1. Come è cambiata e sta cambiando l’Anatomia Patologica
Dal GIST al K-RAS
OVVERO
Dall’ E.E. al D.N.A.
Come è cambiata e sta cambiando
l’Anatomia Patologica
Gallarate Aprile 2009

2. (also called Gleevec® or STI571) is approved by the U.S. Food and Drug
Imatinib mesylate
(also called Gleevec® or STI571)
is approved by the U.S. Food and Drug
Administration (FDA) for the treatment
of some forms of adult and pediatric chronic
myelogenous leukemia (CML), and for the
treatment of a rare form of cancer called
gastrointestinal stromal tumor (GIST).

3. STI571
Foto cd117

4. GLEEVEC
foto

5. Foto cml

6. Foto gist

9. MIB1

10. Alfa-actina

11. Alfa-actina

12. CD117

13. GastroIntestinal Stromal Tumors
Acivating mutations in c-KIT and PDGFRA 5%
About 80-90% of patients show mutations *
Antonescu RC, Clin Cancer Res 2005; 11:

14. GastroIntestinal Stromal Tumors
Acivating mutations in c-KIT and PDGFRA
About 75% of GISTs shows mutations on exon 11 and are
related to Imatinib sensitivity (>80% clinical response)
About 10%- 15% of GISTs shows mutations on exon 9
and have an aggressive clinical behaviour
Fletcher JA and Rubin BP ; Current Opinion in Genetics & Development, 2007; 17: 3-7

15. GastroIntestinal Stromal Tumors
Imatinib or Sunitinib Resistance
Mutations in exon 9 are generally associated to primary resistance
Mutations in exon 13, 14 and 17 of c-KIT gene are subsequent to
exon 11 mutations and are generally associated to acquired resistance
Antonescu RC, Clin Cancer Res 2005; 11:
Heinrich MC, J.Clin. Oncol. 2006; 24:

16. Mutazioni iniziali e secondarie
Mutazioni secondarie
Exon 9
Exon 11
Exon 13
Exon 17
GLEEVEC
Resistenza
primaria
Resistenza
secondaria
Membrana cellulare
CD117

17. Dr. Lei Chen and others at M.D. Anderson Cancer Center in
Houston have identified a secondary mutation that occurs in
KIT kinase domain 1 (exon 13). This secondary mutation
correlates with resistance to Gleevec.
Among the 130 patients in the M.D. Anderson study, 12 who
had an excellent initial response were chosen for further study.
Seven of these patients originally had exon 11 mutations and
five had exon 9 mutations. Five of these patients developed
resistance in a total of six tumors. In each case, in addition to
the original exon 11 or exon 9 mutation, a new secondary
exon 13 mutation, Val654Ala, was identified. In each of these
cases, the secondary mutation was identical and the resistant
tumors now contained both the primary mutation
(exon 11 or exon 9) and the new exon 13 mutation.
In the seven patients who did not develop resistance no
secondary mutations were found.

18. Dalla diagnosi di LEIOMIOBLASTOMA a quella di GIST CD117 +
Parametri prognostici
Valutazione mutazioni
esoni

19. GIST is a rare form of cancer
BUT
LUNG ?
COLON ?

21. TArceva Italian Lung Optimization tRial
TAILOR AIFA
TArceva Italian Lung Optimization tRial

22. Lo studio deve valutare se i casi di K polmonare non mutati per EGFR siano responsivi al Tarceva dando per acquisito che i mutati lo siano
Siamo sicuri che tutti casi ( o almeno gli adenoK giovanili ) siano studiati per stato mutazionale di EGFR ?
Quanti fanno sistematicamente una valutazione dello stato mutazionale di EGFR nel K polmonare ed in quali casi ?

