1. Das 70-Genprofil MammaPrint beim Mammakarzinom
PD Dr. Michael Knauer PhD
Brustzentrum St. Gallen
19. Bamberger Morphologietage

2. Disclosures Kein finanzieller Conflict of interest
Chirurgischer Onkologe mit Interesse für translationelle Forschung
6 Monate Hobbypathologe während FA-Ausbildung

3. Klinischer Nutzen eines Multigenprofils
Prognose: Abschätzen des Rezidivrisikos
Einteilung in Risikogruppen mit hoher Sensitivität und Spezifität
Low Risk: niedrig genug, um auf Chemotherapie zu verzichten
Prädiktion: Abschätzen des Benefits adjuvanter Chemotherapie
High Risk: Patientinnen mit hohem Rezidivrisiko Signifikanter Nutzen durch Chemotherapie

4. Entwicklung Multigen-Assays
Intrinsic subtypes1
Entwicklung von prognostischen Profilen2
Perou, Nature 2000
Parker et al., JCO 2009

5. 21-gene recurrence score – Oncotype Dx™
Category
RS (0 – 100)
Low risk
RS < 18
Intermediate risk RS
High risk
RS ≥ 31
Paik et al, NEJM 2004 Habel et al, BCRT 2006

6. Entwicklung des 70-Genprofils: 25.000 Gene ohne Vorwissen (unbiased)
78 breast tumors
Age < 55 years, Tumor size < 5 cm
Lymph node negative & No adjuvant therapy
Distant metastases
within 5 years
No distant metastases
for at least 5 years
Supervised analysis:
Stratified for prognosis
van‘t Veer LJ et al., Nature, 2002

7. 70-Genprofil MammaPrint
van t’Veer et al., Nature 2002
van de Vijver et al., NEJM 2002

8. Wachstum und Proliferation
IGFBP5, TGFB3, FGF18, ESM1, RARRES3, PITRM1, EXT1, EXTL3, SCUBE2,
EBF4,CDC42BPA, CDCA7, CDCA7L, GMPS, MELK, RFC4, WISP1, HRASLS,
BBC3, DTL, FBXO31, EGLN1, GNAZ, MTDH, FLT1, ECT2, DIAPH3, NUSAP1,
AKAP2, NDC80, PRC1, ORC6L, CENPA, DCK, CCNE2, MCM6, QSOX2, STK32B 1.
Wachstum und Proliferation 2.
Angiogenese
COL4A2, FLT1, FGF18, MMP9 3.
FLT1, TGFB3, IGFBP5, FGF18, RARRES3,
CDCA7L, WISP1, DIAPH3, AKAP2, CDC42BPA,
PALM2, DCLK2, NMU, NMUR1, NMUR2
Lokale Invasion 5.
Überleben im Kreislauf
COL4A2, FLT1, MMP9, TGFB3, MTDH, DIAPH3,
PALM2, DCLK2, NMU, NMUR1, NMUR2 4.
COL4A2, FLT1, MMP9, TGFB3, MTDH, DIAPH3,
PALM2, DCLK2, NMU, NMUR1, NMUR2
Intravasation 6.
COL4A2, FLT1, MMP9, TGFB3, MTDH, DIAPH3,
PALM2, DCLK2, NMU, NMUR1, NMUR2
Extravasation 6.
Extravasation
COL4A2, FLT1, MMP9, TGFB3, MTDH, DIAPH3,
PALM2, DCLK2, NMU, NMUR1, NMUR2 7.
Adaptierung an Microenvironment bei der Metastase
MMP9, COL4A2
IGFBP5, TGFB3, FGF18, ESM1, RARRES3, PITRM1, EXT1, EXTL3, SCUBE2,
EBF4,CDC42BPA, CDCA7, CDCA7L, GMPS, MELK, RFC4, WISP1, HRASLS,
BBC3, DTL, FBXO31, EGLN1, GNAZ, MTDH, FLT1, ECT2, DIAPH3, NUSAP1,
AKAP2, NDC80, PRC1, ORC6L, CENPA, DCK, CCNE2, MCM6, QSOX2, STK32B

9. 70-Genprofil: Stabilität und Reproduzierbarkeit
Glas et al. BMC Genomics 2006 9

10. 70-Genprofil: Stabilität und Reproduzierbarkeit
Ach et al. BMC Genomics 2007 10

