Loss of Heterozygosity and Possible Loss of Allele Amplification in Homozygous Short Tandem Repeats Loci in Two ALL Patients with Central Nervous System and Bone Marrow Relapses

Introduction: Loss of heterozygosity (LOH) was described for many forms of cancer including leukemia. LOH may contribute to oncogenesis through the deletion of tumor suppressor genes due to appropriate allelic loss. LOH in short tandem repeats loci (STR) has been reported for solid tumors and is widely used as malignant cells population marker. It should be noted that little is known yet about possible loss of one of certain homozygous alleles in tumour cells and STR analysis might be a promising approach to investigate this phenomenon. STR analysis is used as a routine test for chimerism monitoring after bone marrow transplantation. It could not also be excluded that recipient's chimerism estimation may be distorted in some cases due to LOH issues.

The aim: To investigate possible allele loss in homozygous loci in tumour cells and to assess the contribution of LOH to the error in mixed chimerism estimation.

Patients and Methods: This study includes analysis of STR DNA-profiles of two ALL patients after allogeneic stem cells transplantation. Patient 1 was 54 years old female with B-ALL, she underwent allogenic BM transplantation but after it she had four central nervous system (CNS) relapses and three bone marrow relapses. Patient 2 was 23 years old male with preT-ALL, two CNS relapses: first before transplantation and second after it. The DNAs were isolated from bone marrow samples of patients in remission before transplantation, from donors blood samples, from cerebrospinal fluid during neuro relapse and from bone marrow samples. The presence of blast cells in bone marrow samples and cerebrospinal fluid was confirmed morphologically and cytogenetically. STR-profiles were assessed by polymerase chain reaction with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on ABI3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4-0 software.

Results: The STR profile analysis for Patient 1 showed complete recipient chimerism in cerebrospinal fluid with LOH in 10 from 15 heterozygous loci (Fig.1a,b) comparative to control sample of DNA (bone marrow before transplantation). The same LOH pattern was detected in archival DNA sample from first spinal relapse. Mixed chimerism in bone marrow samples estimated by STR analysis was equal to blast cell percentage assessed by cytogenetics. Loss of non-informative allele in D16S539 (Fig.1b) didn't distort chimerism estimation. However decreased levels of mixed chimerism were calculated in two homozygous loci (Fig.1a,c). We suspected that this decrease could be caused by the loss of one allele amplification in homozygous locus. Therefore, we proposed the mathematical model f(x), where x - is donor percentage in chimera, f(x) - difference between real recipient percentage (100-x) and calculated for locus with loss of allele amplification ((100-x)/2:((100-x)/2+x))*100% (Fig.1c) to test this hypothesis. The highest difference in chimerism calculation (about 17%) is expected in about 40% of donor and 60% recipient percentage. For Patient 2 the STR analysis detected only one LOH locus in DNA samples from cerebrospinal fluid during CNS relapse before and after BMT. However this patient has eight homozygous STR loci and complete donor chimerism in bone marrow. To find possible "loss of allele amplification" we used the artificial chimera, the mix of Patient 2 and unrelated random healthy donor DNA (Fig.1d). We used control DNA of Patient 2 (bone marrow before BMT) to check chimerism level deviations in loci of interest. The decreased level of mixed chimerism was detected in two homozygous loci from cerebrospinal fluid DNA-sample. The decreased estimate for chimerism correlated with proposed math model. For the same STR loci in control DNA no chimerism deviations were detected in mixed artificial chimera (Fig.1e).

Conclusions: The LOH loci pattern might be preserved during long period of disease for certain malignant clone. Mixed chimerism or artificial mixed chimera can be useful in finding of homozygous loci with loss of one allele amplification. We suppose homozygous loci are the preferable subject for investigating LOH mechanisms. Loci with "loss of allele amplification" might be the used as malignant cells population marker.

This study was supported by the Russian Foundation for Basic Research (RFBR) grant 18-015-00399a.

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