the mhc is a genomic region located on the short arm of chromosome 6 ( 6p ) , encoding hla molecules .
loss or down - regulation of mhc class i expression in cancer cells is a major immune escape route used by a large variety of human tumors to evade anti - tumor immune responses mediated by cytotoxic t lymphocytes [ 1 - 3 ] .
multiple mechanisms are responsible for such hla class i alterations [ 4 - 11 ] . among them , loss of heterozygosity ( loh ) of chromosome 6p has been reported in a number of solid tumors and some hematologic malignancies , mainly in all [ 12 , 13 ] . in patients with leukemia ,
loh at the mhc region can give false homozygosity results in hla typing when the typing is performed using peripheral blood containing a high proportion of leukemic cells .
however , such a problem has rarely been reported because hla typing is not usually carried out on a blood specimen containing a high proportion of neoplastic cells . a case of a cll patient
has been reported involving loh at the mhc region at the initial hla typing , which was then detected at confirmatory typing for hematopoietic stem cell transplantation . here
we report false homozygosity results in hla genotyping due to loh associated with the loss of whole chromosome 6 in an adult korean patient with all .
a 33-yr - old korean female was admitted to the hemato - oncology ward of seoul national university hospital on october 4 , 2010 for the evaluation of leukocytosis found during a workup for headache .
a complete blood cell count showed a hemoglobin level of 7.8 g / dl , a platelet count of 1510/l , and a white blood cell count of 336.910/l , of which 84% were blasts .
the blasts were homogeneously small sized with high nuclear / cytoplasmic ratio and inconspicuous nucleoli .
flow cytometric analysis revealed that the neoplastic cells were positive for tdt , cd19 , cd34 , and cytoplasmic cd79a , and a diagnosis of precursor - b all was made .
on october 7 , 2010 ( the third day of hospitalization ) , before initiation of induction chemotherapy , high - resolution hla dna typing of the patient was performed using the peripheral blood sample . for future hematopoietic stem cell transplantation , hla class i ( a , b , and c ) serological typing of her 2 siblings
high - resolution hla typing was performed by sequence - based typing ( sbt ) using the alleleseqr hla sequencing kit ( atria genetics , south san francisco , ca , usa ) .
hla genotyping of the patient revealed homozygosity at class i and ii loci , except for the drb1 locus : a02:01 , b54:01 , c08:01 , drb104:05 , drb111:01 , dqb104:01 ( table 1 , fig .
1 ) . one of the deduced hla haplotypes ( a02:01-b54:01-c08:01-drb111:01-dqb104:01 ) of the patient was a fairly unusual one for korean ethnicity
. moreover , homozygosity at the hla - b locus ( b54:01 ) in the patient could not be explained , considering the hla - b types of her 2 siblings ( b51 , b54 in one and b60 , b62 in the other ) .
retyping of the patient 's dna for hla - a , -b , and -dr loci by the sequence specific oligonucleotide ( sso ) method using dynal reli sso hla - b , -drb , and -dqb1 kits ( invitrogen , wirral , uk ) also revealed homozygosity at the following loci : hla - b54:01 group , drb104:05 group ( drb401:01 group ) , and dqb104:01 group .
a retrospective review revealed that the patient 's hla genotyping had been requested when she had a high blast count in the peripheral blood , with a white blood cell count of 91.710/l , of which 87% were blasts .
conventional karyotyping performed on a bone marrow aspirate sample obtained on october 11 , 2010 ( the seventh day of hospitalization ) revealed chromosomal abnormalities with loss of multiple chromosomes , including chromosome 6 , and reduplication of the remaining chromosomes : 29,x,+x,+8,inv(9)(p11q13),+10,+14,+18,+21/58,idemx2/46,xx , inv(9 ) ( fig .
2 ) . retrospective analysis of short tandem repeat ( str ) markers revealed loh in the str markers on chromosomes 3 , 4 , 5 , 7 , 12 , and 13 ( table 2 ) , which was in accordance with conventional karyotyping results .
minor alleles due to loss of pertinent chromosomes in neoplastic cells comprised on average 4.1% ( range 2.9 - 6.9% ) .
hla genotyping was repeated on december 13 , 2010 , with the peripheral blood in a remission state , 67 days after the initiation of induction chemotherapy .
a complete blood cell count showed a hemoglobin level of 10.0 g / dl , a platelet count of 16010/l , and a white blood cell count of 2.310/l , of which 0% were blasts .
all of the hla - a , -b , -c , -drb1 , and -dqb1 loci were typed as heterozygous : a02:01 , a02:06 , b40:01 , b54:01 , c03:04 , c08:01 , drb104:05 , drb111:01 , dqb103:01 , dqb104:01 ( table 1 , fig .
