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Traditional pathology Molecular pathology
Molecular/Predictive Pathology
Comprehensive molecular tumor profiling
Pathologists provide personalized medicine /predictive biomarker information
Proteomic and genomic data in the context of morphology
Morphologic Pathology
Morphologic classification of cancer
Pathologists provide diagnostic and prognostic information
Hematoxylin and eosin is ‘primary stain’ for all cases
Cellular Pathology
Leeuwenhuek (1858-1950): Developed 1st microscope
Virchow (1821-1905): Recognized that diseases arise from alterations within tissues and cells
Evolution of Pathology
Gross Pathology
Antonio Benivieni (1443-1502): First autopsy
Giovanni Morgagni (1682-1771): Correlated patient symptoms
to autopsy findings
John Hunter (1728-1793): Devised method for
preserving tissue
Bichat (1771-1802): “Father of modern pathology”
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The pathologist as diagnostic
oncologist John R. Srigley Pathology,2009;41:513-514
The role of pathologists in the era
of personalized medicine Eric E. Walk Arch Pathol Lab Med, 2009;133:605-610
Resection specimen:
Process immediately after surgery
Fix for 6-72hrs
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Post-analytic Analytic Pre-analytic
Interpretation Assay validation Time to fixation
Image analysis Equipment calibration Method of tissue
processing
Reporting Standard Laboratory
Procedure
Time of fixation
QA procedures:
Staff training
assessment
Method of fixation
-lab accreditation Antigen retrieval
-Proficiency tests Test reagents
-pathologists… Standard controls
Automation 6
7 Adenocarcinoma, well to moderately differentiated
http://www.google.co.il/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=TVtCgEWgWN8TpM&tbnid=Z0u4NkHd1OJLQM:&ved=0CAUQjRw&url=http://www.flickriver.com/photos/jian-hua_qiao_md/8027979330/&ei=-EeOUczvBsastAbywICgDA&psig=AFQjCNFIKNh_hU2JbDJ6wAyuEyQU2QGvCA&ust=1368365295558174
8 Poorly differentiated carcinoma
http://www.google.co.il/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=vz8JyHbfbiqvAM&tbnid=C0r6pkeDwtZ1uM:&ved=0CAUQjRw&url=http://plaza.umin.ac.jp/pathol2/photo-library/index.php?first_category_id=1&second_category_id=1&ei=N0uOUbvoJIiVswbYlICgDA&psig=AFQjCNGgtjtSrxRuUkJwjrQ3RrRxhfBWAA&ust=1368366094854717
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We are involved in the whole continuum of
cancer care:
Screening
Diagnosis
Identification of risk factors
Monitoring of cure
And now:
Our role in targeted therapies.
(Prediction of therapeutic response).
Pharmacopathology – Targeted
Therapy
Biological therapies that are based on specific
attachment of medications to specific sites on
tumor cells (receptors), or within loci in tumor
cells.
…by targeting specific somatic alterations
present in these tumors.
This attachment causes specific damage to the
tumor cells.
Only minimal damage to non neoplastic cells. 10
Detection of translocations, amplifications by ISH.
Sequence based detection of genes for detection
of somatic mutations in tumors.
Detecting mutations by RT-PCR.
Circulating tumor DNA is relevant only relatively.
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New roles for “old” pathologists
Targeted/ Personalized Therapy
Paradigm shift in Pathology
(Letting the gen(i)e out of the bottle)
Genomics
Genomic driven diagnoses
Genomic driven therapies
Epigenetics
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Eric E Walk Arch Pathol Lab Med, 2009; 133:605-610 14
http://www.archivesofpathology.org/action/showFullPopup?doi=10.1043/1543-2165-133.4.605&id=_e1
Tamoxifen - Anti Estrogen receptor/Breast Ca
Herceptin - Anti HER2/neu in over-expressing
tumors/Breast Ca, Gastric ca.
Mabthera - Anti CD20/B cell lymphomas
STI571(Gleevec) - Tyrosine kinase inhibitor – CML, GIST
Zelboraf (Vemurafenib) – BRAF inhibitor - Melanoma
Avastin - Anti angiogenic factor
Anti growth factor receptors – Erlotinib/Tarceva (lung Ca),
Cetuximab (Erbitux) (colon and head and neck Ca.)…
Xalkori - Anti ALK-EML (lung)…
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Tamoxifen
(Anti
Estrogen
receptors)
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Breast carcinoma
Herceptin
anti Her2/neu Protein
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+1
+2 +3 +3
Immunohistochemistryt
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FISH-HER2/amplified Non amplified 19
Gene Expression Profiling
Measurement of expression of thousands of genes simultaneously.
