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miRNA-ISH for Cancer of Unknown Primary

miRNA-ISH for Cancer of Unknown Primary


World-wide approximately 5% of all cancers are determined to have an occult or unknown primary location, upon initial assessment, prior to completing a full pathology report. [Oien] Unfortunately in 20-50% of CUP cases the primary tumor is never found. These cancers of unknown primary (CUP) result from metastasis. Occult cancer is most frequent in individuals around 60 years of age and an estimated 31,480 cases of CUP will be diagnosed in the US in 2019, making up around 2% of all US cancers. [Siegel] People with a family history of lung, kidney, or colorectal cancer are more likely than average to develop occult cancer. [Hemminki] Median survival time is 8-12 months, 80% of patients live only 6 months after diagnosis. [Losa]



Patients with metastatic cancer are often recruited to clinical trials using investigational drugs due to a lack of effective market approved treatment options. CUPs are most often found in the liver, lung, bone, brain; lymph nodes; and peritoneal and pleural serous cavities. [Oien] Once a tumor has been discovered, in order to provide a working diagnosis for the treating oncologist, a detailed pathology report must be prepared. A crucial component of this report is a microRNA in situ hybridization (miRNA-ISH) based classification of the tumor to aide categorization and for determining the site of origin.


The pathologist will initially grade the tumor, based on a core needle biopsy or fine-needle aspiration that has been fixed, with the aid of hematoxylin and eosin (H&E) and cytogenetic stains. This grading process may lead the pathologist to suspect that the tumor is a CUP, for example, it is grossly histologically different from the surrounding tissue. This suspicion can be confirmed or rejected by the technique of immunohistochemistry, using panels of antibodies against specific tumor markers. For example, pan-keratin antibodies can determine if the tumor is a carcinoma. This method can be a powerful way to classify cancer and determine the metastatic status but is not very location specific. The pathologist still may not be confident of the location of the primary tumor, particularly if it is an adenocarcinoma. At this point techniques such as in situ hybridization (ISH), that provide information about the tumor DNA and RNA, can help the pathologist confidently predict the location of the primary tumor. Tumor sequencing is, explicitly, not currently recommended by the National Comprehensive Cancer Network (NCCN) clinical practice guidelines on CUPs.

MicroRNA, as a class of evolutionarily conserved small RNA, is 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 conditions such as cancer. [Bhaskaran]

A CUP is a clonal derivative of the primary tumor which has migrated and established itself in a new location within the body. The genetic makeup of the CUP is therefore similar to that of the primary tumor. [Gevaert] This provides a vital clue for where to look for the primary. Developmental/ differentiation genes and their products, such as microRNA (miRNA) highly tissue specific and their activity/ expression can help establish the origin of a particular tumor. A caveat of this is that the primary tumor location of a poorly differentiated CUP will be particularly difficult to predict. In such cases, imaging of the lung, kidney, colon, and rectum should be prioritized as these are known likely sites of origin for CUP. [Hemminki]

The most common form of CUP is adenocarcinoma (60%), and poorly/undifferentiated carcinoma (25-30%). Although IHC is adequate for determining the tumor type, it is not powerful in the prediction of tumor origin. Gene profiling including genes that encode miRNA, has been shown to be superior for tumor origin prediction in poorly differentiated cancers. [Handorf] That said, sequencing is neither cost-effective nor recommended by NCCN clinical guidelines for CUP.

On the other hand, ISH is an ideal cost-effective approach that the pathologist can use in the clinical setting to detect miRNAs, as well as other important DNA markers, for cancer diagnosis. [Song] MicroRNA detected using ISH can complement, but not replace IHC in the classification of the CUP tumor type. Further, it can be used to predict the origin of the tumor better than is currently possible with IHC alone. For this purpose formalin-fixed, paraffin-embedded (FFPE) samples are suitable for the preservation of tumor miRNA and subsequent analysis by ISH. [Xi]

Relative to normal tissue, the levels of miRNA in tumors may increase or decrease in a way that is characteristic of particular cancer types. This property can be used to classify cancers. For example, researchers from the University of Kerala, India, used microRNA transcriptome data to rank the differential expression of miRNA in lung, liver, and bladder cancers compared to matched normal tissue. [Salim] Individual tumor differential miRNA expression profiles were used to train a support vector machine, a type of statistical algorithm (computer program) used to classify inputs, such as the miRNA expression profile of a CUP. This profile could be determined by ISH. Under the experimental conditions the researchers were working with, their classifier had an accuracy of over 90% for lung, bladder and liver tumors. Although lung cancer is a common origin of CUPs the classifier was not specifically designed for use in CUPs, but it demonstrated clear potential for determining the tumor type of a CUP.

