colorectal cancer - university...

Colorectal cancer

Chapelle, J Clin Oncol, 2010

Asit Paul, MD, PhD

11/24/15

Early-Stage Colorectal cancer:

Microsatellite instability, multi-gene assay & emerging molecular strategy

Mr. X:

A 50 yo asymptomatic male, Mr X, underwent staging

colonoscopy. Colonoscopy showed a 3 cm-mass in the

sigmoid colon. Staging scans showed no adenopathy or

distant metastases. CEA was 2.0 ng/mL (normal <3 ng/mL).

Patient underwent sigmoid colonic resection. Pathology

showed moderately differentiated adenocarcinoma,

invading upto muscularis propria (pT2). 20 lymph nodes

were retrieved and negative for metastasis.

What is the next step to do?

1. Observation

2. Adjuvant chemotherapy

3. Testing for MMR protein

4. Testing for RAS and BRAF status

Mr. Y:

A 45 yo M presented with 3 episodes of FBPR within a month.

Colonoscopy with EUS showed a 4-cm ulcerating-mass

obstructing the sigmoid colon, but no enlarged LN. Staging scans

showed no adenopathy or distant metastases. CEA was 4.2 ng/mL

(normal <3 ng/mL). Patient underwent R hemicolectomy with

diverting colostomy. Pathology showed a mucinous

adenocarcinoma with involvement of serosa (pT4). 27 lymph

nodes were retrieved and negative for metastasis.


1. Observation


3. Testing for MMR-protein

4. Multi-gene expression analysis

Current standard of care for

non-metastatic CRC

• Stage I (T1/T2, Node negative): • Surgery alone

• Stage II (T3/T4, Node-negative): • Surgery -> ??

• Stage III (any N, Node positive)• : Surgery + adjuvant chemotherapy

Adjuvant chemotherapy is

stage II CRC could be

beneficial, but the absolute

benefit is small (5.4% high-

risk, 3.6% low risk, over 5

years)

Lancet, 2007, 370:2020-29

Adjuvant Chemotherapy

Guidelines for stage II CRC

• pT4

• Tumor perforation

• Obstructing tumor

• Inadequate node sampling (<12

nodes)

High-risk

stage II Colon

cancer

Beyond the traditional measures

of risk stratification….

Risk of recurrence in Stage II CRC patient in QUASAR study (n=1436)

Gray RG, J Clin Oncol, 2011

T4 status, MMR protein status & Recurrence score by multigene assay are the significant prognostic factors for

recurrence

• Basics

• Testing strategy

• Clinical relevance

MMR & MSI

CRC carcinogenesis pathways

Villar & Gruber, Nat Rev Oncol, 2010

Microsatellites

Microsatellites are short (1-7 bp) repetitive

nucleotide sequences scattered throughout

human genome.

There are estimated 500,000 microsatellites in

human genome. Most common MS in human

genome is a dinucloide-repeats of A & C

Length of microsatellites varies from person-

to-person, but have a set length for an

individual person (‘DNA finger print’).

• Microsatellites are prone to replication error

• Erroneous replication process leads to increase or

decrease in number of repeats in MS known as

microsatellite instability (MSI)

Microsatellite Instability (MSI)

Gruber, J Nat Cancer Inst, 2003

In most cases, MSI may not have any

consequences. If MSI occurs in an important

gene, it leads to disease. MSI was first

described in XP.

MSI in an oncogene or tumor suppressor gene

increases the risk of cancer. Lynch syndrome is

the classic example of MSI as a result of germ-

line mutation of MMR gene

Consequence of MSI

Mismatched Repair Proteins (MMR)

• MMR proteins are responsible for

surveillance & correction of replication-

errors

• MMR proteins are products of 4 genes:

MLH1, MSH2, MSH6, PMS2

• Deficiency of MMR protein (MMR-d), due to

mutation of MMR gene (germ line or

sporadic), leads to replication error & MSI

Testing of MMR/MSI

• IHC of tumor tissue for MMR protein expressions

• MSI testing of tumor tissue

A panel of microsatellite

markers, is compared in

tumor tissue and normal

tissue. Core panel of

five markers are

generally used: BAT25,

BAT26, D2S123, D5S346,

and D17S250

Chapelle, J Clin Oncol, 2010

• DNA-based testing, PCR, gene-sequencing analysis

(blood or tumor tissue)

