Race/Ethnicity-Specific Molecular

Cancer Biomarkers: What’s Next?

Partnerships to Advance Cancer Health Equity (PACHE) Investigators Workshop, July 28-29, 2014

Upender Manne, M.S., Ph.D.

Professor of Pathology Senior Scientist of Comprehensive Cancer Center,

University of Alabama at Birmingham Lead PI of the UAB Component of the MSM/TU/UAB CCC

Partnership-U54

MSM/TU/UAB-CCC Partnership Leadership

UAB Comprehensive Cancer Center

Lead PI: Upender Manne, MS, PhD

PI: Isabel Scarinci, PhD

PI: Edward Partridge, MD

Program Manager: Suzanne Byan-Parker, BS

Tuskegee University

Lead PI: Timothy Turner, PhD

PI: Roberta Troy, PhD

Prog. Manager: Chiquita Lee, MBA

Morehouse School of Medicine

Lead PI: James Lillard, PhD

Prog. Managers: Rene Jackson, MPH

Sonja Warner

Cancer Incidence and Mortality by Race/Sex

ACS-2014 Cancer Facts & Figures

Premise

Biological differences in demographic (racial/ethnic, age and sex) groups have implications on cancer incidence (new cases), presentation (stage at diagnosis) and outcomes (mortality/response to Rx).

Approaches to reduce cancer health disparities should also include understanding of their molecular basis.

NODULE

OF BPH

Products from Dying

Cells and Duct

Contents e.g., PSA

tumor

tissue

reaction

Venous and

lymphatic fluids

containing tumor

and tissue-reaction

products and their

metabolites

liver

Urine with tumor and tissue-

reaction products and metabolites

kidney

Molecular Cancer Biomarkers

Cancer biomarkers are present in malignant tissues or serum

They encompass a wide variety of molecules, including DNA, mRNA, miRNA, transcription factors, cell surface receptors, and secreted proteins.

These molecules are produced by tumors or by normal tissues in response to tumor.

Biomarkers will aid in evidence-based patient care and in predicting the clinical outcomes

Brennan DJ, et al. Cancer Genomics Proteomics. 2007;4(3):121-34.

Tumor Development and Progression: Cancer Biomomarkers

Early Detection (population/individual level)

Diagnosis (symptomatic/asymptomatic)

Prognosis (survival/recurrence/relapse)

Prediction (Therapy Efficacy)

Surrogates (Therapy Monitoring)

Surveillance (Risk

Assessment)

Biomarkers of Cancer: Usefulness Depends on Patient Race/Ethnicity

Prognostic Value of p53 Depends on Tumor Location and Patient Race/Ethnicity

Manne U, et al. Cancer 83:2456-2467, 1998

Afr

ica

n A

me

rica

ns (

n=

20

3)

Ca

uca

sia

ns (

n=

30

5)

Shanmugam C/Manne U et al. In Review

Validation of p53 Prognostic Value in an Independent Cohort of Colon Cancers

Ca

uca

sia

ns

Proximal tumors Distal tumors

Afr

ica

n A

me

rica

ns

Proximal tumors Distal tumors

p53 nuclear accumulation is a poor prognostic marker only for non-Hispanic Caucasians when CRCs are located in the proximal colon.

UAB-Caucasians (n=346) and non-UAB-African Americans (n=241)

0

10

20

30

40

50

60

70

Fre

qu

ency

%

Wild type Missense Point-

Mutations

Polymorphic Point-

Mutations

p53 gene status

African Americans (N=63)

Caucasians (N=121)

p53 Mutational Profile of Proximal Colonic Adenocarcinomas

from African-Americans and Caucasians

Survival Analysis of Caucasian Colon Cancer Patients Based

on the Type of p53 Mutations (L 3 Domain Vs. Wild-Type)

0 20 40 60 80 100 120 140 160 180 200

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Wild type p53

Mutations in L 3 Domain

Post-surgical Survival in Months

Su

rviv

al P

rop

ort

ion

Log-rank, Over all P = 0.0291

Log-rank, 10-yr P = 0.0291

Log-rank, 5-yr P = 0.0377

(N = 17)

(N = 61)

