Intrachromosomal Amplification Research Articles

17. Intrachromosomal amplification of chromosome 21 as the sole chromosomal aberration in a primary AML patient

17. Intrachromosomal amplification of chromosome 21 as the sole chromosomal aberration in a primary AML patient

Targeted treatment options for paediatric B-cell precursor acute lymphoblastic leukaemia patients with constitutional or somatic chromosome 21 alterations

BackgroundChromosome 21 is affected in ∼60% of paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients and includes somatic and constitutional gains, intrachromosomal amplification of chromosome 21 (iAMP21), and the translocation t(12;21) resulting in the ETV6::RUNX1 gene fusion. MethodsSince these numeric and structural chromosome 21 alterations are not targetable, we studied the type and frequency of yet-proven targetable events co-occurring with chromosome 21 alterations. ResultsAmong 307 primary paediatric BCP-ALL cases, JAK/STAT pathway lesions were most frequent in patients with constitutional gain of chromosome 21 (Down syndrome ALL; 35/71, 49%) and iAMP21 (9/22, 41%). RAS pathway lesions were most frequent in high hyperdiploidy (62/108, 57%) and FLT3 lesions were most frequent in iAMP21 (7/22, 32%). Virtually all cases expressed CD19 and CD22 at the cell surface. Positivity for CD20 surface expression ranged from 67% in iAMP21 (8/12) to 20% in ETV6::RUNX1 (26/129). ConclusionActivated JAK/STAT, RAS or FLT3 signalling, and CD marker surface expression may provide targetable treatment options for the majority of chromosome 21-altered BCP-ALL cases.

Improved detection of clinically relevant fusion transcripts in cancer by machine learning classification

BackgroundGenomic rearrangements in cancer cells can create fusion genes that encode chimeric proteins or alter the expression of coding and non-coding RNAs. In some cancer types, fusions involving specific kinases are used as targets for therapy. Fusion genes can be detected by whole genome sequencing (WGS) and targeted fusion panels, but RNA sequencing (RNA-Seq) has the advantageous capability of broadly detecting expressed fusion transcripts.ResultsWe developed a pipeline for validation of fusion transcripts identified in RNA-Seq data using matched WGS data from The Cancer Genome Atlas (TCGA) and applied it to 910 tumors from 11 different cancer types. This resulted in 4237 validated gene fusions, 3049 of them with at least one identified genomic breakpoint. Utilizing validated fusions as true positive events, we trained a machine learning classifier to predict true and false positive fusion transcripts from RNA-Seq data. The final precision and recall metrics of the classifier were 0.74 and 0.71, respectively, in an independent dataset of 249 breast tumors. Application of this classifier to all samples with RNA-Seq data from these cancer types vastly extended the number of likely true positive fusion transcripts and identified many potentially targetable kinase fusions. Further analysis of the validated gene fusions suggested that many are created by intrachromosomal amplification events with microhomology-mediated non-homologous end-joining.ConclusionsA classifier trained on validated fusion events increased the accuracy of fusion transcript identification in samples without WGS data. This allowed the analysis to be extended to all samples with RNA-Seq data, facilitating studies of tumor biology and increasing the number of detected kinase fusions. Machine learning could thus be used in identification of clinically relevant fusion events for targeted therapy. The large dataset of validated gene fusions generated here presents a useful resource for development and evaluation of fusion transcript detection algorithms.

The 3D Chromatin Landscape of B-Cell Precursor Childhood Acute Lymphoblastic Leukemia