DNA microarray analysis.
Gene chips (up to >20,000 genes), commercial, are hybridized to probes from tumor samples, labeled with fluochromes.
Computerized analysis.
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Microarray Analysis
Pioneered by Perou/Sorlie et al, 2001,
established that breast cancer can be
classified into distinct molecular groups.
Measurement of quantities of individual
mRNAs.
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Unsupervised Classification “cluster analysis”
Samples “clustered” by expression similarity
Closer together the samples the more similar the
gene expression
Sorlie T, et al. Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10869-74.
Expression Dendrogram
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Well diff. AC
Mod. diff. AC Poorly diff. AC 24
TRIPLE NEGATIVE/BASAL-LIKE CARCINOMAS
Low grade High grade
secretory
adenoid cystic
medullary metaplastic
no special type 25
Colorectal Adenocarcinoma (CRC)
Anti EGFR therapy (TKIs) (Erbitux, Cetuximab)
was found to be effective in CRC.
However ~50% of all patients with CRC have
RAS mutations and do not respond as well to
anti EGFR therapy.
Patients harboring RAS mutations receiving anti EGFR treatment , may do worse than those without RAS mutations.
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ASCO recommends that all patients with metastatic CRC should be tested for RAS mutational status, at least at codons 12/13.
FDA - Cetuximab (Erbitux) is not recommended for CRC patients with RAS mutations.
No FDA approved test yet.
Tests principles:
-RT-PCR
-Direct sequencing
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Pathologist’s role
The KRAS mutations are detected on
DNA from tumor sections.
The pathologist has to choose the right
area with enough viable tumor.
Perform the molecular test.
Or select a reference lab.
Select the technology.
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Lung Cancer / New Paradigm
Histology versus molecular diagnosis in lung
cancer.
Primary v’ metastatic.
If primary Small Cell Lung Ca. v’ Non SCLC.
If NSCLC Squamous Cell Ca (p40) v’ AdenoCa
(TTF1).
Clinical correlation of paramount importance.
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Although there is only one sequence of
morphological changes that is characterized to-
date for the development of invasive lung AC from
AAH, there is evidence to suggest at least 2
molecular pathways:
The KRAS and EGFR pathways in smoker and
never-smoker AC subpopulations:
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EGFR and KRAS… EGFR mutations (exons 19, 21), more common in never
smokers (17/30 v 1/39).
KRAS mutations, more common in smokers (24/39 v 3/30).
Lung adenocarcinomas in Asians had more EGFR mutations than Caucasian tumors (15/23 v 3/45).
Caucasians had more KRAS mutations than Asians (25/45 v 2/23).
No difference in mutation rates between women and men.
Mutually exclusive. Lung adenocarcinoma of never smokers and smokers harbor differential regions of genetic
alteration and exhibit different levels of genomic instability.
Kelsie L. Thu et al
PLoS ONE March 2012, vol 7, issue 3 32
Continued…
Smoking status – most strongly associated clinical variable observed with Proportion of Genomic Alterations in a multivariate analysis, compared with other clinical variables (stage, gender…).
When clinically applicable, EGFR testing is performed on lung tumor tissue (mainly on AC).
~25% of lung adenocarcinomas harbor mutations in the EGFR gene (28 mutations are investigated).
If EGFR negative ALK (3-5%) mutation tested.
Lung adenocarcinoma of never smokers and smokers harbor differential regions of genetic
alteration and exhibit different levels of genomic instability.
Kelsie L. Thu et al
PLoS ONE March 2012, vol 7, issue 3
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Lung Cancer Therapy Algorithm
If adenocarcinoma, TTF1(+), p40 (-) and if clinically applicable EGFR testing
Positive ~25% negative
Anti EGFR therapy
ALK testing
+ve ~3-5% Xalkori
-ve CT
TTF1
p40
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EML4–ALK fusion gene
Generally mutually exclusive with EGFR mutations and
KRAS mutations; indicating that these molecular defects
function as drivers of pathogenesis.
Concurrent mutations have been reported1–3
Despite correlations observed between ALK status
histology/smoking history, it is possible for any patient to
have ALK-positive NSCLC
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EML4-ALK testing
Up to recently, FISH study demonstrating the
rearrangement was gold standard.
Now, since ESMO 2012, Immunohistochemistry is
becoming part of the algorithm validation.
EML4-ALK +ve
IHC and FISH
EML4-ALK –VE
IHC and FISH
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Importance of adequate tissue
collection
Tissue management starts with interaction
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DCIS
This is just the beginning of a great friendship….
Morphology
Genomics
Proteomics
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