MicroRNA in situ hybridization for cancer diagnosis

Summary: MicroRNAs (miRNA) are approximately 19–24 nucleotides in length and base-pair to complementary sites within messenger RNA (mRNA). Aided by the “RISC” complex this binding functions to promote down-regulation of the mRNA’s protein product. MicroRNAs are an important biomarker of cancer as a result of their involvement in multiple biological processes including development, differentiation, proliferation, metabolism, and apoptosis. Cancer is now diagnosed by two different but complementary modalities, liquid biopsy, and tumor tissue biopsy. Tumor tissue biopsy is still the “gold standard” for cancer diagnosis by pathologists. ISH is ideally suited for detecting miRNA in the “gold standard” of fresh, frozen or formalin-fixed paraffin-embedded (FFPE) biopsy samples due to its sensitivity, specificity and the spatial information on tumor heterogeneity it provides, available at relatively low cost. BioGenex ISH system has the ability to generate a robust chromogenic signal while preserving the spatial context of the tissue sample. This provides a powerful tool for precise tumor characterization and a breakthrough for clinical research and analysis of cancer of unknown primary (CUP), poorly differentiated or undifferentiated tumors, and cancer staging.

Conclusion: Only the ISH techniques give the full spatial picture of the tumor which is crucial to aid the pathologist in diagnosis and for initiating tumor treatment decisions.

Introduction

MicroRNAs (miRNAs) were discovered in 1993 by Rosalind Lee and Rhonda Feinbaum in the laboratory of Victor Ambrost at Harvard University. [Lee] They were studying the development of the nematode worm Caenorhabditis elegans. MicroRNA, as a class of evolutionarily conserved small RNA are approximately 19–24 nucleotides in length that base-pair to sites within messenger RNA (mRNA), targeting these transcripts for down-regulation. MicroRNA target the full spectrum of cellular activities including development, differentiation, proliferation, metabolism, and apoptosis. As a result, they play a prominent role in infectious diseases and cancer. [Bhaskaran]

MicroRNA are important biomarkers for cancer screening, diagnosis, including cancers of unknown primary location, and prognostication. Tumor tissue biopsy is still the “gold standard” for cancer diagnosis and in situ hybridization is a particularly suitable miRNA detection method for diagnosis and prognostication. ISH is optimal due to its sensitivity and specificity, particularly the spatial tumor heterogeneity data it can provide. Compared to other techniques for miRNA analysis only in situ hybridization cost-effectively provides the spatial information routinely needed by pathologists for tumor grading, staging, and assessment of heterogeneity.

miRNA as a biomarker for cancer diagnosis

Next generation sequencing studies have shown that certain miRNAs, termed oncomiRNAs, are often upregulated, while others are downregulated in cancer, meaning that they can potentially serve as useful biomarkers for diagnosis and monitoring of disease progression. [Li J] [Wua]

Many miRNA can act as tumor suppressors, such as miR-340, miR-320a, miR-215,miR-143-5p,miR-18a. Their tumor suppressor potential is defined by the absence in tumor but presence in matched control tissue. It is possible for a given miRNA to be both a tumor suppressor and an oncomiRNA depending on the tumor tissue. As an example, miR-215 is reported to be an oncomiRNA in glioma, but a tumor suppressor in non-small cell lung cancer. [Meng] [Hou]

The field of cancer diagnostics is moving towards earlier detection with cancer screens at the systemic level, meaning blood, urine, and saliva “liquid biopsy”tests. [Wub] Blood circulating biomarkers are of particular interest and these include miRNA.

The expression signature of miRNAs isolated from urinary exosomes can also be used. [Gheinani] These approaches involve workflows for cancer research that involve non-invasive extraction and purification of ribonucleic acid (RNA).

Microarray, PCR, and next generation sequencing are able to quantify cell-free miRNA circulating in the blood. These circulating miRNAs are often located within cancer cell-derived vesicles known as exosomes. Circulating miRNA signatures for many types of solid tumors have been determined including lung, colorectal, gastric, breast, prostate, and pancreas cancers. [Zhang] [Baek] [Zhou] [Zou] [Sita-Lumsden] [Joshi] The detection of these miRNA signatures in the blood indicates the presence of cancer. The technique of ISH is a better option for assaying this cancer tissue than the aforementioned techniques.

The composition of circulating miRNA can predict cancer response to treatments such as checkpoint immunotherapy, kinase inhibitors, chemotherapy, and radiotherapy. [Sudo] [Lin] [Baek]These signatures can also be used to predict the risk of metastasis of particular cancer. [Pi] These same signatures can be ascertained in cancer tissue by ISH.

The abundance of miRNA in exosomes seems to be dependent on the biofluid from which they are isolated. Exosomes isolated from the blood appear to contain more miRNA than exosomes isolated from urine. [Li M]Blood exosomes may also contain considerably more miRNA compared to mRNA, making blood miRNA a better cancer biomarker. The absolute numbers of specific miRNAs, for example,miR-200c, may be quite low, on the order of one or less per exosome, on average, even from exosomes isolated from blood. [Chevillet] This necessitates very sensitive miRNA detection methods for samples from liquid biopsies.

