acute leukemia of ambiguous lineage ( alal ) is a rare diagnosis associated with complex clinical , cytogenetic , immunophenotypic , and/or molecular genetic features and with adverse clinical outcomes . according to the world health organization 2008 classification ,
alal includes several rare leukemia subtypes , such as acute undifferentiated leukemia ( leukemia without lymphoid or myeloid markers ) and mixed phenotype acute leukemia ( mpal ; leukemias with co - expression of dual lineage antigens or with two distinct clonal monolineage blast populations ) . representing approximately 2% of all acute leukemias
patients with mpal have poor responses to lymphoid- and myeloid - directed chemotherapy and experience high rates of relapse .
recent analyses estimate that 1030% of patients with mpal exhibit complex clonal chromosomal abnormalities , including trisomies of chromosomes 4 and 8 or occasional etv6-runx1 , mll , and bcr - abl rearrangements .
the prognostic significance of such cytogenetic abnormalities remains unknown in mpal given its rarity and the lack of unifying genetic alterations .
hypodiploid chromosome number is typically associated with children with b acute lymphoblastic leukemia ( b - all ) with event - free survival ( efs ) estimates ranging from 2040% [ 79 ] .
outcomes appear to be associated with degree of hypodiploidy , as worse event - free survival occurs with decreasing number of chromosomes in the leukemias .
hypodiploid leukemias may undergo chromosomal doubling such that the total chromosome number appears falsely hyperdiploid , leading to misclassification as high hyperdiploid all with favorable prognosis .
recent data demonstrate that low hypodiploid clones may be associated with tp53 deletions ( somatic or germline ) and high rates of early medullary relapse [ 7 , 10 , 11 ] . despite recent improvements in understanding hypodiploid b -
all biology , co - occurrence of hypodiploidy and alal remains a novel finding to our knowledge .
we describe the first known case of a pediatric patient with an mpal with masked duplicated low hypodiploidy and a somatic tp53 deletion .
a previously healthy 16 year - old female of indian ethnicity presented initially to an indian hospital with a 3 month history of bone pain , fever , weight loss , menorrhagia , and rash .
she had an initial white count of 4.0  109/l , and no central nervous system involvement with leukemia .
flow cytometric analysis of her bone marrow demonstrated blasts positive for cd45 , cd34 , tdt , hla - dr , cd13 , cd33 , dim for cd79a , and negative for cd10 , cd19 , cd20 , and cmpo .
bone marrow aspirate immunostaining was positive for tdt , cd24 , and pax-5 and negative for cd10 , cd20 , and mpo .
she was thus diagnosed with pro - b all with aberrant cd13 and 33 expression .
reverse transcriptase polymerase chain reaction ( rt - pcr ) testing was negative for bcr - abl1 and mll rearrangements and for 26 other common acute myeloid leukemia ( aml)- and all - associated translocations .
she underwent a 4-drug induction with dexamethasone , vincristine , daunorubicin , and l - asparaginase and triple intrathecal chemotherapy .
the patient transferred to a singaporean institution where review of initial leukemia pathology was interpreted as more consistent with a diagnosis of mpal given a subset of mpo+ blasts .
fluorescence in situ hybridization assays performed there on initial diagnostic marrow slides revealed additional runx1 and runx1t1 signals , but no runx1-runx1t1 fusion resulting from a t(8;21 ) ( figure 1a ) .
repeat bone marrow completed 6 weeks from diagnosis showed no evidence of disease by morphologic and flow cytometric testing .
she subsequently received consolidation therapy with cyclophosphamide and cytarabine given her favorable response to lymphoid - directed induction chemotherapy . due to the conflicting diagnoses at these two institutions , however
, the patient sought a second opinion at the children s hospital of philadelphia for further treatment recommendations .
based solely upon the available written reports from both institutions , our hematopathologists deemed her diagnosis more consistent with aml not otherwise specified ( nos ) .
repeat bone marrows performed here at that time were negative for aml and b - all by morphology and flow cytometric minimal residual disease ( mrd ) testing . given our diagnosis of aml nos , we recommended myeloid - directed therapy with several cycles of cytarabine , anthracyline , and etoposideshe returned to her treating singaporean institution and received 3 cycles of therapy with a first cycle of cytarabine , daunomycin , and etoposide , a second cycle of attenuated high - dose cytarabine with l - asparaginase , and a third cycle of high - dose cytarabine .
four months after chemotherapy completion , the patient re - presented to our institution due to worsening thrombocytopenia .
bone marrow aspirate , biopsy , and extensive leukemia genetic testing ( cytogenetics , whole genome single nucleotide polymorphism ( snp ) microarray , targeted hematologic malignancies dna sequencing mutation analysis ) were performed given the initial complexity of her leukemia diagnosis and potential leukemic evolution upon relapse .
bone marrow morphology from the relapse specimen contained 83.5% large , vacuolated leukemic blasts and no visible auer rods .
