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Molecular and Genetic

Tumor Testing: Why is it Important?

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Dr. Tanios Bekaii-Saab, MDSection Chief , Gastrointestinal Oncology Program

Associate Professor of Medicine and Pharmacology

The Ohio State University – James Cancer Hospital

Molecular and Genetic Tumor Testing:

Why is it Important?

Tanios Bekaii-Saab, MD

Section Chief , Gastrointestinal Oncology Program

Associate Professor of Medicine and Pharmacology

The Ohio State University – James Cancer Hospital

Colorectal Cancer as Worldwide Health Problem

– 3rd highest incidence rate (~ 1,200,000/yr)

– 4th highest mortality rate (~ 608,000/yr)

CA: A Cancer Journal for Clinicians 2011;61:69-90

Worldwide Developed Developing

Sporadic

Lynch Syndrome

Familial

Hereditary

FAP; AFAPMixed Polyposis SyndromeAshkenazi I1307KCHEK2 (HBCC)MYHTGFBR1

PJSFJPCDBRRS

= as yet undiscovered hereditary cancer variants

HamartomatousPolyposis

Syndromes

AC-1 without MMR(Familial CRC of

syndrome “X”)

Colorectal Stage Distribution at Diagnosis (%)

19 % patients have Stage IV disease on diagnosis5 year-survival of Stage IV disease is 12%

Altekruse SF, Kosary CL, Krapcho M, et al. SEER Cancer Statistics Review, 1975-2007, National Cancer Institute. Bethesda, MD, http://seer.cancer.gov/csr/1975_2007/, based on November 2009 SEER data submission, posted to the

SEER web site, 2010.

10

Years

No benefit of chemotherapy

Cured bychemotherapy

FluoroP + oxali

Already cured by surgery

Adjuvant Therapy for Colon Cancer Stage III

0

20

40

60

80

100

0 1 2 3 4 5

expo

sed to

toxicity

Surgery alone

Surgery plus Chemotherapy

20%

%

Dis

ease

Fre

e S

urv

ival

60%

20%

20%

20%

Moertel CG, N Engl J Med 1990 IMPACT investigators, Lancet 1995

André T, J Clin Oncol. 2009Yothers G, J Clin Oncol 2011Haller D, J Clin Oncol 2011

Moertel CG, N Engl J Med 1990 IMPACT investigators, Lancet 1995

André T, J Clin Oncol. 2009Yothers G, J Clin Oncol 2011Haller D, J Clin Oncol 2011

High-risk features Stage II colon cancer should be

considered for adjuvant therapy

• According to clinico-pathologic features:

• Obstruction/perforation

• T4 tumors

• Less than 10/12 LN examined

• Lymphatic or vascular invasion

• Poorly differentiated histology

• Molecular Biomarkers ?

• Microsatellite instability (MSI)

– Deficient Mismatch Repair as a Predictive Marker for Lack of Benefit from 5-FU

based Chemotherapy in Adjuvant Colon Cancer (Sargent et al JCO 2010)

Microsatellite Instability

• 4 MMR (mismatch repair) proteins: MLH1, MSH2, MSH6, and PMS2

• MSI is due to defects in MMR, resulting in the accumulation of nucleotide

mutations and alteration in microsatellite length.

• Genotyping: MSI-high versus MSS or MSI-low

• IHC: deficient MMR versus proficient MMR

• MSI tumors are due to:

• Germline mutation of MMR proteins secondary to Lynch syndrome (5%)

• Sporadic hypermethylation causing gene silencing of hMLH1

• Lynch syndrome screening:

• IHC of MMR proteins

• If missing MMR protein check BRAF mutation

• If BRAF wild-type genetic testing for Lynch syndrome

Risk of recurrence

• Adjuvant online:

– Age

– Co-morbidity

– Tumor depth

– # nodes positive

– # nodes examined

– Tumor Grade

• Oncotype Dx – 12 gene recurrence score (8-24%)

