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Immunophenotype: <= /span>Correlates to Cytogenetics and Prognostic Markers in Leukemia
Marc Golightly, Ph.D
Professor of Pathology
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Topic to be considered
Cytogenetics and Immunophenotypes
–Can Immunophenotype predict cytogenetics
–Details of common associations
3D"*"t(1;19)(q23;p13)
3D"*"t(4;11)(q21;q23)
3D"*"t(12;21)(p13;q22) =
Prognostic markers by Flow
New Antigen density/ Fluorochrome issues in prognostic/diagnostic Flow Cytometry
–<= /span>
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Cyto= genetics t(1;19)(q23;p13) positive ALL
3D"*"= 25% of childhood pre-B ALL, = significantly lower in adults = 3D"*"E2A-PBX1 fusion product portents increased resistance = to therapy, poor prognosis = 3D"*"t(1;19) rarely seen in early precursor B-ALL
 *Jean-Luc Lai; Christiane Charrin, JL Huret
c-E2A at 19p13.3 in normal cells**
c-PBX1 at 1q23 in normal cells **
der(19) t(1;19)(q23;p13)*
Normal gene - PBX1a and PBX1b Proteincontains a homeodomain to binds to DNA; nuc= lear localisation; transcription regulation
E2A coded Protein contains transcriptional activation domains and a basic helix-loop-helix DNA binding site; binds specifically to an immunoglobulin enhancer; nuclear localization; transcription factor


5' E2A from 19p13 fused to 3' PBX1; breakpoints are clustered on both genes (bet= ween exons 13 and 14 in E2A); the reciprocal 5' PBX1 - 3' E2A is not transcrib= ed
Fusion Protein Description
550 amino acids; 85 kDa; N-term transcriptional activation domains from E2A f= used to the Hox cooperative motif and homeodomain of C-term PBX1; potent transcriptional activator
Oncogenesis
pleiotropic transforming activity



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Immu= nophenotype t(1;19)(q23;p13) positive ALL
Pre B ALL Blasts
–Brightly positive CD19, CD10, and CD9
–CD34 negative, CD20 negative to dim
60% with this phenotype demonstrate molecular evidence of the E2A-PBX1 fusion product
Rearrangement never seen with other pre-B phenotype
Useful to help screen for molecular studies
------=_NextPart_01C70A56.66672750 Content-Location: file:///C:/91145D83/HBP401L17flowprognostic&cytogenetic_files/slide0031.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Immune Response
Cyto= genetics t(4;11)(q21;q23) positive ALL
3D"*"3-4% childhood ALL, 50%
of infancy ALL harb= or t(4;11)
(q21;q23) or other 11q= 23
involving the MLL gene
3D"*"Poor prognosis and survival
–<= /span>CR can be obtained, but followed by relapse
–<= /span>Median survival; 7 months adults, 9 months childre= n
3D"*"Bone marrow transplant highly indicated
3D"*"11q23 translocations also found in treatment related AML M4/M5- very poor prognosis =
–
–
*Diane H. Norback, Eric B. Johnso= n, and Sara Morrison-Delap, Cytogenetics at the Waisman = Center, J. Huret & Christiane Charr= in
t(= 4;11)(q21;q23) *
http://www.infobiogen.fr/services/chromcancer/
Mll =3D mixed lineage leukemia gene
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Immu= nophenotype t(4;11)(q21;q23) positive ALL
3D"*"Precursor B ALL  t(4;11)(q21;q23) <= /span>
–<= /span>Bright CD19 & DR
–<= /span>Myelomonocytic antigen CD15 positive
–<= /span>CD10 negative (normally pos), CD24 +/- =
–<= /span>Bright CD45 (>75% percentile) independent <= /span>
3D"*"Very specific >95% for MLL (11q23) abnormality
3D"*"Very rare for typical common ALL to show a = 11q23 abnormality
3D"*"Only 66% sensitive (only 2/3rds of 11q23 rearrangements show above phenotype)
–
------=_NextPart_01C70A56.66672750 Content-Location: file:///C:/91145D83/HBP401L17flowprognostic&cytogenetic_files/slide0033.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Immune Response
Cytog= enetics t(12;21)(p13;q22) positive ALL