11. Unabhängige Information
Knauer et al., ASCO 2010, abstract 561

12. Mehrere Antworten aus einem Test
MammaPrint
70 x 9
231 x 5
Molecular subtypes BluePrint
80 x 5
mRNA readout ER, PR and HER2
TargetPrint
3 x 5
Normalization
465 x 3
Control probes
536
Research Gene Panel TheraPrint
56 x 3
Drug response profile
Drug response profile

13. 1. Validierung 70-Genprofil
n= 151, N0, ohne adj. Therapie, 40% low risk, 60% high risk
metastases-free
metastases-free
HR 5.5 ( ), P<0.001
Van de Vijver MJ et al., NEJM, 2002

14. Validierungsstudien 70-Genprofil
MammaPrint Studies
Validation
Details
Results/Comments
Van de Vijver et al, (2002)
NEJM;347,
First validation in consecutive series*
295 patients
151 patients LN-, 5% adj treatment, 7.3 yrs follow-up
10 year DMFS:
Low Risk: 87%
High Risk: 44%
Buyse et al, (2006)
JNCI;17,
Independent validation
302 patients
no adj treatment
13.6 yrs follow-up
Low Risk: 88%
High Risk: 71%
Wittner et al. (2008)
CCR;14,
Validation in US patients*
100 patients
45% adj treatment
11.3 yrs follow-up
Low Risk: 100%
High Risk: 90%
Mook et al, (2009)
BrCResTr;116(2):
LN+ patients (1-3)
241 patients
91% adj treatment
7.8 yrs follow-up
Low Risk: 91%
High Risk: 76%
Bedard et al ,(2009)
SABCS
Postmenopausal (>65 yrs)
330 patients
35% adj ET
7.4 yrs follow-up
5 year DMFS:
High Risk: 77%
Bueno-de-Mesquita et al. (2009) BrCResTr;117(3):
123 patients
37% adj treatment
5.8 yrs follow-up
Low Risk: 98%
High Risk: 78%
Mook et al. (2010)
Ann Onc;21(4):
Postmenopausal (>55-70 yrs)
148 patients
18% adj treatment
11.6 yrs follow-up
Low Risk: 81%
High Risk: 68%
Ishitobi et al (2010)
Jpn JCO;40(6):
Japanese patients*
102 patients
92% adj treatment
7.1 yrs follow-up
High Risk: 94%
Ann Surg Onc; 17(5):
T1 tumors
964 patients
Pooled analysis
High Risk: 72%
Knauer et al. (2010)
Br J Cancer;103(12):
HER2+ tumors
168 patients
5.4 yrs follow-up
Low Risk: 89%
High Risk: 64%
Mittempergher Let al. (2013)
Mol Oncol;7/5):987-99
LN+ patients (4-9)
173 patients
97% adj treatment
15.9 yrs follow-up
High Risk: 63%

15. Update 2013: N0-Patientinnen (n=151)
Drukker C et al., BCRT 2014

16. Update 2013: Alle Patientinnen (n=295)
Drukker C et al., BCRT 2014

17. Späte Rezidive: abhängig vom ER-Status
Subtypes
MammaPrint
Oncotype Dx
National Cancer Institute's Surveillance, Epidemiology, and End Results 13 Registries Databases (1992 to 2007) for invasive female breast cancer; n≈ ;
Jatoi I et al. JCO 2011;29:
Fan et al. NEJM 2006

18. EndoPredict (EP) Validation I Validation II Training Multicenter
BIRC5
RBBP8
UBE2C
IL6ST
AZGP1
DHCR7
MGP
STC2
CALM2
OAZ1
RPL37A
Member 1
Member 2
Member 3
Member 4
Reference genes
Multicenter
TAM Monotherapy (n=964)
ABCSG 6
TAM
(n=378)
ABCSG 8
TAM vs. TAM/ANA
(n=1.324)
The Endopredict prognostic test is a multigene signature trained in a large cohort of tamoxifen treated women with ER+/Her negative tumors. The signature comprises 8 genes and three reference genes and has been succesfully validated in two phase iii trials from ABCSG. Both trials included postmenopausal women treated with adjuvant endocrine therapy in the absence of adjuvant chemotherapy. In the same cohort we have furthermore been able to demonstrate that Endpredict correctly reassignes approximately 60% of women classified as high-/intermediate-risk (according to clinical guidelines) to low risk. Currently this test can be assigned an Evidence Level of Ib according to Simon and colleagues,
Validated in two independent prospective-retrospective studies
Evidence level: Ib (according to Simon et al., J Natl Cancer Inst, 2009)
Filipits et al., Clin Cancer Res. 2011
Dubsky et al., Ann Oncol. 2012

19. EndoPredict (EP) 0 – 5 years > 5 years
The EndoPredict low-risk group comprises approximately half of all patients and had a significantly improved clinical outcome before and after 5 years of follow-up. 96.3% were free of distant metastases between 5 and 10 years in the EP low risk group
The EndoPredict low-risk group (49%) had a significantly improved clinical outcome before and after 5 years of follow-up. 96.3% were free of distant metastases between 5 and 10 years in the EP low risk group.

