AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow...
Transcript of AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow...
![Page 1: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/1.jpg)
AIEOP-BFM consensus guidelines 2016flow cytometric immunophenotypingin pediatric acute leukemias
Michael N. Dworzak
St. Anna Children‘s Cancer Research Institute
Vienna, Austria, EU
![Page 2: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/2.jpg)
• assure quality
• make results comparable
• foster collaborative research
Coordination: Michael N. Dworzak
iBFMFLOW network
n=56 labs• AIEOP-BFM
• ALL-IC
• UKALL
• NOPHO
• Moscow-Minsk
• AIEOP-BFM
• ALL-IC
• UKALL
• NOPHO
• Moscow-Minsk
![Page 3: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/3.jpg)
Cytometry B Clin Cytometry, 2017
Harmonization of leukemia immunophenotyping
• multi-color (≥ 6) • single panel recommended for all ALs• immunological gate: „Bermude“-area of
CD45 plus Lin marker• in-sample cross-lineage negative controls• semi-quantitative expression rating• blast heterogeneities – subclone
resolution• dominant lineage assignement• MPAL distinction• refined ALL subclassification
![Page 4: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/4.jpg)
Consensus antibody panel
Extensive single-platform panel for acute leukemia in children
Compatible with:
WHO 2008/2016
EGIL score
„New“ ALL subtypes
o ETPo Switch ALLo CRLF2+ ALL
![Page 5: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/5.jpg)
New entity: ETP
![Page 6: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/6.jpg)
New entity: CRLF2r-ALL – CRLF2+
![Page 7: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/7.jpg)
In-sample cross-lineage negative control populations
Expression rating: neg / weak / strong
![Page 8: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/8.jpg)
![Page 9: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/9.jpg)
![Page 10: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/10.jpg)
WHO-style expression ranking
A B
C D
![Page 11: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/11.jpg)
![Page 12: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/12.jpg)
Dominant lineage concept
![Page 13: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/13.jpg)
Gerr et al., BJH 2010
Therapy: BFM
Mejstrikova et al., Haematologica 2010
Therapy: BFM
Al-Seraihy et al., Haematologica 2009
Therapy: TTX 13
Steiner et al., JPHO 2010
Therapy: BFM
MPAL in pediatrics
![Page 14: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/14.jpg)
MPAL – trial inclusion & treatment
![Page 15: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/15.jpg)
Dominant lineage: ALL or MPAL?
![Page 16: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/16.jpg)
Dominant lineage: ALL or MPAL?
![Page 17: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/17.jpg)
Simple co-expressing MPAL
Bi-lineal MPAL
![Page 18: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/18.jpg)
Heterogeneous blasts: MPAL - bilineal
Genetics: 46XY; t(4;11)
• Diagnosis: MPAL B/M• Complex immunophenotype
• Separate blast subsets with differentiation drift into opposing lineage directions
• No common antigenic denominator of lineage
• Dominant lineage cannot be determined
• No inclusion into AIEOP-BFM trial
![Page 19: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/19.jpg)
• Diagnosis: MPAL B/M• Complex immunophenotype
• Branched, interconnected blast subsets with differentiation drift into opposing lineage directions
• No common antigenic denominator of lineage
• Dominant lineage cannot be determined
• No inclusion into AIEOP-BFM trial
Heterogeneous blasts: MPAL - complex
Genetics: MLLr & BCR/ABL negative
![Page 20: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/20.jpg)
New entity: ETP
![Page 21: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/21.jpg)
Dgn.: T-ALL EGIL: T-IIETP: yesMPAL: yesBAL: yesBlast clone heterogeneity: yes
Inukai-Score
![Page 22: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/22.jpg)
![Page 23: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/23.jpg)
“heterogeneous blast populations”
immunophenotypic subclone formation
may lead to divergent interpretation and reporting
characterized by partial expression of certain markers:
B-ALL: iIgM (in 40% of cases), CD10, K/λ-chain
T-ALL: sCD3 (in 50% of cases), CD1a, TCR
differentiation may even drift in a single case from
B-I till B-IV
T-I/II till T-IV
cumulative designation adapted fromEGIL: e.g. TII/III/IV
Heterogeneous blasts: ALL subtype drift
T-III
T-I/II
T-IV
B-III
B-I/II
B-II
B-I
![Page 24: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/24.jpg)
MPAL - BIL
![Page 25: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/25.jpg)
Refined subclassification: prec-B-IV (non-Burkitt)
![Page 26: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/26.jpg)
Reporting
![Page 27: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/27.jpg)
Blast cell heterogeneities in the sense of immunophenotypic subclone formation shouldnot be overlooked even if discriminative antigen-positivity occurs in less than 10% ofblasts, i.e. the usual threshold to claim an antigen as being positive on a relevantpopulation of cells. Of note, this threshold is artificial and derives from the necessity toseparate unambiguous expression from background which is influenced by a plenitude offactors like sample quality, cell viability, autofluorescence, antigen of interest,fluorochrome, antibody combination, compensation matrix, negative control strategy etc.Hence, as shown in this BCP-ALL case with a minor CRLF2-positive blast sub-clone of 5%,the case result for CRLF2 expression is “negative” (for the major blast population).However, the existence of the small CRLF2-positive blast subclone must be captured in theitem “heterogeneous blast population” and described in the report conclusion.
Heterogeneous blasts: clones <10%
![Page 28: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/28.jpg)
ACKNOWLEDGMENTS
A. Schumich
S. Suhendra
D. Printz
M. Reiter
F. Kleber
M. Diem
G. Basso, B. Buldini (Padova)G. Gaipa, O. Maglia, S. Sala (Monza)O. Hrusak, E. Mejstrikova (Prague)L. Karawajew, R. Ratei, S. Groeneveld-Krentz, J. Hoffmann (Berlin)
![Page 29: AIEOP-BFM consensus guidelines 2016 flow cytometric … · AIEOP-BFM consensus guidelines 2016 flow cytometric immunophenotyping in pediatric acute leukemias Michael N. Dworzak St.](https://reader035.fdocuments.in/reader035/viewer/2022062307/5fc8f07bccedf023b96f03ea/html5/thumbnails/29.jpg)