3D"=Most common translocation in childhood precursor B ALL
3D=Results in cryptic TEL-AML1  rearrangement
&#= 8211;ETV6 (TEL oncogene) 12p13
&#= 8211;CBFA2 (AML1 gene) 21q22
3D=Often undetectable without FISH
3D"=CR in all cases and associated with excellent prognosis
------=_NextPart_01C70A56.66672750 Content-Location: file:///C:/91145D83/HBP401L17flowprognostic&cytogenetic_files/slide0034.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Immune Response
Immuno= phenotype t(12;21)(p13;q22) positive ALL
3D=Precursor B ALL t(12;21)(p13;q22)
&= #8211;Bright CD19, CD10, & DR
&= #8211;CD9 & CD20 negative or partial expression
&= #8211;In study of >200 precursor B ALL
3D=Phenotype had 70% specificity and 90% sensitivity for TEL rearrangement and 92-93% sensitivity for TEL-AML1 translocation (Borowitz et al = Leukemia 1998 12, 1764-1770)
&#= 8211;CD66c (KOR-SA3544) consistently absent
–
Korsa 80 kda glycoprotein on grans
------=_NextPart_01C70A56.66672750 Content-Location: file:///C:/91145D83/HBP401L17flowprognostic&cytogenetic_files/slide0035.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Immune Response
Other cytogenetic/phenotypic correlations
3D"*"AML – M2 with t(8;21)
–<= /span>80% of cases - CD34 Blasts express dim CD19
–<= /span>Leukemic cells with more mature phenotype <= /span>and higher SSC express CD56 and dim CD33
3D"*"AML – M4e with inv(16)
–<= /span>Normal AML M4 phenotype
plus CD2 expressi= on – highly
suggestive
3D"*"T lineage ALL
–<= /span>Many T cell cytogenetic
abnormalities hav= e been found
–<= /span>To date – phenotypic
correlations have not
–
AML M4EO
From G Flandrin, CD-ROM AML/MDS
Cr in 90% but freq relapse- AML-m2
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quoted-printable Content-Type: text/html; charset="us-ascii" Immune Response
Indi= vidual Prognostic Markers in Flow Cytometry
= 3D"*"History and theory of prognostic markers
–I= nitial studies generally looked at single markers and absence/presence
<= img src=3Dimage004.gif alt=3D"*" style=3D'position:absolute;top:16.66%;left:-3= .12%; width:2.64%;height:61.11%'>Few have held up as useable prognostic indicator by themselves
–R= ecent studies with multiple color capabilities have looked at multiple antigens and levels of expression
3D"*"Correlates with cytogenetic abnormalities
3D"*"Unknown or weak cytogenetic correlates
–R= ole of chosen Fluorochrome
l
------=_NextPart_01C70A56.66672750 Content-Location: file:///C:/91145D83/HBP401L17flowprognostic&cytogenetic_files/slide0037.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Immune Response
CD2 in T-ALL
3D"*"= Level of CD2
expression
correlates with
better prognosis
before & after
treatment (n=3D651) 3D"*"Correlation held in both NCI stratified groups
Blood, Vol 88, No 1 1 (December 1), 1996: pp 4286-4295
NCI poor risk group age >10 or wbc >50,000; trisomy of both 4 and 10:or bad translocation t(4;11, t(9;22), or t1;19)
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Quanti= tative CD20/CD45 in B-ALL
3D"*"CD20 > 50th percentile fl intensity
–<= /span>Did not correlate with any other known adverse predictor
3D"*"CD45 Bright > 75th percentile fl intensity
–<= /span>Did correlate with t(4;11), however, remained prognostic after removing t(4;11) patients
3D"*"Risk Factor for recurrence independent
N=3D1231, Borowitz etc al Blood 89:3960-66, 1997
Doted line is both – unfortunely they didn’t present data looking at the NCI poor prognostic groups like the last one, this would have been important since the relative risk of recurence was similar to the other r= isk factors, nor did they present the data for combination – they state= it is much better but don’t show it
