Come e quando utilizziamo la biologia...

Come e quando utilizziamo la biologia molecolarein pratica clinica?

Di quali metodiche non possiamo fare più a meno?

Andrea Mancuso, MDDepartment of Medical Oncology,

San Camillo and Forlanini Hospitals, Rome, Italy

A New Paradigm in Cancer Treatment

Haber, Gray, Baselga Cell 2011

A New Paradigm in Cancer Treatment

Molecular Biology in choice of Cancer Treatment

Breast Cancer

Lung Cancer

Colorectal Cancer

Gastric Cancer

Sarcoma

Brain tumors

Breast Cancer


Pre‐1980 1980s 2000

Single‐gene predictors

21st century (?)

DNA arraysSNP analysisMultiplex PCR

Proteomics

Not a single disease!

Histology Multi‐gene predictors (?)

Breast cancer: assessment

Histological FeaturesOther Morphometric Indexes

MenopausalStatus

GeneticBackground

Axillary NodeStatus

HER2/neuStatus

ER/PRStatus

Multi‐factorial characterization of each tumor is needed to assess the most appropriate strategy in different moments of the disease!!

Breast cancer: role of hormonal status

Breast cancer is a hormone‐dependent tumor and estrogens play amajor role in the development and progression of the disease. Nearly70%of breast tumors express the ER and/or the PgR.

The presence of ERs and PRs is predictive for response to endocrinetherapy , with an improvement of the DFS [1, 2].

[1] Katzenellenbogen BS, Frasor J. Semin Oncol 2004[2] Robertson JFR et al. Int J Biol Markers 1996

Amplification of HER2 occurs in about 20‐25% of invasivebreast cancers

It is associated with G3 cancers, high rates of cellproliferation and N+ tumors

Most clinical studies have shown that patients with HER2 +tumors have a poorer prognosis

The prognostic value is strongest in patients with nodepositive disease while there is no consensus in its value innode negative patients

HER2 as a prognostic factor

Menard et al; Oncogene. 2003; 22, 6570

HER2 as a predictive factor

Anthracycline based chemotherapy appears to beparticularly beneficial

HER2 overexpression may be associated withresistance to alkylator based therapy

HER2 may also predict response to endocrine therapy

Ellis et al; JCO 2006; 24 (19): 3019‐25

Trastuzumab

Trastuzumab is a humanized monoclonal antibodyanti‐ HER‐2

The beneficial effects on morbidity and/or mortalityare seen in patients with HER2‐positive breastcancer with HER2 overexpression at the 3+ level(IHC analysis) and/or HER2 gene amplification(FISH‐positive).

Plosker GL and Keam SJ: Drugs 2006

Molecular Class and clinicopathological features

C Sotiriou & L Pustzai, NEJM 2009


Lung Cancer

NSCLC: therapeutic strategy

THE OLD WAYNSCLCSCLC

SEER Cancer Statistics Review 1975‐2004. National Cancer Institute. 2007.

NSCLC: therapeutic strategyTHE INTERMEDIATE WAY: DECISION BASED ON HISTOLOGY

NSCLC‐ Adenocarcinoma: 40% of cases

• Includes bronchioalveolar subtype‐ Squamous: 20% of cases

‐ Large cell: 5% of cases

‐ NOS (not otherwise specified) (35%)

SCLC


NSCLC: therapeutic strategyTHE NEW WAY: MOLECULAR BIOLOGY

Extracellular targets• EGFR/HER (cetuximab, panitumumab, trastuzumab)

• VEGF (bevacizumab)

Intracellular targets• EGFR (erlotinib, gefitinib)• ALK (crizotinib)


KRAS 23%

No Mutation 42%

EGFR 15%

TP53 5%

IDH1 <1%

NRAS 1%BRAF 2%

HER2 2%

PIK3CA 4%ALK 6%

CTNNB1 2%

AKT 1%

Mutational profiling in lung cancers

EGFR

.

K K Grb‐2

MAPK

MEK

Raf

RasSOS

mTOR

PTEN Akt

PI3K

STAT 3/5

Survival Proliferation

Tyrosine kinase inhibitors(ATP‐binding cleft)

Ligand‐binding domain

Ligand Receptor antibodies

Adapted from: Pao W and Miller VA. J Clin Oncol. 2005;23:2556‐2568.

