FFPE) cases of
different grades of prostate cancer, including paired normal prostate and BPH (1). Prostate cancer
tissues and BPH were differentiated using the BioGenex Xmatrx®
automated system and miRNA ISH
Prostate panel probes.
Results and conclusion:
As shown in figure 1, miR-17 expression levels were strongly overexpressed in 50% (13/26) of prostate
cancer IV compared with normal cases, while miR-125b was strongly upregulated in over 77% (20/26)
of cases of prostate cancer IV and III (Table 1, Figure 2). However, miR-17 and miR-125b did not show
any significant variation in expression pattern in BPH cases, suggesting oncogenic role of these miRNAs
in prostate cancer tumorigenesis and differentiation (1).
Application Note
Table 1: In situ expression profile of miRNAs in normal, BPH and different grade of prostate cancer (PCa).
miR-17
Pattern
BPH
(n=20)
P Normal
(n=18)
PCa II
(n=18)
P Normal
(n=29)
PCa III
(n=29)
P Normal
(n=26)
PCa IV
(n=26)
Negative 9 6 3 10 4 10 2
Weak 3 2 1 4 1 4 0
Moderate 4 8 7 13 14 10 11
Strong 4 2 7 2 10 2 13
miR-125b
Negative 3 3 1 1 0 1 0
Weak 3 5 3 17 5 14 2
Moderate 7 6 3 3 3 6 4
Strong 7 4 11 8 21 5 20
Application Note
Datasheets:
The BioGenex miRNA probe datasheets provide additional information on the recommended usage
guidelines and storage. Refer to the datasheets below before use:
• HM125B-100 • HM017-100
Disclaimer:
The research group and authors have expressed no conflict of interest. BioGenex has optimized the
protocols for optimal staining results, using positive tissue controls. Due to complex ISH procedures
care should be taken in each step. Variations in tissue embedding and fixation and tissue nature should
be taken into account for variation in results. Reagents and probes must be prepared and handled
according to the manufacturer's instructions.
Refer to the user manual for the automated detection kit and manual kit
1. DF400-YADE: XISH™ One-Step Polymer-HRP ISH Detection Kit (Automation)
2. DF400-50KE: Super Sensitive One-Step Polymer-HRP ISH Detection Kit (Manual)
PCa II PCa III PCa IV
Paired Normal Paired Normal Paired Normal
Figure 2. miR-125b ISH expression profile in different grade of prostate cancer.
These findings underscore the importance of visualizing miRNA expression profile in situ on a cellular
level. Previous studies have indicated that miR-125b is usually downregulated in prostate cancer
and its low expression is associated with poor survival (2-4), In another study, overexpression of
miR-17 in prostate cancer biopsies provided evidence for a stem-cell-like miRNA profile in high
grade/high stage tumors (5).
In summary, miRNA ISH probes can be successfully used for differentiation of prostate cancer and
BPH, thereby providing the feasibility of using these miRNAs in the diagnosis, prognosis, and
therapy regime of prostate cancer patients. BioGenex SSNA miRNA ISH probes give consistent,
reproducible, and reliable outcomes. Adaptation of automated processing using Xmatrx®
in ISH
procedure eliminates error-prone manual steps and greatly increases reproducibility, accuracy and
sensitivity of the test results.
Normal Pca IV (miR-17)
Figure 1. ISH expression profile of miR-17 in normal and high-grade prostate cancer.
Accelerating the pace of precision medicine
In the U.S., call +1 (800) 421-4149
Outside the U.S., call +91-40-27185500
For Research Use Only (RUO). Not for use in diagnostics procedure
Doc. No. 937-4118.0 Rev A
© 2018 BioGenex Laboratories, Inc. All Rights Reservedwww.biogenex.com
Application Note
References:
1. Thakur S et al. In situ expression profiling of microRNA in different grades of prostate cancer.
Presented as Poster in Annual Meeting of the United States & Canadian Academy of Pathology (USCAP),
2015.
2. Giangreco AA et al. Tumor suppressor microRNAs, miR-100 and -125b, are regulated by 1,25-
dihydroxyvitamin D in primary prostate cells and in patient tissue. Cancer Prev Res (Phila).
2013;6:483-94.
3. Yu J et al. miR-200b suppresses cell proliferation, migration and enhances chemosensitivity in
prostate cancer by regulating Bmi-1. Oncol Rep. 2014;31:910-8.
4. Wang N et al. miR-205 is frequently downregulated in prostate cancer and acts as a tumor suppressor
by inhibiting tumor growth. Asian J Androl. 2013;15:735-41.
5. Gaston SM et al. Overexpression of miR-17 family miRNAs in prostate cancer biopsies: Evidence for
a stem-cell-like miRNA profile in high grade/ high stage tumors. Cancer Research 2010;70:3049.
