Download Molecular Diagnostics in Clinical Pathology

Molecular Diagnostics in Clinical Pathology
Cancer Biology PhD Program
Clinical Cancer Research 2012 – Tumor biology
Wednesday, September 26, 2012
Achim Weber
Institute of Surgical Pathology
University and University Hospital Zurich
Pathology - Clinical / Surgical Pathology
cases / year
autopsies
cytologic specimens
biopsy / resction specimens
molecular tests
~ 400
(gynecologic) ~ 12.000
(non-gynecologic) ~ 14.000
~ 66.000
~ 2.000
> tour through the institute by Holger Moch, 1 p.m.
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Clinical / Surgical Pathology – tumor diagnosis
1946
Squamous cell carcinoma of the lung
Virchow C et al. (1997) DMW 46:1432-1437
Diagnostic Molecular Pathology - tumor pathology
2012
pathology report
molecular pathology report
lung adenocarcinoma
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Diagnostic Molecular Pathology - tumor pathology
*
numeric and structural changes of chromosomes
(point) mutations
epigenetic changes
Taylor S (2006) Cellscience Reviews Vol.2 No.3
Hanahan D, Weinberg RA (2000) Cell 100: 57-70
Hanahan D, Weinberg RA (2011) Cell 144(5):646-74
Diagnostic Molecular Pathology - tumor pathology
http://www.sgmp.uzh.ch/
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Diagnostic Molecular Pathology - tumor pathology
Examples - Diagnostic Molecular Pathology in daily practice
Colorectal cancer
Breast cancer
GIST
Lymphoma
Molecular pathology - colorectal carcinoma
Vogelstein sequence:
genetic changes in colorectal carcinogenesis
Fearon, ER and Vogelstein B (1990) Cell 61, 759-767
normal colon mucosa
loss of apcgene
(early) adenoma
genetic
instability
mutation of kas gene
(late) adenoma
loss of
p53
(invasive) carcinoma
loss of
smad4
?
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Molecular pathology - colorectal carcinoma
KRAS
- proto-oncogene, ras family
- Epidermal growth factor receptor (EGFR)-signal
transduction
- kras mutaten in ~ 40-50% of colorectal cancers
- antibody therapy (Cetuximab – Erbitux®,
Panitumumab – Vectibix®)
loss of apcgene
mutation of
kras gene
genetic
instability
loss of
p53
loss of
smad4
?
Bode P et al. (2008) UZL News 18:10-12
Molecular pathology - colorectal carcinoma
prediction
kras mutational analysis
N
sequencing of kras gene (exon 2, codon
12, 13; exon 3, codon 61)
T
therapy
T
wild type sequence
PCR kras gene
*
no therapy
kras mutation
(p.G12V)
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Molecular pathology - colorectal carcinoma
Molecular pathology - colorectal carcinoma
Mismatch Repair (MMR)
DNA repair for replication-associated errors
proteins
hMSH2, hMSH6, hMLH1, hPMS2
Defects in mismatch repair
- hereditary (Lynch/HNPCC-Syndrome)
- sporadic (hypermethylation)
Alberts Molecular Biology of the Cell, 4th edition
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Molecular pathology - colorectal carcinoma
A
hMSH2 (N)
B
C
hMLH1 (N)
D
hMSH2 (T)
hMLH1 (T)
immuno staining for MMR proteins: loss of nuclear staining specifically in the tumor (D)
Molecular pathology - colorectal carcinoma
microsatellites
regions of repetitive DNA of small (16 nts) repeats
gi|23087|emb|Z16551.1|HS093XH3 H. sapiens (D2S123) DNA segment containing (CA)
repeat; clone AFM093xh3; single read
AGCTTGAGAATTCCAGCAGCATTTAAACAAGGTTATCAAGCTAACATCATACTCAATAATGAATGA
CCAAAAGCATTTCTCTTATGATAATGGACAAAAACAGGATGCCTGCCTTTAACAGTGCTATTCAAC
ATTGCTGGAAGTTCTGGCCAGAGAAATTAGACACAGTGATACACACACACACACACACACACACAC
ATACACACACACACACACACACACACACACACATATTTTATAGATAGATAGATGGTATCCAGTCAG
AAAGGGGAAGTAAACTATCCCTATTGTAGATGCACAGTCCCATAGGTGGAAAGTCCCCTCCAATTC
microsatellite instability (MSI)
change of the length of a
microsatellite in a tumor compared
to normal tissue
expected
product
‚stutterbands‘
Berg KD et al. J Mol Diagn (2002) 2(1):20-28
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Molecular pathology - colorectal carcinoma
microsatellite instabilität (MSI): Surrogate marker for a functional inactivation of DNA
mismatch repair (MMR) genes
N
T
Molecular pathology - colorectal carcinoma
mikrosatellite instability (MSI):
diagnosis of Lynch syndrome
Lynch syndrome:
•hereditary nonpolyposis colorectal cancer (HNPCC)
•autosomal dominant vererbtes Leiden
•~5% of CRC
•mean age: ~45 years
•diagnosis: for counseling patients and their families
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Molecular pathology - colorectal carcinoma
mikrosatellite instability (MSI):
prognosis of CRC
conclusion:
MSI-H is an independent favourable prognostic
factor for patients with CRC
