Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clones.

The unique structure of the T cell receptor (TCR) enables molecular identification of individual T cell clones and provides an unique opportunity for the design of molecular diagnostic tests based on the structure of the rearranged TCR chain e.g., using the TCR CDR3 region. Initially, clonal T cell malignancies, including T cell large granular lymphocyte leukemia (T-LGL), mucosis fungoides and peripheral T cell lymphoma were targets for the TCR-based analytic assays such as detection of clonality by T-gamma rearrangement using y-chain-specific PCR or Southern Blotting. Study of these disorders facilitated further analytic concepts and application of rational methods of TCR analysis to investigations of polyclonal T cell-mediated diseases. In hematology, such conditions include graft versus host disease (GvHD) and immune-mediated bone marrow failure syndromes. In aplastic anemia (AA), myelodysplastic syndrome (MDS) or paroxysmal nocturnal hemoglobinuria (PNH), cytotoxic T cell responses may be directed against certain antigens located on stem or more lineage-restricted progenitor cells in single lineage cytopenias. The nature of the antigenic targets driving polyclonal CTL responses remains unclear. Novel methods of TCR repertoire analysis, include VB flow cytometry, peptide-specific tetramer staining, in vitro stimulation assays and TCR CDR3-specific PCR. Such PCR assay can be either VB family-specific or multiplexed for all VB families. Amplified products can be characterized and quantitated to facilitate detection of the most immunodominant clonotypes. Such clonotypes may serve as markers for the global polyclonal T cell response. Identification of these clonotypes can be performed in blood and tissue biopsy material by various methods. Once immunodominant clonotypes corresponding to pathogenic CTL clones are identified they can serve as surrogate markers for the activity of the pathophysiologic process or even indicate the presence of specific antigens. The relevance of the individual clonotypes can be ascertained from clinical correlations with the activity of the disease. Quantitative clonotypic assays such as sequencing of multiple CDR3 clones or clonotypic Taqman PCR can be applied for the monitoring of the immunosuppressive therapy and prediction of relapse. Future technologies may allow for the design of clonotypic microarrays or other more clinically applicable methods of clonotypic diagnostics. Similarly, identification of immunodominant clonotypes may facilitate targeting of autoimmune or malignant clones with vaccination and induction of anti-idiotypic responses

SNP Array Karyotyping Allows for the Detection of Uniparental Disomy and Cryptic Chromosomal Abnormalities in MDS/MPD-U and MPD

We applied single nucleotide polymorphism arrays (SNP-A) to study karyotypic abnormalities in patients with atypical myeloproliferative syndromes (MPD), including myeloproliferative/myelodysplastic syndrome overlap both positive and negative for the JAK2 V617F mutation and secondary acute myeloid leukemia (AML). In typical MPD cases (N = 8), which served as a control group, those with a homozygous V617F mutation showed clear uniparental disomy (UPD) of 9p using SNP-A. Consistent with possible genomic instability, in 19/30 MDS/MPD-U patients, we found additional lesions not identified by metaphase cytogenetics. In addition to UPD9p, we also have detected UPD affecting other chromosomes, including 1 (2/30), 11 (4/30), 12 (1/30) and 22 (1/30). Transformation to AML was observed in 8/30 patients. In 5 V617F+ patients who progressed to AML, we show that SNP-A can allow for the detection of two modes of transformation: leukemic blasts evolving from either a wild-type jak2 precursor carrying other acquired chromosomal defects, or from a V617F+ mutant progenitor characterized by UPD9p. SNP-A-based detection of cryptic lesions in MDS/MPD-U may help explain the clinical heterogeneity of this disorder

Deleterious Heteroplasmic Mitochondrial Mutations are associated With an increased Risk of Overall and Cancer-Specific Mortality

Mitochondria carry their own circular genome and disruption of the mitochondrial genome is associated with various aging-related diseases. Unlike the nuclear genome, mitochondrial DNA (mtDNA) can be present at 1000 s to 10,000 s copies in somatic cells and variants may exist in a state of heteroplasmy, where only a fraction of the DNA molecules harbors a particular variant. We quantify mtDNA heteroplasmy in 194,871 participants in the UK Biobank and find that heteroplasmy is associated with a 1.5-fold increased risk of all-cause mortality. Additionally, we functionally characterize mtDNA single nucleotide variants (SNVs) using a constraint-based score, mitochondrial local constraint score sum (MSS) and find it associated with all-cause mortality, and with the prevalence and incidence of cancer and cancer-related mortality, particularly leukemia. These results indicate that mitochondria may have a functional role in certain cancers, and mitochondrial heteroplasmic SNVs may serve as a prognostic marker for cancer, especially for leukemia

Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation.