Whilst the molecular pathogenesis of childhood B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) has been studied extensively, its 3D chromatin landscape remains poorly explored. Genome-wide chromosome conformation capture methods have provided the tools to investigate the different units of chromatin organization, such as transcriptionally active (A) and inactive (B) compartments, topologically associating domains (TADs), and fine-scale chromatin loops and enhancer-promoter interactions. The aim of this study is to elucidate the chromatin architecture and topological gene regulation in childhood BCP ALL. To date, 29 primary patient samples were included, comprising the high hyperdiploid (HeH) (n=11), ETV6:: RUNX1-positive (n=8), BCR:: ABL1-positive (n=2), TCF3:: PBX1-positive (n=2), DUX4-rearranged (n=2), intrachromosomal amplification of chromosome 21 (iAMP21) (n=1), KMT2A-rearranged (n=1), near-haploid (n=1) and near-triploid (n=1) genetic subtypes. Leukemic blast cells obtained at diagnosis were analyzed using Micro-C, a high-resolution variation of Hi-C (average number of total reads = 1.4 billion, highest resolution = 5 kb) combined with pair-end sequencing. Chromatin contact heatmaps were generated for each case using Juicer and Cooler. A/B compartments were identified using FanC at 500 kb resolution and visualized in the software IGV, while TAD calling was performed by Juicer, Domaincaller and Insulation Score. Differential chromatin interaction loop calling was made using Pareidolia and Mustache, and structural variant (SV) calling was carried out by EagleC. Preliminary principal component analysis of the 29 cases, based on the first two eigenvectors of the contact matrix (500 kb resolution), showed that HeH, ETV6:: RUNX1-positive, TCF3:: PBX1-positive and DUX4-rearranged cases each clustered based on their chromatin 3D organization. Furthermore, the A/B compartments of 11 HeH and 8 ETV6:: RUNX1-positive cases were analyzed at 500 kb resolution and compartment shifts among the two subtypes were annotated. A total of 390 shifts were detected, where activating shifts (from B to A compartment) happened more often in HeH (263 shifts) than in ETV6:: RUNX1-positive cases (127 shifts). Analysis of TAD boundary strength at 25 kb resolution revealed that HeH cases displayed significantly weaker boundaries compared to ETV6:: RUNX1-positive cases. TAD boundary strength showed no bias towards the frequently gained or non-gained chromosomes in HeH ALL. By merging individual heatmaps of all HeH and ETV6:: RUNX1-positive cases using Cooler, we created subtype-specific profiles and compared the intensity of chromatin interactions between the two genetic subtypes. Chromatin interaction intensity analysis was then combined with previously published RNA-sequencing data to identify transcriptional dysregulation events that could be associated with chromatin interaction changes. Preliminary results show that there was a chromatin loop missing close to the well-known leukemia-related gene IKZF1 in HeH compared to ETV6:: RUNX1-positive cases; this gene also showed lower expression in the RNA-sequencing data. FLT3 was associated with weakened chromatin interactions and down-regulated in ETV6:: RUNX1-positive cases compared to HeH, in agreement with its known high expression in HeH. Finally, we performed screening of SVs using EagleC and Micro-C heatmaps in HeH and ETV6:: RUNX1-positive samples. Out of the 19 included cases, previous whole-genome sequencing (WGS) data were available for 16. We detected 75 SVs, of which 50 were intrachromosomal rearrangements and 25 were translocations. Micro-C heatmaps allowed visual detection of SVs smaller than 1 Mb and permitted identification of the type of SVs. WGS detected 61% of the SVs found in the HeH samples with Micro-C and 51% of those in the ETV6:: RUNX1-positive cases. In summary, we present the first high-resolution genome-wide map of chromatin 3D organization in pediatric ALL. Our results indicate that different subtypes of childhood BCP ALL have distinct 3D chromatin landscapes and that abnormal chromatin architectures affect the regulation of leukemia-related genes.

Association of Latino Ethnicity with Cytogenetic Subtype in Pediatric Acute Lymphoblastic Leukemia: A Report from the Reducing Ethnic Disparities in Acute Leukemia Consortium