1 ) . karyotyping performed on a peripheral blood sample obtained on the same date ( in a remission state ) revealed a normal karyotype : 46,xx , inv(9)(p11q13 ) .
str analysis performed on the same blood sample also revealed heterozygosity results in all 9 markers .
thus the heterozygosity results of hla - a , -b , -c , -dr , and -dq loci were confirmed and finally used to search for an unrelated donor for bone marrow transplantation .
hla class i antigens are vital in the recognition of tumor cells by tumor - specific cytotoxic t lymphocytes .
loss or down - regulation of hla class i antigens , therefore , represents a way by which tumors can escape t - cell surveillance , adversely affecting the course of disease and the outcome of t - cell - based immunotherapy [ 1 - 3 ] .
this can have several causes : absence of 2-microglobulin or transporter associated with antigen processing ( tap ) expression [ 4 - 6 ] ; loss of heterozygosity , large deletions , or mitotic recombination in chromosome 6 [ 4 , 8 , 9 ] ; transcriptional down - regulation ; or point mutation , partial deletion , or somatic recombination [ 4 , 7 , 11 ] .
loh in chromosome 6p has been detected in about 10 - 55% of solid tumors such as those of the colon or larynx , melanoma , and breast cancer [ 12 , 15 ] .
previously reported frequencies of loh in 6p in hematologic malignancies are variable according to the detection method and definition of loh .
masuda et al . reported loss or down - regulation of hla class i expression in only 4 out of 43 freshly isolated adult hematologic malignancy samples and no haplotype loss in the hla genome . in childhood
all , the frequency of loh in 6p has been reported to be 7 - 8% [ 17 , 18 ] .
in contrast , mcevoy et al . reported a high frequency of mhc loh in all patients ( 19/56 ) , which was usually associated with whole chromosome 6 loss ( 13/19 ) .
loh in chromosome 6p in hematologic malignancies can cause false homozygosity results in hla genotyping when the typing is performed with leukemic blood samples . this can give rise to a serious problem in hematopoietic stem cell transplantation in that the patient would be at a risk of being transplanted from a falsely matched donor if the false typing results are not properly recognized .
serologic typing of hla antigens with blood samples containing high blast counts is difficult , and therefore , typing is usually not attempted . nowadays
dna typing is increasingly used for hla typing , and such problems may occur if clinicians inappropriately order hla genotyping with leukemic blood samples , and the hla typing laboratory is not aware of the problem in the blood sample requested for typing .
false homozygosity results for hla class i due to loh at the mhc region has been reported in a cll patient , which was detected at confirmatory typing for hematopoietic stem cell transplantation . in this patient ,
loh in chromosome 6p was confirmed by microarray - based comparative genomic hybridization of tumor cells . in our case ,
whole chromosome 6 loss in leukemic cells caused false homozygosity results in hla - a , -b , -c , and -dqb1 sbt and hla - b , -drb , and -dqb1 sso typing . retesting with a peripheral blood sample in a remission state using sbt revealed heterozygosity results in all of the 5 hla loci tested . in hla - drb1 typing in the initial sample ,
a sequencing electropherogram showed amplification peaks of the minor allele ( drb111:01 , present only in a small fraction of normal leukocytes ) which were much lower than those of the major allele ( drb104:05 , present in both leukemic blasts and normal leukocytes ) ( fig .
the discrepancy in hla - drb1 typing results between the 2 typing methods , differences in the primers used for these methods might be the cause .
for hla - drb1-specific amplification , different hla - drb1 alleles can not be amplified by a single pair of primers and thus several different primer pairs amplifying different groups of drb1 alleles are used in hla - drb1 sbt .
the primer pairs that can amplify each of the 2 drb1 alleles ( drb104:05 , 11:01 ) in this patient are expected to be different and the minor allele could thus be amplified in sbt . in hla - drb sso typing , using a dynal reli sso kit , single primer pairs amplifying drb1/drb3/drb4/drb5 genes are used , and the minor allele in this patient seems not to have been effectively amplified to be detected .
likewise , minor alleles in hla - a , -b , -c , and -dqb1 loci in this patient seem not to have been effectively amplified to be detected by sbt .
thus , when hla typing is performed on blood samples containing a high proportion of blasts with loh in chromosome 6p , typing results may differ between different typing methods according to differences in their detection sensitivity . in the pcr - sequence specific primer ( ssp )
method , different primer pairs amplify different hla alleles , and this method is expected to detect minor alleles with higher sensitivity than the sso or sbt methods .