• Coloprint- 18 gene profile

Oncotype DX: Primary Analysis: Recurrence Score Predicts Recurrence Risk in Stage II Colon Cancer

Patients in QUASAR

Oncotype DX: Primary Analysis: Recurrence Score Predicts Recurrence Risk in Stage II & III Colon

Cancer Patients in NSABP C-07 (n=892)

Solid: 5FU Dashed: 5FU+Ox

Stage III C

Stage III A/B

Stage II

• With similar relative benefit of oxaliplatin added to adjuvant 5FU across the range of RS, absolute benefit of oxaliplatin increases with increasing RS, most apparently in stage II and stage IIIA/B patients

p<0.001

Solid: 5FUDashed: 5FU+Ox

O’Connell, ASCO 2012

Summary: Stages II and III

• Stage II colon cancer

– look at high-risk features, MSI status to help

decide whether to offer adjuvant chemotherapy

• Stage III colon cancer

– Offer adjuvant 5FU/Xeloda or FOLFOX

• Stage II and III rectal cancer

– Neoadjuvant chemoradiation surgery adjuvant

chemotherapy

Stage IV Colorectal Cancer (mCRC):

Which biologic agent and for whom?

A high number of agents is currently available for the treatment of mCRC

5-FU Capecitabine Irinotecan

Oxaliplatin Bevacizumab

Panitumumab

Cetuximab

AfliberceptRegorafenib

Concept of “All-3-Drugs” : 11 Phase III Trials; 5,768 Patients

OS (mos) = 13.2 + (% 3 drugs x 0.1), R^2 = 0.85

0 10 20 30 40 50 60 70 80

Infusional 5-FU/LV + irinotecan

Infusional 5-FU/LV + oxaliplatin

Bolus 5-FU/LV + irinotecanIrinotecan

+ oxaliplatinBolus 5-FU/LV

LV5-FU2

FOLFOXIRI

CAIRO

22

21

20

19

18

17

16

15

14

13

12

Med

ian

OS

(m

os)

Patients With 3 Drugs (%)

p = .0001

First-Line Therapy

LV = leucovorin.Grothey et al, 2004; Grothey & Sargent, 2005; Koopman et al, 2007; Falcone et al, 2007.

Murine Ab“momab”

ChimericMouse-Human Ab

“ximab”

Humanized Ab“zumab”

Fc

Fab

Human Ab“mumab”

Biologic Agents in CRC =MoAbs

(17-1A) Cetuximab Bevacizumab

PanitumumabEGFR

VEGFMoAbs = monoclonal antibodies.McRee & Goldberg, 2011; Eng, 2010.

Extracellular

Intracellular

Ligand

EGFR

PI3K

Akt

Raf

MEK

MAPK

Cell Motility

MetastasisAngiogenesisProliferation

Cell SurvivalDNA

PTEN

Ras

Fakih & Wong, 2010; Williams & Lockhart, 2009.

KRAS WT = Wild TypeKRAS MT = Mutant ( codons 12 and 13)

VEGF-A

VEGF-R1(Flt-1)

MigrationInvasionSurvival

VEGF-R3(Flt-4)

Lymphangiogenesis

VEGF-R2(KDR/Flk-1)Proliferation

SurvivalPermeability

PlGFVEGF-B

VEGF-C, VEGF-D

Fu

nc

tio

ns

Bevacizumab

Ramucirumab

Aflibercept (VEGF Trap)

PIGF = placental growth factor. Holash et al, 2002; Roy et al, 2006; Ghosh et al, 2000.

