How to count, what to count and why to count Ki-67 immunoreactive cells Beppe Viale IEO & UNIMI
Ki-67: son of a lesser God
Can we do it better? Ki67 www.google.com
Mitsubishi Ki-67 Hiryu (Peggy) Though classified as a medium bomber by the Japanese, by Western standards, the Ki-67 Peggy...
Ki-67 <ul><li>Nuclear protein with 2 isoforms (345 & 395 kDa) named after its immunoreactivity for the Ki-67 MAb  </li></u...
The biological meaning <ul><li>Ki-67 labeling index is a measure of the proliferative  fraction  of the tumours </li></ul>...
A reassuring starting point <ul><li>Plenty of data on the prognostic/predictive value of KI-67 </li></ul><ul><li>Despite: ...
 
 
 
STEPP Ki-67 LI by Treatment  in the PACS01 trial
 
 
BIG 1-98 Trial Design <ul><li>Postmenopausal women with endocrine-responsive early breast cancer  </li></ul><ul><li>BIG 1-...
DFS by Ki-67 LI
STEPP Ki-67 LI by Treatment  in the BIG1-98 trial
 
Contributions to Composite Risk Points: 0 ~0.25 ~0.5 ~0.75 ~1.5 Lymph nodes 0 1-3 4-9 10+  ER %  50+ 30-49 <30% PgR % 20+ ...
STEPP 5-year DFS by Composite Risk Increasing Composite Risk
Chemotherapy in ER+ve HER2-ve Relative Indications for chemotherapy: any of Factors not useful in decision* Relative Indic...
St Gallen Consensus 2011 <ul><li>For practical purposes tumour subtype can be ascertained by non-genetic tests for ER, PgR...
Intrinsic Subtypes  Clinicopathological correlates (1) Intrinsic Subtype  Clinico-pathologic definition Notes Luminal A ‘ ...
Intrinsic Subtypes  Clinicopathological correlates (2) Intrinsic Subtype Clinico-pathologic definition Notes Erb-B2 overex...
St Gallen Consensus 2011 <ul><li>Is Ki-67 >14% in endocrine-responsive tumours arguing  for inclusion of ChT  ? </li></ul>...
‘ Subtype’ Type of therapy Notes ‘ Luminal A’ Endocrine therapy alone Few require cytotoxics (e.g. high nodal status). ‘ L...
A reassuring starting point <ul><li>Plenty of data on the prognostic/predictive value of KI-67 </li></ul><ul><li>Despite: ...
Work in progress: Fostering standardisation  Ki67 in breast cancer: recommendations from the International Ki67 in Breast ...
What & How to count <ul><li>( Invasive)Tumour cells only </li></ul><ul><li>Avoid normal cells (lymphocytes!) </li></ul><ul...
Gradient of Ki67 labelling index
Gradient & hot spot
Gradient & hot spot ?
Heterogeneity-related issues <ul><li>Comparison of Ki-67 LI in core biopsies vs surgical specimens </li></ul><ul><li>Use o...
Scoring/Reporting <ul><li>Negative/Positive </li></ul><ul><ul><li>“ Negative” cancers do not exist </li></ul></ul><ul><li>...
Take home <ul><li>Ki-67 is a useful prognostic marker in breast cancer </li></ul><ul><li>It may have predictive value -at ...
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Gene Profiling in Clinical Oncology - Slide 8 - G. Viale - Classic pathology: why is proliferation difficult to determine? Why is Ki-67 not a standard? What was decided at St. Gallen last week!

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  • BIG 1-98 is a large, international, randomized, double-blind Phase III trial Altogether 8101 patients were randomized to four arms between March 1998 and May 2003 This study is limited [point to oval around top 2 arms] to patients randomized to one of the two monotherapy arms, to receive letrozole or tamoxifen for 5 years.
  • Overall, pooling the two treatment arms, higher Ki-67 LI was confirmed to be a highly significant adverse prognostic factor for DFS in these patients, all of whom received some adjuvant endocrine therapy. The hazard of a disease-free survival event with higher Ki-67 LI was 1.8, corresponding to four-year DFS estimates of 86% versus 92% for high versus low Ki-67 LI. A significant difference was maintained in multivariate analyses allowing for other prognostic factors. Thus, Ki-67 LI is an independent prognostic factor in our hands.
  • Summary of approximate number of &apos;points&apos; for each factor;
  • OK – how does composite risk interact with treatment – does it have predictive value? Tamoxifen alone deviates (solid red line) from others earlier in the risk spectrum. Finally [click to show red oval] separation among letrozole-containing treatments is apparent particularly at higher risk.
  • Gene Profiling in Clinical Oncology - Slide 8 - G. Viale - Classic pathology: why is proliferation difficult to determine? Why is Ki-67 not a standard? What was decided at St. Gallen last week!

