institute of pathology - hss · 2016. 12. 19. · 09.10.2013 1 institut für pathologie –...
TRANSCRIPT
09.10.2013 1Institut für Pathologie – Charité Berlin
M. Dietel
Institute of Pathology(Rudolf-Virchow-Haus)
Humboldt University, Berlin
Fortschritte in der personalisierten Medizin
e-mail: [email protected]
Rudolf Virchow
1821 - 1902 1710 - 2010
Allgemeine Definition
predictivetests based
on reliable BM
Es geht nicht darum, für einen Patienten ein
individuell maßgeschneidertes Medikament zu
entwickeln und dann zu verabreichen.
09.10.2013 3Institut für Pathologie – Charité Berlin
Unter personalisierter Medizin in der Onkologie wird die durch
die Arzneimittelzulassungsbehörde bindend
vorgeschriebene prädiktive Biomarker-basierte
Diagnostik als Voraussetzung zur Applikation eines
zielgerichteten Arzneimittels
verstanden.
Es wird also vor Gabe eines Medikamentes der Nachweis
seiner Wirksamkeit gefordert (compagnon diagnostic).
Definition in der Onkologie
FDA: Drug-Diagnostic Co-development Initiative
09.10.2013 4Institut für Pathologie – Charité Berlin
Prediction is difficult, especially about the future
Niels Bohr, 1885-1962
In oncology, the predictive power of tissue
based analyses is underestimated.
2010
Thinking back to infectious diseases and seeing the current develop-
ment in cancer – HER2, KRAS, BRAF, EGFR etc. - we shouldn’t be too
pessimistic.
09.10.2013 5Institut für Pathologie – Charité Berlin
Predictive tissue-based biomarkers for targeted therapies
FDA / EMA-approved drugs associated with eligibility tests* (selection)
• Trastuzumab → metastatic breast cancer, overexpression/amplification of HER-2
• Cetuximab → metastatic colorectal cancer, overexpressing EGFR/wild-type KRAS
• Panitumumab → colorectal cancer with wild-type KRAS (mutation excluded)
• Gefitinib → non-small cell lung cancer with mutated EGFR
• Erlotinib → non-small cell lung cancer with mutated EGFR
• Crizotinib → non-small cell lung cancer with mutated EML4-ALK
• Nimotuzumab → metastatic colorectal cancer (still experimental)
• Lapatinib → metastatic breast cancer overexpression HER-2/neu (?)
• Vemurafenib (PX4032) → malignant melanoma with mutated B-RAF
• Imatinib → CML, bcr/abl–positive (activated PK),
• Imatinib → GIST with activated c-kit receptor tyrosine kinase/CD117, exon 9 mut
• Rituximab (+ CHOP), Y90-Ibritumomab, I131-Tositumomab → NHLymphoma with CD20
• Gemtuzumab-Ozogamicin → AML with CD33 ( > 60 yrs.), mal. melanoma
• Tamoxifen+/- chemo → ER+/HER2 - breast cancer, mutation pattern - multigene assays
*Strongly suggested by FDA’s Drug-Diagnostic Co-Development Initiative
Already now, in 35% of all tumors a predictive
molecular test is appropriate. Notably, prediction of
tumour response is exclusively tissue-based.
All these substances have been developed on the
basis of histologically characterised human tissue.
This underlines the importance of biobanks.
09.10.2013 6Institut für Pathologie – Charité Berlin
Recommendations for sample preparation and molecular analysis
detectiondetection
DNA isolationDNA isolation
manual microdissectionmanual microdissection
histological evaluationhistological evaluation
grossingparaffin embedding
grossingparaffin embedding
tumour resectiontumour resection
SurgicalPathology
MolecularPathology
Oncologicaltreatment
Oncologicaltreatment
Surgery
amplificationamplification
manual microdissectionmanual microdissection
09.10.2013 7Institut für Pathologie – Charité Berlin
Sequencing
AutomatedDNA extraction
Without manual microdissection the error rate can be up to 8%* due only to inadequate samples,independent of any technical problems
Weichert W et al. J Mol Diagn;12:35-42
Molecular pathology report
Incoming
Name BirthXXXXX
XXXXX
Mol. analysis:
KRAS mutation -GGTGGC to GATGGC (ca. 40%)
Combined Report on Anatomic and Molecular Pathology
Material: external colon biopsy, FFPE - block no. ##### -10
Clinical Data: Met. ### cancer ###
Histopath.: Malignant epithelial ……….