Introduction: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, and is a heterogeneous group of bone marrow diseases with multiple cytogenetic subtypes leading to distinct clinical outcomes. Despite advances in treatments that improve survival, racial and ethnic disparities continue to persist. Latino children have a higher incidence of ALL and poorer overall survival compared with non-Latino white children. Although several studies conducted have identified cytogenetic differences in Latinos, larger studies are needed to confirm and further elaborate these findings. The aim of our study was to analyze cytogenic profiles of a large, diverse cohort of children with ALL to assess the associate between cytogenic subtypes in Latino ethnicity. Methods: We analyzed the distribution of ALL subtypes and self-reported race/ethnicity in the Reducing Ethnic Disparities in Acute Leukemia (REDIAL) Consortium retrospective registry, which includes patients (age 0-24 at diagnosed) with newly diagnosed ALL from six pediatric cancer centers in the southwest region that were diagnosed between 2017 - 2021. The demographic and clinical factors included self-reported race/ethnicity, sex, age at diagnosis, immunophenotype, and cytogenetic aberration. Cytogenetic subtypes evaluated included double trisomies (DT; trisomies chromosomes 4 and 10), BCR::ABL (Ph+), ETV6::RUNX1 KMT2A ( MLL) rearrangements, hypodiploidy, intrachromosomal amplification of chromosome 21 ( iAMP21) TCF3::PBX1, IGH, and CRLF2 rearrangements. Multivariable logistic regression models were used to estimate adjusted odds ratios (aORs) and 95% confidence intervals (95% CIs), accounting for sex and age at diagnosis. Results: Of the 2,388 ALL patients evaluated, 90.2% had B-cell ALL and 64.4% self-identified as Latino and the mean age at diagnosis was 6.6 years. Compared with non-Latino white patients, the cytogenetic findings associated with favorable prognosis, DT and ETV6:: RUNX1 t(12;21), were less frequent in Latino patients (aOR: 0.80, 95% CI: 0.65-0.98 for DT and aOR: 0.67, 95% CI: 0.53-0.85 for ETV6:: RUNX1). Overall, favorable cytogenetics were less frequent among Latino children compared with non-Latino white children (aOR: 0.82, 95% CI: 0.67-0.99). The neutral cytogenetic subtype TCF3:: PBX1 was also less frequent clinically among Latino patients (aOR: 0.39, 95% CI: 0.18 - 0.85). Among unfavorable cytogenetic alterations, iAMP21 was significantly less frequent among Latino children compared with non-Latino white children (aOR: 0.57, 95% CI: 0.32-0.99). There was no significant association between other unfavorable cytogenetic subtypes and Latino ethnicity, either individually or for the overall unfavorable group. Conclusions: Overall, our findings indicate that Latino children are significantly less likely to have favorable cytogenetic subtypes, TCF3:: PBX1 and iAMP21 and prognostically neutral cytogenetics. Further evaluation of cytogenetic subtypes and race/ethnicity may elucidate their contribution to outcome disparities.

Comprehensive Characterisation of the Copy Number Landscape in Acute Lymphoblastic Leukemia Using Digitalmlpa

Introduction: Acute lymphoblastic leukemia (ALL) is a heterogeneous disease affecting various signaling pathways including cell differentiation and cell cycle control. ALL subtypes are defined by their genetic abnormalities, encompassing gross chromosomal aberrations, ploidy changes, translocations or rearrangements, regional or focal (intragenic) copy number alterations (CNAs), gene fusions and point mutations. Novel biomarkers for risk stratification are still being discovered, further mapping the diverse genetic ALL landscape. Testing for these newly identified genetic biomarkers and characterising the copy number (CN) landscape in ALL samples can provide useful new insights for risk stratification and patient-tailored treatment regimens. Methods: For multiplex detection of CNAs in ALL samples, a new test version of SALSA® digitalMLPA™ Probemix D007 Acute Lymphoblastic Leukemia was developed. Hereafter, this new test version will be referred to as D007 Acute Lymphoblastic Leukemia. This assay contains probes for CNA detection of 70 key genes and regions frequently altered in B-cell ALL and T-cell ALL ( Figure 1). In addition, this probemix contains digital karyotyping probes covering each chromosomal arm for the detection of gross chromosomal aberrations (except the p-arm of acrocentric chromosomes). A selection of these digital karyotyping probes also serve as reference probes. Finally, a set of single nucleotide polymorphism (SNP) probe pairs, targeting both alleles of each of the SNPs, was included for sample identification and guiding the correct CN baseline determination, facilitating optimal data normalisation in samples with complex karyotypes. To test the performance of this new test version of the probemix, DNA samples from 29 ALL cell lines were analysed. digitalMLPA-derived PCR amplicons were sequenced on a NextSeq 1000 Sequencing System (Illumina). FastQ files were analysed using Coffalyser digitalMLPA™ (MRC Holland) and a custom-built algorithm was used for copy number determination using SNP probe pairs. Results: digitalMLPA data of all 29 cell lines was analysed and compared to Cancer Cell Line Encyclopedia (CCLE) copy number data (Depmap, Copy Number Public 23Q2 download). CNAs were detected with D007 Acute Lymphoblastic Leukemia, including clinically relevant biomarkers such as the IKZF1 plus profile, PAX5alt, TCF3 fusions, focal 22q11.22 deletion (incl. VPREB1), intrachromosomal amplification of chromosome 21 (iAMP21), hypo- and hyperdiploidy. Other CNAs of emerging significance in ALL risk stratification included 6q15-q16 deletions, DNMT3B, CDKN1B and KRAS alterations. SNP probe pairs were effectively used to facilitate correct CN baseline determination, and results were confirmed using CCLE data (Depmap, CCLE_ABSOLUTE_combined 2019 download). Correct CN baseline detection is particularly important for samples with complex karyotypes, such as the P12-ICHIKAWA cell line. In this cell line, our data revealed that most chromosomal regions targeted by the SNP probe pairs had a copy number ≥3 ( Figure 2), consistent with the hypotetraploid karyotype previously described for this cell line. Conclusions: Using this new test version of SALSA® digitalMLPA™ D007 Acute Lymphoblastic Leukemia, numerous CNAs can be detected in a single reaction. Results obtained with this probemix showed a high correlation with CN data from the CCLE database in a panel of 29 ALL cell lines. digitalMLPA provides valuable insights into the CN of key target sequences relative to reference sequences in the genome. Moreover, our novel approach using pairs of probes that target both SNP alleles provides an additional layer of information, guiding CN and ploidy status determination in highly complex karyotypes. The SNP probe pair based approach for detection of CN levels in complex samples shows promising results and will be further validated in an independent cohort of 140 ALL patient samples. These results will be presented at the ASH meeting.