23. La famiglia HER (erbB) ed i suoi ligandi
EGF
TGFa
Amphiregulin
b-cellulin
HB-EGF
Epiregulin
NRG2
NRG3
Heregulins
b-cellulin
Heregulins
Cysteine-rich
domains
100 44 82 33 36 59 24 48 79 28
Tyrosine kinase
domain
C-terminus
ErbB-1 Her1
EGFR
ErbB-2
Her2
neu
ErbB-3
Her3
ErbB-4
Her4

24. Location of mutations in TK domain of EGFR gene
del ELREA (E746-A750)

25. Mechanisms of resistance to TK inhibitors
Exon 19 del E745-A750
Exon 20 T790M

26. PARLIAMO DI K COLON E DI K-RAS
CANCRO COLON-RETTO
90 CASI x 105 ANNUI
IN LOMBARDIA 8000 CASI ANNUI
RICADUTA CIRCA 4000 Pz

27. La famiglia HER (erbB) ed i suoi ligandi
HERCEPTIN
EGF
TGFa
Amphiregulin
b-cellulin
HB-EGF
Epiregulin
NRG2
NRG3
Heregulins
b-cellulin
CETUXIMAB
Heregulins
Cysteine-rich
domains
100 44 82 33 36 59 24 48 79 28
Tyrosine kinase
domain
C-terminus
ErbB-1 Her1
EGFR
ErbB-2
Her2
neu
ErbB-3
Her3
ErbB-4
Her4

29. NESSUN RISULTATO ATTENDIBILE
DIFFICOLTA’ TECNICHE DI RECUPERO ANTIGENICO
NON RESPONSIVITA’ CASI POSITIVI ALLA IIC
RESPONSIVITA’ CASI NEGATIVI ALLA IIC

30. Centr
EGFR

31. COLON NORMALE
CARCINOMA -- POLISOMIA

32. Perdita materiale nucleare
2 green 2 red
4 green 4 red
Perdita materiale nucleare
2 green 2 red
CELLULA NORMALE
CELLULA TUMORALE

33. CARCINOMA – AMPLIFICAZIONE

34. CARCINOMA – AMPLIFICAZIONE

35. APPROCCIO AUTOMATICO DI LETTURA

38. Gene copy number for epidermal growth
factor receptor (EGFR) and clinical
response to antiEGFR treatment in
colorectal cancer: a cohort study
M.Moroni, S.Veronese,S.Benvenuti,
G.Marrapese,A. Sartore-Bianchi,
F.Dinicolantonia,M.Gambacorta,S.Siena,
A.Bardelli.
Lancet Oncology 2005

39. EGFR gene copy number analysis
Disomy
Polysomy
Focal amplification
> 40% polysomic cells with
> 3 EGFR copies
EGFR/CEP 7 > 2,50 copies per cell

40. PFS and OS according to the proposed cut off values
Sartore-Bianchi A et al. J Clin Oncol 2007

41. Clinical usefulness of EGFR gene copy number as a predictive marker in colorectal cancer patients treated with Cetuximab : A FISH study
Personeni N. et Al
Clin Cancer Res 2008
E’ in corso uno studio pluricentrico per
standardizzazione tecnica e interpretazione
risultati F.I.S.H. Her1 su sezioni

42. SEQUENZA E G F R : ASSENZA DI MUTAZIONI
ESONI 18 , 19 , 20 , 21

43. TK signaling cascade

44. Mutated KRAS is prevalent in many different tumor types
Cancer Type
Reported Incidence of Mutated KRAS
Pancreatic
72 – 90%
Colon
32 – 57%
Lung
15 – 50%
Ovarian
5 – 50%
Gall bladder
14 – 38%
Multiple myeloma
16 – 33%
Adapted from: Friday BB and Adjei AA. Biochim. Biophys. Acta. 2005; 1756:

45. K-RAS Mutations G12D Gly12Asp GGT>GAT G12A Gly12Ala GGT>GCT
G12S WT
…… GGA GCT GGT GGC GTA GGC ……
Wild-type
G12D Gly12Asp GGT>GAT
G12A Gly12Ala GGT>GCT
G12V Gly12Val GGT>GTT
G12S Gly12Ser GGT>AGT
G12R Gly12Arg GGT>CGT
G12C Gly12Cys GGT>TGT
G13D Gly13Asp GGC>GAC

