In situ hybridization (ISH)

- theory and practice

- a molecular tool in cancer diagnosis and treatmentexample : the Her-2 story in breast cancer(targeted therapy)

JPEMS – Histological methods – 13/09/2013 Anne Jarry, EA4273 Biometadys, Faculté de Médecine, Nantesajarry@univ-nantes.fr

Immunohistochemistry (IHC) In situ hybridization (ISH)

• ISH techniques provide informations complementary to those provided by IHC : detectnuclei acid sequences coding for the expression of specific proteins

• combination of IHC and ISH : detailed analysis of gene exp ression in situ

proteindetection

mRNAdetection

DNAdetection

DNA structure (Watson and Crick, 1953)

units : nucleotides (ATP, TTP, GTP, CTP)

nucleic acid structure

DNA : A always pairs with T, and G with C

RNA : U pairs with T, and G with C

In situ hybridization – how it works• Hybridization with a labeled probe (DNA or RNA probe)

complementary to a specific DNA or RNA sequence of interest in cellsor tissue sections : target DNA or RNA

• The hybrid formed in situ is revealed by a method permitting its detectionby fluorescent microscopy or standard microscopy

Probe labelled …Radioactively ( 35S, 3H)

autoradiography

Needs special facilitiesTime-consumingExpensiveCannot be routinely used

Fluorochromes : FISH (fluorescent ISH)

Chemically

Fluorescence microscopy Ex. : Fluorescein isothiocyanate(FITC) (λ exc 494 – λ em 518 nm);

texas red (596 – 615nm)• advantages : Multicolor detection of simultaneouly hybridized probes

Sensitive and specific method ; can be coupled to automated morphometric image analysis• inconveniences : reduced stability of the fluorescent detection signals (decay over time –«bleaching»)

limited morphological assesment of overall histology

Probe labelled …

CISH (chromogenic ISH)

Chemically

Hapten – enzymatic reactionEnzyme Metallography : SISH (silver ISH)

Visualized by standard, bright field microscopy

• staining stable for long periods• simultaneous morphological assessment• rapid to perform and analyze ; can be automated• commercially available kits

Hapten(digoxigenin)

first antibody

second antibody Horseradish peroxidase(HRP) + chromogen : DAB

Chromogen deposition at target(brown for DAB)

In situ hybridization : applications

• Detection of expression of mRNA in situ (research)

• Localization of viral DNA : EBV or HPV

• Analysis of genes in chromosomes (FISH)� localization and exploration of chromosomal, genetic and genomicaberrations that can be directly correlated to disease causation andprogression (cancer)

FISH successful for :

• mapping single copy and repetitive DNA sequences usingmetaphase and interphase nuclei

• evaluation for ploidy and gene amplification (cancer)

• detecting targeted chromosome translocation (lymphoma, leukemia),

• localizing large repeat families to aid in chromosome identification and karyotype analysis (� multicolor FISH)

In situ hybridization : applications

Applications of FISH and CISH in :• research• clinically : has been proven invaluable in

the– diagnosis– pronostication, and

– pharmacogenomic assessment of manydiseases, especially in cancer

In situ hybridization : an essential tool in research and also clinically

In situ hybridization : stages of thetechnique (ex : FISH)

Material used : • paraffin sections(retrospective studies)high throughput analysis : tissue microarrays (TMA)• frozen sections• cytologic specimens• smears (haematology)

choice of probelabel probedenature probe (heat)

pretreatment of tissue(permeabilization,proteolysis)

Stringent washes to removeunbound probe

Fluorescence in situ hybridization (FISH)

ISH in clinical diagnosis

in cancer (breast, stomach)

the Her2 story

Her-2 (Erb2, Her2 neu)

HER-2 (human epidermal growth factor receptor 2 : EGFR2) gene :

• located on chromosome 17q

• encodes a transmembrane tyrosine kinase receptorprotein, member of the EGFR family, involved in cell-cell and cell-stroma interactions through «signal transduction

Hudis CA, N Engl J Med 2007;357:39-51

Signal transduction by the HER family

Her-2 (Erb2, Her2 neu)

• HER-2 gene amplification and/or protein overexpressionhave been identified in breast carcinoma, lung carcinoma, gastrointestinal tumors, melanoma …

• HER-2 gene amplification in breast cancer has been associated with increased cell proliferation, cell motility, tumorinvasiveness, metastasis, accelerated angiogenesis, reducedapoptosis.

• Abnormalities in HER-2 expression at the gene (ISH), message (mRNA levels by quantitative RT-PCR), and protein(IHC) levels have been associated with poor prognosis in breast and gastric cancer (correlation with clinical outcome).

Hudis CA, N Engl J Med 2007;357:39-51

Potential mechanisms of action of Trastuzumab(Herceptin) : a humanized anti-Her2 antibody

Development of humanized anti-Her-2 antibodies : Trastuzumab (= Herceptin ; Hoffman-La Roche, Basel )

• confers a significant survival benefit in the treatment of Her2+ advanced or metastatic breast cancer, as monotherapy or in combination withchemotherapy (Slamon DJ et al., N Engl J Med 2001)

• therapeutic benefit from treatment with trastuzumab only in patients withHer2+ disease

� accurate determination of Her2 by IHC and/or ISH (FISH, CI SH) iskey in identifying patients eligible for trastuzumab th erapy

Hanna and Kwok, Mod Pathol 2006 - Breast cancer

Her2IHC3+

Her2CISH

Her2FISH

Her2 gene amplification

Nitta H et al, Diagnostic Pathology 2008;3:41-53 – breast cancer

Nitta H et al, Diagnostic Pathology 2008

Breast cancer – double bright field ISH for Her2 (black ) and chromosom 17 (CEN17 - red)

Normal Her2 amplified Her2

Her-2 gene not amplified Her-2 gene amplified

CISH

Penault-Llorca F et al., Am J Clin Pathol 2009

SISH

JF Mosnier, submitted

Gastric cancer – intestinal type E cadherin - upregulat ion

Her2 overexpression (3+) – IHC Her2gene amplification - CISH

Her2 gene amplification and protein overexpression associated with a poor prognosis in gastric cancer ; Patients with Her2 amplification eligible for trastuzumab therapy

The recommended human Her 2-testing algorithm. Adapted from Hanna andKwok (Mod Pathol 2006). Recommended by the FDA

Take home message

• ISH : a morphological molecular techniquea «clinical» laboratory tool

• detects genetic aberrations of medical significance

• can help in :– the diagnosis of a genetic disease– in suggesting pronostic outcomes– in prediciting the response to therapies (anti-Her2

therapies), in association with IHC (close collaboration between pathologists and molecular biologists / geneticists)� targeted therapy in cancer management (personalisedmedicine)