Molecular pathology - colorectal carcinoma
mikrosatellite instability (MSI):
prediction of CRC therapy outcome
recommendation: no 5-FU based therapy for patients
with MSI-H stage II CRC (still a matter of debate)
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Diagnostic Molecular Pathology - tumor pathology
Examples - Diagnostic Molecular Pathology in daily practice
Colorectal cancer
Breast cancer
GIST
Lymphoma
Molecular pathology – breast carcinoma
HER2 (also: neu or ErbB-2)
member of the epidermal growth factor receptor (EGFR/ErbB) family )
membrane-bound receptor tyrosine kinase
signaling pathways activated by HER2:
•
•
•
•
•
mitogen-activated protein kinase (MAPK)
phosphoinositide 3-kinase (PI3K/Akt)
phospholipase C γ
protein kinase C (PKC)
signal transducer and activator of transcription (STAT)
amplification or over-expression of this gene occurs in ~30% of breast
cancers and crucial in the pathogenesis and progression of certain aggressive
types of breast cancer
biomarker and target of therapy
HER2 is the target of the monoclonal antibody trastuzumab (Herceptin®).
Trastuzumab is effective only in cancers with HER2 is over-expression
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Molecular pathology – breast carcinoma
Her2 immuno stain
Her2 FISH
FISH negative
score
0
1
chromosome 17 centromer probe
Her2 gene
FISH positive
2
3
Diagnostic Molecular Pathology - tumor pathology
Examples - Diagnostic Molecular Pathology in daily practice
Colorectal cancer
Breast cancer
GIST
Lymphoma
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Molecular pathology - GIST
gastrointestinal stromatumor (GIST)
mesenchymal tumor
molecular biology
mutations in c-kit gene (~ 90%; exon 9 and 11 >>13, 17 or PDGFR gene)
c-kit comprises a extracellular domain, a transmembrane segment, and an
intracellular part acting as a tyrosine kinase
macroscopy / microscopy
-well-defined lesion with contact to Muscularis propria
-stomach (~ 60%), small bowel (~ 30%), colorectum (~ 5%)
-mostly uniform spindle cell morphology, can be epitheloid
-immuno phenotype: CD117 / c-kit: positive, DOG1: positive
prognosis
variable, criteria: location (organ), size, mitotic count
therapy
surgical, drugs: tyrosine kinase inhibitors, Imatinib (Glivec®)
Molecular pathology - GIST
polypoid GIST of the stomach
GIST of the stomach with ulceration
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Molecular pathology - GIST
GIST of the small bowel with ulceration
Molecular pathology - GIST
GIST (rectum): well-defined submucosal tumor with c-kit (CD117) expression (insert)
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Molecular pathology - GIST
GIST (stomach) with epitheloid (upper left) and spindle cell (lower right) morphology
Molecular pathology - GIST
mutational analysis for prognosis and prediction of therapy response
GIST mutational
analysis, c-kit gene:
-deletion in exon 11
-predictive of response to
Glivec therapy
Rubin BP et al. Lancet 2007; 369: 1731–41
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Molecular pathology - GIST
prognostic factors in GIST:
established parameters:
-size
-location
-proliferation
further parameters :
-tumor rupture
-moleculare markers (type of ckit- or PDGFR mutation)
Rubin BP et al. Lancet 2007; 369: 1731–41
Molecular pathology – infectious diseases in tumor pathology
Kaposi sarcoma
HHV8
HHV8 immuno stain
1
2
3
W
PC
160 bp
HHV8 minor capsid protein gene
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Diagnostic Molecular Pathology - tumor pathology
Examples - Diagnostic Molecular Pathology in daily practice
Colorectal cancer
Breast cancer
GIST
Lymphoma
Molecular pathology - lymphoma
Routine molecular testing in lymphoma diagnostics
Clonality testing
-
takes advantage of a specific event in the maturation of lymphocytes
-
discriminates polyclonal versus monoclonal cell populations
-
no (!) absolute criterium for disriminating bengin versus malignant
lymphoma-“specific“ test
-
lymphoma / entity-“specific“ molecular changes are detected, e.g.
translocations, viral infections
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Molecular pathology - lymphoma
changes during lymphocyte maturation in
immunglobulin (B-cells) or T-cell receptor
(T-cells) genes
somatic deletionen, mutations, insertions and
specific splicing required for
diversification
the IgH gene has three highly variable region
(complementarity determining regions,
CDR) in between four framework regions
(FR)
resulting in variable length of gene fragments
in different lymphocytes
1.