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers

FOLIA HISTOCHEMICA ET CYTOBIOLOGICA

Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clone

Clonal Hematopoiesis and the Risk of Hematologic Malignancies after Curative Therapies for Sickle Cell Disease

Sickle cell disease (SCD) is associated with severe morbidity and early mortality. Two large population studies found an increased risk for leukemia in individuals with SCD. Notably, while the relative risk of leukemia development is high, the absolute risk is low in individuals with SCD who do not receive cell-based therapies. However, the risk of leukemia in SCD is high after graft rejection and with gene therapy. Clonal hematopoiesis (CH) is a well-recognized premalignant condition in the general population and in patients after high-dose myelotoxic therapies. Recent studies suggest that CH may be more common in SCD than in the general population, outside the cell-based therapy setting. Here, we review risk factors for CH and progression to leukemia in SCD. We surmise why patients with SCD are at an increased risk for CH and why leukemia incidence is unexpectedly high after graft rejection and gene therapy for SCD. Currently, we are unable to reliably assess genetic risk factors for leukemia development after curative therapies for SCD. Given our current knowledge, we recommend counseling patients about leukemia risk and discussing the importance of an individualized benefit/risk assessment that incorporates leukemia risk in patients undergoing curative therapies for SCD

Overexpression of MPG Is Associated with Distinct Defects in Base Excision Repair in a Subgroup of Patients with MDS

Abstract Dysfunction of the DNA repair machinery may be one of the potential mechanisms leading to DNA damage and subsequent evolution of chromosomal abnormalities in MDS. However, the complexity of the possible mechanisms that could result in increased propensity to chromosomal breaks constitutes one of the obstacles in the rational targeting of deficient repair pathways for future investigations. Our study involved 37 patients with MDS (19 RA/RARS, 16 RAEB/AML, 2 CMML) and 10 controls. As expression arrays constitute a suitable hypothesis-forming tool, we have applied a specialized expression array to interrogate the most important DNA repair mechanisms in highly purified CD34+ progenitor cells from controls (N=10) and MDS patients (N=12). Based on the expression cut off value of (>1.5 x background) we detected 22/113 genes present on the array in all samples tested. 16/22 DNA repair genes were concordantly upregulated (> 2 fold above the control values established in pooled control reference). As a defective base pair excision repair (BER) pathway could play a role in the generation of chromosomal breaks, we focused our investigation on enzymes involved in this pathway. Expression array analysis showed that the MPG gene was significantly overexpressed, and we have validated this finding using TaqMan PCR in original samples and additional cohort of patients; 21/37 cases showed significantly elevated MPG transcripts. Overexpression of MPG may be in response to increased alkylation of DNA or may serve as a marker of CpG island methylation. While the latter alternative has been the subject of intense study, increased level of MPG is consistent with the presence of chemically modified bases incorporated into DNA during a toxic insult. Polymerase b (POLB) is a downstream enzyme in BER pathway, and its upregulation could indicate increased activity of this repair mechanism. Consequently, to test the integrity of the BER pathway, we set out to determine POLB expression levels by TaqMan PCR; two distinct subsets of patients (>2 and ≤ 2 fold of the expression in controls) were identified (5/21 and 16/21). While 4/5 vs. 7/16 patients had chromosomal abnormalities in high vs. low POLB group respectively, these patient groups were otherwise clinically indistinguishable. Hence, we devised our next set of experiments that would discern the differences between these patients at a molecular level. Increased POLB would be consistent with the adequate BER activity. Here, we chose to further investigate the consequences of POLB upregulation in order to distinguish whether BER function results in appropriate DNA repair (upregulation of POLB and MPG) or whether it constitutes response to the deficiency of the AP endonuclease (upregulation of MPG and normal or downregulation of POLB) located immediately downstream of MPG. The content of apurinic sites per genome constitutes a suitable parameter to distinguish the aforementioned possibilities. An apurinic site-specific genomic ELISA assay clearly identified patients (6/19) in whom low POLB expression coincided with an elevated number of apurinic sites (>0.50 apurinic sites per 1x105bp). We conclude that such a constellation is reflective of altered function of AP endonuclease or inadequate expression of MPG that could predispose to single and double-stranded breaks, and consequently lead to evolution of abnormal clone