20. EndoPredict: Prognose NACH 5 Jahren
C-Index * (> 5 years)
The EndoPredict Score improves the prognostic performance of all common clinical parameters.
The EPclin – a predefined score of EndoPredict and the clinical parameters nodal status and tumor size – showed the best performance to predict late metastases.
P < 0.001
The C index is a statistical measure for the prognostic performance of a test. Similar to a likelyhood ratio test or to a ROC analysis It allows assumptions concerning the fit of a model- or in our case the actual occurrence of late distant metastasis. The c- index was calculated for the time interval after 5 years using clinico-pathologic factors and an algorhythm thereof, specifically Adjuvant online. This data confirms the independence of Ep but also indicates that EP clearly adds information from what is gained form even a combination of factors.
P < 0.001
P = 0.001
*C-index = Measure of prognostic performance

21. ATAC Study: Tamoxifen or Anastrozole, NO chemotherapy
Signature
Information included
Clinical Treatment Score (CTS)
Nodal status, grade, tumour size, age, treatment
Immunohistochemical markers (IHC4)
ER, PgR, Ki-67, Her2
Oncotype Recurrence Score (RS)
21 genes (oestrogen, proliferation, invasion, HER2 genes)
Breast Cancer Index (BCI)
H/I and 5 proliferation genes (Molecular Grade Index)
Prosigna (ROR)
46 genes, proliferation score, tumour size
(EU cut-offs from transATAC for N- and N+)
EndoPredict (EPclin)
12 genes (proliferation, differentiation, oestrogen);
nodal status and tumour size

22. Prognostic value years 0-10 – node-negative
% Improvement
53.8%
70.8%
33.3%
74.5%
47.8%
CTS
IHC4
IHC4
BCI
BCI RS RS
ROR
ROR
EPclin
EPclin
Likelihood Ratio χ2
Likelihood Ratio ∆χ2

23. Prognostic value years 0-10 – node-positive
% Improvement
11.9%
12.9%
12.3%
14.9%
21.1%
CTS
IHC4
IHC4
BCI
BCI RS RS
ROR
ROR
EPclin
EPclin
Likelihood Ratio χ2
Likelihood Ratio ∆χ2

24. DMFS (%) in years 0-10 – node-negative
DR risk %
Patients % in risk groups
100 80 60
BCI
3.9%
61.8% patients
19.3%
24.2% patients
27.3%
14.0% patients
100 80 60 RS
5.9%
63.3% patients
16.7%
26.4% patients
Distant recurrence free (%)
27.2%
10.3% patients
100 80 60
ROR
3.0%
53.8% patients
14.1%
30.1% patients
33.4%
16.1% patients
EPclin
100 80 60
6.6%
72.6% patients
22.1%
27.4% patients 2 4 6 8 10
Follow-up time [years]

25. DMFS (%) in years 0-10 – node-positive
DR risk %
Patients % in risk groups
100 80 60
BCI
23.8%
49.3% patients
33.1%
32.6% patients
50.6%
18.1% patients
100 80 60 RS
26.2%
57.7% patients
34.7%
31.7% patients
Distant recurrence free (%)
48.8%
10.6% patients
100 80 60
ROR
0.0%
6.6% patients
20.7%
25.6% patients
39.1%
67.8% patients
100 80 60
EPclin
5.6%
18.9% patients
37.2%
81.1% patients 2 4 6 8 10
Follow-up time [years]

26. EP score vs. EPclin score
0 – 5 years
Inclusion of tumor size
and nodal status
> 5 years
EPclin low: 64% with>98% >5 years
When tumor size and nodal status are included into the model there seems to be an increase in the sensitivity of the test. 64% of all patients are now contained in the low risk group- 98% of these women remain free of distant metastasis at 10 years after diagnosis.
0 – 5 years
> 5 years

27. Risikoeinschätzung AdjuvantOnline: ein Drittel diskordant
High clinical risk
Adjuvant online N=222
Low clinical risk
Adjuvant online N=80
29% discordant
27%
Low Risk
35%
High Risk
30 % diskordante Risikoeinschätzung führte in 20% zur Therapieänderung
Buyse et al. JNCI, 2006
Bueno-de-Mesquita et al. Lancet Oncol, 2007