MMR Proteins

• MMR proteins make heterodimers

• MLH1 & MSH2 are the dominant partner of their pair

• MSH6 & PMS2 proteins are unstable in absence of their dominant partners

IHC patterns

of MMR

protein

expression

Richman, Int J of Oncol, 2015

Mr. Z:

A 51 yo M underwent staging colonoscopy. Colonoscopy

showed a non-obstructing 3 cm-mass in the ascending

colon. Staging scans showed no adenopathy or distant

metastases. CEA was 3.0 ng/mL. Patient underwent colonic

resection & anastomosis. Pathology showed mucinous

adenocarcinoma, invading through muscularis propria to

adjacent pericolonic tissue (pT3). 27 lymph nodes were

retrieved and negative for metastasis.


1. Observation


3. Testing for MMR proteins

4. Testing for RAS and BRAF status

Who should get testing for MMR/MSI

Testing for MMR protein and/or MSI should be done for all patients with newly-diagnosed colorectal cancer

ASCO, ESMO & ACG guidelines, 2014

Patients who are diagnosed with colorectal cancer <70 years, stage II tumors, or who meet the Bethesda Criteria

NCCN, 2015

Infiltrating Mucinous differentiation

Poorly differentiated

with medullary

growth pattern

Crohn-like lymphocytic

reaction

Phenotype of d-MMR/ MSI

Early age of onset, R sided tumors,

synchronus or metachronus tumors, high-grade/low-stage

Mr. ZMr. Z with pT3pN0M0 (stage II) colon cancer. His tumor resected tumor was tested for MMR-protein expression by IHC. Staining showed absence of MLH1 & PMS2-proteins, staining for MSH2 & MSH6 are positive.

What to do next?

1. Observation2. Adjuvant chemotherapy3. MSI-testing of tumor tissue4. Testing for BRAF mutation

IHC pattern in MMR deficiency

MMR-intact: (MMR-i)

All MMR proteins are present ->MSS

MMR-deficient (MMR-d):

1 or more MMR protiens are deficient -> MSI

• 15% of CRCs are MMR-d (MSI)

• More commonly, MMR-d is

sporadic & is due to

hypermethylation of MLH1

gene promoter. Sporadic

tumors often carry the BRAF

V600E somatic mutation.

• Others have germ line

mutation of MMR gene

(MLH1,MSH2, MSH6, PMS2) i.e

Lynch syndrome

• In case of MLH1-loss by IHC,

BRAF testing should be done

prior to testing for Lynch

Syndrome

Majority of MMR deficiency are

sporadic, not Lynch Syndrome

• Loss of MLH1: Check BRAF mutation first. If wt,

genetic testing for LS

• Loss of MSH2, MSH6, PMS2: genetic testing for LS

Prognostic value ofMMR/MSI status in CRC

Gryffe, NEJM, 2000

• N=607, 50 y or younger

• 17% patients have MSI

• MSI has independent survival

advantage over other risk factors

• MSI tumors tend to metastasize

to LNs less

Patients with stage II & III CRC & MSI-status

have better prognosis

n outcome HR CI Data type

Dienstmann R, J Clin Oncol. 2015

MSI as a predictor of response to 5FU based chemotherapy

Villar & Gruber, Nat Rev Oncol, 2010

Mr. Z

Mr. Z with pT3pN0M0 (stage II) colon cancer. Patient’s resected

tumor was tested for MMR-protein expression by IHC.

Staining showed absence of MLH1 & PMS2-proteins,

staining for MSH2 & MSH6 are positive. BRAF mutation

testing of the tumor was done, which came out to be

mutated for BRAF 600E

Based on the molecular markers, who has the best & worst prognosis

1. MMR-i (MSS), BRAF wt, R sided tumor

2. MMR-i (MSS), BRAF V600 E mutated, L sided tumor

3. MMR-d (MSI), BRAF wt, R sided tumor

4. MMR-d (MSI), BRAF V600E mutated, R sided tumor

MSS- All site MSS- Left side MSS- Right side

Popovici, BMC cancer, 2013

In stage II-III colorectal cancer, BRAF mutation was confirmed a marker of

poor survival only in subpopulations involving microsatellite stable and

left-sided tumors (n=1423, PET-ACC3 cohort). KRAS status had no

prognostic value.