0 20 40 60 80 100 120 140 160 180 200

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Wild type p53

0 20 40 60 80 100 120 140 160 180 200

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Wild type p53

Mutations in L 3 Domain

Post-surgical Survival in Months

Su

rviv

al P

rop

ort

ion

Log-rank, Over all P = 0.0291

Log-rank, 10-yr P = 0.0291

Log-rank, 5-yr P = 0.0377

(N = 17)

(N = 61)L 3 Domain =

Codons 236 – 251

Exon 7 = Codons

225 - 261

Manne U, Future Med (Reviews in oncology), 3(3);235-241-2007

Distribution of p53 Mutations in CRCs

Based Patient Race/Ethnicity

3 (5 %)10 (8 %)Other codons

11 (18 %)5 (4 %)Polymorphisms

(Codon 72)

4 (6 %)13 (11 %)LSH-Domain

(Codons 273-286)

2 (3 %)17 (14 %)L 3-Domain

(Codons 236-251)

4 (6 %)15 (12 %)L 2-Domain

(Codons 163-195)

0.0180

39 (62 %)61 (51 %)Wild Type p53

χ2

P - Value

African-

Americans

N = 63 (34%)

Caucasians

N = 121 (66%)p53 Status

3 (5 %)10 (8 %)Other codons

11 (18 %)5 (4 %)Polymorphisms

(Codon 72)

4 (6 %)13 (11 %)LSH-Domain

(Codons 273-286)

2 (3 %)17 (14 %)L 3-Domain

(Codons 236-251)

4 (6 %)15 (12 %)L 2-Domain

(Codons 163-195)

0.0180

39 (62 %)61 (51 %)Wild Type p53

χ2

P - Value

African-

Americans

N = 63 (34%)

Caucasians

N = 121 (66%)p53 Status

Clinical Implications of p53 Codon 72 Polymorphisms in African

American and Caucasian CRCs

Katkoori VR/Manne U, Clin Can Res, 15: 2406-2416, 2009

miR-181b in CRCs

Combined miR-181b (N=344)

0 60 120 180 240 300 3600.0

0.2

0.4

0.6

0.8

1.0

Low (N=234)

High (N=110)

p=0.004

Survival (months)

Su

rviv

al

Pro

po

rtio

n

Blacks miR-181b (N=106)

0 60 120 180 240 300 3600.0

0.2

0.4

0.6

0.8

1.0

Low (N=72)

High (N=34)

p=0.003

Survival (months)

Su

rviv

al

Pro

po

rtio

n

Whites miR-181b (N=238)

0 60 120 180 240 300 3600.0

0.2

0.4

0.6

0.8

1.0

Low (N=162)

High (N=76)

p=0.120

Survival (months)

Su

rviv

al

Pro

po

rtio

n

Whites miR-181b: Stage III (N=70)

0 60 120 180 240 300 3600.0

0.2

0.4

0.6

0.8

1.0

Low (N=42)

High (N=28)

p=0.502

Survival (months)

Su

rviv

al

Pro

po

rtio

n

Blacks miR-181b: Stage III (N=35)

0 60 120 180 240 300 3600.0

0.2

0.4

0.6

0.8

1.0

Low (N=24)

High (N=11)

p=0.001

Survival (months)

Su

rviv

al

Pro

po

rtio

n

__High (N=27)

……Low (N=43)

(N=70)

Bovell L. et al/Manne U. Clin Can Res; 19(14); 1–11, 2013

Jones J et al /Yates C, The A Journal Pathol, 181; 5, 2012 ***Patent pending

Kaiso is Biomarker for African American Prostate Cancer Patients

Kaiso is Biomarker for African American Breast Cancer Patients

Jones et al/Yates C Clin Exp Metastasis. 2014 Feb 26. ***Patent pending

Biomarkers of Cancer: Useful to both African American and

Caucasian Patients

Bcl-2 Expression: Prognostic Markers of Stage II CRC

(Manne/Grizzle, et al. Int. J. Cancer, 74:346-358, 1997)

Prognostic Value of Bcl-2/p53: Multivariate Analysis

0 40 80 120 160 200 240 2800.0

0.2

0.4

0.6

0.8

1.0

Bcl-2 High

Bcl-2 Low

log-rank, Over all P = 0.0003log-rank, 10 year P = 0.0006log-rank, 5 year P = 0.0015

Time to Recurrence(months after surgery)

