SNP Karyotyping Can Detect Clonality of Stem Cell Compartment and Early Malignant Evolution in Aplastic Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 443-443
Author(s):  
Marcin Wlodarski ◽  
Sanjay Mohan ◽  
Lukasz Gondek ◽  
Jungwon Huh ◽  
Michael Clemente ◽  
...  
Keyword(s):  
Stem Cell ◽  
Aplastic Anemia ◽  
Chromosomal Abnormalities ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Multiple Time ◽  
Cell Compartment ◽  
Monosomy 7 ◽  
Stem Cell Compartment ◽  
Trisomy 12

Abstract Aplastic anemia (AA) is characterized by pancytopenia due to contraction/destruction of stem cell compartment. Most investigators consider the presence of clonal cytogenetic abnormalities as incompatible with the AA diagnosis. Despite excellent response rates to immunosuppression (IS) in the majority of AA patients, clonal malignant evolution to myelodysplasia (MDS) can occur in 10–15% in 10 years. Among such cases monosomy 7 (mono7) is the most commonly reported cytogenetic abnormality. Routine metaphase cytogenetics (MC) depends upon high numbers of dividing cells with inducible mitosis and is therefore often noninformative in AA. The inability to early detect AA patients at risk for clonal evolution constitutes a significant clinical problem. We hypothesized that high resolution SNP-array technology (SNPA) that allows for the analysis of interphase genomes will improve detection of chromosomal abnormalities in MDS evolving from AA. In addition to MC, we applied Affymetrix chips to study whole genomes of AA patients (N=100; 69 and 67 were investigated using 50K/250K and 6.0 arrays, respectively; 25 patients were studied at multiple time points prior and post IS). Data was analyzed using CNAG v3.0 and Genotyping Console v2.0 and unbalanced lesions were detected, including regions of genomic gain, loss and copy number neutral loss of heterozygozity. Clonal malignant evolution was observed in 13 patients resulting in a conversion rate of 13%. We focused on longitudinal analysis of these patients. Abnormal endpoint MC was detected in a total of 69% of transformed patients (mono7 in 8/13, t(10;18)(q11.2;q21) in 1/13 and trisomy 12 in 1/13 patients, respectively). Remarkably in 5/13 (38%) of evolving AA patients, numerical aberrations were detected earlier by SNPA than MC. Mono7 (N=4) and trisomy 12 (N=1) were detected by SNPA in aspirates that showed normal or noninformative MC, while in subsequent analysis bone marrow exams concurred with the early diagnosis by SNPA. In 3 patients, MC and SNPA results were concordant and in 1 patient SNPA failed to early identify mono7,. Acquired uniparental disomy of various chromosomes was detected in a total of 4 patients and the analysis of nonmyeloid CD3+ cells revealed somatic nature of these lesions: 7q31.31–31.33(4.9mb), 17q11.2-qter(56mb), 6p12.1-pter(56mb) and 3q12.2-qter(97mb). Interestingly, 2 patients with UPD had normal concurrent MC and the clonal lesions were detected before clinical diagnosis of MDS was established. It is likely that depletion of stem cell compartment might favor detection of pseudoclonality manifested as non-pathogenic random chromosomal lesions. In our cohort, in 2 AA patients with normal MC, SNPA analysis prior to IS therapy revealed clonal UPD as confirmed by comparison of myeloid lineage with germ line configuration in CD3+ cells. These lesions disappeared post IS in both patients and nearly 2 years later a newly recruited pathogenic clone with mono7 was detected as a sole abnormality by SNPA and MC. These patients are very illustrative as they may point towards genomic instability as well as depletion of available stem cell pool resulting in frequent recruitment of defective SC. Our study demonstrates that SNPA is a powerful tool to early identify AA patients harboring clonal defects consistent with the diagnosis of MDS, and thus it potentially might have strong clinical implications.

