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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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