3D Genome Organization and Single-Cell Characterization of Pediatric Intrachromosomal Amplification of Chromosome 21 (iAMP21) B-Cell Acute Lymphoblastic Leukemia

B-cell Acute Lymphoblastic Leukemia (B-ALL) is the most frequent childhood cancer, and around 2% of these cases are associated with intrachromosomal amplification of chromosome 21 (iAMP21). The iAMP21 cases represent one of the high-risk but least understood sub-group of B-ALL. The sub-group also has a significantly low survival rate and a higher risk of relapse than non-iAMP21 cases. Recent studies suggest that structural variations in iAMP21 are linked to complex rearrangements such as a chromothripsis event on chromosome 21. The consequences of chromothripsis by which it drives the tumorigenesis are still unclear. Given the limited knowledge of chromothripsis and its impact on the epigenetic landscape in pediatric iAMP21 cases, we aim to systematically provide a critical understanding of the causes and consequences of such complex genomic rearrangements. We conducted an in-depth analysis of 9 iAMP21 and 10 non-iAMP21 pediatric patient samples associated with B-ALL using different sequencing techniques. These included whole genome sequencing (WGS), long-read sequencing, RNA-sequencing, and 3D genome organization. The WGS analysis revealed that iAMP21 cases had more genomic rearrangements compared to the non-iAMP21 group. Interestingly, iAMP21 subtypes displayed a unique feature - the presence or absence of the chromothripsis signature on chromosome 21. However, all patients shared a common characteristic - amplified and frequently deleted regions on chromosome 21, suggesting these genetic changes are a key aspect of the iAMP21 subtype, regardless of chromothripsis. Genomic rearrangements, a crucial element in oncogenesis, also modify the 3D structure of the human genome, and iAMP21 is no exception. Our Hi-C data analysis showed the devastating effect of chromothripsis on the 3D organization of chromosomes in iAMP21 patients. We observed a significant reconfiguration of the genomic compartments, along with changes in cis- and trans-acting regulatory elements such as enhancers and promoters. These changes disrupt the delicate balance of gene expression profile. To better understand the impact of these genomic rearrangements on the transcriptional landscape, we examined global gene expression patterns in iAMP21 subtypes, identifying distinct upregulated and downregulated genes. Our comparison between iAMP21 and non-iAMP21 groups identified a unique set of 186 upregulated and 174 downregulated genes in iAMP21 patients. Further, tumor diversity can greatly influence cancer diagnosis and treatment responses. Therefore, analyzing gene expression at the single-cell level is essential to uncover this diversity and identify unique cellular characteristics. Our analysis provided clear evidence of iAMP21 subclones within individual patient samples, resulting from differences in genomic rearrangements in each cancer cell. The iAMP21 cases with complex chromosomal rearrangement make it a high-risk and poorly characterized subtype of pediatric ALL. We hypothesize that even though related to worse outcomes, investigating the chromosomal rearrangement and the epigenetic landscape in iAMP21 may expose specific vulnerabilities of cancer cells in these patients allowing for targeted therapies and devising new therapeutic strategies. While our findings so far are preliminary, they offer valuable insights that can guide the formulation of hypotheses for further detailed experiments and data analysis. The project has already amassed a wealth of genomic data and a consistent approach to data analysis, addressing the challenging task of identifying relevant prognostic and predictive molecular factors in patients battling aggressive forms of cancer.