Clonal Evolution in Aplastic Anemia

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 90-95 ◽  
Author(s):  
Manuel G. Afable ◽  
Ramon V. Tiu ◽  
Jaroslaw P. Maciejewski
Keyword(s):  
Aplastic Anemia ◽  
Chromosomal Abnormalities ◽  
Clonal Evolution ◽  
Immunosuppressive Treatment ◽  
Snp Array ◽  
Term Survival ◽  
Strong Argument ◽  
Monosomy 7 ◽  
Definitive Therapy

Abstract Current immunosuppressive treatment (IST) induces remissions in 50%-70% of patients with aplastic anemia (AA) and result in excellent long-term survival. In recent years, the survival of refractory patients has also improved. Apart from relapse and refractoriness to IST, evolution of clonal diseases, including paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome (MDS), are the most serious long-term complications and constitute a strong argument for definitive therapy with BM transplantation if possible. Consequently, the detection of diagnostic chromosomal abnormalities (mostly monosomy 7) is of great clinical importance. Newer whole-genome scanning technologies such as single nucleotide polymorphism (SNP) array–based karyotyping may be a helpful diagnostic test for the detection of chromosomal defects in AA due to its precision/resolution and lack of reliance on cell division.

scholarly journals A severe and consistent deficit in marrow and circulating primitive hematopoietic cells (long-term culture-initiating cells) in acquired aplastic anemia

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 1983-1991 ◽  
Author(s):  
JP Maciejewski ◽  
C Selleri ◽  
T Sato ◽  
S Anderson ◽  
NS Young
Keyword(s):  
Stem Cell ◽  
Aplastic Anemia ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Cell Compartment ◽  
Long Term Culture ◽  
Cell Numbers ◽  
Stem Cell Compartment ◽  
Limiting Dilution

We examined the stem cell compartment of patients with acquired aplastic anemia (AA) using the long-term culture-initiating cell assay (LTC-IC), in parallel with measurements of CD34+ cells and mature hematopoietic progenitors. Secondary colonies from cells surviving 5 weeks of long-term bone marrow culture (LTBMC) were determined for the peripheral blood (PB) of 68 AA patients and 13 normal controls and for BM of 49 AA patients and 14 controls; because of low cell numbers, formal limiting dilution analysis could only be performed in 10 patients. The relationship of cell input in LTBMC and the output of secondary colonies was linear, allowing quantification of LTC-IC number from bulk cultures. Secondary colony formation was markedly abnormal in severe AA. In contrast to 7.8 colony-forming cells (CFC)/10(5) mononuclear cells in normal BM and 0.14 CFC/10(5) normal PB mononuclear cells, patients with severe disease showed 0.024 CFC/10(5) in BM and 0.0068 CFC/10(5) in PB. Under limiting dilution conditions, patients' cells also showed markedly lower colony-forming ability. In contrast to 4.3 +/- 1 colonies/normal LTC-IC, we obtained only 1.27 +/- 0.09 and 2.0 +/- 0.35 colonies from BM of acute and recovered cases, respectively. These values were used to extrapolate LTC-IC numbers from secondary colony formation in suspension cultures. In PB, calculated LTC-IC were decreased 7.4-fold in new and relapsed severe AA and 2.8- fold in recovered AA. In BM, LTC-IC were decreased 10-fold in new and relapsed AA and sixfold in recovered cases. Compared with measurements obtained on presentation, LTC-IC were lower in post-treatment samples from patients who had failed to recover after intensive immunosuppression and relatively higher in cases at relapse. In recovered patients, LTC-IC number increased but remained below the normal range in 20 of 25. In patients studied serially for 3 to 12 months after treatment, LTC-IC numbers remained stable but low. LTC-IC number correlated with concurrently determined CD34+ cell number and primary hematopoietic colony formation. These results indicate that stem cell numbers, as quantitated by the LTC-IC assay, are markedly diminished in number in all severe AA. Additionally, the function of the stem cell or the stem cell compartment in AA is also abnormal, as inferred from the low clonogenic potential in secondary colony assays. Early hematologic improvement in some patients occurs without increasing numbers of LTC-IC, and a minority of recovered cases show apparent repopulation of the LTC-IC compartment years after treatment.