Mutations are associated with “Never‐Smokers”, gender and Histological subtypes

Mutations are more common in East Asians (30.6% than in Caucasians (7.6%) p<0.0001

Mutations are more common in non‐smokers (34.8%) than in smokers (7.8%) p<0.0001

Mutations are more common in women (26.4%) than in men (9.3%) p<0.0001

Mutations are more common in adenocarcinomas (23.2%) or BAC (17.9%) than in other histologies (2.2%)

Lynch et al, NEJM 2004; 350:2129‐39

EGFR TK Inhibitors: Molecular predictors

Molecular predictors of response• EGFR TK mutations:

Highly predictive• EGFR overamplification by FISH:

Predictive• EGFR expression by IHC:

not predictive

EGFR Inhibitors: Summary and future directions

Gefitinib/Erlotinib is approved for 1st line therapy in patients with Mutation of EGFR

Erlotinib: approved also for 2nd line therapy

Acquired resistance may occur with secondary mutations – novel agents needed to overcame these

Rapid integration of FISH : ALK Rearrangements in NSCLC

Crizotinib: Potent & selective ATP competitive oral inhibitor of MET and

ALK kinases and their oncogenic variants

Timeline for Crizotinib and ALK in NSCLC

Identification of PF2341066

PF2341066 Inhibits ALK activity

PF2341066 demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house)

PF2341066 activity in cells exhibiting ALK fusion in broad screen (MGH‐McDermott)

Discovery of EML4‐ALK fusions in NSCLC (CREST) Japan Science & Technology Agency)

2007

PF2341066 FIPMay

2005 2006 2008 2009

Objective responses demonstrated in ALK fusion positive NSCLC and IMT

Phase III study of “Crizotinib” in ALK positive NSCLC starts


Colorectal Cancer

The evolution of drugs in mCRC treatment …

1940 1950 1960 1970 1980 1990 2000 2010

CapecitabineIrinotecanOxaliplatin

5‐FU/LV5‐FUBevacizumabCetuximabPanitunumab

Molecular evaluation in choice of anti‐EGFR therapy for mCRC

Krasinskas M .A. ‐ Path. Res. International 2011

Respon

se ra

te (%

)

59

37

0

10

20

30

40

50

60

70

CRYSTAL(n=540)

OPUS1(n=233)

43

61

FOLFIRI FOLFOXCetuximab + FOLFIRI

Cetuximab + FOLF0X

CRYSTAL ‐ KRAS wild‐type: HR=0.68

p=0.017

32% risk reductionfor progression

OPUS ‐ KRAS wild‐type: HR=0.57

p=0.016

43% risk reductionfor progression

1Bokemeyer C et al, Abst # 4000; Proc. ASCO 2008

0.00.10.20.30.40.50.60.70.80.91.0

0 2 4 6 8 10 12 14 16 18Months

PFS estim

ate

0.00.10.20.30.40.50.60.70.80.91.0

0 2 4 6 8 10 12Months

PFS estim

ate

Cetuximab + CT in KRAS Wild‐type

KRAS MUTATION: MAIN RETROSPECTIVES STUDIES

Author N pts Mutated/Resp Mutated/Unresp P

Moroni et al. 31 2/10 8/21 0.43

Lievre et al. 76 0/24 27/52 NR

Romagnani et al. 27 2/11 8/16 NR

Di Fiore et al. 59 0/12 22/47 0.0005

Benvenuti et al. 48 1/11 15/37 0.073

De Roock et al. 37 0/8 17/29 0.01

Finocchiaro et al. 85 3/11 29/74 0.02

Juan et al. 37 0/4 12/33 NR

Khambata‐Ford et al. 80 3/27 27/53 0.0003

POOLED DATA 400 8/91 (8.8%) 138/309 (44.7%) <0.0001

Future directions: mutation of KRASG13D

Teipar S et al, ASCO 2011

Caveat: Should Oncologists Be Aware inTheir Clinical Practice of KRAS

Molecular Analysis?Different methods for KRAS mutation analysis have been used: direct sequencing analysis and real‐time PCR

Pyrosequencing has recently emerged as a new powerful sequencing methodology

Among 29 patients, we identified three (10.3%) KRAS mutant by pyrosequencing whereas all of them were reconfirmed KRAS wild‐type by real‐time PCR

Prospective studies using pyrosequencing are needed to evaluatethe role of different percentages of mutated alleles in terms of

responseto anti‐EGFR treatment in larger number of patients.