46. Gastrointestinal (Colorectal) Cancer
2008 ASCO Annual Meeting
Gastrointestinal (Colorectal) Cancer
KRAS status and efficacy in the first-line treatment of patients with mCRC
treated with FOLFIRI with or without Cetuximab : the CRYSTAL experience
Van Cutsem E et al; JCO 26; 2008 (May 20 Suppl; abstr 2)
540 archived tumor material
WT Patients (64.4%)
Progression Free survival (PFS) p=0.0167
Overall response (OS) p=0.0025
KRAS mutated Patients (35.6%)
Progression Free survival (PFS) p=0.75
Overall response in (OS) p=0.46
As far as KRAS mutational status is concerned, recently at the ASCO meeting the Crystal study reported the efficacy of cetuximab treatment only in KRAS WT patients both in term of progression-free and overall survival by concluding that KRAS mutation status is a valuable predictive marker for treatment with FOLFIRI plus cetuximab in the first-line treatment of metastatic colorectal cancers.
Conclusion : KRAS mutation status has a predictive value for treatment
with FOLFIRI + Cetuximab in the first-line treatment of mCRC

47. Gastrointestinal (Colorectal) Cancer
2008 ASCO Annual Meeting
Gastrointestinal (Colorectal) Cancer
KRAS status and efficacy of first-line treatment of patients with mCRC with
FOLFOX with or without Cetuximab : the OPUS experience
Bokemeyer C et al; JCO 26; 2008 (May 20 Suppl; abstr 4000)
233 tumor samples 99/233 (42.5%) KRAS mutated
134/233 (57.5%) KRAS WT
PFS and overall Response Rate (RR) by KRAS mutation status
KRAS status
Median PFS
(mo)
Cetuximab + FOLFOX
FOLFOX
Overall RR
(%)
Wild-type
7.7 (n=61)
7.2 (n=73)
HR = 0.57 p=0.02
61 (n=61)
37 (n=73)
p=0.01
Mutation
5.5 (n=52)
8.6 (n=47)
HR = 1.83 p=0.02
33 (n=52)
49 (n=47)
p=0.11
HR : Hazard Ratio
In the same meeting also the OPUS study confirmed the predictive role of KRAS mutational status and the efficacy of cetuximab in KRAS WT population
Conclusions: The benefit from addiction of Cetuximab to standard treatment is
higher for KRAS WT population.

48. Best response Ras/Raf mutations - + Total SD+PD 15 21 (58.3%) 36 PR 10
Total
SD+PD 15
21 (58.3%) 36 PR 10
1 (9.0%) 11 25 22 47
p =0.005 (Fisher's exact test)
Time To Progression according
KRas and BRaf mutational status
Logistic regression
Odds Ratio = (CI95%=(0.008, 0.619); p=0.017)

49. Constitutive activation of EGFR effectors
MoAb
RAS
RAF
Protein kinase BRaf mutations

50. PTEN inactivation or loss of expression
(Phosphatase and Tensin homolog deleted on chromosome 10)
MoAb
pAkt

51. KRAS gene v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
12p12.1
v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
The KRAS gene is located on the short (p) arm of chromosome 12 at position 12.1.
More precisely, the KRAS gene is located from base pair 25,249,446 to base pair
25,295,120 on chromosome 12. 1 2 3 4 5 6
gene
ENSG
Exons: 6 Transcript length: 5,419 bps Protein length: 189 residues

52. KRAS gene
5' upstream sequence ………………… cccggccccgaactcatcggtgtgctcggagctcgattttcctaggcggc
Exon 1 (170)GGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCGGCGAAGGTGGCG
GCGGCTCGGCCAGTACTCCCGGCCCCCGCCATTTCGGACTGGGAGCGAGCGCGGCGCAGG
CACTGAAGGCGGCGGCGGGGCCAGAGGCTCAGCGGCTCCCAGGTGCGGGAGAGAG
Intron (5355) gtacggagcg ttattataag
Exon 2 (122)GCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGG
CAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAA
CAATAGAG
Intron (17.861) gtaaatcttg cccttctcag
Exon 3 (179)GATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTC GACACAGCAGGTCAAGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGG
AGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTCACCATTATAG
Intron (1.460) gtgggtttaa tctttcccag
Exon 4 (160)AGAACAAATTAAAAGAGTTAAGGACTCTGAAGATGTACCTATGGTCCTAGTAGGA
AATAAATGTGATTTGCCTTCTAGAACAGTAGACACAAAACAGGCTCAGGACTTAGCAAGA
AGTTATGGAATTCCTTTTATTGAAACATCAGCAAAGACAAGACAG
Intron (10.053) gtaagtaaca tacaatgcag
Exon 5 (124)AGAGTGGAGGATGCTTTTTATACATTGGTGAGGGAGATCCGACAATACAGATTGA
AAAAAATCAGCAAAGAAGAAAAGACTCCTGGCTGTGTGAAAATTAAAAAATGCATTATAA
TGTAATCTG
Intron (5.525) gtaagtttaa tgtatttcag
Exon 6 (4.664)GGTGTTGATGATGCCTTCTATACATTAGTTCGAGAAATTCGAAAACATAAAGAAAAGATG
………………………………………………………………………………………………………………………
3' downstream sequence actatgagtgtgtatttattcatgaaatttgaactgtttgccccgaaatg