Re

cu

rre

nc

e f

ree

pro

po

rtio

n

Patients with Stage II CRCs (n=210)

0 40 80 120 160 200 240 280

0.0

0.2

0.4

0.6

0.8

1.0

Bcl-2 High

Bcl-2 Low

log-rank, Over all P = 0.6063log-rank, 10 year P = 0.6063log-rank, 5 year P = 0.6058

(months after surgery)

Recu

rren

ce f

ree

pro

po

rtio

n

Patients with Stage III CRCs (n=306)

Chatla /Manne . et al. Cancer Biomarkers, 1 ; 17-27, 2006

Kennedy R D/Manne U et al. JCO 2011;29:4620-4626

(A) Receiver operating characteristic curve of 10 cross-validation repeats from the 215-sample training set.

Development of a Multiple-Gene Expression Prognostic Signature Stage II CRCs

(A) Receiver operating characteristic (ROC) curve of the 144-sample independent validation set.

The 634 –probe set signature identified high-risk patients with a hazard ratio (HR) of 2.62 (P=0.001) in the training set.

In an independent validation set of 144 samples, this signature identified high-risk patients with an HR of 2.53 (P=0.001) for recurrence and an HR of 2.21 (P .0084) for cancer-related death.

The signature was shown to perform independently from known prognostic factors (HR=2.55; P=0.001).

Stage I

0 60 120 180 2400

20

40

60

80

100

P = 0.500

(n=15)

(n=27)

Post-Surgery Survival in Months

Su

rviv

al

Pro

bab

ilit

y

0 60 120 180 2400

20

40

60

80

100

P = 0.159

Stage II

(n=50)

(n=19)

Post-Surgical Survival in Months

Su

rviv

al

Pro

bab

ilit

y

Stage III

0 60 120 180 2400

20

40

60

80

100

P = 0.014

(n=37)

(n=16)

Post-Surgery Survival in Months

Su

rviv

al

Pro

bab

ilit

y

Stage IV

0 10 20 30 40 500

20

40

60

80

100

(n=11)

(n=31)P = 0.553

Post-Surgery Survival in Months

Su

rviv

al

Pro

bab

ilit

y

Figure 1

A B

C D

Prognostic Significance of p27kip-1 Based on Tumor Stage

______ p27kip-1 high expressors

------- p27kip-1 low expressors

p27kip-1 Expression Biomarker of Stage III CRCs

Manne et al. Clin Cancer Res. 10, 1743-1752, 2004

Bax Expression Predicts Efficacy 5-FU-Based Therapy in CRCs

0 30 60 90 120 150 180 210 2400.0

0.2

0.4

0.6

0.8

1.0

Bax - High

Bax - Low

A

Log-rank, Over all P = 0.0006Log-rank, 10-yr P = 0.0006Log-rank, 5-yr P = 0.0055

Post-surgical Survival in Months

Su

rviv

al

Pro

po

rtio

n

Surgical resection only

0 30 60 90 120 150 180 210 2400.0

0.2

0.4

0.6

0.8

1.0

Bax - High

Bax - Low

Post-surgical Survival in MonthsS

urv

iva

l P

rop

ort

ion

Log-rank, Over all P = 0.0343Log-rank, 10-yr P = 0.0343Log-rank, 5-yr P = 0.0343

B

Surgical resection plus

5-FU based therapy

Age, race, gender, tumor location and stage matched 5-FU treated (Rx) (N=56) and untreated (only surgical resection) (N=56) CRC patients.

( Shanmugam CKet al /Manne U J. GI Oncol, 1(2):76-89, 2010)

Serum CXCL13 & PSA Correlates of Prostatic Disease

CXCL13/CXCR5 as Biomarkers of Prostate Cancer

( Singh S et al /Lillard J. Int J Cancer 15;125(10):2288-95, 2009)

overweight and obese TNBC patients have higher levels of leptin and develop chemo-resistance.

ObR and Leptin levels higher in African Americans than Whites.

Leptin secreted by adipose and breast cancer tissue activates the Notch pathway, increasing tumor growth, angiogenesis and breast cancer stem cell numbers.