PCR analysis using oligos located in less
variable regions
B-cells: immunoglobulin heavy
chain gene (14,q32.3)
2.
T-cells: T-cell receptor (e.g.
TCR)
Molecular pathology - lymphoma
resulting PCR fragment
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Molecular pathology - lymphoma
resulting PCR fragment
Molecular pathology - lymphoma
reactive process:
polyclonal lymphatic population
lymphoma:
monoclonal lymphatic population
FRIII-CDRIII-FRIV:
PCR amplificate of the
FRIII-CDRIII-FRIV region:
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Molecular pathology - lymphoma
gel electrophoresis
fragment analysis
1. PCR using fluorescence-labeled
oligos
2. separation by capillaryelectrophoresis
Arber DA, J Mol Diagn (2000) 2(4), 178-190
Molecular pathology - lymphoma
gastritis versus MALT lymphoma
case 1
CD 20
CDR II
case 2
X
1
2
3
4
gel interpretation:
lane 1, 2: monoclonal
lane 3, 4: polyclonal
c/w B- cell NHL in lane 1 and 2
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Molecular pathology - lymphoma
detection of ‚tumor-specific‘ molecular markers
translocations
-
t(14;18)
follicular lymphoma
IgH / bcl-2
apoptosis
-
t(11;14)
mantel cell lymphoma (MZL)
bcl-1 / IgH
cell cycle
-
t(8;14)
Burkitts lymphoma
c-myc / IgH
proliferation
-
t(8;22)
Burkitts lymphoma
c-myc / IgH
proliferation
-
t(8;2)
Burkitts lymphoma
c-myc / IgH
proliferation
-
t(2;5)
anaplastic large cell lymphoma
npm / alk kinase
tyrosine kinase
viral infections
-
Epstein-Barr-Virus (EBV)
endemic Burkitt lymphoma (90%)
Hodgkin lymphoma (40%)
nasal NK/T-vell lymphoma
-
HTLV-1
adult T cell leukemia / lymphoma (ATLL)
-
HHV-8 (KSHV)
primary effusion lymphoma,
multicentric Castleman‘s disease
Molecular pathology - lymphoma
t(14;18)- translokation in follicular lymphoma
PCR: fusion product
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Molecular pathology - lymphoma
morphology
1
molecular tests for follicular
lymphoma
1.
clonality (IgH, CDR II, CDR III)
2.
PCR for t(14;18) translokation
3.
FISH for t(14;18) translokation
2
3
4
immuno stain for BCL-2
gene specific
oligonucleotide primers:
FISH for t(14;18)
P
W
PC
M
bcl-2 / JH Fusionsprodukt
Molecular pathology – infectious diseases
EBV- driven post transplantation lymphoproliferative disease (PTLD)
case
•57-years old female, 6 months after
liver transplantation
EBNA1 gene
X
X
1
2
3
4
W
PC
203 bp
Real-time PCR for quantification of viral load
(TaqMan-System)
CD 20
EBER
Jebbink J et al. (2003) J Mol Diagn 5(1):15-20
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Clincial Molecular Pathology – Outlook, Perspectives
cutting edge 2012
cutting edge at the benning
of the millenium
The Cancer Genome Atlas Network.
Nature 2012, 487:330-337
high troughput techniques (next generation
sequencing) are currently evaluated for applicability
in routine test settings
> presentation by Markus Rechsteiner on NGS, 2 p.m.
Alizadeh et al. Nature 2000, 403:503-511
Summary and take home message – Clincial Molecular Pathology
molecular testing is an integral part of daily practice in Clinical Pathology
molecular tests are performed for diagnostic, prognostic and predictive
purposes
tests and techniques are subject to change and constantly updated – e.g.
high troughput techniques (next generation sequencing) are currently
evaluated for applicability in routine test settings
molecular testing is a supplemental diagnostic approach, does not replace
morphology, results have to be interpretated in the context of morphology
(integrative report)
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