DNA Repair Gene Expression in CD34+ Bone Marrow Cells in MDS Patients with Mutiple Chromosomal Abnormalities by Array-Based Comparative Genomic Hybridization (A-CGH)

Abstract Based on the high rate of chromosomal defects in MDS, inherent chromosomal instability (CIN) has been hypothesized as a key pathophysiologic factor of clonal evolution. Predisposition to DNA damage may be primarily due to acquired/inherited weakness in DNA repair machinery; such insufficiency may become manifest after a long latency following cumulative exposure to genotoxic agents. Subsequent changes in chromosomal structure and stepwise acquisition of neoplastic features could lead to leukemic progression. Previously, a number of allelic polymorphisms in DNA repair genes were observed. These variants may lead to altered expression of corresponding proteins. Conversely, if DNA damage is a primary defect, upregulation of specific DNA repair enzymes may be compensatory. Irrespective of the initial pathogenetic defect, we theorized that broad analysis of DNA repair machinery in MDS may point towards specific lesions that could be a subject of more targeted studies. Therefore, we examined levels of DNA repair enzymes using gene expression arrays. For proper comparisons, CD34 cells from 10 MDS patients (4 RA, 6 RAEB/RAEBt) and healthy controls were used. Expression array results were confirmed by Taqman PCR. Reference expression was established by pooling RNA from 12 controls. For more targeted analysis, A-CGH based genomic scan was used to better assess the extent of DNA damage in patients. The expression of 22 out of 113 DNA repair genes tested was detectable at levels >1,5X background; 2-level normalization of gene expression was performed according to variation of mRNA input (housekeeping gene-ACTB) and inter-assay variation in the signal intensity (biotinylated artificial sequence -BAS2C). Our combined standard sample was validated against individual controls; signals <1,5X pooled expression were obtained. Using expression levels of normal CD34 cells as a reference we found that 19 genes were upregulated in concordant fashion. The most dramatically increased genes included APEX, ATM, XRCC1, XRCC5 and MPG. This finding favors the theory that overexpression of the DNA repair machinery is a compensatory event to cope with a primarily increased level of DNA damage. When we subgrouped MDS patients according to FAB criteria, the expression of DNA repair genes (e.g., CIB1, ERCC1, SUMO1) increased with the malignant progression. For further analysis we have defined CIN phenotype by the presence of large or multiple small defects as determined by A-CGH. When patients with CIN vs. those with normal karyotype were compared, we found that chromosomal damage was not accompanied by a higher expression of DNA repair genes. MPG was most dramatically upregulated in all MDS patients. This gene involved in excision of methylated bases can induce single stranded breaks (SSB) and increase sensitivity to alkylating agents. Our finding suggest that either increased purine methylation induces a compensatory mechanism (MPG upregulation) or that overactivity of MPG itself results in increased base excision. Alternatively, overexpression of MPG may lead to SSB especially because downstream genes (e.g. XRCC3 or DNA ligase III) were not accordingly upregulated. In conclusion, our studies form a basis for further analysis of clinical phenotypes associated with upregulation of specific DNA repair genes and may indicate possible therapeutic targets in molecularly defined subtypes of MDS

Chromosomal lesions and uniparental disomy detected by SNP arrays in MDS, MDS/MPD, and MDS-derived AML