For additional Application Notes, visit www.biogenex.com

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prostate cancer classification - BioGenex

  • 1. Keywords: Benign prostatic hyperplasia, In situ hybridization, miRNA, Prostate cancer, Xmatrx® Application Highlights: • Both benign prostatic hyperplasia (BPH) and prostate cancer remain the most prevalent urologic health concerns affecting elderly men in their lifetime, frequently coexist and therefore share common symptoms. • Distinguishing nonaggressive BPH from aggressive prostate cancer is crucial for effective treatment and better clinical outcome. • Several techniques for diagnosing prostate cancer have evolved over the past decades; however there are concerns on spatial resolution of these diagnostic tools. • BioGenex Xmatrx® automated systems and BioGenex miRNA ISH Prostate panel probes were used to successfully differentiate prostate cancer and BPH. • Downregulation of miRNAs involved in cellular regulation and upregulation of miRNA in disease progression were observed. • The in situ experimental conditions for hybridization were optimized for both BioGenex manual and automated systems. BioGenex Products Used: • #HM125B-100: miR-125b • #HM017-100: miR-17 • #DF400-YADE: XISH™ One-Step Polymer-HRP ISH Detection Kit (Automation) • #DF400-50KE: Super Sensitive One-Step Polymer-HRP ISH Detection Kit (Manual) Accelerating the pace of precision medicine Introduction: Prostate cancer and benign prostatic hyperplasia (BPH) are the most frequent pathologies of the prostate gland that are responsible for morbidity in men. Unlike prostate cancer, BPH is nonmalignant and nonfatal. BPH and prostate cancer frequently coexist and therefore share common symptoms; over 20% of men with prostate cancer have BPH. There remains an important clinical challenge in prostate oncology to distinguish the aggressive from the nonaggressive form, without requiring all patients to undergo a painful tissue biopsy. In the United States, prostate cancer is the second leading cause of cancer death affecting one in nine men. This year, an estimated 164,690 men in the United States will be diagnosed with prostate cancer and 29,430 will die from it. Therefore, early diagnosis and timely detection of disease progression is a very important step for effective treatment and a beneficial clinical outcome. Although several techniques for diagnosing prostate cancer have evolved over the past decades, lack of specificity and sensitivity of these diagnostic tools hinder their application in clinical practice. Additionally, the existing methods to utilize serum prostate-specific antigen (PSA) does not provide enhanced survival rates nor do these techniques offer absolute results for the differentiation of prostate cancer and BPH. Elevated levels of PSA have also been reported in non-malignant conditions of the prostate, including BPH, which is commonly misdiagnosed as prostate cancer, leading to unnecessary biopsies. Furthermore, metastatic and advanced prostate tumors types respond very poorly to chemotherapy. Accumulating data suggest that small noncoding RNAs such as microRNAs (miRNAs) can be utilized as potential biomarkers for differentiation of prostate cancer and BPH. Application Note Ready-to-Use fully optimized SSNA miRNA in situ hybridization (ISH) Kit Prostate Cancer (PC) and Benign Prostatic Hyperplasia (BPH) Differentiation by New miRNA Biomarker Panel
  • 2. miRNAs are small, evolutionarily conserved, noncoding RNAs that negatively regulate gene expression in divergent cellular processes including development, differentiation, proliferation, cell cycle control, and apoptosis. Owing to their stability in biological fluids and resistance to various storage conditions, miRNAs are considered as useful biomarkers for cancer diagnosis, prognosis, and prediction of treatment efficacy. Previous reports have shown that miRNAs have the potential to improve current clinical practice to distinguish aggressive from the nonaggressive prostate cancer. Adopting BioGenex Super Sensitive Nucleic Acid ISH-based microRNA (SSNA miRNA ISH) detection, the expression pattern of miRNAs in prostate cancer and BPH can be successfully differentiated. Super Sensitive Nucleic Acid (SSNA) miRNA probes: BioGenex has developed proprietary SSNA miRNA probes that are specially designed to enhance signals from the intrinsically low populated miRNAs. These probes have high melting temperatures enabling stringent washes at elevated temperatures to remove non-specific binding. BioGenex miRNA probes are dual-end labeled with a fluorophore that amplifies the signal, giving intense stains. Overall, SSNA miRNA probes aid in studying the lowly expressed miRNA populations to assess the physiological function of miRNA. This Application Note addresses the feasibility of using BioGenex SSNA miRNA ISH probes for differentiation of prostate cancer and