بسم ا الرحمن الرحيم
ACUTE LEUKEMIA WITH AMBIGUOUS PHENOTYPE IN CHILDREN Are v ie w Akram Al-Hilali 2012
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DEFINITION A rare entity of acute leukemia identified by the expression of certain level of surface and/or cytoplasmic markers that belong to two or more of the broad classes of acute leukemia, either on the same leukemic blasts or on two separate populations of blasts.
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OTHER NAMES
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Biphenotypic Acute Leukemia (BAL) Mixed Phenotype Acute Leukemia (MPAL) Acute mixed lineage leukemia (AML) Acute bilineal leukemia Hybrid leukemia
ACUTE LEUKEMIA WITH AMBIGUOUS PHENOTYPE
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Rare type of acute leukemia. Few series of 20 or more cases reported. It represents 1-8 % of total acute leukemias in the latest literature. Almost equally distributed between adults and children. Condition can represent two separate populations of blasts or one only that is biphenotypic permanently or in the process of changing from one phenotype to another. The biphenotypic malignant clone probably arises from one early multipotent precursor. Can be therapy-related.
WHO CLASSIFICATION
Mixed Phenotype acute leukemia with t(9;22) MPAL with t(v;11q23); MLL rearrangement MPAL, B/Myeloid, NOS MPAL, T/Myeloid, NOS MPAL, NOS
PROVISIONAL FEATURES RAISING SUSPICION Ambiguous morphology and cytochemistry of the blasts. More commonly M1 or M5 features.
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AMBIGUOUS “FAB” PICTURE
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Myeloid features can almost always be noted. More often diagnosed as AML than ALL on study of marrow and blood film by morphology and cytochemistry. May be reported as having “granular” features. or “mixed”. Occasional cases show Auer rods in Blasts.
FAB MORPHOLOGY CLASS * St. Judes collection: 22/35 labeled as AML and provisionally treated as such. M/TL: M/BL 19:11
*13/23 in Owaidah et al collection labeled as “M”, mostly M2. 11 were labeled myeloid and 6 as mixed All cases labeled as M1 or M0 (5) were children.
*14/23 in Legrand et al collection were labeled as myeloid by morphology, majority M1
Cytochemistry is usually done, but is not very helpful.
IMMUNOPHENOTYPING This is an inevitable step in any AL, even if the biphenotypic nature is not suspected. Myeloid markers are usually encountered in an otherwise B or T lymphoid flowcytometry picture. B markers are commoner than T in most series, but T is commoner in St. Judes’ series of childhood ALAP. Trivial deviations may not make the case biphenotypic, and score needs to be used.
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EGIL SCORE
SCORE
B-LINEAGE
T-LINEAGE
MYELOID
2
cCD79a
cCD3 or sCD3
MPO
cIgM
Anti-TCR
cCD22 1
0.5
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CD19
CD2
CD117
CD10
CD8
CD33
TdT
TdT
CD14
CD24
CD7
CD15
CD20
CD5 CD10
CD1a
CD13 CD65
CD64
IMMUNOPHENOTYPING 

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Criteria for scoring by the European Group for the Immunological Classification of Leukemia (EGIL) is still the more widely used system. A case of acute leukemia expressing a score of 2 or more for two phenotypes is to be considered biphenotypic for them. However, difference of opinion is not unexpected in this difficult topic.
COMMENTS ON THE SCORING
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Some centers may leave out the total score of the EGIL system and put emphasis on those markers which are strong indicators of lineage and carry a score of 2 in that system. There is still a difference of opinion on what to consider positive expression in immunophenotyping. Most workers in the field take 20% marker expression as cut off for positivity. Positive MPO is more decisive as myeloid marker in T-ALL cases.
OTHER COMMENTS ON EGIL SCORE
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CD79 positivity CD10 positivity WHO considers that some cases of precursor T cell leukemia can express these 2 markers and therefore one should not rely on them alone to give B-lineage co-expression label to an otherwise a case of precursor T cell leukemia.
PREVALENCE OF M/BL & M/TL Ref
M/BL (%)
M/TL (%)
M/B/T (%)
1
65
26
9
2
64
40
4
3
38
23
23
4
69
23
4
5
100
-
-
6
70
26
4
7
34
57
0
8
69
17
0
9
44
41
-
1-Owaidah et al. Leukemia (2006)20: 620-626 2.Killick et al. Haematologica (1999)84: 699-706 3.Buccheri et al. Leukemia (1993)7: 919-927 4.Carbonell et al. Leukemia (1996), 10: 1283-1287
5. Sulak et al.AmJClinPathol (1990), 94: 54-58 6. Legrand et al.BrJHaematol (1998);100: 147-155 7. Jeffry et al.Blood (2009);113 (21)5083-5089 8.Majestrikova et al. (2010), Haematologica;95(6): 928-935 9.\\\\gujral et al. (2009) Indian J. of Cancer;46 (2):160-168
EARLY SWITCH CASES
Sometimes an acute leukemia case switches from ALL to AML early in the course, as shown by FCM. These are not exactly biphenotypic and can be considered as an ordinary leukemia having the phenotype at which they eventually settle. They may have been biphenotypic with double blast populations, one of which gets eliminated.