Prognostic value of BRAF V600E mutation

• BRAF V600E is considered poor prognostic factor in CRC

• Interpretation of BRAF V600E mutation should always be done

in the context of MSI status in early CRC

• The prognostic effect of MSI in early CRC overrides poor

prognosis determined by BRAF

• Worst prognosis is seen in patient with MMR-i (MSS) & L sided

tumorDienstmann R, J Clin Oncol. 2015

Mr. Q

A 51 yo M underwent staging colonoscopy. Colonoscopy showed

a non-obstructing 3 cm-mass in the sigmoid colon. Staging scans

showed no adenopathy or distant metastases. CEA was 3.0 ng/mL.

Patient underwent sigmoid colonic resection. Pathology showed

mucinous adenocarcinoma, invading through muscularis propria

to adjacent pericolonic tissue (pT3). 27 lymph nodes were

retrieved and negative for metastasis. IHC showed expression of

all 4 MMR proteins (MMR-i). 27 lymph nodes were retrieved and

negative for metastasis.


1. Observation


3. Molecular/genetic testing for MSI-status

4. Multi-gene assay

Multigene assay in CRC


Risk of recurrence in Stage II CRC patient who were treated with edrecolomab or observation in CALGB

9851 study (n=162)

Venook, J Clin Oncol. 2013

Recurrence score

&

5 year risk of of recurrence

Incorporating RS & MMR-status in

decision making of stage II CRC

• T4 tumor: – High risk, independent of RS & MMR status

– Gene expression analysis in not needed

– Patient should receive adjuvant treatment

• T3 Tumor, MMR-deficient (MSI)– Good outcome, adjuvant chemotherapy is not

indicated

• T3 Tumor, MMR-intact (MSS)– Standard risk patient

– Multigene assay can identify high risk patient

• IHC of MMR proteins is recommended for all new

patients with CRC

• MLH1 loss by IHC can be associated with germ-line

mutation or sporadic mutation.

• Loss of MLH1 should have BRAF testing to rule out

sporadic mutation

• Loss of MLH2, MSH2, PMS2 are due to germ line

mutation.

• Germ line mutation of MMR-gene is the hall-mark of

Lynch Syndrome.

• Patients with MMR-d/ MSI have better prognosis

• Patients with BRAF V600E mutation has poor

prognosis, when combined with MSS state

Adjuvant chemotherapy in stage II CRC


Adjuvant chemotherapy

• Stage III (node-positive), CRC patients are at high risk &

should receive adjuvant chemotherapy.

• In Stage II standard risk patients, MMR status & Multigene

assay should be taken into consideration in deciding

chemotherapy

• MMR-d (MSI) has good prognosis without adjuvant

treatment in stage II CRC.

• Stage II, MMR-i(MSS) can be risk stratified using

multigene assay

• To date, none of the multi-gene assay are predictive of

treatment benefit & should always be used in conjunction

with MMR status & clinico-pathologic variables

Emerging Molecular Strategy in

CRC

Herzig & Vassiliki, J Surg Onc, 2015

Hereditary Colorectal Neoplasms

• The Genetics of Colorectal Cancer

– Jasperson K & Burt RW, Surg Oncol Clin N Am,

2015: 24:683–703

• ACG clinical guideline

– Syngal et al, Am J Gastroenterol, 2015; 110: 223-

262

• Genetic/familial high risk assessment, NCCN

• GeneReviews@ NCBI

Hereditary CRC

• 5-6% of all CRCs are associated with

germ-line mutations, causing

hereditary predisposition

• Lynch syndrome is the most common,

accounting for 2-3% of all CRCs

• FAP patients account for <1% of CRCs

• Other hereditary CRCs are very rare

Jasperson, Sur Clin NA, 2015

Life-time Risk of Cancers in Lynch Syndrome

Kohlmann & Gruber, GeneReviews, 2011

Kastrinos, JAMA, 2009

• 6342 individuals from 147 families• 31 families (21%) had at least 1

pancreas CA

Indications of MSI testing to rule out Lynch

Syndrome

Jasperson, Sur Clin NA, 2015

Questions?

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