Multiplexed Imaging, Pathology and Personalized Medicine: Keeping Sight of the Forest

Richard Levenson, MD

Dept. of Pathology and

Laboratory Medicine

UC Davis Medical Center

Themes

1.The stroma (not the epithelium) may be key (…“STROMICS”)

2.“Systems biology” is most relevant at structural, intercellular and tissue levels

3.Bonus: glimpse of novel structural (target: collagen) and multiplexed (target: heterogeneity) imaging technologies

Are we looking in the right places?

How might a focus on micro-environment and host factors affect diagnosis and treatment strategies?

Normal breast

Invasive squamous cell carcinoma

Gabriella dental x-ray and CT: loss of bone

Nov, 2011 Feb, 2012

14 months later…

Implications:

New therapies will need new companion diagnostics—or perhaps none…

Molecular phenotypes in spatial context can be informative

Coming up:

Multiplexed imaging

Selection of targets

12

How well could we understand the battle of Waterloo…if we blenderized the battlefield?

Spectral imaging variants

PARISS

NUANCE/VECTRALCTF

HSi-440cAOTF

RebellionIMS

Vectra: Evaluating EMT in FFPE-Tissue Sections

C. Hoyt, PerkinElmer, Inc.; C. Gagen & R. Wetzel, Cell Signaling Technology

DAPI E-cadherin vimentin

Raw color image

Immunofluorescence with quantum dots

DAPIPR (585-nm QDot)ER (655-nm QDot)Convert to brightfieldDAPI and autofluorescence

16

From Mouse to Microscope: Breast cancer and Cy5-anti-Her2 antibody

Courtesy Roche, Penzberg, Germany

17

Four nuclear signals can be distinguished using chromogens in brightfield

Hematoxylin

Ki67—red cell cycle

pHH3—brown cell cycle

CC3—gray antiangiogenesis met. suppressor

Great tools and reagents…

So what compartments and components should we be looking at?

(hint: stroma)

Nature Medicine, 2008

Top 200 gene expression levels most different between microdissected normal and tumor-associated stroma.

Automatic clustering gave these recurrence-free survival curves

26-gene stromal signature is independent of all major clinical and pathological variables:

• ER • HER2• lymph node status • age• grade• tumor size

NOT PREDICTIVE!

C-Path: ‘Omic’s approach to the analysis of tumor morphology

AH Beck, …, D Koller. Systematic analysis of breast cancer morphology uncovers stromal features associated with survival. Science Translational Medicine 2011. 3, 108ra113.

Seventy-gene prognosis signature Size Invasiveness gene signature Mastectomy Wound response signature ERBB2 molecular subtype Grade Basal molecular subtype ER Lymph node Genomic grade index Luminal A molecular subtype Luminal B molecular subtype Chemotherapy

NOTSIGNIFICANT:

Classic tumor characteristics are not predictive

Andy Beck

• C-Path score • Hypoxia • Age

What is significant:

Just 3 stromal features predicted outcome better than 8 epithelial features

Moreover:

Andy Beck

Untreated Treated

2009

Tumor heterogeneity: Stromal environment affects stemness and HER2-positivity

(Stem cell marker)

In this case, HER2- induction NOT due to gene amplification

Implications for HER2 IHC and ISH assays?

Wicha 2013

What about structural properties of the stroma?

Can we detect them in tissue slices?

Boyd et al. Breast Cancer Research 2011 13:223

OR 1 1.7 2.1 2.4 4.7

Mammographic density affects the risk of breast cancer

0 <10 <25

<50 <75 >75

28

Stromal rearrangement around cancer can reflect biological properties

Linear collagen strands oriented tangentially (0 degrees)

Linear collagen strands oriented at 90 degrees

Non-invasive tumor

Locally invasive tumor

(Second harmonic generation imaging)

Provenzano et al., BMC 2008

TACS-2 TACS-3

Locker and Segal commentary on Conklin et al, AJP, 2011

Risk independent of:GradeSizeAgeER/PR/HER-2node status

Conklin et al, AJP, 2011

DISEASE-FREE SURVIVAL

SHG: complex, expensive

Inexpensive collagen detection

Tissue microarray sample: low magnification

Orientation of fibers viewed with color map

Bile duct in cirrhotic liver

Sneak preview: Subcellular-resolution multiplexed MS

Conventional imaging mass spec: spatial resolution ~35 microns

(Images by Richard Caprioli, keynote speaker at a previous Tucson Symposium)

Sneak preview: Subcellular-resolution multiplexed MS

Hematoxylin and Ki-67 imaged via mass spec

Spatial resolution < 1 micron

Large field of view, high-resolution mass-spec image

So what’s coming?

•Stromics •“Companion diagnostics” regime not

going away …(so ER/PR/Her2 etc. will be around for a while)

•… but disruptive therapies will alter the diagnostic landscape

•Spatially resolved, highly multiplexed subcellular-scale molecular and structural profiling is feasible  

•New tools--and new paradigms—should allow us to see the leaves, the trees and the forest.

Prognosis: encouraging

The Venezuelan Poodle Moth is a possible new species of moth discovered in 2009 by Dr. Arthur Anker of Bishkek, Kyrgyzstan.

Snapshot collagen birefringence image of an H&E-stained breast cancer sample

Notes

• Instead of using canonical cell surface markers to identify leukemia stem cells (LSCs), Lagadinou et al. apply a functional approach… of ROS measurements: …both novel and potentially of great utility.

• Cell Stem Cell. 2013 Jan 15.

• BCL-2 Inhibition Targets Oxidative Phosphorylation and Selectively Eradicates Quiescent Human Leukemia Stem Cells.

• The Bcl-2 gene has been implicated in a number of cancers, including melanoma, breast, prostate, chronic lymphocytic leukemia, and lung carcinomas.

• “We demonstrate here that it is feasible to eradicate resistant LSC populations by targeting their unique metabolic dependencies.”

Simple antigen-positivity measurements in bulk tumors may be simplistic

Yellow = HER2(+) stem cells (not bulk population)

Wicha, 2013

FT-SHG: tissue-scale featureschange optics, then:

P-SHG: molecular organization(6 measurements required)

N.B.: Half the malignant stroma is “normal,” and localized features important, so imaging is necessary

-1

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70 80 90 100

Tumor Transplantation (days)

Tu

mo

r B

urd

en (

cm3 )

Left Inguinal (Control rat)Right Axillary (Control rat)Left Axillary (Control rat)Right Axillary (Cured rat)Left Axillary (Cured rat)

Death

Effect of immunophotonic therapy on primary and metastatic tumors in rats

Primary

Mets

CD8 cell attacking a tumor cell

Tumor Immunotherapy: Stage 4 breast cancer

Biology of tumor progression meets biology of aging