Santini D and Tonini G J Clin Oncol 2011

No evidence of EGFR testing

K‐Ras is necessary for a decision inmetastatic colorectal cancer for anti‐EGFRtherapy

Colorectal cancer: techniques of molecular biology


Gastric Cancer

Molecular Targets: Gastric Cancer

KRAS mutation: < 5% to 10%[1,2]

BRAF mutation: < 5%[1,2]

EGFR overexpression: ~ 50% to 80%[3,4]

• TKIs inactive[4]

• Cetuximab monotherapy inactive[5]

EGFR mutation: very low[4,6]

HER2 overexpression: 10% to 25%[7]

1. Lee SH, et al. Oncogene. 2003;22:6942‐6945. 2. Kim IJ, et al. Hum Genet. 2003;114:118‐120. 3. Galizia G, et al. World J Surg. 2007;31:1458‐1468. 4. Dragovich T, et al. J Clin Oncol. 2006;24:4922‐4927. 5. Chan JA, et al. Ann Oncol. 2011;[Epub ahead of print]. 6. Mammano E, et al. Anticancer Res. 2006;26:3547‐3550. 7. Yano T, et al. Oncol Rep. 2006;15:65‐71.

HER2 Positivity Histological Type

Bang YJ, et al. ASCO 2009. Abstract 4556.

Subtype HER2 Positivity (%)

P Value

Histological type

IntestinalDiffuseMixed

32.26.120.4

< .001

Localization GEJGastric

33.220.9

< .002

Histological subtype and tumor sublocalization are important factors for HER2 expression/amplification in gastric cancer

HER2 Positivity GEJ vs Gastric Cancer

Gastric cancerGEJ cancer

100

80

60

40

20

0

Samples (%)

N = 2759; *P < .001 Gastric Cancer GEJ Cancer Total

HER2 positive, % 20.9 33.2* 21.3

Bang YJ, et al. ASCO 2009. Abstract 4556.

HER 2 neu expression in GC leaded to TOGA trial

Van Cutsem et al. – JCO, 2009

Efficacy: OS by HER 2 neu

Van Cutsem et al. – JCO, 2009


Sarcoma & GIST

Molecular diagnosis of sarcomas

Now possible for about 50% of cases

Based on elementary structural genomic lesions (translocations, mutations, amplifications and deletions)

Potential target for new treatments

Coindre JM. Bull Cancer. 2010

Molecular classification of sarcomas

Gene profiling

Simple genomic profile:

• translocations (Ewing family tumors)• inactivating mutations• amplifications

Complex genomic profile:

• leyomiosarcoma• angiosarcoma• pleomorphic liposarcoma


Molecular Diagnosis of SarcomasTumor Translocation Fusion Gene

Ewing/PNET t(11;22)(q24;q12) EWS/FLI1

t(21;22)(q22;q12) EWS/ERG

Alveolar Rhabdomyosarcoma t(2;13)(q35;q14) PAX3/FKHR

t(1;13)(p36;q14) PAX7/FKHR

Desmoplastic small round cell tumor

t(11;22)(p13;q12) EWS/WT1

Synovial Sarcoma t(X;18)(p11.2;q11.2) SYT/SSX1+2

Congenital Fibrosarcoma t(12;15)(p13;q25) ETV6/NTRK3

Clear Cell Sarcoma t(12;22)(q13;q12) EWS/ATF1


Detection of translocations insarcomas

RT – PCR (real time)FISHFrozen tissueFormalin‐fixed tissueCytology


GIST

Most frequent sarcomas of GI tractSpindle and/or epithelioid cell tumorC‐kit + (IHC) required for diagnosisActivating mutations of KIT or PDGFRAPrognosis dramatically changed by imatinib

Cassier PA, Blay JY. Target Oncol. 2010

Mutations of GISTand response to imatinib


Detection of mutations in GISTClinical applications

Prediction to imatinib response

Therapeutic choices?

Resistance to imatinib


Sarcomas with amplifications

MDM2 and CDK4 genes• Well-differentiated and

dedifferentiated liposarcomas• Intimal sarcoma• Parosteal osteosarcoma

Crago AM, Singer S. Curr Opin Oncol. 2011

Detection of MDM2 and CDK4

Immunohistochemistry for the daily practice

FISH for problematic cases

Crago AM, Singer S. Curr Opin Oncol. 2011


Brain Cancer

MGMT Promoter Methylation Predicts Better Outcome in Glioblastoma Patients

(O6-methylguanine–DNA methyltransferase) DNA-repair gene

MGMT Promoter Methylation PredictsBenefit from TMZ Treatment

Breast Cancer: RER, PGR, Ki 67, HER‐2

Lung Cancer: EGFR, ALK

Colorectal Cancer: k‐Ras

Gastric Cancer: HER‐2

Sarcoma: Kit, PDGFRA, MDM2, CDK4

Brain tumors: MGMT

Molecular technology necessary in clinical practice

Come e quando utilizziamo la biologia...

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