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MBdwK8Y9j09hW35MX/PJP++RR5MX/PJP++RQBXXUtOVQovrbAGB++X/Gnx39lK4jivIHduirICTU vkxf880/75FKI41OQig+oFAHmPxM/wCRjt/+vRf/AEN6KPiZ/wAjHb/9ei/+hvRQB//Z ------=_NextPart_01C70A56.66672750 Content-Location: file:///C:/91145D83/HBP401L17flowprognostic&cytogenetic_files/slide0038.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Immune Response
Others
3D=CD7+CD56+ AML-M0  (n=3D105) =
&= #8211;Int. J. Hematol. 77:482-9, 2003
R= 11;Extramedullary involvement, less circulating blasts, poor prognosis, n= o 5q abnormalities
l
------=_NextPart_01C70A56.66672750 Content-Location: file:///C:/91145D83/HBP401L17flowprognostic&cytogenetic_files/slide0040.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Immune Response
CD38 and CLL
3D"*"CD38 expression on CLL was des= cribed in 1999 as poor prognostic indicator
–<= /span>Significantly shorter survival <= /div>
–<= /span>Poorer response to therapy
–<= /span>Independent of mutational status of IgH genes
3D"*"In patient’s requiring treatment the antigen density appears to be more importan= t than percent positive
3D"*"Issue of Fluorochromes
–APC, PE should be used
l
Hsi et al  Br. J Hemato= l. 2003, 120:1017-1025
Suvival bad for unmutated IgH genes, antigen density translates to fl intensity
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charset="us-ascii" Immune Response
Fluorochromes
3D"*"FITC < APC < PE
3D"*"For detection of Ags with low surface density <= span style=3D'font-size:88%'>– fluorochrome choice is important
3D"*"Example: CD5 FITC-, PE+
–<= /span>10/2002 CD38+
(31%) low int = ;
–<= /span>9/2003 CD38+
(84%) low int
CD5 PE now neg
Tran= sformation
to PLL
–
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Summary
3D"*"Several good phenotypic correlations to cytogenetic abnormalities exist =
–<= /span>They are rarely absolute – SUGGESTIVE
–<= /span>No additional markers are required
–<= /span>May help direct effort towards genetic studies = ;
3D"*"Prognostic markers should continue to improve
–<= /span>Quantitation of actual number of molecules is easily done in the clinical lab =
–<= /span>Multicolor analysis will define unique combination= s
3D"*"Fluorochrome choices will need to be standardized
–
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Second level
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Fourth level
Fifth level
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‹#›
Normal gene - PBX1a and PBX1b Proteincontains a homeodomain to binds to DNA; nuclear localisation; transcription regulation E2A coded Protein contains transcriptional activation domains and a basic helix-loop-helix DNA binding site; binds specifically to an immunoglobulin enhancer; nuclear localization; transcription factor
5' E2A from 19p13 fused to 3' PBX1; breakpoints are clustered on both gene= s (between exons 13 and 14 in E2A); the reciprocal 5' PBX1 - 3' E2A is not transcribe= d
Fusion Protein Description
550 amino acids; 85 kDa; N-term transcriptional activation domains from E2= A fused to the Hox cooperative motif and homeodomain of C-term PBX1; potent transc= riptional activator
Oncogenesis
pleiotropic transforming activity
 
 
Mll =3D mixed lineage leukemia ge= ne
Korsa 80 kda glycoprotein on gran= s
Cr in 90% but freq relapse- AML-m= 2
NCI poor risk group age >10 or= wbc >50,000; trisomy of both 4 and 10:or bad translocation t(4;11, t(9;22),= or t1;19)
Doted line is both – unfort= unely they didn’t present data looking at the NCI poor prognostic groups l= ike the last one, this would have been important since the relative risk of recurence was similar to the other risk factors, nor did they present the = data for combination – they state it is much better but don’t show = it
Suvival bad for unmutated IgH gen= es, antigen density translates to fl intensity
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