Gain of chromosome 21 increases the propensity for P2RY8::CRLF2 acute lymphoblastic leukemia via increased HMGN1 expression.

Acute lymphoblastic leukemia (ALL) patients with a gain of chromosome 21, intrachromosomal amplification of chromosome 21 (iAMP21), or Down syndrome (DS), have increased expression of genes in the DS critical region (DSCR) of chromosome 21, including the high-mobility group nucleosome-binding protein 1, HMGN1. Children with DS are predisposed to develop hematologic malignancies, providing insight into the role of chromosome 21 in the development of leukemias. A 320-kb deletion in the pseudoautosomal region of the X/Y chromosome in leukemic cells, resulting in a gene fusion between the purinergic receptor and cytokine receptor-like factor-2 (P2Y Receptor Family Member 8 (P2RY8)::CRLF2), is a common feature in ~60% of DS-ALL and ~40% of iAMP21 patients, suggesting a link between chromosome 21 and P2RY8::CRLF2. In an Australian cohort of pediatric B-ALL patients with P2RY8::CRLF2 (n = 38), eight patients harbored gain of chromosome 21 (+21), and two patients had iAMP21, resulting in a significantly increased HMGN1 expression. An inducible CRISPR/Cas9 system was used to model P2RY8::CRLF2 and investigate its cooperation with HMGN1. This model was then used to validate HMGN1 as an influencing factor for P2RY8::CRLF2 development. Using Cas9 to cleave the DNA at the pseudoautosomal region without directed repair, cells expressing HMGN1 favored repair, resulting in P2RY8::CRLF2 generation, compared with cells without HMGN1. CRISPR/Cas9 P2RY8::CRLF2 cells expressing HMGN1 exhibit increased proliferation, thymic stromal lymphopoietin receptor (TSLPR) expression, and JAK/STAT signaling, consistent with cells from patients with P2RY8::CRLF2. Our patient expression data and unique CRISPR/Cas9 modeling, when taken together, suggest that HMGN1 increases the propensity for P2RY8::CRLF2 development. This has important implications for patients with DS, +21, or iAMP21.

Intrachromosomal amplification 21: A driver of acute myeloid leukemia?

A 68-year-old woman was referred for anemia and circulating peripheral blood blasts. Bone marrow aspirate confirmed the diagnosis of acute myeloid leukemia (AML) with a background of variable dysplasia that was not otherwise sufficient to subclassify as AML with myelodysplasia-related changes (AML-MRC) (panel A; original magnification × 1000; Wright-Giemsa stain). By flow cytometry, the blasts were positive for dim/partial CD7, CD13, CD34, CD117, HLA-DR, partial MPO and TdT, and were negative for other B/T cell associated antigens. G-banding karyotype showed the presence of tandem duplication at the long arm of chromosome 21 (panel B; 46,XX,dup(21)(q21q22)). Single color fluorescence in situ hybridization probes targeting the 21q22.13-q22.2 region showed a homogeneously staining region on chromosome 21 containing >5 copies of RUNX1 (panels C and D; metaphase-interphase FISH), further confirmed by comparative genomic hybridization. Intrachromosomal amplification 21 (iamp21) is a well-recognized event in B lymphoblastic leukemia; however, it is rare in AML with <20 published cases, variably associated with morphologic dysplasia although it uniformly occurred in the setting of complex karyotypes. It was thus postulated to be a late event resulting from genome instability and chromothripsis. In our case, iAMP21 occurred as a sole abnormality, suggesting the possibility of a rather early role in leukemogenesis. N.A wrote the manuscript and compiled the genetics images. Z.C designed the cytology and flow cytometry images. Z.C revised the manuscript. The study received no funding from internal or external sources. The authors disclose no conflict of interests. The Institutional Review Board at the American University of Beirut exempted our single case report from review. Informed consent was explained to and signed by the patient. Data sharing is not applicable to this article as no new data were created or analyzed in this study.