28. MINDACT Study Design 41% 27% 9% 23% Abbreviations
C-low= Clinical Risk assessment low
C-High= Clinical Risk assessment high
G-Low= MammaPrint Low (MP Low)
G-High= MammaPrint High (MP High)
Cardoso, F., et al. N Engl J Med

29. 2% gain in survival counterbalanced by treatment associated risks
Definition of clinical low risk: Why a 92% threshold for 10y OS w/o adjuvant chemotherapy using Adjuvant!Online?
Helps with the definition of Low Risk….add to the title
-10 year overall survival was selected to determine the appropriate cut-off that would eliminate the harm vs benefit for adding chemotherapy. With such a good prognosis, ACT would not be associated with a favorable benefit vs harm ratio.
-Gain in survival counterbalanced by treatment associated risks in clinically low patients
-2% ER negative in the primary test group
2% gain in survival counterbalanced by treatment associated risks

30. Endpoints and events in MINDACT Population (First Event Analysis)
&
Contralateral breast vs 2nd primary
DMFS definition changed in 2009 to include deaths by other causes
Excerpt from Hudis, JCO 2007
Distant DFS
Of all the nondeath events listed above, it is generally accepted that distant recurrence (metastasis) trumps other events because it is a threat to patient survival. Indeed, it is the main predictor of death in all end point definitions. Our proposed definition for distant DFS (DDFS) preserves this focus. Therefore, it excludes ipsilateral breast tumor recurrence, regional invasive recurrences, contralateral breast cancer, and all in situ carcinomas, as these events are potentially nonlethal. The separation of distant as a specific end point is also very important for ancillary studies involving microarray analysis and for developing genetic panels for use in determining prognosis and/or response to treatment. In these situations, distant disease recurrence is often used as a marker for survival to increase statistical power because there is such a strong correlation between these end points and because there will be more distant events than deaths. Using a combined regional/distant end point would dilute the correlation
End Points are assessed based on the time to date of the first event as listed above, whichever occurs first.

31. MINDACT Trial Primary Test Analysis:
Cardoso, F., et al. N Engl J Med

32. MINDACT Trial Primary Test Analysis: C-high / G-low group- No CT
5-Year DMFS for the C-high / G-low (MP Low) group with no CT= 94.7% (CI: 92.5 – 96.2%)
Cardoso, F., et al. N Engl J Med

33. Intent to treat group: Chemo efficacy in C-high / G-low (DMFS)
No statistical difference between CT vs no CT
∆1.5%
Cardoso, F., et al. N Engl J Med

34. Intent-to-Treat Population: Chemo efficacy in C-low / G-high (DMFS)
Summarize who were the clin Low/MP High group… dive into what MammaPrint is looking for (pr
∆0.8%
Cardoso, F., et al. N Engl J Med

35. Zusammenfassung 70-Genprofil ist ein validierter prognostischer Test
Zusätzliche unabhängige prognostische Information von Multigenprofilen (mehr bei N0 als bei N1)
Mindact Phase III Studie zeigt >95% DMFS nach 5 Jahren für 70- Gen low risk Patientinnen – keine Chemo sinnvoll
70-Genprofil ist kein prädiktiver Test für Nutzen von Chemotherapie bei diskordanter Risikoeinschätzung: low risk = low risk, egal ob klinisch oder genomisch
Verschiedene Tests für unterschiedliche Fragen

36. Algorithmus für das Tumorboard
Subtypen des Mammakarzinoms (klinisch oder mittels Genprofil bestimmt)
Luminal A
Luminal B
Her2 +
TNBC
60% % % 15%
keine Chemo (auch LN+) meistens Chemo
Luminal B
1. klinisch: AdjuvantOnline, CTS, IHC4, composite risk index...
2. Genomisch: Multigentest

37. Herzlichen Dank ABCSG SAKK NKI St. Gallen Breast Center
© / V e
St. Gallen Breast Center
Rahel Hiltebrand
Christina Heinl
Tina Egli
Ljiljana Zuder
Patrik Weder
Salome Riniker
Ursula Hasler
Thomas Ruhstaller
Beat Thürlimann
ABCSG
Michael Gnant
Peter Dubsky
Florian Fitzal
Rupert Bartsch
SAKK
Walter Weber
Christoph Tausch
NKI
Emiel Rutgers
Laura van t`Veer

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39. Titel