come e quando utilizziamo la biologia...
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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
Molecular Biology in choice of Cancer Treatment
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
Molecular Biology in choice of Cancer Treatment
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
SEER Cancer Statistics Review 1975‐2004. National Cancer Institute. 2007.
NSCLC: therapeutic strategyTHE NEW WAY: MOLECULAR BIOLOGY
Extracellular targets• EGFR/HER (cetuximab, panitumumab, trastuzumab)
• VEGF (bevacizumab)
Intracellular targets• EGFR (erlotinib, gefitinib)• ALK (crizotinib)
SEER Cancer Statistics Review 1975‐2004. National Cancer Institute. 2007.
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
Molecular Biology in choice of Cancer Treatment
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
Molecular Biology in choice of Cancer Treatment
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
Molecular Biology in choice of Cancer Treatment
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
Coindre JM. Bull Cancer. 2010
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
Coindre JM. Bull Cancer. 2010
Detection of translocations insarcomas
RT – PCR (real time)FISHFrozen tissueFormalin‐fixed tissueCytology
Coindre JM. Bull Cancer. 2010
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
Cassier PA, Blay JY. Target Oncol. 2010
Detection of mutations in GISTClinical applications
Prediction to imatinib response
Therapeutic choices?
Resistance to imatinib
Cassier PA, Blay JY. Target Oncol. 2010
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
Molecular Biology in choice of Cancer Treatment
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
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