we later performed pcr - ssp typing ( allset gold ssp hla typing kit , invitrogen , brown deer , wi , usa ) for hla - b ( 48 primer pairs ) and -dr loci ( 24 primer pairs ) using the dna sample from our patient at the time of diagnosis .
as expected , the pcr - ssp method detected heterozygosity for both hla - b and -dr loci , although the minor alleles showed much weaker amplification bands on the electropherogram than the major alleles . to avoid false hla typing results in patients with hematologic malignancies , clinicians as well as laboratory personnel need to be aware of such problems and take appropriate precautions .
in leukemia patients , hla typing is usually performed at the time of diagnosis or during the first complete remission .
clinicians should not request hla typing using blood samples at the time of diagnosis if patients have blasts in the peripheral blood .
there is no data available on what percentage of blasts or neoplastic cells in the white cell differential count of a peripheral blood sample can be present without affecting the hla molecular typing results in patients with loh in 6p . because blasts generally have considerably larger nuclei than normal leukocytes , the dna fraction from blasts would comprise a much larger portion of total dna extracted from leukocytes than that simply calculated from the percentage of blasts in the blood sample
this is evidenced in our case ; whereas the blood sample used for hla dna typing contained 87% blasts , the minor alleles detected by str analysis comprised a much lower percentage ( average 4.1% ) than expected ( above 10% ) .
if hla typing is required in such patients before remission is obtained , alternative samples , such as a buccal swab or hair follicles , should be considered for hla typing .
hla typing laboratories involved in the typing of patients with hematologic malignancies should be aware of such problems and should check patients ' hematologic data whenever they have unusual hla typing results or discrepancies between different typing methods .
in addition , the patients ' hla typing results should be reconfirmed by confirmatory typing .
for hematopoietic stem cell transplantation , confirmatory hla typing using newly collected samples should be performed not only for donors but also for patients [ 20 , 21 ] .
however , confirmatory hla typing of patients is not reimbursed by the national medical insurance system and is not routinely carried out as a standard clinical practice in this country .
we have described a case of false homozygosity results in hla genotyping in a patient with precursor - b all due to the loss of whole chromosome 6 in the neoplastic cells .
to avoid false hla typing results in patients with hematologic malignancies , clinicians and hla typing laboratory personnel need to be aware of the problem and take appropriate precautions .
hla typing should not be performed with blood samples containing a high proportion of neoplastic cells , and alternative samples of buccal swab or hair follicles should be used if needed .
in addition , confirmatory hla typing should be routinely performed for hematopoietic stem cell transplantation .
 loss of heterozygosity ( loh ) in chromosome 6p has been reported in a number of tumors and some hematologic malignancies , including all . 
 loh in chromosome 6p , on which the hla genes are located , can give rise to false homozygosity results in hla genotyping of patients with hematologic malignancies . 
 here we report false homozygosity results in hla genotyping due to the loss of whole chromosome 6 in the neoplastic cells of a patient with all . 
 a 33-yr - old korean female patient was admitted for the evaluation of leukocytosis detected during a workup for headache . 
 her initial white blood cell count was 336.9109/l with 84% of blasts in the differential count . 
 precursor - b lymphoblastic leukemia was diagnosed from a subsequent bone marrow study . 
 hla high - resolution genotyping of the patient was requested at the time of diagnosis for possible hematopoietic stem cell transplantation . 
 homozygosity results ( a*02:01 , b*54:01 , c*08:01 , dqb1 * 04:01 ) were obtained , except for the drb1 locus ( drb1 * 04:05 , drb1 * 11:01 ) , in sequence - based typing . 
 conventional karyotyping of bone marrow metaphase cells revealed chromosomal abnormalities , with loss of multiple chromosomes including chromosome 6 , and reduplication of the remaining chromosomes : 29,x,+x,+8,inv(9)(p11q13),+10,+14,+18,+21[15]/58,idemx2[3]/46,xx , inv(9)[2 ] . 
 loh at the hla region was suspected and hla genotyping was repeated with the peripheral blood in remission state after induction chemotherapy . 
 all 5 hla loci were typed as heterozygous ( a*02:01 , a*02:06 , b*40:01 , b*54:01 , c*03:04 , c*08:01 , drb1 * 04:05 , drb1 * 11:01 , dqb1 * 03:01 , dqb1 * 04:01 ) . to avoid false hla typing results in patients with hematologic malignancies , clinicians , as well as laboratory personnel , need to be aware of such problems and take appropriate precautions . 