The primary tumor of a CUP is not found in up to 50% of cases using conventional IHC techniques alone. Fortunately, research and development have uncovered new ways of locating the primary tumor using miRNA panels. Researchers from the Comprehensive Cancer Center of Ohio State University in the USA and Universita di Ferrara in Italy identified a 47-miRNA signature in FFPE samples to estimate tissue-of-origin probabilities for CUPs. Primary site predictions had probabilities success higher than 90% in most cases. [Ferracin] An independent group of researchers also developed a tumor origin classifier using miRNAs that had a similar 88% positive identification rate. [Søkilde] This is a clear improvement over current practice.

Further research could focus on generating classifiers based solely on miRNA expression profiles of CUPs determined by ISH, as this data could be readily generated by a pathologist. It will also be important to develop positive control reference standards for miRNAs preserved in FFPE samples from different cancers, to allow the pathologist to verify that their ISH assay systems for miRNA are working as expected and to compare miRNA expression levels in a standardized fashion, optimally in an automated fashion. [Boisen]


As miRNA panels are integrated into routine pathology work-ups on CUPs, this will increase the proportion of CUPs where the primary is resolved. ISH is ideal for identifying the primary site of a tumor, as, in addition to miRNA, DNA rearrangements classically detected by ISH are also useful for classifying the tumor site of origin. [Pantou]. Identification of the primary site is crucial in order for these patients to be eligible for the best possible treatment in clinical trails using investigational drugs. Any other treatment is usually merely palliative, as there are rarely effective market approved drugs for patients with metastatic disease.

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 SensitiveTM 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.

More Information on our Biomarker Panels!

Get miRNA Catalog

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.


  1. Bhaskaran M. and Mohan M. MicroRNAs: history, biogenesis, and their evolving role in animal development and disease. 2014. Vet Pathol.
  2. Boisen MK. MicroRNA Expression in Formalin-fixed Paraffin-embedded Cancer Tissue: Identifying Reference MicroRNAs and Variability. 2015. BMC Cancer.
  3. Ferracin M. et al. MicroRNA profiling for the identification of cancers with unknown primary tissue-of-origin. 2011. J Pathol.
  4. Gevaert O. et al. A taxonomy of epithelial human cancer and their metastases. 2009. BMC Med Genomics.
  5. Handorf CR. et al. A multicenter study directly comparing the diagnostic accuracy of gene expression profiling and immunohistochemistry for primary site identification in metastatic tumors. 2013. Am J Surg Pathol.
  6. Hemminki K. et al. Familial risks in cancer of unknown primary: tracking the primary sites. 2011. J Clin Oncol.
  7. Losa F. et al. SEOM clinical guideline on unknown primary cancer (2017). 2018. Clin Transl Oncol.
  8. Oien KA. and Dennis JL. Diagnostic work-up of carcinoma of unknown primary: from immunohistochemistry to molecular profiling. 2012. Ann Oncol.
  9. Pantou D. et al. Cytogenetic profile of unknown primary tumors: clues for their pathogenesis and clinical management. 2003. Neoplasia.
  10. Salim A. et al. An approach to forecast human cancer by profiling microRNA expressions from NGS data. 2017. BMC Cancer.
  11. Siegel RL. et al. Cancer statistics, 2019. CA Cancer J Clin.
  12. Søkilde R. et al. Efficient identification of miRNAs for classification of tumor origin. 2014. J Mol Diagn.
  13. Song R. et al. In situ hybridization detection of microRNAs. 2010. Methods Mol Biol.
  14. Xi Y. et al. Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. 2007. RNA.

Topics: in situ hybridisation, ISH, miRNA, miRNA biomarkers, Cancer diagnostics, cancer of unknow primary

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