Application of Automated RNA in situ Hybridisation in Routine … · 2015. 12. 7. · Biomarker...

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Application of Automated RNA in situ Hybridisation in Routine Clinical Practice: Targeting Albumin, Kappa/Lambda and High Risk HPV

Biomarker Meeting, Munich, Dec 2015

Paul Turner, PhD

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ViewRNA ISH: Dopamine Receptor D1A (Drd1a)

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Examples ISH IHC View RNA ISH (red) IHC (green): Rat Testes Automated

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ViewRNA Assays

Permeabilize cells and unmask RNA RNA-1

RNA-2

1. Pretreat: FFPE/Frozen tissue sections are pretreated to allow access to target RNA

2. Hybridize: Gene specific “II” (double I) probe pairs and blocking probes hybridize to target RNA

3. Amplify: A signal amplification tree is created with a series of sequential hybridization steps or PreAmp, Amp and Labeled Probes

4. Detect: Target RNA is detected chromogenically or fluorescently and visualized with a brightfield or fluorescent microscope

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ViewRNA ISH 2-plex ISH detection in dog kidneys

Data provided courtesy of customer. Target identifications not disclosed.

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RNA ISH Workflow –Automated

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RNA ISH in the Clinical Laboratory

3 RNA Biomarker Assays

• Albumin

• Kappa/Lambda immunoglobulin light chains

• High Risk Human Papilloma Virus (HPV)

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Markers of Hepatocellular Carcinoma

Sensitivity (all grades)

Sensitivity (poorly differentiated)

Specificity

Albumin ISH 99% 99% Excellent

Arginase 84% 71% Excellent

HepPar1 83% 64% Poor

Shahid M, Mubeen A, Tse J, Kakar S, Bateman AC, Borger D, Rivera MN, Ting DT, Deshpande V. Branched chain in situ hybridization for albumin as a marker of hepatocellular differentiation: evaluation of manual and automated in situ hybridization platforms. Am J Surg Pathol. 2015 Jan;39(1):25-34.

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Liver Carcinoma

Hepatocellular Carcinoma

Intrahepatic Cholangiocarcinoma

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Liver primary

Metastasis

Cholangiocarcioma Metastatic carcinoma

Hepatic resection

Chemotherapy

Management of Primary and Secondary Liver Cancers

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Albumin ISH

• 66 of 69 IHCCs (96%) positive for albumin expression

• 8 of 8 well-differentiated (100%)

• 41 of 43 moderately-differentiated (95%)

• 17 of 18 poorly-differentiated (94%)

• 42 of 42 HCCs (100%) were positive for albumin expression

• 0 of 351 non-hepatic carcinomas were positive for albumin expression • Group included adenocarcinomas from lung (N =22), esophagus (N =40),

stomach (N =72), colon (N =40), gallbladder (N=10), pancreas (N =95), urogenital tract (N=8), ovary (N=8), and endometrium (N=8)

• Additionally, 22 carcinomas metastatic to the liver from known primary tumors of the colon, breast and lung were evaluated

Ferrone CR, Ting DT, Shahid M, Konstantinidis IT, Sabbatino F, Goyal L, Rice-Stitt T, Mubeen A, Arora K, Bardeesey N, Miura J, Gamblin TC, Zhu AX, Borger D, Lillemoe KD, Rivera MN, Deshpande V. The Ability to Diagnose Intrahepatic Cholangiocarcinoma Definitively Using Novel Branched DNA-Enhanced Albumin RNA In Situ Hybridization Technology. Ann Surg Oncol. 2014 Dec 18.

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Albumin in the Clinical Laboratory

• 44/M

• Liver transplant

• 7 cm mass in the liver

• ?PET avid lesion in small bowel

• Clinical impression- Metastatic carcinoma!

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H&E Albumin ISH

Intrahepatic Cholangiocarcinoma

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• Lymphoma = Cancer clone of Lymphocytes

• Lymphocytes are either Kappa or Lambda

• Current ISH assay can ONLY detect signal in plasma cells

2. ISH for Kappa (IGKC) and Lambda (IGLC) for the Diagnosis of Lymphoma

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Normal Lymphocytes

Kappa

Lambda

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Kappa Lymphoma (malignant)

Kappa

Lambda

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Lambda Lymphoma (malignant)

Kappa

Lambda

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Type 1 Kappa Type 6 Lambda

Normal Reactive Lymph Node

?

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Type 1 Kappa Type 6 Lambda

Lymphoma Kappa clonal

?

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Kappa Lambda

Vocal Cord Automated

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Lamda Positive: Automated

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Interpretive Algorithm for K/L ISH

Probe Design

IGLL-5

IGLC

IGLC probe = IGLC+IGLL-5

Kshitij Arora, Ivan Chebib, Lawrence Zukerberg, Manoj Gandhi, Miguel Rivera, David Ting, Vikram Deshpande. Branched-Chain in situ Hybridization for κ and λ Light Chains: A Powerful Ancillary Technique for Determining B-Cell Clonality in Cytology Samples. Cancer Cytopathology. 2015 Aug 31.

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3. High Risk (HR) HPV detection in HNSCC

• HR-HPV present in subset of primary HNSCC (mainly non-keratinizing SCC)

• p16 IHC positivity not indicative of transcriptionally active HPV (HR)

• IHC followed by PCR or DNA ISH

• HR-HPV - 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, & 68

• HPV16 – ~90%, • HPV18 – ~ 5% • HPV33 – ~ 2%

• Low risk HPV – 6, 11 (not as important)

• RNA ISH allows determination of the variant of HPV, which is important for patient eligibility towards clinical trials

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HPV16 – E6/E7 RNA ISH

ViewRNA Manual ISH 2-plex assay HPV16 – Fast Red Keratin 7 – Fast Blue

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HPV 16 TMA Head and Neck Cancer

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HPV 16 TMA Head and Neck Cancer

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Darcy A. Kerr, Kshitij S. Arora, Krishnan K. Mahadevan, Jason L. Hornick, Jeffrey F. Krane, Miguel N. Rivera, David T. Ting, Vikram Deshpande, William C. Faquin. Performance of a Branch Chain RNA In Situ Hybridization Assay for the Detection of High-risk Human Papillomavirus in Head and Neck Squamous Cell Carcinoma. Am J Surg Pathol. Dec 2105.

Sensitivity of RNA ISH vs DNA ISH for hr HPV

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ViewRNA™: Integration of gene expression and morphology

Traditional pathology (H&E)

• Cell type and tissue

structure

• No molecular information

Traditional molecular pathology (IHC)

• Cell type and tissue structure

• Limited by antibody availability & quality

Traditional molecular assays (bind and grind)

• Quantitative assays

• Lack cellular resolution

ViewRNA Assays Molecular Detection and Morphological Context

Molecular detection for any target gene

Morphological context of gene expression

Amenable to Quantitation