Large Molecule VEGF Inhibitors

Regorafenib (BAY 73-4506), an oral multikinase inhibitor targeting multiple tumor

pathways1-3

KIT

PDGFR

RET

1. Wilhelm SM et al. Int J Cancer 2011.2. Mross K et al. Clin Cancer Research 2012.

3. Strumberg D et al. Expert Opin Invest Drugs 2012.

PDGFR-βFGFR

VEGFR1-3TIE2

Regorafenib

Inhibition of neoangiogenes

is

Inhibition of neoangiogenes

is

Inhibition of tumor

microenvironment signaling

Inhibition of tumor

microenvironment signaling

Inhibition of proliferation Inhibition of proliferation

Biochemicalactivity

Regorafenib IC50 mean ± SD nmol/l

(n)

VEGFR1 13 ± 0.4 (2)

Murine VEGFR2

4.2 ± 1.6 (10)

Murine VEGFR3

46 ± 10 (4)

TIE2 311 ± 46 (4)

PDGFR-β 22 ± 3 (2)

FGFR1 202 ± 18 (6)

KIT 7 ± 2 (4)

RET 1.5 ± 0.7 (2)

RAF-1 2.5 ± 0.6 (4)

B-RAF 28 ± 10 (6)

B-RAFV600E 19 ± 6 (6)

Treatment Paradigms for mCRC

• NCCN guidelines are a great resource to help see the “trees in the

forest”

• Some patients with stage IV disease are cured by an interdisciplinary

approach

• FOLFOX = CAPOX = FOLFIRI

(XELIRI has problems with toxicity)

• Most patients tolerate a chemotherapy doublet (but not all need it)

• The addition of biologics to chemotherapy has improved outcomes

• We are on the verge of individualized therapy based on

molecular predictive factors

mCRC = metastatic CRC; FOLFOX = oxaliplatin, 5-FU, leucovorin; CAPOX = capecitabine, oxaliplatin; FOLFIRI = irinotecan, infusional 5-FU; XELIRI = capecitabine, irinotecan.

Aranda et al, 2011; Eadens & Grothey, 2011; NCCN, 2011.

Current Biologic Landscape in mCRC

• Bevacizumab and EGFR mAbs competing for first-line

patients in KRAS WT CRC

• Bevacizumab (TML) and ziv-Aflibercept(VELOUR)

competing for second-line and with EGFR mAbs in KRAS Wt

CRC

• Best sequence of therapies (VEGFi vs EGFRi) still to be

established

• Regorafenib as salvage therapy option (CORRECT)

mab: 40 mg m i.v. 120min init ial dose250 mg/m2 i .v. 60min q 1w

Bevacizumab: 5 mg/kg i .v. 30-90min q 2w

/0i

FIRE-3 Phase III study design

Cetux 2

FOLFIRI + Cetuximab

FOLFIRI + BevacizumabBevacizumab: 5 mg/kg i.v. 30-90min q 2w

mCRC1st-line therapy KRAS wild-type

N= 592

Randomize 1:1

Cetuximab: 400 mg/m2 i.v. 120min initial dose

250 mg/m2 i.v. 60min q 1w

• Primary objective: Overall response rate (ORR) (inv assessed)

• Designed to detect a difference of 12% in ORR induced by FOLFIRI + cetuximab (62%) as compared to FOLFIRI + bevacizumab (50%)

• 284 evaluable patients per arm needed to achieve 80% power for an one-sided Fisher‘s exact test at an alpha level of 2.5%

FOLFIRI: 5-FU: 400 mg/m2 (i.v. bolus); folinic acid: 400mg/m2

irinotecan: 180 mg/m2 5-FU: 2,400 mg/m2 (i.v. 46h)

Heinemann et al., ASCO 2013

Predictive Biomarkers For the Use of Anti-EGFR Antibodies in mCRC

79%

18%

3%

MT BRAF 5-10%

Other- PTEN LOE- NRAS MT- PIK3CA ex 20 MT

~40%

Adapted from multiple sources: 1- Wheeler DL, et al Nat Rev Clin Oncol 2010 7(9) : 493-507;2- De Roock et al . Lancet Oncology, 2010 11: 753-762; 3- Frattini M , BJC 2007 97; 1139-1145;

4- Di Nicolantonio F et al . Journal of Clin Oncol 2008 26(35); 5705-5712;5- Loupakis F et al. BJC 2009 101;715-721; 6- Tran B . Cancer 2011 ; 1-10.