flow cytometric immunophenotyping confirmed a b / myeloid mpal with blasts brightly positive for cd45 , cd34 , and cd19 , variably positive for cd9 , cd38 , cd24 , and cd15 , and subset positive for scd22 , ccd79a , tdt , and mpo .
cd10 , cd2 , cd3 , cd5 , cd7 , and cd8 staining was negative .
snp array analysis demonstrated loss of heterozygosity and two copies of chromosomes 3 , 4 , 5 , 7 , 9 , 13 , 15 , 16 , 17 , 20 , and x , trisomies of chromosomes 1 , 2 , 10 , 18 , and 19 , and tetrasomy of chromosomes 6 , 8 , 11 , 12 , 14 , 21 , and 22 ( figures 1 and 2 ) .
these results are consistent with doubling of a low hypodiploid clone with 35 chromosomes , with subsequent loss of one copy of chromosomes 1 , 2 , 10 , 18 and 19 . mutation analyses for aml - associated internal tandem duplication of the fms - like tyrosine kinase receptor 3 ( flt3-itd ) ,
germline tp53 mutation testing was recommended to evaluate for possible li - fraumeni syndrome , but was not performed to our knowledge .
following our evaluation , the patient transferred care to another american institution , where she was treated with two cycles of salvage therapy , including one with dexamethasone , thiotepa , topotecan , and vinorelbine .
the patient is currently in an mrd negative remission with plans to undergo matched unrelated donor bone marrow transplant .
to date , only one other case of mpal with hypodiploidy has been reported . tallents et al . described a 37 year - old man who initially presented with b - all , relapsed 4 months later on therapy , and
was found to have a hybrid mpal with masked duplicated low hypodiploidy ( 70 chromosomes ) . to our knowledge
, our patient is the first case of childhood alal with hypodiploidy reported in the literature .
a variety of chromosomal abnormalities have been associated with mpal ; however , true hypodiploidy has not been reported .
chromosomal abnormalities associated with mpal include monosomies of chromosomes 5 or 7 , trisomies of chromosomes 4 , 19 and 21 , polysomies of chromosome 8 , and hyperdiploidy .
deletions associated with mpal include those in 1p32 , 5q , 6q , 7q , and 12p .
other fusion genes found in patients with alal include bcr - abl1 , etv6-runx1 , nup98-iqcg , or mnx1-etv6 , and mll rearrangements [ 1 , 3 , 4 ] .
this patient had trisomy 19 and tetrasomy 8 , which have been reported in children and are also associated with aml . while polysomy 8 has been reported in a case of myeloid / b or t lymphoid mpal and may have a more favorable prognosis , trisomy 19 is associated with myeloid / t lymphoid mpal and is of uncertain significance .
despite these similarities , the hypodiploidy observed in this patient remains a novel finding in a patient with mpal .
this patient s leukemia hypodiploidy also occurred in the context of a somatic tp53 mutation , a recently characterized genetic finding in low hypodiploid all .
also described 29 cases of low hypodiploid all that were characterized by frequent duplication of the leukemic chromosomes , losses of chromosomes 3 , 7 , 13 , 15 , 16 , and 17 , tp53 and cdkn2a / b deletions , as well as poor event - free and overall survival . in this patient , the hypodiploid clone doubling , somatic mutations , and poor response to chemotherapy are consistent with these previous descriptions of hypodiploid all with tp53 mutation .
hypodiploidy is associated with poor prognosis in both childhood all and adult aml [ 8 , 14 ] . while hypodiploid all comprises approximately 2% of childhood all ,
a recent meta - analysis reported that 22% of de novo aml cases in adults are hypodiploid [ 9 , 10 , 15 ] .
this patient s early medullary relapse suggests that her mpal demonstrates a similar poor prognosis to those of both hypodiploid all and aml .
although the patient s de novo leukemia cytogenetics were reported as normal at the original treating institution , subsequent testing of her diagnostic material reveal some evidence of chromosomal number abnormality by fish .
it remains unknown whether her leukemia evolved at time of relapse with a predominant hypodiploid clone and if her original cytogenetics data represented normal cells and not leukemia .
interestingly , her leukemia flow cytometric immunophenotyping and immunohistochemical data were identical from specimens at diagnosis and relapse , suggesting that her mpal was likely the same disease with minimal genetic evolution .
in summary , the combination of mpal with masked doubling of a hypodiploid clone and a tp53 mutation demonstrates an unusual genetic finding in a childhood leukemia patient that is likely associated with high risk of relapse and poor outcome .
 we describe the case of a 16 year - old female with mixed phenotype acute leukemia b / myeloid , nos ( formerly biphenotypic leukemia ) with masked hypodiploidy and somatic tp53 and cdkn2a / b deletions . 
 she achieved morphologic remission with lymphoid - directed multi - agent chemotherapy , but experienced an early medullary relapse 11 months from initial diagnosis . 
 her case details the unusual finding of hypodiploidy in a patient with ambiguous lineage leukemia and highlights the complexity of therapy selection for these high - risk patients . 
1. INTRODUCTION
2. RESULTS
3. DISCUSSION