PROGNOSTIC AND THERAPEUTIC IMPACT OF THE EGIL SCORE
There are no clear figures, because there is no consensus on type of therapy to be used and because of the small number of patients involved in the reported series. Some authors now recommend using lymphoid leukemia therapy to start with, in spite of the myeloid expression. As with ordinary AL, response is relatively better in children and young adults than in older patients (>60) Achievement of CR with initial therapy is a good prognostic sign for the long term survival.
CYTOGENETIC FINDINGS 

Acute leukemia is classified BY WHO on the basis of morphology, IPT, cytogenetics and molecular genetics. Mixed phenotype acute leukemia as an entity picked by immunophenotyping is also sub-typed according to cyto- and molecular genetics. AML and ALL gene fusions arising from translocations used for placing these cases in the WHO classification: t(8;21),inv (16), t(16;16), t(15;17), t(11;17), t(9;11), t(6;9), t(1;22) and t(8;16). These can show in the biphenotypic AL and confer their respective prognostic effects, as well as help decision on treatment choice. Ploidy (>50 chr)- Incidence ranged from 0 to 14% in reports. May confer a relatively better prognosis as in precursor B ALL of children.
MOLECULAR GENETICS FINDINGS
Commonly found, but there is no consistent abnormality specific for biphenotypic leukemia. MLL gene (q11:23, or t; 4:11, q21:q23) is not different from AML as such, in which it causes poorer prognosis in infants M4/M5 in particular. 12/23 done in KFSH series. 6 negative and 6 positive. C-kit positivity (CD117) is a strong sign in favor of a myeloid identity. Some add an additional point in the FCM score for this element. It is a receptor for SCF. It is, however, not an absolute marker of AML. BCR-ABL gene status and the possible use of kinase inhibitors. Positivity ranged from 9 to 35% in various series published. Some hematologists consider these as CML in blast transformation with multilineage and treat accordingly. Gujral et al series 7 of 32 were positive. Done in 6/23 cases by Owaidha et al. Positive result in 2 cases.
IMMUNOGLOBULIN AND TCR GENE REARRANGEMENT
Non-malignant B cells can be found to have rearrangement of at least one Ig gene. They also can rearrange TCR gene. However, in both cases it is not a monoclonal rearrangement. Monoclonal rearrangement was found in B-ALL, B cell lymphoma and most cases of T-ALL Biphenotypic AL with AML picture and treated as such mostly were negative for rearrangements. Owaidah et al. Ig gene arrangement: 17/23 done. 11 negative. All M/TL were negative. TCR rearrangement: 18/23 done. 7 negative. 4 M/TL negative. Not very helpful in therapy decision or prognosis.
RESPONSE TO THERAPY
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AL with ambiguous phenotype usually does not respond well to AL therapy, with lower CR and higher relapse rates. Criteria for allocation to myeloid or lymphoid leukemia therapeutic regimens is still not settled and an individual case allocation may be difficult. Many cases have therapy initiated before the type is settled. St. Judes’s cases that did not go into CR by initial AML regimen responded to ALL therapy by prednisolone, asparaginase and vincristine.
RESPONSE TO THERAPY- Contd.
4 year DFS is also less favorable than for single phenotype leukemia [0 against 30% in one study]1 5year EFS of PBC/AML was 60% vs 78 in the AL of single phenotype in another study. 2 5 year event-free survival was 53% against 76% in ALL, when receiving ALL therapy2 25% early death when combined AML-ALL therapy was used in Killick et al series3. Those treated with B-ALL regimens apparently did worse than with T-ALL 1.Cherie Dunphy- Medscape updates- Feb 2010 2.Mejstrikova (2010) Haematologica.95(6): 928-935 3. Killick et al (1999). Haematologica;84:699-706
RESPONSE TO THERAPY-Contd
B-lymphocyte lineage in the condition seems to confer less favorable prognosis. Childhood ALAP responds better to therapy than adult cases and particularly those of old age. Because of high rate of relapse stem cell transplantation should be considered after achieving CR in ALAP.
TREATMENT DECISIONS
Because of difference of opinion, rarity of the condition and variability in presentation we do not have a standard rule on how to treat such cases. AML therapy is selected by certain centers when their patients express on their blasts two markers of: CD13, CD33, CD65, CD117 and MPO. Some of these cases did not get CR on AML therapy but ALL therapy achieved it. No statistically significant difference in achievement of CR between cases labeled as lymphoid or myeloid. This may be due to small numbers.
CONCLUSIONS
Ambiguous morphology to be noted. Give a provisional FAB class if possible. Go by EGIL score but take other findings into consideration. Always perform cytogenetics and molecular studies, when available. Start with AML regimen but shift to ALL therapy if CR is not achieved. Never give the two therapies simultaneously. Consider HSCT and arrange for it. Follow up closely after CR, as relapse is more likely than in mono-phenotypic leukemias.