MicroRNA are also useful for traditional cancer diagnoses and prognosis using tissue biopsies. Panels of miRNA are potentially associated with tumor formation, tumor stage and grade [Nam] [Zakrzewska], and cancer patient survival. [Wong]

Exosomes may mediate oncogenic properties such as drug resistance by fusing with cells and releasing their contents,including miRNA, within the cytoplasm. It is thought that cancer cells can spread drug resistance throughout the tumor via exosomes. This means that, potentially, only a limited number of tumor cells may act as drug resistance exosome factories, releasing “doses” that confer resistance upon the entire tumor. The presence of exosomal miRNA within tumor cells are markers of this process and can be detected with fluorescence ISH (FISH). [Zhao]

Tumor tissue biopsy is the “gold standard” needed to accurately grade the tumor and initiate patient treatment algorithms.Although useful for initial diagnosis, the results of liquid biopsies are binary, that is they only predict the presence of cancer, or the likelihood of a particular property of cancer being present in the tumor, such as resistance to therapy, or the ability to metastasize. They provide no information on the heterogeneity of the tumor or indeed its grade. Liquid biopsies can predict metastasis, but this is often graded independent, that is metastasis can occur early, with only a few of the cells in the tumor mass possessing the capacity to metastasize. Alternatively,metastasis can occur late, as traditional models of cancer progression predict. The grade of the primary tumor remains unknown without direct biopsy and analysis by a pathologist, which can be greatly aided by the technique of FISH for miRNA detection.

The in situ hybridization approach vs other techniques

Techniques such as digital droplet PCR, microarray hybridization, and RNA extraction-based next-generation sequencing are more appropriate for the detection of miRNA in liquid biopsies due to their sensitivity and multiplex capacity, however, FISH comes into its own for pathological tumor tissue grading. FISH can provide spatial and low-cost cell distribution (tumor heterogeneity) data which is not possible by microarray or traditional next-generation sequencing workflows. Single-cell sequencing can provide spatial and cell distribution data;however, it is currently prohibitively expensive for routine diagnostic use. Therefore, chromogenic in situ hybridization (CISH) or FISH is currently the most viable, cost-efficient options for the practicing tumor pathologist to provide quantitative and spatial miRNA data from tumor tissue biopsies, fresh, frozen or formalin-fixed paraffin-embedded (FFPE).

CISH is the most cost-effective ISH with a very stable signal requiring only basic laboratory equipment and uses very well-established peroxidase or alkaline phosphatase reactions viewed under a standard

bright-field microscope.It is compatible with FFPE, blood or bone marrow smears, and other types of fixed cells. The CISH methodology may be used to evaluate the presence of a particular miRNA, however, in contrast to FISH, it is not suitable for multiplexing and is not effective for defining the sub cellular localization of the miRNA. The lack of multiplex capacity is a major limitation, however for some diagnostic miRNA applications, single miRNAs such as miR-200c are associated with drug response and therefore CISH is a suitable tumor assay. [Lin] However, most diagnostic assays involving miRNAs require panels, for which CISH is inadequate. FISH, on the other hand, is highly compatible with multiplexing.

FISH involves the hybridization of a fluorescently labeled probe designed to hybridize with a specific target DNA or RNA of interest, such as miRNA, inside fixed cells. The resulting cells containing the probes can be analyzed by digital microscopy or flow cytometry. The requirement for such equipment makes FISH more expensive than CISH. FISH probes are also subject to photobleaching making it important to rapidly capture as much digital information as possible from FISH slides as they cannot be stored indefinitely.

The clinical advantage of using miRNA ISH

Cancers of unknown primary (CUPs) are metastatic tumors that have disseminated from unknown sites. Traditionally tumors are classified by the sites in which they originate, making treatment decisions difficult if the location of the primary tumor is unknown. Approximately 3%–5% of all cancer presentations are CUP. Determining the location of the primary tumor is essential for both surgical and targeted drug interventions. Patients with metastases of unknown primary origin have a poor prognosis and short survival. Fortunately, miRNA has certain properties that make it useful as a diagnostic tool for identifying the origin of CUPs.

In normal tissues, miRNA expression is highly specific. This property has been used to train tumor origin classifiers with an accuracy of between 88-92% for identifying the tumor site of origin when applied to unknown metastases. [Søkilde] [Pentheroudakis] This was accomplished by profiling metastasis of known origin using microarrays. These same miRNAs can be profiled by multiplex FISH, at low cost, by pathologists seeking to identify the primary site of a CUP.