Heterozygous Germ Line Deletion of a 2Mb Interval in Mice That Models Loss of 7q22 in Human Myeloid Malignancies Results in Defective Hematopoietic Stem Cell Function Reminiscent of Premature Aging

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2340-2340
Author(s):  
Jasmine C. Wong ◽  
Kelley Weinfurtner ◽  
Yan Zhang ◽  
Pilar Alzamora ◽  
Eugene Hwang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Compartment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Myeloid Malignancies ◽  
Cell Lineages ◽  
Stem Cell Compartment ◽  
Lineage Distribution

Abstract Abstract 2340 Monosomy 7 and deletion 7q [del(7q)] are among the most common cytogenetic alterations found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, little is known regarding how chromosome 7 deletions contribute to the pathogenesis of myeloid malignancies. We harnessed chromosome engineering in mice to investigate the in vivo consequences of deleting a contiguous 2 Mb interval on chromosome 5A3 that is syntenic to a segment of chromosome band 7q22 that is commonly deleted in human myeloid malignancies (Blood 88:1930–5, 1996). This region contains 14 known genes, and homozygous deletion is embryonic lethal. Heterozygous 5A3+/del mice are grossly normal and have normal peripheral blood counts, but they have a 20% reduction in spleen weight and a 25% reduction in bone marrow (BM) cellularity per femur. We did not observe a significant difference in the frequency of phenotypic long-term hematopoietic stem cells (LT-HSC) (c-kit+, Sca-1+, Lin−, CD150+, CD41−, CD48−) between 5A3+/del mice and their wild-type (WT) littermates; however, there is a ∼30% reduction in the frequency of multipotent progenitors (MPP) (c-kit+, Sca-1+, Lin−, CD150−, CD41−, CD48−), suggesting a defect in the transition from LT-HSC to MPP. Since 5A3+/del mice did not spontaneously develop evidence of hematologic malignancy in >2 years of observation, and transplantation enforces enhanced HSC cycling which may reveal subtle HSC phenotypes not obvious in the steady state, we performed competitive repopulation assays to directly assess the function of 5A3+/del HSC in vivo. Unfractionated test BM from WT or 5A3+/del animals (CD45.2) were mixed with WT competitor BM (CD45.1) at a 1:1 or 1:2 ratio, followed by transplantation into lethally irradiated WT recipients (CD45.1+2). Although 5A3+/del BM supported reconstitution of all hematopoietic lineages 6 months after adoptive transfer, reductions of 46% (p=0.0003) and 45% (p=0.0010) in comparison to WT were observed in the repopulation of B and T cell lineages, respectively. Reconstitution of the myeloid lineage was reduced to a lesser extent (25% reduction; p=0.0921), and analysis of the lineage distribution of myeloid, B, and T cell lineages within 5A3+/del derived cells revealed a myeloid lineage skewing reminiscent of the pattern of repopulation exhibited by aged HSC. Interestingly, a progressive decline in the repopulating ability of 5A3+/del BM upon cell differentiation was observed: when 5A3+/del test BM were transplanted in a more stringent 1:2 test:competitor ratio, we observed a 29% reduction (p=0.1657) in contribution to the K+L−S+ (c-kit+, Lin−, Sca-1+) stem cell compartment in comparison to WT, but a 39% reduction (p=0.0498) in the more mature progenitor K+L−S− (c-kit+, Lin-, Sca-1−) population. To investigate if the marked deficit in repopulation is intrinsic to the LT-HSC population and to assess if haploinsufficiency of the 5A3 region in the microenvironment modulates the phenotype, we purified and transplanted 15 WT or 5A3+/del LT-HSC together with 250,000 unfractionated WT BM competitors into WT or 5A3+/del recipients respectively. Transplantation of 5A3+/del LT-HSC demonstrated a similar trend of global repopulating deficit and overall pattern of altered lineage distribution regardless of the genotype of the recipient, which infers a dominant cell intrinsic mechanism of action of the 5A3 deletion. Meanwhile, transplantation of WT LT-HSC into 5A3+/del recipients did not significantly modulate repopulating potential. To determine if 5A3+/del LT-HSC is defective in self-renewal, we analyzed reconstitution in the LT-HSC compartment. 5A3+/del LT-HSC repopulated the LT-HSC compartment as efficiently as WT controls regardless of the recipient genotype, and subfractionation of the 5A3+/del derived KLS population to LT-HSC and MPP subsets revealed a significantly biased distribution towards LT-HSC by 1.39-fold (p=0.0146), suggesting increased self-renewal. Together, these data demonstrate that haploinsufficiency of the 5A3 interval leads to defective HSC function including a perturbed stem cell compartment, diminished overall repopulating potential and a myeloid-biased differentiation pattern. The phenotypic resemblance to aged HSC is intriguing given the increased incidence of MDS with monosomy 7 in elderly individuals. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document