First International Conference on Clinical Metagenomics October 14, 2016

2 µm

Using biofilms (and metagenomics) to diagnose colon cancer Cynthia L Sears, M.D.

Johns Hopkins University School of Medicine

Microbial:Colon Cancer Disease Paradigms

ETBF Escherichia coli (pks island) Fusobacterium nucleatum

Single species Microbiome

Keystone Alpha-Bugs

& Consorts

(Driver:Passenger)

ETBF = Enterotoxigenic Bacteroides fragilis

Control

ETBF

ETBF Germ free

Microbiota BFT, IL-17-mediated

Sears CL, Pardoll DM J Infect Dis 203:306, 2011 Sears CL, Garrett WS Cell Host Microbe 15:317, 2014

Hypothesis The microbiota and likely specific members of the

microbiota are important to the initiation and progression of colon cancer.

Christine Dejea

Johns Hopkins School of Medicine

Tumor Mucosa Paired normal mucosa

Individuals with colon cancer Colonoscopy control subjects

without colon cancer

Vogelstein group Pathology Suite Surgery Department, Dr. Liza Wick Colonoscopy Suite, Drs. Ellen Stein and Mark Lazarev

Human Sample Collection

Fix tissues in Carnoy’s

Figure1.

Spatial mucosal microbiota geography: defining CRC bacterial biofilms

Dejea et al. PNAS, December 2014

Descending(le1colon)

Paire

dDistalnormal

Ascending(rightcolon)

Tumor

Colon Cancer Host Universal Bacterial 16S FISH

Bf+ R CRC Bf+ R adenoma Bf- L CRC

Gunnar Hansson

Figure1.

Bacterial biofilms in CRC or colonoscopy hosts are polymicrobial &, in CRC, mucosa invasive.

Dejea et al. PNAS, December 2014

Colon Cancer Host

Right tumor polymicrobial

bacterial invasion (100%)

Paired normal polymicrobial

bacterial invasion (50%)

Rightcolonoscopybx

Le1colonoscopybx

Healthy Colonoscopy Host

Throughout colon

~15% of healthy

hosts bf+

Bacterial invasion not seen

N=142

Figure1.

Sporadic right colon tumors are defined by bacterial biofilms Johns Hopkins & University of Malaya cohorts

Diet, colon prep, other demographics do not correlate

with findings

When biofilms are present, CRC and normal tissues are nearly

always 100% concordant for biofilms.

Transverse

Splenic Flexure

Descen

din

g

Sigmoid

Cecum

Asc

end

ing

Hepatic Flexure

LEFT

RIG

HT

Biofilm

No Biofilm

P<0.0001 P=0.015 P=0.005

Julia Drewes

Dejea et al. PNAS, December 2014 Dr. Jamuna Vadivelu

Biofilms alter normal colon epithelial biology: colonic epithelial cell proliferation (Ki67)

Colon Cancer Patient Distal Normal

Colonoscopy Control Biopsy

Biofilm negative mucosa

Biofilm positive mucosa

Biofilm positive mucosa

Biofilm negative mucosa

Base Crypt top Base Crypt top

*P<0.05 **P<0.01

P<0.0001 P<0.01

All left samples

Also changes E-cadherin, IL-6, pStat3

Dejea et al. PNAS, December 2014

Principle coordinates analysis of bacterial clustering

.

.

Decreased diversity

of microbiome

PNAS, December, 2014

16S rRNA sequencing Hopkins cohort

Limited number specific

species identified as differentially abundant between biofilm+ and

biofilm- tissues.

USA cohort, Johns Hopkins, 454 pyrosequencing

Left biofilm+ colon cancer

N1, N12-diacetylspermine also increased in biofilm+

normal tissues.

More detailed studies suggest produced both by biofilm & host cells

Scripps Institute

Cell Metab 21:891, 2015

Metabolomics: Biofilm presence correlates with increased N1, N12-diacetylspermine

(untargeted à targeted mass spectroscopy pipeline)

Gary Siuzdak Caroline Johnson

Hypothesis Biofilms in healthy individuals increase

the risk of developing adenomas or colon cancer

Biofilm formation

Decreased barrier function

Immune activation (IL-6)

Activation (phosphorylation) Stat3

CEC proliferation

Carcinogenesis

Polyamine metabolism

Biofilm+ colon cancer is driven by carcinogenic mechanisms induced by invasive polymicrobial bacterial biofilms. Biofilm negative colon cancer: work in progress

Sporadic Colon Cancer

Hereditary Colon Cancer Work in progress

Working Postulates

By defining the putative microbial drivers of colon carcinogenesis, we can identify microbial-based biomarkers to test as new

approaches to the prevention of human colon cancer.

Goal

Acknowledgements

Funding: NIH (NIDDK, NCI), Institut Mérieux

Sears Laboratory Ki-Jong Rhee Shervin Rabidazeh Emilia Albesiano Florencia McAllister Augusto Franco

Microbiology Brandon Ellis Karen Carroll

Casero Laboratory Christina Destefano-

Shields Baylin Laboratory Heather O’Hagan

JHU/SKCCC David Huso Bert Vogelstein Kathy Romans-Judge University of Malaya Jane Wanyiri Jamuna Vadivelu April Roslani Ausama Malik Khean Goh Ambiga Iyadorai

April 2012