Using metaphase cytogenetics (MC), chromosomal abnormalities are found in only a proportion of patients with myelodysplastic syndrome (MDS). We hypothesized that with new precise methods more cryptic karyotypic lesions can be uncovered that may show important clinical implications. We have applied 250K single nucleotide polymorphisms (SNP) arrays (SNP-A) to study chromosomal lesions in samples from 174 patients (94 MDS, 33 secondary acute myeloid leukemia [sAML], and 47 myelodysplastic/myeloproliferative disease [MDS/MPD]) and 76 controls. Using SNP-A, aberrations were found in around three-fourths of MDS, MDS/MPD, and sAML (vs 59%, 37%, 53% by MC; in 8% of patients MC was unsuccessful). Previously unrecognized lesions were detected in patients with normal MC and in those with known lesions. Moreover, segmental uniparental disomy (UPD) was found in 20% of MDS, 23% of sAML, and 35% of MDS/MPD patients, a lesion resulting in copy-neutral loss of heterozygosity undetectable by MC. The potential clinical significance of abnormalities detected by SNP-A, but not seen on MC, was demonstrated by their impact on overall survival. UPD involving chromosomes frequently affected by deletions may have prognostic implications similar to the deletions visible by MC. SNP-A–based karyotyping shows superior resolution for chromosomal defects, including UPD. This technique further complements MC to improve clinical prognosis and targeted therapies

Detection of Recurrent Uniparental Disomy and Cryptic Chromosomal Abnormalities in MDS/MPD-U and MDS/MPD-Derived Secondary AML

Abstract The WHO classification distinguishes MDS/MPD as a distinct entity. The JAK2 V617F mutation is present in a minority of these patients (pts). UPD9p characterizes pts homozygous for the V617F mutation. Other chromosomal abnormalities can also be detected in pts with typical MPD and MDS/MPD, and it is likely that cytogenetic methods with higher resolution could detect additional defects. We applied 250K SNP-arrays to examine genomic composition and identify previously cryptic defects and molecular abnormalities in pts with MDS/MPD-U and secondary AML (sAML) arising from MDS/MPD-U both in pts wild-type for V617F and those with the mutation. Any deletions, duplications, and/or UPD found by SNP-A in 76 controls or on available internet databases were considered copy number variants (CNV) and non-pathogenic. First, we used pts with typical MPD to assess the ability of SNP-A to detect UPD. All pts homozygous for V617F showed UPD9p by SNP-A. In pts with MDS/MPD, several additional cryptic lesions were detected, including segmental micro-deletions on chr 1, 5, 9, and 12. UPD was common, occurring on chromosomes other than 9 in 9/28 patients (32%, i.e. on chr 1,11,12). Shared/overlapping lesions (in >2 pts) included small segmental lesions on chr 7 (N=3), and a small cytoband (q14.1) of chr 11 (N=3). Overall, clonal lesions including segmental UPD were found in 23/28 (82%) pts by SNP-A in comparison to 17/28 (61%) by metaphase cytogenetics (MC). Pts with a history of MDS/MPD-U with (N=14) and without the JAK2 V617F mutation (N=14) were also analyzed. MC revealed chr aberrations in 10/14 (71%) of V617F+ pts, including common lesions such as +8 and del5q. With SNP-A, 1 additional pt with normal MC was found to have an abnormal karyotype (UPD 7 and 9), and 9/10 pts with abnormal MC had additional lesions previously undetected, including UPD on chrs other than 9 in 3/14 pts (21%). Examples of deleted regions include segmental losses within chrs 2, 7, 8 and 13, and UPD on chrs 1p, 7q, and 11. Likewise, additional lesions were identified in MDS/MPD-U pts negative for V617F. 8/14 pts (57%) showed abnormal MC; however SNP-A showed lesions in 12/14. In addition, 6/8 pts with abnormal MC had lesions in addition to those detected by MC. UPD was also common in V617F- pts, occurring in 6/14 (43%), predominantly on chr 11 (in 3/6 pts). No significant difference was found between the number or type of lesions found in pts with and without V617F mutation. SNP-A can also be used to identify lesions acquired during AML evolution. In 1 MPD pt at diagnosis, SNP-A showed UPD9p as a sole abnormality consistent with a homozygous V617F mutation. Upon transformation, repeated SNP-A showed a V617F- leukemic clone (normal chr 9) possessing microdeletions on chr 4 and 19. Similar evolution of a V617 negative leukemic clone was also observed in an MDS/MPD pt in whom a new UPD6 was detected. However, in 3 other MDS/MPD patients, SNP-A showed the presence of a V617+ leukemic clone (showing UPD9) in AML blasts. In summary, SNP-A-based karyotyping complements MC and allows for precise definition of chr aberrations in pts with MDS/MPD, including copy-neutral LOH. UPD is common in both JAK2 V617F+ and V617F- disorders, and is not restricted only to chr 9p, indicating other potential causative genes