10%

8%

• European Consortium:

– Refractory CRC

patients treated with

irinotecan + cetuximab

– 1022 tumour samples

– 773 samples of quality

De Roock, W et al. Lancet Oncol 2010;11:753-62

Beyond KRAS: BRAF, NRAS, PIK3CA mutations

Are All KRAS Mutations Created Equal? – p.G13D

Pooled analysis of OPUS and CRYSTAL

Tejpar et al, 2011.

KRAS G13D: Treat Similar to 12- Pooled analysis of panitumumab studies showed no difference between codon 13, and codon 12, KRAS mutations

Peeters M, et al. J Clin Oncol. 2012; 30(Suppl 4). Abstract 838.

PanitumumabStudies-’181-’203-’408

CetuximabStudies- CRYSTAL- OPUS

Response Rate

Overall Survival

De Roock, W et al. Lancet Oncol 2010;11:753-62

Beyond KRAS: BRAF, NRAS, PIK3CA mutations

Updated Analysis of PRIME study

KRAS exon 2codon 12/13

40%

KRAS exon 3codon 61

4%

KRAS exon 4codon 117/146

6%

NRAS exon 2codon 12/13

3%

NRAS exon 3codon 61

4%

BRAF exon 15codon 600

8%

17%

Oliner et al., ASCO 2013

Oliner et al., ASCO 2013

HR 0.83

(KRAS wt cod 12/13)

HR 0.78(all RAS wt)

OS

Detriment!

Detriment!

Ir vs IrCsnon-inferior efficacy

primary endpoint PFS at 12 wks

IrPanirinotecan + pan’mab

IrCsirinotecan + c’sporin

Iririnotecan alone

Ir vs IrPansuperior efficacy

primary endpoint OS

Iririnotecan alone

KRAS mutated or unknown KRAS-wt (c.12/13 & 61)

PICCOLO studytarget total n = 1200

(including 494 accrued under previous design)eligibility as before

Seymour et al. Proc ASCO 2011:A3523

PICCOLO Study

IrPan better Ir better

*Adjusted HRs, 95% CIs

KRAS wt: 460 pts, 312 eventsHR=0.91 (0.73, 1.14), p=0.44

Double wt : 348 pts, 230 events; HR=0.87 (0.67, 1.13), p=0.30

BRAF mut: 63 pts, 53 events; HR=2.03 (1.13, 3.64)

NRAS mut: 21 pts, 15 eventsHR=4.59 (1.19, 17.67)

KRAS146 mut: 17 pts, 14 eventsHR=1.32 (0.30, 5.81)

Any mut: 99 pts, 80 eventsHR=2.03 (1.26, 3.28)

All wt: 264 pts, 171 events; HR=0.86 (0.63, 1.16), p=0.32

Seymour et al. Proc ASCO 2011:A3523

OS

PICCOLO Study

BRAF Mutations in mCRC

• BRAF is primary effector of

KRAS signaling

• BRAF mutations

– Occur most frequently in exon

15 (V600E)

– Found in 4%–14% of patients

with CRC

– Mutually exclusive with KRAS

mutations

Raf

MEK

Erk

P

Tumor cellproliferationand survival

EGF

Tumor cell

Ras

Di Nicolantonio et al, 2008; Yarden & Sliwkowski, 2001; Artale et al, 2008.

P

PP

CRYSTAL : Outcome in KRAS WT/BRAF MT mCRC

KRAS WT(n = 566)

KRAS WT/BRAF MT (n = 59)

FOLFIRI 

(n = 289)

FOLFIRI +Cetuximab(n = 277)

FOLFIRI 

(n = 33)

FOLFIRI +Cetuximab

(n = 26)

mOS (mos) 21.6 25.1 10.3 14.1HR [95% CI]p Valuea

0.83 [0.687–1.004].0549

0.91 [0.507–1.624].7440

mPFS (mos) 8.8 10.9 5.6 8.0HR [95% CI]p Valuea

0.68 [0.533–0.864].0016

0.93 [0.425–2.056].8656

RR (%)[95% CI]

42.6[36.8–48.5]

61.0[55.0–66.8]

15.2[5.1–31.9]

19.2[6.6–39.4]

p Valueb < .0001 .9136

aStratified log-rank test.bCochran-Mantel-Haenszel test.