Differentiation refers to the process of cell maturation from an undifferentiated stem cell to a mature morphologically stable or differentiated cell. Many cancer cells possess the property of reverting from a differentiated state to a more stem-like or undifferentiated state. The prognosis of patients with undifferentiated cancers is generally poor. Once again, due to the fact that miRNA expression is normally highly tissue-specific, it is possible to infer the degree of cancer cell de-differentiation by comparing the degree of divergence of the miRNA profile from that expected towards that present in stem cells. This can aid in cancer prognosis. Generally, the lower the differentiation score the worse the prognosis.

As an example, in undifferentiated gastric cancer 3 miRNAs (mir-34b, mir-34c, and mir-128a) were significantly upregulated and 3 miRNAs (mir-128b, mir-129 and mir-148) were downregulated compared to normal gastric tissue. The probability of survival was significantly lower in patients with a high expression level of mir-20b or 150 and there was a correlation between mir-27a and lymph node metastasis. [Katada] With validation in larger studies this could potentially be integrated into prognostic algorithms for undifferentiated gastric cancer.

Staging is an algorithm that pathologists use in order to classify cancer based on its spread within the body. The staging takes account of the size of the primary tumor, whether cancer has metastasized and where the secondary tumors are located. The stages are influenced by specific factors for each cancer type. The TNM staging system classifies as Tumor (T); lymph node involvement (N) and metastatic spread (M).

Grading is also an algorithm for classifying cancers. The pathologist gives cancer a grade based on its differentiation or stem-ness, its doubling time, and the likelihood of metastasis, although as previously mentioned tumor grade does not always predict tumor stage due to heterogeneity.

Both tumor stage and tumor grade can potentially be predicted by the miRNA profile of the tumor, which can be determined by FISH. [Pi] [Katada]

Cancer from each anatomical location can be divided into different types and sub-types. Taking breast cancer as an example, the different types include ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), invasive ductal carcinoma, invasive lobular carcinoma, metaplastic breast, and inflammatory breast cancer. Depending on the stage and grade of cancer it can sometimes be difficult to determine its subtype.

Due to the specific expression of miRNAs within certain tissues in healthy individuals, it is possible to use miRNA signatures within the tumors to give an indication of its origin and subtype. The detection of the signature can be accomplished by FISH.

Conclusion

Tumor diagnosis is greatly complicated by heterogeneity, the cells within a tumor can be very different from each other. This impacts grading, staging, subtyping, and determining the differentiation state. A tumor can be a mixture of cells with different stages and grades. This property of cancer is missed by most analytical techniques such as standard PCR, microarray, and next-generation sequencing which give the average readout of the tumor composition. This may not accurately reflect the true nature of the tumor. Only the in situ hybridization techniques give the full spatial picture of the tumor which is crucial to aid the pathologist in diagnosis and for initiating tumor treatment decisions.

BioGenex miRNA solutions for cancer diagnosis

BioGenex has pioneered the development of miRNA research and diagnostics tools with leading-edge products. Currently, over 240 BioGenex ready-to-use (RTU) Super Sensitive^TM Nucleic Acid (SSNA) miRNA ISH probes are available for accurate and early diagnosis of the tumor. These probes are sensitive enough to detect low-abundance miRNA(s), which is often required for biomarker discovery. They have a high melting temperature enabling stringent washes to remove non-specific binding. BioGenex miRNA probes are dual-end labeled with an anti-fluorophore to amplify the signal, and yielding a clean intense staining.

miRNA specific probes and in situ hybridization kit, developed by BioGenex, allow rapid, sensitive detection of miRNAs with high specificity while preserving tissue morphology. Each kit includes easy-to-follow protocols and ready-to-use (RTU) reagents. Due to high sensitivity and specificity of the probes, hybridization kits can be used for either manual or high-throughput automated staining. BioGenex SSNA miRNA probes combined with the automated processing using Xmatrx® platforms greatly increases the reliability of the test results through the elimination of error-prone ISH procedure. BioGenex fully-automated molecular pathology workstations are the most advanced system globally.

In addition, BioGenex provides the Xmatrx® MINI, a compact, complete, manual slide staining system for miRNA-ISH, CISH and in situ PCR that greatly optimizes and simplifies the workflow. BioGenex also offers solutions for automation, which reduce labor and require fewer reagents. Xmatrx® NANO is an “efficiency” microfluidic system that automates miRNA-ISH processing. The all-in-one small-footprint system is economical, and ideal for FISH, as well as in situ PCR, and CISH assays. For FISH applications, the scientist or pathologist simply needs to load the slides, select the application, pipette the micro-reagents when prompted and digitally capture the images.This can reduce at least 33 manual steps to only six with the resulting cost saving.

BioGenex also offers the new NanoVIP® a fully automated system for miRNA-ISH, FISH, CISH, and in situ PCR assays. It may be particularly suitable for multiplex miRNA FISH. Reliable automation combines with eXACT™ temperature modules and liquid level sensors for accurate liquid handling to guarantee robust and reproducible results. Ten different protocols can be run simultaneously, ideal for protocol optimization. NanoVIP® can reduce 33 step manual FISH to 3 simple steps a) load slides, b) select protocol, and c) capture images.

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