    1. 1. How to count, what to count and why to count Ki-67 immunoreactive cells Beppe Viale IEO & UNIMI
    2. 2. Ki-67: son of a lesser God
    3. 3. Can we do it better? Ki67 www.google.com
    4. 4. Mitsubishi Ki-67 Hiryu (Peggy) Though classified as a medium bomber by the Japanese, by Western standards, the Ki-67 Peggy was seen as a light bomber
    5. 5. Ki-67 <ul><li>Nuclear protein with 2 isoforms (345 & 395 kDa) named after its immunoreactivity for the Ki-67 MAb </li></ul><ul><li>Gene size approx 30,000 bp, with 15 exons and 14 introns on chromosome 10 </li></ul><ul><li>Expressed in all proliferating cells during late G1,S,G2 and M phases of the cell cycle, peaking in the G2-M. </li></ul><ul><li>Rapid decline after mitosis </li></ul><ul><li>During interphase it is only detected within the nucleus; during mitosis most of the protein is relocated to the surface of chromosomes </li></ul><ul><li>MIB-1 has been raised against a recombinant fragment of the protein, and it reacts with a formalin-resistant epitope </li></ul>
    6. 6. The biological meaning <ul><li>Ki-67 labeling index is a measure of the proliferative fraction of the tumours </li></ul><ul><li>It is not a measure of the proliferative rate (e.g. BrdU incorporation, S-phase fraction) </li></ul><ul><li>Actual tumour growth is determined by </li></ul><ul><ul><li>Proliferative fraction </li></ul></ul><ul><ul><li>Proliferative rate </li></ul></ul><ul><ul><li>Extent of apoptosis </li></ul></ul>
    7. 7. A reassuring starting point <ul><li>Plenty of data on the prognostic/predictive value of KI-67 </li></ul><ul><li>Despite: </li></ul><ul><ul><li>Different materials (TMAs vs cores vs whole sections) </li></ul></ul><ul><ul><li>Different staining protocols </li></ul></ul><ul><ul><li>Different evaluation and scoring of the results </li></ul></ul><ul><li>Could we improve standardisation and reproducibility? </li></ul>
    8. 11. STEPP Ki-67 LI by Treatment in the PACS01 trial
    9. 14. BIG 1-98 Trial Design <ul><li>Postmenopausal women with endocrine-responsive early breast cancer </li></ul><ul><li>BIG 1-98 showed letrozole significantly improved disease-free survival compared with tamoxifen </li></ul>N=4922 monotherapy N=3088 sequential N=8010 randomized Tamoxifen Letrozole Letrozole Letrozole Tamoxifen R A N D O M I Z E 0 2 5 YEARS A B C D Tamoxifen
    10. 15. DFS by Ki-67 LI
    11. 16. STEPP Ki-67 LI by Treatment in the BIG1-98 trial
    12. 18. Contributions to Composite Risk Points: 0 ~0.25 ~0.5 ~0.75 ~1.5 Lymph nodes 0 1-3 4-9 10+ ER % 50+ 30-49 <30% PgR % 20+ <20 Ki-67 % <14 14-33 34+ HER2 Neg Pos PVI No Yes Grade 1 2 3 T size ≤ 2 2.1-4.9 5+
    13. 19. STEPP 5-year DFS by Composite Risk Increasing Composite Risk
    14. 20. Chemotherapy in ER+ve HER2-ve Relative Indications for chemotherapy: any of Factors not useful in decision* Relative Indications for endocrine therapy alone Grade 3 Grade 2 Grade 1 High proliferation Intermediate proliferation Low proliferation Lower ER and PgR level Higher ER and PgR level N 4+ N 1-3 Node negative Peritumoral vascular invasion (PVI) No PVI T size > 5cm T size >2 – 5 cm T size <= 2cm*** Patient preference to use all available treatments Patient preference to avoid side effects Multigene assays: High Intermediate Low score
    15. 21. St Gallen Consensus 2011 <ul><li>For practical purposes tumour subtype can be ascertained by non-genetic tests for ER, PgR, Her2 and Ki67? </li></ul><ul><ul><li>Yes: 82% </li></ul></ul><ul><ul><li>No: 12.2% </li></ul></ul><ul><ul><li>A: 4.9% </li></ul></ul>
    16. 22. Intrinsic Subtypes Clinicopathological correlates (1) Intrinsic Subtype Clinico-pathologic definition Notes Luminal A ‘ Luminal A’ ER and/or PgR positive HER2 negative Ki-67 low (<14%)* Optimal cut point for Ki-67 labelling index was established by comparison with PAM50 intrinsic subtyping. Local quality control of Ki-67 staining is important. Luminal B** ‘ Luminal B (HER2 negative)’ ER and/or PgR positive HER2 negative Ki-67 high  ‘ Luminal B (HER2 positive)’ ER and/or PgR positive Any Ki-67, HER2 over-expressed or amplified [5] Genes indicative of higher proliferation are poor prognostic markers in multiple genetic assays [7].   Useful to distinguish ‘luminal B HER2 positive’ as both endocrine and anti-HER2 therapy indicated