Combined patho-report: Metastasized adenocarcinoma of
the ##### with ### mutation as indicated.
Pathologist…………………..
Institut für Pathologie – Charité Berlin
ToDr. ########## #############
Director: Prof. M. Dietel
Charitéplatz 1
10117 Berlin
Tel. 0049 30 450 536 001
Fax 0049 30 450 536 900E-mail: [email protected]
Forward
Reverse
Nerv
Manual microdissection
BRAFV600mut
Sind die Ergebnisse so überzeugend, dass der enorme
Aufwand und die exorbitanten Kosten der onkologsch
orientierten PM gerechtfertigt sind?
•Kosten der F&E ca. 2 Mrd.€ pro neue Substanz
•Kosten pro Therapiezyklus ca. 30-40 T€
Die aktuell immer wieder gestellte Frage …
Die aktuell immer wieder gestellte Frage
Was sind überzeugende Ergebnisse?
Einige Beispiele ………..
Ulzeriertes Dickdarmkarzinom
Dieser Tumor bzw. seine Metastasen können mit
einem zielgerichteten Medikament (therapeutischer
AK) behandelt werden, wenn mittels molekularer
Analysen ein KRAS-Wildtyp nachgewiesen wurde.
Warum?
09.10.2013 11Institut für Pathologie – Charité Berlin
mutant KRAS
Mutant KRAS constitutively active –40% of patients*
KRAS (Wild-type)
Panitumumab and cetuximab inhibit ligand binding, dimeri-
sation, activation of the receptor and the signalling pathway
EGFR
Ligands
RAF
MEK
ERK
ELK
Activation of• Proliferation• Angiogenesis• Malignant phenotype Nucleus
KRAS-MAPK signalling pathway
Y Y
Schubbert S et al. Nat Rev Cancer 2007;7:295-308;
*Friday BB, Adjei AA. Biochim. Biophys. Acta. 2005; 1756:127-144.
Institut für Pathologie – Charité Berlin, Campus Mitte
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Weeks
36 38 40 42 44 46 48 50 52
7 7 6 5 5
10 9 9 6 6 6 5 4 3 3 2 2 2 2 1
124
115/124 (93) 12,3
114/119 (96) 7,3
Pmab + BSC WT
BSC alleine
Events N (%)Median(weeks)
HR = 0,45 (95% CI: 0,34–0,59)
Stratified log-rank, P < 0,0001
Pmab + BSC
BSC alleine
Patients at Risk
119 112 106 80 69 63 58 50 45 44 44 33 25 21 20 17 13 13 13 10
119 109 91 81 38 20 15 15 14 11 6
Pro
po
rtio
n E
ve
nt
Fre
e (
%)
Metastasierte Colon-Ca: Wild-type/mut KRAS/BSC
Amado R, et al. ESMO 2007;a0007; JCO, 26 (2008 1626-1634
76/84 (90) 7,4Pmab + BSC Mut
16 weeks
09.10.2013 13Institut für Pathologie – Charité Berlin
Treatment efficacy - according to mut status
In CRC ca. 55% are KRAS wild type.
Out of these only ca. 50% (i.e. 25% of all CRC) respond
to EGFR antibodies.
Why?
How can they be detected or stratified?
09.10.2013 14Institut für Pathologie – Charité Berlin
Mutations in KRAS and NRAS genes in colorectal cancer
KRAS
NRAS
1 2 3 4 5 6 exon
12/13 59/61 117/146 mutation position
40% 4% 6% frequency
1 2 3 4 5 6 7 exon
12/13 59/61 117/146 mutation position
3.5% 4% 0% frequency
non-coding exon
coding exon
When the rare mutations are added
they represent 17.5 % of all CRC and
they are associated with resistance!
20020408 Trial RAS (Exon 4) Analysis PFS in Patients with WT RAS* mCRC
Patterson SD, et al. J Clin Oncol 31, 2013 (suppl; abstr 3617).