BPH. In situ visualization of miRNA provides a benefit of localization of miRNA inside a tumor cell, which is essentially lost during miRNA isolation used in the high-throughput screening methods. The original study and the results were presented as posters in USCAP (1). Study samples and detection methods: miRNA expression profiles were evaluated in 166 formalin-fixed paraffin-embedded (FFPE) cases of different grades of prostate cancer, including paired normal prostate and BPH (1). Prostate cancer tissues and BPH were differentiated using the BioGenex Xmatrx® automated system and miRNA ISH Prostate panel probes. Results and conclusion: As shown in figure 1, miR-17 expression levels were strongly overexpressed in 50% (13/26) of prostate cancer IV compared with normal cases, while miR-125b was strongly upregulated in over 77% (20/26) of cases of prostate cancer IV and III (Table 1, Figure 2). However, miR-17 and miR-125b did not show any significant variation in expression pattern in BPH cases, suggesting oncogenic role of these miRNAs in prostate cancer tumorigenesis and differentiation (1). Application Note Table 1: In situ expression profile of miRNAs in normal, BPH and different grade of prostate cancer (PCa). miR-17 Pattern BPH (n=20) P Normal (n=18) PCa II (n=18) P Normal (n=29) PCa III (n=29) P Normal (n=26) PCa IV (n=26) Negative 9 6 3 10 4 10 2 Weak 3 2 1 4 1 4 0 Moderate 4 8 7 13 14 10 11 Strong 4 2 7 2 10 2 13 miR-125b Negative 3 3 1 1 0 1 0 Weak 3 5 3 17 5 14 2 Moderate 7 6 3 3 3 6 4 Strong 7 4 11 8 21 5 20
  • 3. Application Note Datasheets: The BioGenex miRNA probe datasheets provide additional information on the recommended usage guidelines and storage. Refer to the datasheets below before use: • HM125B-100 • HM017-100 Disclaimer: The research group and authors have expressed no conflict of interest. BioGenex has optimized the protocols for optimal staining results, using positive tissue controls. Due to complex ISH procedures care should be taken in each step. Variations in tissue embedding and fixation and tissue nature should be taken into account for variation in results. Reagents and probes must be prepared and handled according to the manufacturer's instructions. Refer to the user manual for the automated detection kit and manual kit 1. DF400-YADE: XISH™ One-Step Polymer-HRP ISH Detection Kit (Automation) 2. DF400-50KE: Super Sensitive One-Step Polymer-HRP ISH Detection Kit (Manual) PCa II PCa III PCa IV Paired Normal Paired Normal Paired Normal Figure 2. miR-125b ISH expression profile in different grade of prostate cancer. These findings underscore the importance of visualizing miRNA expression profile in situ on a cellular level. Previous studies have indicated that miR-125b is usually downregulated in prostate cancer and its low expression is associated with poor survival (2-4), In another study, overexpression of miR-17 in prostate cancer biopsies provided evidence for a stem-cell-like miRNA profile in high grade/high stage tumors (5). In summary, miRNA ISH probes can be successfully used for differentiation of prostate cancer and BPH, thereby providing the feasibility of using these miRNAs in the diagnosis, prognosis, and therapy regime of prostate cancer patients. BioGenex SSNA miRNA ISH probes give consistent, reproducible, and reliable outcomes. Adaptation of automated processing using Xmatrx® in ISH procedure eliminates error-prone manual steps and greatly increases reproducibility, accuracy and sensitivity of the test results. Normal Pca IV (miR-17) Figure 1. ISH expression profile of miR-17 in normal and high-grade prostate cancer.
  • 4. Accelerating the pace of precision medicine In the U.S., call +1 (800) 421-4149 Outside the U.S., call +91-40-27185500 For Research Use Only (RUO). Not for use in diagnostics procedure Doc. No. 937-4118.0 Rev A © 2018 BioGenex Laboratories, Inc. All Rights Reservedwww.biogenex.com Application Note References: 1. Thakur S et al. In situ expression profiling of microRNA in different grades of prostate cancer. Presented as Poster in Annual Meeting of the United States & Canadian Academy of Pathology (USCAP), 2015. 2. Giangreco AA et al. Tumor suppressor microRNAs, miR-100 and -125b, are regulated by 1,25- dihydroxyvitamin D in primary prostate cells and in patient tissue. Cancer Prev Res (Phila). 2013;6:483-94. 3. Yu J et al. miR-200b suppresses cell proliferation, migration and enhances chemosensitivity in prostate cancer by regulating Bmi-1. Oncol Rep. 2014;31:910-8. 4. Wang N et al. miR-205 is frequently downregulated in prostate cancer and acts as a tumor suppressor by inhibiting tumor growth. Asian J Androl. 2013;15:735-41. 5. Gaston SM et al. Overexpression of miR-17 family miRNAs in prostate cancer biopsies: Evidence for a stem-cell-like miRNA profile in high grade/ high stage tumors. Cancer Research 2010;70:3049. For additional Application Notes, visit www.biogenex.com