Global microRNA profiling using novel miRCURY LNATM microarrays enables identification of tumors of unknown primary originRolf Søkilde*, Poul Erik Høiby*, Boye Schnack Nielsen*, Kim Bundvig Barken*, Alastair Hansen**, Søren Møller *, Thomas Litman*

*Exiqon A/S, Skelstedet 16, DK-2950 Vedbæk, Denmark. **Department of Pathology, Herlev University Hospital, DK-2730 Herlev, Denmark.

IntroductionMicroRNAs (miRNAs) constitute a recently discovered class of tissue specific, small, non-coding RNAs, which regulate the expression of genes involved in many biological processes, including development, differentiation, apoptosis and carcinogenesis.

Around 5% of all newly diagnosed metastatic cancers are carcinoma of unknown primary (CUP, Figure 1) origin, where the site of the primary tumor cannot be determined, despite the use of advanced immunohistochemical or radiological techniques. Because effective cancer treatment depends on early identification of the primary tumor, CUP patients have poor prognosis with a median survival of 3-6 months and a 1-year survival rate of less than 25%.

By applying a novel microarray platform based on locked nucleic acid (LNATM) modified detection probes, which enable highly sensitive and specific detection of miRNAs, we are able to identify tumor-specific microRNA signatures for each cancer histology. Our vision is to identify the primary tumor in all cancer patients, without delay, in order to offer the most relevant treatment. As part of this effort we are developing a classifier tool based on miRNA profiles for diagnosis of CUP, enabling individualized treatment of this otherwise intractable group of cancers.

Figure 1

MethodsTissueMore than 500 tumor and normal adjacent tissue samples were collected from both fresh frozen and formalin fixed, paraffin embedded sections. Table 1 lists 18 tissues that are included in our analysis and which represent the most common tissues of origin for CUP.

Table 1

RNA isolationTotal RNA was isolated by guanidinum isothiocyanate/phenol: chloroform extraction.

Microarray profilingOne microgram total RNA was analyzed for miRNA expression on miRCURY LNA™ Discovery arrays containing Tm normalized capture probes for 2090 miRNAs, including 110 human miRPlus sequences not yet annotated in miRBase, +200 miRNAs discovered by 454 high throughput sequencing, as well as the corresponding pre-miRNAs (Figure 2). All hybridizations were made against a common reference pool.

Data analysisA miRNA expression database was established for identification of miRNAs with high discriminatory power between tumors. Both unsupervised hierarchical clustering and supervised analysis was performed to generate a multiclass classifier.

Properties of the LNA™ capture probes and microarraysFigure 3

Using LNA™, the Tm is increased significantly and the Tm range is narrowed significantly, com-pared to DNA probes. This results in increased stringency and optimal hybridization conditions for the LNA™ capture probes.

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Figure 5. It is clear that Supplier A and B have difficulties in designing optimal probes spanning the entire region of GC%.In contrast, the miRCURY LNA™ Array offers optimal performance of all probes usingTm-normalized probes.The unmatched sensitivity and specificity is partly explained by the optimal design of the LNA™ capture probes.Method: Each of 557 synthetic microRNAs were used at a concentration of 50 amol (~500 copies per cell if using 1 μg total RNA).

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ResultsFigure 6

Figure 7. Case story: Identification of the origin of a primary cancer, which has metastasized to a regional lymph node. A biopsy from a lymph node metastasis was profiled on the miRCURY LNA™ microRNA Array. Based on a simple cluster analysis, the lymph node metastasis miRNA profile is shown to belong to the colorectal miRNA cluster and is thus, identified as a colorectal cancer (CRC). This patient did have a primary tumor identified in the colon, from which we also received a sample, as well as from the rectum and from a normal lymph node (LN). All the colorectal samples, including the metastasis, cluster together, and clearly define the origin of the metastasis, namely colon. Therefore, in this case, one would – based on the microRNA profile alone – be able to direct the treatment against a colon cancer without having to perform a full body PET scan searching for the primary tumor. A colonoscopy would be sufficient, as was the case for this patient.

Validation and Localization of miRNAsWith LNA™ technology, tissue specific markers can by validated with in Situ hybridization (ISH). This can potentially increase the strength of CUP classification. Below is shown an ISH experiment, which allows localization of specific miRNAs.

Figure 8

Figure 8: MicroRNA in Situ hybridization in formalin fixed, paraffin embedded tissue sections. A shows staining with a nucleus specific probe, while B shows staining of a microRNA with an LNA™ probe. The normal colon tissue has intact villus structure while the cancer tissue is less differentiated. Upregulation of the microRNA in the cancer tissue is seen compared to the normal tissue. The staining pattern suggests that this particular microRNA is located in the fibroblast like cells adjacent to the tumor cells. Little background staining is observed in the normal tissue.

Conclusions• We have generated a miRNA based classifier, which can identify the origin

of the primary tumor in CUP patients with metastatic disease.• The classifier is based on a limited number of microRNAs and may be improved by including spatial

information on miR expression. • To expand the classifier to include histological subtypes, we are currently sampling Exiqon’s

tumorbank of +150.000 specimens.

BreastBladder/Ureter

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GenitalsBowel

Kidney/AdrenalsLiver/Bile ductsOther

PancreasLungNot Found

Primary site found at autopsy Figure 1. Primary cancer site found at autopsy.The chart depicts the relative proportion of sites of origin found at autopsy in 884 CUP patients in 12 different follow-up studies(from Pentheroudakis et al. Oncologist 2007; 12: 418-425).

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Acknowledgments: We wish to thank Stine Jørgensen and Gitte Friis for excellent technical assistance.

Poster: #5038 - Job: 913866

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Figure 6. The bar diagrams illustrate the differential expression of 7 different microRNAs (the miR numbers are arbitrary) in the 18 tissues. The y-axis is the log-ratio between the tissue microRNA expression and the average expression across all tissues

Figure 2. Procedure for miRNA profiling with miRCURY LNATM-based microarrays.1. Prepare total-RNA2. Label RNA with Hy5 / Hy3 dyes3. Hybridize overnight 4. Scan and analyze

Differential expression of 7 miRNAs across 18 tissues

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Figure 5 Figure 7

Normal colon profile

CRC: Colorectal cancerLNM: Lymph node metastasis

PlacentaHeartAdrenalLymph nodeProximal colonColonRectumCRC LNMCRCKidneyBreastLungCervixBladderPancreasDuodenumSmall intestine

Metastasis from lymph node

Primary tumor profile

Step 1 Metastasis identified in the lymph

Step 2 Biopsy taken: miRNA profile conducted

Step 3 Profile matched normal colon tissue

Step 4 Primary tumor identified in colon

Step 5 Focused and optimized treatment

Normal colon Colon cancer

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Experimental set-up:A dilution series of 557 synthetic microRNAs was hybridized, and for each array the percentageof capture probes detecting microRNAs was plotted.At 50 amol only 40% of the probe of Supplier B and 66% of Supplier A detect their targets. Exiqon capture probes detect 96% of their targets.All arrays were processed according to the manufacturers’ protocols.

Figure 3. The miRCURY LNA™ enhanced microarray is Tm normalized ensuring all miRNA targets hybridize to the array with equal affinity under high stringency hybridization conditions.