54. KRAS mutations in adenocarcinomas of large intestine
Gene Name
Sample Number
Positive Samples
Percent Mutated
KRAS
2657
995
37%
APC
937
365
38%
BRAF
824
114
13%
CTNNB1
894 51 5%
PIK3CA
254 41
16%
COSMIC
Wellcome Trust SANGER Institute

55. K-RAS Mutations SnaPShot Sequencing Electropherograms
…… GGA GCT GGT GGC GTA GGC ……
Wild-type
SnaPShot
G12D
G12S WT
Sequencing
About KRAS mutations we know seven different activating mutations that are reported here.
G12D

56. KRAS Testing Characteristics of Selected Assays
Methodology
Kit
(Manufacturer)
Mutations
detected
Features
Direct sequencing
Individual reference laboratory
All
Direct sequence information
Independent of allele present
Allele-specific
real-time PCR
TaqManTM KRAS-specific primer + probe sets
(Applied Biosystems)
Any designed
Result depends on individual
assay design
Single-tube amplification +
detection
Real-time PCR detection using
allele-specific
primer-probes
TheraScreenTM
K-RAS Kit
(DxS)
G12D
G12A
G12V
G12S
G12R
G12C
G13D
High sensitivity and specificity
First CE-labelled kit
ELISA microplate
DNA hybridisation
Invigene® K-ras Genotyping Kits
(Invitek)
G13S
G13R
G13C
G13A
G13V
Includes sample preparation
module
Detection includes manual steps
Universal DNA extraction kits are used for sample preparation prior to PCR to increase sensitivity, specificity and reproducibility of mutation detection. They can be used in combination with all amplification methods.
The TheraScreenTM K-Ras Kit (DxS Ltd, Manchester, UK) is the first CE* marked kit for KRAS mutation testing and includes the reagents for amplification and detection. The PCR is allele-specific for mutations in codon 12 and 13. Amplification and detection are carried out using ARMS®/Scorpions® primer-probes and a real-time PCR instrument. The assay detects <1% mutant DNA in a background of wild-type genomic DNA and has a detection limit of ≤10 copies.
The Invigene® K-ras Genotyping Kit includes modules for DNA extraction from paraffin tissue, amplification with exon-specific PCR and detection with mutation-specific probes using microplates (DNA-ELISA) and detects mutations in codon 12 and 13. The procedure includes several manual steps in the detection process. The exact sensitivity has not been determined by the manufacturer.
Applied Biosystems offers primer/probe sets and reagents for real-time PCR carried out using the TaqManTM instrument and uses allele-specific, target-specific fluorescent probes as designed by the user; the assays must be optimized individually.
*The CE marking (also known as CE mark) is a mandatory conformity mark on many products placed on the single market in the European Economic Area. By affixing the CE marking, the manufacturer, its authorized representative, or person placing the product on the market or putting it into service asserts that the item meets all the essential requirements of the relevant European Directive(s).
References

57. KRAS Mutation Testing Histopathological evaluation is essential
Paraffin-embedded, formalin-fixed tissue (biopsies, surgical samples)
Macro/microdissection can enhance sensitivity
Various test methods available, all adequate
Direct sequencing
- Sensitivity of standard sequencing techniques is sufficient in most cases
- Sequencing provides direct information about the type of mutation
- Detects all mutations in region sequenced
Allele-specific amplification and detection
- Provides high sensitivity
- Appropriate for high-throughput or multiplex applications
- Only pre-defined mutations can be detected
Detection can be performed both on primary tumor and metastasis

58. DISSEZIONE
MICROSCOPICA

59. CETUXIMAB SI USA IN CASI IV° STADIO RAS W. T. ora
Circa il 40 % di tutti i casi di K del grosso intestino sono o diventano IV° stadio
Ma circa il 50 % sono stadio II°
e su questi ?