*WT KRAS and NRAS exons 2, 3, and 4
Weeks
20
40
60
80
100
90
70
50
30
10
Pro
po
rtio
n E
ve
nt-
Fre
e %
4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96
0
0 100
HR=0.36 (95% Cl: 0.25–0.52)p<0.001
Eventsn/N (%)
Median weeks (95% CI)
Panitumumab + BSC(n=73) 70 (96) 14.1 (10.3–23.3)
BSC (n=63) 61 (97) 7.0 (6.0–7.4)
22 weeks
NSCLC - Macroscopy
central squamous cell carcinoma
peripheraladenocarcinoma
aeno carcinoma
broncho-alveolar type
Für diese Tumoren kommt eine Therapie mit
Tyrosinkinase-Inhibitoren in Betracht, wenn im
Einzelfall bestimmte Mutationen nachgewiesen
werden können.
09.10.2013 17Institut für Pathologie – Charité Berlin
Kris MG, et al. Presented at ASCO 2011; Abstract CRA7506LCMC, Lung Cancer Mutation Consortium
NSCLC: Past and Current Landscape
Actionable driver mutations identified in 54% of lung adenocarcinoma tumours
1999Histology-driven
selection1
Adenocarcinoma
Squamous-cell carcinoma
Large cell carcinoma
2012Targeting oncogenic
drivers
NO MUTATION DETECTED
EML4-ALK7%
DOUBLE MUTANTS 3%
BRAF 2%
PIK3CA
HER2
MET AMP
MEK1
NRAS
AKT1
KRAS22%
EGFR17%
09.10.2013 18Institut für Pathologie – Charité Berlin
Rapid Responses Seen In Some PatientsRapid Responses Seen In Some Patients
Ou et al. J Thoracic Oncol 2010;5:2044–2046 Camidge RD et al.: ASCO 2011
09.10.2013 19Institut für Pathologie – Charité Berlin
100
Tumour responses to crizotinib by patient
*Mature population, excluding those with early death, indeterminate response and non-measurable disease
Study A8081001N=1161
PD SD PR CR
Best objective response according to RECIST:
PROFILE 1005N=2402*
De
cre
ase
or
incre
ase
fro
m b
aselin
e (
%)
80
60
40
20
0
–20
–40
–60
–80
–100
100
80
60
40
20
0
–20
–40
–60
–80
–100
1. Camidge DR, et al. Lancet Oncol 2012;10:1011−9;2. Kim DW, et al. Presented at ASCO 2012; Abstract 7533
Institut für Pathologie – Charité Campus Mitte
Modified according to
Soda et al. nature 448:561 (2007).
EML4-ALK Fusion in NSCLC
Chromosom 2
Anaplastic
lymphoma kinase
ALK EML4
Alk EML4
Exon20 Exon13
20 13
20 20
20 6a, b
PF02341066
EML4-Alk
EGFRwt
KRASwt
11 variants
constitutiveactivation
09.10.2013 21Institut für Pathologie – Charité Berlin E23002-11_ALK-BA
Anaplastic
lymphoma kinase
ALK EML4
Alk EML4
Exon20 Exon13
09.10.2013 22Institut für Pathologie – Charité Berlin
*Total V600 mutation rate for BRIM-3 (cobas® 4800 BRAF V600 Mutation Test); 9.9% of the cobas-positive cases subjected to retrospective Sanger sequencing had V600K mutations
50% of all malignant melanomas exhibit a BRAF-Mutation
Malignant Melanoma
09.10.2013 23Institut für Pathologie – Charité Berlin
Vemurafenib phase I overall survival: Updated KM estimates (08. 2011)
Median OS (month)
Dose escalation 25.2
Extension 13.8
WT or sub-therapeutic. 4.18
Extension cohort landmarkEstimated survival: 1 year = 50%, 2 years = 38%
— V600E dose escalation (n=16)
— Extension (n=32)
— WT or sub-therapeutic exposure (n=33)
1 year 2 years
0 3 6 9 12 15 18 21 24 27 30 33 360
10
20
30
40
50
60
70
80
90
100
Time since first dose (months)
Ove
rall
su
rviv
al
(%)
wild-type
BRAF-mutated
09.10.2013 24Institut für Pathologie – Charité Berlin
Response to
BRAF-inhibitors is
given only if a
BRAF mutation is
present
This has to be
tested prior to the
therapy.
