Review&Session&#3&

Deni&ons)
Pathology:&The&scien0c&study&of&the&nature&of&
disease&and&its&causes,&processes,&
development,&and&consequences.&&
&
Molecular)Pathology:)aims&to&achieve&a&more&
detailed&classica0on&of&tumors&by&adding&
molecular&annota0on&to&exis0ng&pathologic&
and&clinical&data.&&
&
 Heterogeneity
Solid tumors should be thought of ecosystems
with their heterogeneity and their ability to
adapt for survival

Morphologic
Cell Biology: Hierarchical and Stochastic
Molecular
Clinical
Morphologic heterogeneity
Tissue heterogeneity
Laser Capture Microdissection

DNA
RNA
Proteins

MOLECULAR ANALYSIS
EGFR)kinase)domain)muta&ons)

Paez&Science&2004&
EGFR)TKIs:)eect)in)EGFR)mutated)lung)
ACA)

Large&right&lung&adenocarcinoma,& Following&six&weeks&of&therapy&
refractory&to&chemotherapy.& on&ge0nib.&

Lynch&et&al.&&NEJM&2004.&
Mok&et&al.&&NEJM&2009.&
 ALK)transloca&ons)in)NSCLC:)
3)years)(!))from)bench)to)bedside)
 ROS1)translocated)
lung)tumors)
Never&to&light&smoking&history&
Adenocarcinomas&
Can&be&detected&by&FISH,&RTSPCR&
and&IHC&
Promising&results&in&largest&Phase&I&
clinical&trial&of&crizo0nib&in&ROS1&
rearranged&tumors&

Sholl)et)al.)Am)J)Surg)Path)2013;)September)37(9))
Shaw)AT)et)al.)N)Engl)J)Med)2014;371:1963X1971.)
 Next&Genera0on&Sequencing&Basics&
Tradi0onal&sequencing&is&Analog&
All&DNA&from&a&sample&mixed&together&
See&changes&present&in&majority&of&cells&
Analyze&one&sec0on&of&the&genome&at&a&0me&
NGS&is&digital&
Individual&DNAs&separated&spa0ally&,&sequenced&in&parallel.&&&
Computer&recombines&individual&sequences&to&give&big&picture&
can&s0ll&zoom&in&on&individual&molecules&
Enables&
Assessment&of&many&dierent&targets&in&one&test&
Iden0ca0on&of&subSpopula0ons&
Analysis&of&heterogeneous&or&very&small&samples&
Rela0ve&quan0ta0on&of&muta0ons&

Courtesy&of&Neal&Lindeman&
 Next&Genera0on&Sequencing&
PROS)
High&throughput&sequencing&of&as&
many&genes&as&you&like,&
simultaneously&
Much&more&sensi0ve&than&
tradi0onal&sequencing&
Requires&very&li]le&input&DNA&
CONS)
Expensive&
Requires&batching&to&be&cost&
eec0ve,&so&long&turnaround&0me&
Major&informa0cs&requirements&
 Next&Genera0on&Sequencing&basics&
Design&ques0on&1:&How&much&to&sequence?&

Design Findings Capacity Analysis

Whole genome Everything One Weeks
Whole exome Potentially Dozens Days
actionable
Targeted exome Plausibly Scores Minutes to
actionable hours

DECISION:&extent&of&ac0onability&vs.&cost&and&throughput&
 Next&Genera0on&Sequencing&basics&
Design&ques0on&2:&What&kind&of&Library?&

Hybrid&Capture& Amplicon&Sequencing&

Quan0ta0ve& Not&quan0ta0ve&
~1&week& 1S2&days&
Lower&coverage& Higher&coverage&
More&targets& Fewer&targets&

Ku, et al., Mod Path, 2012 Beadling, et al., JMD, 2013

DECISION&2:&Turnaround,&coverage&vs.&quan0ta0on,&#&of&targets&
 Fluorescence in situ Hybridization (FISH):
Overview

www.mtsinai.on.ca/pdmg/Genetics/cytogenetics.html
 Molecular Cytogenetics:
Denaturation of Target DNA

Target&cell) Denature&doubleSstranded&
chromosomal&DNA&into&&
single&strands)
 Hybridization of DNA Probe to
Chromosomal Target

Add&labeled,&denatured&probe&& DNA&reSannealing&allows&probe&&
complementary&to&chromosomal&& binding&to&target&region)
region&of&interest)
 &
Detection of Fluorescent Signals

Wash off unbound probe Evaluate by fluorescence microscopy

 Metaphase vs. Interphase FISH

Metaphase FISH requires cell culture

Chromosome morphology retained, therefore
Hybridization signals can be localized

Interphase FISH
Chromosome morphology unavailable
Signals cannot be reliably mapped to chromosomes