61. 18q allelic loss in CRC
The short arm of the chromosome 17 (17p) and the long arm of the chromosome 18 (18q) are frequently loss in CRC
Inactivation of the p53 gene (on 17p) and DCC gene (on 18q) probably contributes to neoplastic transformation
Distant metastasis of CRC is associated with deletions of 17p and 18q, and loss of 17p and 18q has prognostic value

62. 18q deletion and outcome in untreated CRC
The status of chromosome 18q has strong prognostic value in patients with stage II colorectal cancer. The prognosis in patients with stage II cancer and chromosome 18q allelic loss is similar to that in patients with stage III cancer, who are thought to benefit from adjuvant therapy. In contrast, patients with stage II disease who do not have chromosome 18q allelic loss in their tumor have a survival rate similar to that of patients with stage I disease and may not require additional therapy.
Jen et al. NEJM 1994

63. Info sul trial

64. EGFR-targeted therapies1 4 2 3

65. Molecular conditions for anti-EGFR efficiency
I. Activation of EGFR in the tumor: EGFR gain of copies, ligands overexpression
at the receptor level
at the intracellular level
II. No downstream oncogenic activation:
KRAS (40%)
BRAF (10%)
PTEN (loss, 30%)
PIK3CA? (20%)

66. A sequential strategy:
downstream signaling
KRAS
Cancer Res 2007
BRAF
J Clin Oncol 2008
PTEN
PIK3CA
Cancer Res 2009

67. ?

68. The role of PIK3CA/PTEN (I)
Available data of the predictive role in CRC:
Preclinical: Jawher, Cancer Res 2008
Clinical:
-Frattini et al. Br J Cancer 2007
-Perrone et al. Ann Oncol 2009

69. 32 pts: -24% KRAS mut (p=0.03) -10% BRAF (p=ns) -13% PIK3CA mut (p=ns)
-10% PTEN mut
-13% PTEN loss
(p=0.02)
Perrone F et al. Ann Oncol 2009

71. 110 metastatic colorectal cancers patients
treated with the EGFR-targeted monoclonal
antibodies panitumumab or cetuximab
KRAS mutational status
(evaluable n=109)
*p<0.05
(p=0.019)
Mutated KRAS
32/109 (29%)
Wild-Type KRAS
77/109 (71%)
Responders 2*
20*
Non Responders
30*
57*
PIK3CA/PTEN evaluation in wild-type KRAS tumors
(evaluable n=59)
**p<0.01 (p= )
Altered
PIK3CA/PTEN
27/59 (46%)
Normal
32/59 (54%)
Responders
1**
17**
Non Responders
26**
15**

72. Frequency of mutations in PIK3CA and KRAS, and loss of PTEN protein expression
15 (13.6%) PIK3CA mutations, both in exon 9 (4 cases) and in exon 20 (11 cases);
32 (29.0%) KRAS mutations, occurring at codon 12 in 23 cases (71.9%), and at codon 13 in 8 cases (25.0%); a double point mutation involving both codons was detected in 1 case (3.1%)
Concomitant PIK3CA and KRAS mutations were observed in 2 samples
PTEN protein assessment was performed by immunohistochemistry analysis. Among the 81 evaluated tumor specimens, 32 (39.5%) showed loss of PTEN protein

74. 168 pts

75. BRAF mutation predict for poor prognosis regardless the line of treatment

76. Molecular determinants of response in CRC: what is reasonable to look for in the clinical practice?
Stage II (prognostic)
experimental
18q LOH
clinical practice
Stage IV (predictive)
KRAS
BRAF
PIK3CA
PTEN

77. GRAZIE Silvio Veronese : SS Patologia Molecolare H Niguarda Milano
Andrea Sartore Bianchi : Oncologia Falk
Salvatore Siena H Niguarda Milano
Alberto Bardelli : IRCC Candiolo