Fig$13.$PEG+LPrA2$reduces$BC$growth$in$lean$and$obese$mice.$

m4T1$cells$

BC+4T1$

%BC$free$obese$DIO+$mice$

PEG+LPrA2$

DMBA$

months$

control$

PEG+LPrA2$

%$of$mE0771+Tumors$

2

(A)LPrA2reduced4T1-BCgrowthinBalb/csyngeneicmice[4];(B)humanBCxenogra s[5];DMBA(a

carcinogenic)-inducedBCinC57Bl/6Jobesemice(diet-induced-obesity,DIO[6],and(C)E0771-BC

hostedbyDIO-syngeneicmice[7].

Leptin peptide receptor antagonist (LPrA2):

LPrA2 is a small peptide (~3,000 Da) derived from Helix 3 of leptin that shows high binding affinity for OB-R (Ki ≈ 0.6x10-10 M).

LPrA2 significantly decreases leptin-mediated growth of breast cancer and pancreatic cancer cells.

LPrA2 inhibits leptin-induced Notch and cancer stem cells markers, and improves chemotherapeutic effects.

LPrA2 abrogates growth of breast cancer and Notch expression in mice.

Leptin-VEGFR2/Notch Axis in Breast Cancer Progression and Chemoresistance

Gonzalez-Perez RR et al Cell Signal. 2010;22(9):1350-62. Gonzalez-Perez RR et al. Cancers 2013; 6;5(3):1140-62.

Manne U et al. Drug Discovery Targets (review),10 (14); 2005

Phases of Discovery and Validation of Cancer Biomarkers(Developed by the NCI-EDRN)

Academic/Research

Institutions

Potential

Biomarkers

Pre-ClinicalExploratory Studies

Phase I

Phase IVProspective

Validation Studies forDisease Detection

ClinicalAssay/Technique

Validation StudiesPhase II

RetrospectiveValidation Studies for

Disease Detection

Prospective ClinicalUtility Assessment

Studies

Approval byRegulatoryAgencies

Development ofClinical Testing Kits

Clinical Usage forDisease Detection

Phase III

Phase V

Distinguishes differentcancers or/and

normal vs cancers

Estimates false referralrates & disease

detection

Assesses assay ortechnique reproducibility

& portability

Evaluatessensitivity & specificity of

disease detection

Evaluates overallbenefits & risks of the

test

Industry

Discovery

Preclinical Validation

Clinical Validation

Convenient Sample Set

Reference

Sample Set

Steps to Move Biomarkers to the Next-Level

To translate race/ethnicity-specific discoveries into clinical practice:

Initiate larger national-level prospective studies

Conduct academic-community partnership studies by involving community oncologists to better serve the local communities

Needs funding mechanisms specifically to conduct race/ethnicity-specific trials.

Benefits of Academic-Community Partnership

The academic-community based efforts -

will aid in rapid incorporation of molecular test (profiling) into community practice

will facilitate in integrating molecular biomarker profiles with epidemiological, nutritional, and behavioral data

will improve interactions with community oncologists and aid in updating the latest advancements in molecular and medical oncology

will aid in integrating the use of molecular analyses to make treatment decisions both in community and academic practices, and in reducing the cancer burden.

Thanks!!

Bcl-2 (IHC) - increase expression is a good prognostic markers only for Stage II CRC (Int. J. Cancer, 74:346-358, 1997 & Cancer Biomarkers, 1; 17-27, 2006)

p27kip-1 (IHC) – increased nuclear accumulation is a good prognosticator only in Stage III CRCs (Clin Cancer Res. 10, 1743-1752, 2004).

MUC1 (IHC) – increased expression is a poor prognostic indicator (Clin Cancer Res. 6,4017, 2000).

MUC4 (IHC) – increased expression is a poor prognosticator only in early stage (Stages I & II) CRCs (Cancer, 116,15:3577-86,2010).

Bax or Bax/Bcl-2 (IHC) - high Bax is good prognosticator and low Bax/Bcl-2 expressors are candidates for adjuvant chemotherapy (J. GI Oncol, 1(2):76-89, 2010).

Microsatellite Instability (MSI) – MSI-L in Caucasians is a poor prognosticator, behave like MSS, but in African-Americans it is a good prognosticator like MSI-H (in review).

Distinct Prognostic Value of Molecular Marker in CRCs: Tumor Stage, Location, Race/Ethnicity (UAB-Studies)