Touch preparations
Disaggregated single cell suspensions
Fine needle aspirates
Formalin-fixed paraffin sections
50 micron sections via 5 micron sections
Enumeration Probe:
Trisomy 12 in CLL
Gene-specific Probe:
TP53 Deletion in CLL
 PML/RARA Rearrangement:
Dual Color, Dual Fusion Probe
 Synovial Sarcoma: Break-apart Probe

t(X;18)(p11.2;q11.2)
Array Comparative Genomic Hybridization

Co-hybridization of test and reference genomes to detect copy

number changes
aCGH
3 BRAF Duplication Detected by aCGH
 Relative Resolutions

! Aberra&ons)detectable)by)karyotype)
! Structural&aberra0ons&(>5&Mb)&
! Numerical&aberra0ons&&
&
! Aberra&ons)detectable)by)targeted)FISH)
! Aneusomies&(150&kb&to&5&Mb)&and&aneuploidies&
! Cryp0c&(submicroscopic)&rearrangements&
! Targets&must&be&known&
&
! Aberra&ons)detectable)by)microarray)
! Copy&number&changes,&genomic&imbalances&
! Aberra0ons&<5&Mb&(as&small&as&tens&of&kilobase&pairs)&
! a"priori&knowledge&of&chromosomal&loca0on&is&unnecessary&
&
 HTBM%200%
Review%Session%#3%
Amanda%Mar8not%
4/25/16%
 GI#Pathology#

GI%tract%has%large%amounts%of%lymphoid%8ssue%
that%is%in%constant%contact%with%the%outside%
world%which%is%the%bacterial%ora%of%the%gut%
The%GI%tract%epithelium%rapidly%turns%over%%
Over%exuberant%expansion%of%lymphoid%8ssue%
leads%to%inammatory%lesions%
Poten8al%accumula8on%of%muta8ons%in%the%
rapidly%dividing%epithelium%in%areas%of%chronic%
inamma8on%can%predispose%to%neoplasia%
Pathology%of%GI%tract%reects%both%abnormal%
inammatory%inltrates%and%epithelial%turnover%
%
 GI#Histopathology#

Squamous% Glands% Villi% Goblet%cells%

Epithelium%
 Ulcera4on#of#the#GI#epithelium#

Figure%17V11%Robbins%and%Cotran,%8th%Ed%
GI#PathologyDr.#Glickman#
 Celiac#disease#

Fig%17V26%Robbins%and%Cotran,%8th%Ed%
Chrohns@@transmural,#anywhere,#granulamatous#
Ulcera4ve#coli4ssupercial,#colon,#lymphoplasmacy4c#
Hepatobiliary#PathologyDr.#Glickman#
 Responses#of#Liver#to#Injury#

R&C%8th%Ed%
 Acute#vs.#Chronic#hepa44s#

R&C%8th%Ed.%%
 Fibrosis#versus#Necrosis#
Canine%Herpesvirus%
Nodular%regenera8on% associated%hepa8c%necrosis%%
Cardiovascular#pathologyDr.#Padeira#
Pulmonary#pathologyDr.#Padeira#
Pulmonary#Histology#
 Pulmonary#Pathologies#

COMMON#THEME:#Processes#are#interfering#with#gas#exchange#at#the#level#of#the#alveoli#or##
preven4ng#gas#exchange#by#limi4ng#access#of#air#to#alveoli#
 Alveolar#DamageTiming#is#Everything#
DADVFibro8c%Phase%

Hyaline%membranes%
Fibrosis%%
%

PneumoniaV%% PneumoniaV%% PneumoniaV%%

PneumoniaV%Acute% Early%Organizing% Organizing% Resolu8on%
Asthma##
In#vivo#histologyDr.#Tearney#
 Op4cal#coherence#tomography@OCT#

High%resolu8on%
Shallow%penetra8on%
**LumenVrelated%morphologies**%
Can%detect%some%cellular%inltratesmacrophages%and%cholesterol%crystals%
%
%
Kostan8nos%et#al.#Eur%Heart%Journal,%2106%
 BarreSs#esophagussquamous#to#glandular#
metaplasia#
Op4cal#Frequency#
Domain#Imaging:#OFDI#

CrossVsec8onal%imaging%
Requires%contact%with%capsule%via%peristalsis%
Allows%detec8on%of%submucosal%changes%that%
%%%%%may%not%be%evident%from%lumen%as%seen%by%endoscopy%
%
Tearney%et#al.#Nature%Med%2013%
 Review #3!
Biomaterials and Implantable Devices !