a jarry 13.09.13 histological methods - in situ hybridization · anne jarry, ea4273 biometadys,...
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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, [email protected]
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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
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DNA structure (Watson and Crick, 1953)
units : nucleotides (ATP, TTP, GTP, CTP)
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nucleic acid structure
DNA : A always pairs with T, and G with C
RNA : U pairs with T, and G with C
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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
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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
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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)
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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)
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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
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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
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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)
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ISH in clinical diagnosis
in cancer (breast, stomach)
the Her2 story
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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
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Hudis CA, N Engl J Med 2007;357:39-51
Signal transduction by the HER family
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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).
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Hudis CA, N Engl J Med 2007;357:39-51
Potential mechanisms of action of Trastuzumab(Herceptin) : a humanized anti-Her2 antibody
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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
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Hanna and Kwok, Mod Pathol 2006 - Breast cancer
Her2IHC3+
Her2CISH
Her2FISH
Her2 gene amplification
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Nitta H et al, Diagnostic Pathology 2008;3:41-53 – breast cancer
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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
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Her-2 gene not amplified Her-2 gene amplified
CISH
Penault-Llorca F et al., Am J Clin Pathol 2009
SISH
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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
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The recommended human Her 2-testing algorithm. Adapted from Hanna andKwok (Mod Pathol 2006). Recommended by the FDA
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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)