Van Cutsem et al, 2011.

Potential Predictive Biomarkers

MUTATIONS

•KRAS mut

•NRAS mut, BRAF mut

•PIK3CA mut

•TP53 mut, PTEN mut

RECEPTOR EXPRESSION

• EGFR (HER1), HER-2/neu,

HER-3, HER-4, IGF1R

LIGAND EXPRESSION

• EGF, TGF-α, HB-EGF,

amphiregulin (AREG),

betacellulin, epiregulin (EREG)

and epigen

DOWNSTREAM EFECTORS

EXPRESSION

• MPK-1 (DUSP-1), DUSP-4,

DUSP-6

Q: Do EGFR Antibodies Harm KRASMT? A: Yes, especially combined with bevacizumab (VEGF antibody)

Study TreatmentTotal

Patients

KRASMT

PFS

KRASWT

PFS

Tol

NEJM 2009

CAPOX/Bev

+/- C755

8.1 mos

(worst)

10.5 mos

--

Hecht

JCO 2009

5-FU/OX/Bev

+/- P823

10.4 mos

HR=1.25

9.8 mos

HR 1.36

Hecht

JCO 2009

5-FU/IRI/Bev

+/- P230

8.3 mos

HR 1.19

10.0 mos

HR 1.50

Bokemeyer

JCO 2009

FOLFOX

+/- C344

5.5 mos

HR 1.83

7.7 mos

HR 0.57

CAP = capecitabine; OX = oxaliplatin; IRI = irinotecan; Bev = bevacizumab; C = cetuximab; P = panitumumab

Tol J, et al. N Engl J Med. 2009; 360(6):563-572; Hecht JR, et al. J Clin Oncol. 2009;27(5):672-680; Bokemeyer C, et al. J Clin Oncol. 2009;27(5) 663-671.

Q: Is Re-Biopsy Necessary?

A: No

> 96% concordancebetween primariesand metastases

Only 2% clinicallyrelevant

Knijn N et al. Br J Cancer. 2011;104:1020-1026

Conclusions: EGFR mAbs

• Efficacy in KRAS wt CRC well established

• KRAS G13D and BRAF mutations likely have an adverse

prognostic effect in mCRC

• All-RAS wild-type mCRC > 45% of mCRC

• Further molecular refinements in future (PTEN, EGFR

ligands, PIK3CA…) could limit the patient population

suitable for EGFR mAbs down to <35%

– This refined patient population could sustain a marked

benefit from use of first-line EGFR mAbs!

• In KRAS WT, bevacizumab or EGFR antibodies can be added

to chemotherapy in first-line

– Oxaliplatin-based regimens should not be used in combination with

cetuximab (COIN and NORDIC studies negative)

– EGFR antibodies maintain efficacy in later lines of therapy

– Would favor bevacizumab for now ( FIRE 3 and Ras mutations?) in

view of palliative setting and more favorable toxicity profile

• Main toxicities of EGFR inhibitors include rash, diarrhea hypomagnesemia and

hypersensitivity reactions

• Main toxicities for bevacizumab include hypertension and less commonly

arteriothromboembolic events ( in elderly patients with risk factors) and GI

perforations.

• Where to place aflibercept in the continuum? 44

Standard Therapy for Stage IV

median overall survival

Advances in the Treatment of Stage IV CRC

1980 1985 1990 1995 2000 2005

5-FUIrinotecan

CapecitabineOxaliplatin

CetuximabBevacizumab

BSC

Panitumumab

20152010

Aflibercept

Regorafenib

BBP

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