Vemurafenib inhibits V600 mutated BRAF kinase
Vemurafenib inhibits V600 mutated BRAF kinase
CellularProliferation
RTK
RAF
ATP
ATP
ERK
MEK
BRAFV600mut
RAS
50-60%* of melanomas
CellularSurvival
*Total V600 mutation rate for BRIM-3 (cobas® 4800 BRAF V600 Mutation
Test); 9.9% of the cobas-positive cases subjected to retrospective Sanger sequencing had V600K mutations
• Constitutive activation is independent of extracellular factors
• Not responsive to normal regulatory signals
VEMURAFENIB(PLX4032, RG7204, RO5185426)
MEK-IB
Institut für Pathologie – Charité Campus Mitte
Presented in Vienna at ESMO 09/2012:
Flaherty (NEJM, 2012):
OS from 5.8 months with monotherapy to 9.9 months
with combinational targeted therapy.
09.10.2013 27Institut für Pathologie – Charité Berlin
• Multi-gene analyses, predictive molecular pathology and
response to chemotherapy in breast cancer
• Which patient with ER+ and Her2 neg. breast carcinoma
will show a good prognosis when treated by endocrine
therapy only?“
Next Steps in Molecular Pathology –
Multigene Assays in Breast Cancer
09.10.2013 28Institut für Pathologie – Charité Berlin
EPclin: Validation
Martin Filipits, Margaretha Rudas,
Raimund Jakesz, et al.
A new molecular predictor of distant recurrence in
ER-positiveHER2-negative breast cancer adds
independent information to conventional
clinical risk factors.
Clin Cancer Res Published OnlineFirst August 1, 2011.
0 20 40 60 80 100 1200
0.2
0.4
0.6
0.8
1
months
ABCSG-6 – mol. + clin. EP
numbers at risk:
208 200 194 129 120 114 91
170 156 141 87 76 71 57
EPclin low
EPclin high
P < 0.001
HR 7.97 (3.56-17.83)
96%
78%
Following the EPclin-based predictive data 96% of the
low-risk patients do not show-up with metastases
after 10 years.
09.10.2013 29Institut für Pathologie – Charité Berlin
Stratification by EndoPredictclin ®
1702 Patientinnen in ABCSG 6 & 8
1.371 Pat.
„intermed. risk“*
840 EPclin „low risk“
531 EPclin „high risk“
83 Pat. „high risk“*
4,5 % Metastasen
4,5 % Metastasen 19,7 % Metastasen19,7 % Metastasen
44,5 %
Metastasen
44,5 %
Metastasen
248 Pat.
„low risk“*
5,3 %
Metastasen
5,3 %
Metastasen
nach S3-Leitlinien
*nach S3-Leitlinien
w/o Endopredict
these patients may
have recieved CTx
09.10.2013 30Institut für Pathologie – Charité Berlin
A look into the future, exemplified by a current case
09.10.2013 31Institut für Pathologie – Charité Berlin
Up-coming Molecular Diagnostic
histological standard sequential parallel moleculardiagnosis molecular diagnostics diagnostics
KRAS
BRAF EGFR exons 18,19, 21
cKIT
usw.
IonAmpliseq* Cancer
Panel in 46 gene (total 604 loci).
other relevant
mutations
????no mutations
metastasized
neuro-endocrinecarcinoma, grade 3
*Ion Torrent
09.10.2013 32Institut für Pathologie – Charité Berlin
If a nucleotide, for example a C, is added to a DNA template and is then incorporated into a strand of DNA, a hydrogen ion will be released. The charge from that ion will change the pH of the solution, which can be detected by our proprietary ion sensor.
Proprietary Semiconductor Sequencing Technology
Ion Torrent®, the chip is the mashine
09.10.2013 33Institut für Pathologie – Charité Berlin
Up-coming Molecular Diagnostic
histological standard sequential parallel moleculardiagnosis molecular diagnostics diagnostics
KRAS
BRAF EGFR exons 18,19, 21
cKIT
usw.
IonAmpliseq* Cancer
Panel in 46 gene
(total 604 loci).