    17. 23. Intrinsic Subtypes Clinicopathological correlates (2) Intrinsic Subtype Clinico-pathologic definition Notes Erb-B2 overexpression ‘ HER2 positive (non luminal)’ HER2 over-expressed or amplified ER and PgR absent The majority of HER2 positive tumours are endocrine-receptor negative ‘ Basal-like’ ‘ Triple negative (ductal)’ ER and PgR absent HER2 negative   Approximately 80% overlap between ‘triple negative’ and intrinsic ‘basal-like’ subtype   Staining for basal keratins although shown to aid selection of true basal-like tumours, is not considered insufficiently reproducible for general use.
    18. 24. St Gallen Consensus 2011 <ul><li>Is Ki-67 >14% in endocrine-responsive tumours arguing for inclusion of ChT ? </li></ul><ul><ul><li>Yes: 68.8% </li></ul></ul><ul><ul><li>No: 14.6% </li></ul></ul><ul><ul><li>A: 16.7% </li></ul></ul>
    19. 25. ‘ Subtype’ Type of therapy Notes ‘ Luminal A’ Endocrine therapy alone Few require cytotoxics (e.g. high nodal status). ‘ Luminal B (HER2 negative)’ Cytotoxics + endocrine therapy   Inclusion and type of cytotoxics may depend on perceived risk and patient preference. ‘ Luminal B (HER2 positive)’ Cytotoxics + anti-HER2 + endocrine therapy   No data are available to support the omission of cytotoxics in this group. ‘ HER2 positive (non luminal)’   Cytotoxics + anti-HER2   ‘ Triple negative (ductal)’ Cytotoxics Consider DNA disrupting agents. ‘ Special histological types’* A. Endocrine responsive B. Endocrine non responsive   Endocrine therapy Cytotoxics     Medullary and adenoid cysctic carcinomas may not require any adjuvant cytotoxics).
    20. 26. A reassuring starting point <ul><li>Plenty of data on the prognostic/predictive value of KI-67 </li></ul><ul><li>Despite: </li></ul><ul><ul><li>Different materials (TMAs vs cores vs whole sections) </li></ul></ul><ul><ul><li>Different staining protocols </li></ul></ul><ul><ul><li>Different evaluation and scoring of the results </li></ul></ul><ul><li>Could we improve standardisation and reproducibility? </li></ul>
    21. 27. Work in progress: Fostering standardisation Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer Working Group   Mitch Dowsett 1 , Torsten O Nielsen 2 , Roger A’Hern 3 , John Bartlett 4 , R Charles Coombes 5 , Jack Cuzick 6 , Matthew Ellis 7 , Lynn Henry 8 , Tracy Lively 9 , Lisa McShane 10 , Soon Paik 11 , Ljudmila Prudkin 12 , Meredith Regan 13 , Janine Salter 1 , Christos Sotiriou 14 , Ian Smith 15 , Giuseppe Viale 16 , Jo Anne Zujewski 17 , Daniel F Hayes 8 . Meeting in London, March 2010
    22. 28. What & How to count <ul><li>( Invasive)Tumour cells only </li></ul><ul><li>Avoid normal cells (lymphocytes!) </li></ul><ul><li>Percentage of immunoreactive cells (irrespective of staining intensity) over 500-2,000 tumour cells </li></ul>
    23. 29. Gradient of Ki67 labelling index
    24. 30. Gradient & hot spot
    25. 31. Gradient & hot spot ?
    26. 32. Heterogeneity-related issues <ul><li>Comparison of Ki-67 LI in core biopsies vs surgical specimens </li></ul><ul><li>Use of TMA </li></ul><ul><li>Intra- and inter-observer reproducibility </li></ul><ul><ul><li>Very good in core biopsies/TMAs </li></ul></ul><ul><ul><li>Less than optimal in surgical specimens </li></ul></ul>
    27. 33. Scoring/Reporting <ul><li>Negative/Positive </li></ul><ul><ul><li>“ Negative” cancers do not exist </li></ul></ul><ul><li>Thresholds for low/high proliferative fraction? </li></ul><ul><ul><li>Depending on the population </li></ul></ul><ul><ul><li>Median value approx 20% in the overall population </li></ul></ul><ul><ul><li>Median values variable in pre-defined populations </li></ul></ul><ul><li>Continuous variable </li></ul>
    28. 34. Take home <ul><li>Ki-67 is a useful prognostic marker in breast cancer </li></ul><ul><li>It may have predictive value -at least in defined subgroups of patients </li></ul><ul><li>Its prognostic/predictive value is magnified when used in combination with additional clinico-pathological parameters (combined risk scores) </li></ul>

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