ABLAPCALK
KRASBRAF
EGFR mut exon 20ERBB2
FGFR2 mutFGFR3
cKITKDL mut
604 further loci…….
no mutations
metastasized
neuro-endocrine
carcinoma, grade 3
*Ion Torrent
Iressa ⇒⇒⇒⇒
FGFR-inhibitor ⇒⇒⇒⇒
sorafinib/sufitinib ⇒⇒⇒⇒
09.10.2013 34Institut für Pathologie – Charité Berlin
Digital pathologywild type
normal + mutated
Multigene
Assays
Clinical
data
tissue
All test are
done on
formalin fixed
paraffin
embedded
tissuePersonalisierte (Onko-)Medizin basiert auf einer komplexen
diagnostischen Prozedur, die Pathologie und Klinik
gleichermaßen zu erfüllen haben.
Weitere Fortschritte, z.B. neue Substanzen, neue Substanz-
Kombinationen, weitere Tumorentitäten etc., werden im
Verbund von Akademia und forschender Pharma-Industrie in
den kommenden Jahren erreicht werden.
35Institut für Pathologie – Charité, Berlin, Campus Mitte
Institute of Pathology,
Rudolph-Virchow-Haus, CharitéHumboldt-Universität zu Berlin
The Berlin WallBerliner MauerAlexander Ufer
Institut für Pathologie,
Rudolf-Virchow-Haus, Charité
Humboldt-Universität zu Berlin
Berliner Medizin-historisches Museum
09.10.2013 36Institut für Pathologie – Charité Berlin
Patient Oncologist
Tumour board
Radiology
Surgery
EndoscopySamp-
ling
Targeted therapies
Results
Cohorts:
Clinical Trials
Tumour Registries
Predictive biomarkers
I+D+i
QA/QC
Teaching
PublicationsConsensus
IHC
In situ hybridization PC
R
Diagnosis
Multidisciplinary cooperation enables personalised oncology
Conde E, et al. Clin Transl Oncol 2013;15:503–8; AMP Whole Genome Analysis WG. J Mol Diagn 2011;13:249–51
Pathology
pre-analytic
09.10.2013 37Institut für Pathologie – Charité Berlin
Three Cellular RAS Genes Encode Four Highly Homologous 21 kD Proteins
Adapted from Schubbert S, et al. Nat Rev Cancer 2007; 7:295-308.
P loop Switch I Switch II
G domainHypervariable
region
KKKKKK CVIM
1 1881312 61
KRAS4B
*Boxes at the bottom of each isoform representation show the
conserved residues in magenta and the variable residues in pink.
C C CVLS
1 85 165 1891610 32 38 59 671312 61
HRAS
KRAS4A
C CIIM
1 1891312 61
C CVVM
1 1891312 61
NRAS
20020408 Trial RAS (Exon 4) Analysis PFS in Patients with WT KRAS Exon 2 mCRC
Patterson SD, et al. J Clin Oncol 31, 2013 (suppl; abstr 3617); 1. Amado RG, et al. J Clin Oncol 2008; 26:1626-34.
Eventsn (%)
Median weeks(95% CI)
Panitumumab + BSC (n=124) 115 (93) 12.3 (8.3–16.1)
BSC (n=119) 114 (96) 7.3 (7.0–7.7)
HR=0.45 (95% Cl: 0.34–0.59)p<0.001
Weeks
20
40
60
80
100
90
70
50
30
10
Pro
po
rtio
n E
ve
nt-
Fre
e %
0
4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 1000
Overall
Overall Survival Kaplan-Meier Plot in Patients
with Non-Mutated RAS
20020408 Trial RAS (Exon 4) Analysis PFS in Patients with MT RAS* Exon mCRC
Patterson SD, et al. J Clin Oncol 31, 2013 (suppl; abstr 3617).
*MT in any KRAS and NRAS exons 2, 3, and 4
20
40
60
80
100
90
70
50
30
10
Pro
po
rtio
n E
ve
nt-
Fre
e %
4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 800 8684
0
Eventsn/N (%)
Median weeks(95% CI)
Panitumumab + BSC(n=99) 90 (91) 7.4 (7.3–7.7)
BSC (n=114) 108 (95) 7.3 (6.4–7.9)
HR=0.97 (95% Cl: 0.73–1.29)p=0.729
Weeks
A prerequisite of personalized medicine is the
capability to predict pre-therapeutically the
response of individual tumors to certain (targeted)
drug.
For this prediction one needs reliable and
reproducible biomarker and predictive assays.
This is the current challenge of predictive molecular
pathology.
Predictive Molecular Pathology and
Personalized Medicine