The genomic landscape of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21

AbstractIntrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21–ALL ones, including multiple genes implicated in the pathogenesis of acute leukemia (CHAF1B, DYRK1A, ERG, HMGN1, and RUNX1). Using multimodal single-cell genomic profiling, including single-cell whole genome sequencing of 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the acquisition of the iAMP21 chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV-mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.

Intrachromosomal amplification of chromosome 21 in Egyptian pediatric B-lineage acute lymphoblastic leukemia: a hospital-based observational study

Abstract Objective This study was conducted to evaluate the clinicopathologic features as well as the association between the intrachromosomal amplification of chromosome 21 (iAMP21) and the clinical outcome in Egyptian patients with B-cell acute lymphoblastic leukemia (B-ALL) attending/admitted in the Department of Pediatric Oncology of our university hospital. Patients and methods This was a prospective study conducted on 25 newly diagnosed and five relapsed pediatric patients with B-ALL (23 males and seven females), with a mean age of 7.76 ± 5.11 years. Bone marrow aspiration was done to diagnose and evaluate remission of participants, and fluorescence in situ hybridization (FISH) analysis using the ETV6-RUNX1 probes was used to detect RUNX1 amplification. Results iAMP21 was identified in 28% of newly diagnosed patients with B-ALL and in 80% of relapsed cases with B-ALL. There was a significant association between positive iAMP21 cases and failure to attain complete remission at day 28 (P=0.004). A significant relationship among the 14 patients monitored for disease outcome was found between the presence of iAMP21 and occurrence of relapse or death in newly diagnosed, or failure to achieve complete remission or death in relapsed cases (P=0.036). Conclusion There was a higher incidence of iAMP21 among our Egyptian pediatric B-ALL cases and unfavorable effect of iAMP21 on patients’ prognosis even with coexistence of ETV6-RUNX1.

B-Cell Acute Lymphoblastic Leukemia with iAMP21 in a Patient with Constitutional Ring Chromosome 21

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is associated with various specific cytogenetic and molecular markers that significantly influence treatment and prognosis. Intrachromosomal amplification of chromosome 21 (iAMP21) defines a rare distinct cytogenetic subgroup of childhood B-ALL, which is characterized by amplification of region 21q22.12 comprising the RUNX1 gene. Constitutional structural chromosomal abnormalities involving chromosome 21 confer an increased risk for B-ALL with iAMP21. Here, we report the development of B-ALL with iAMP21 in a 9-year-old child with a constitutional ring chromosome 21, r(21)c, uncovered after B-ALL diagnosis. Cytogenetic and microarray analysis of the post-therapy sample revealed an abnormal chromosome 21 lacking a satellite and having a deletion of the terminal 22q22.3 region, consistent with a constitutional ring chromosome 21, r(21)(p11.2q22). On a retrospective analysis, this ring chromosome was observed in the normal cells in the pre-treatment diagnostic specimen. Constitutional ring chromosome 21 may remain undetected in patients with mild or no neurodevelopmental phenotype, posing an unknown lifelong risk of developing B-ALL with iAMP21. Individuals with constitutional structural chromosome 21 rearrangements such as ring 21 require a close surveillance and long-term follow-up studies to establish their risk of B-ALL relapse and possibility of developing other malignancies. Germline analysis is recommended to all pediatric patients with iAMP21-related B-ALL to rule out structural chromosome 21 rearrangements and to elucidate molecular mechanisms of iAMP21 formation.

FLEXIBILITY OF FISH PROBES IN MONITORING IAMP21 ACUTE LYMPHOBLASTIC LEUKEMIA: STUDY OF FOUR BRAZILIAN CHILDREN

Intrachromosomal Amplification of chromosome 21 (iAMP21) is an independent outcome indicator in B-cell Precursor ALL (BCP-ALL). IAMP21 is characterized by multiple RUNX1 copies in blast cells, and comprises 2% of children with BCP-ALL. It was first detected during routine screening for ETV6-RUNX1 fusion by FISH. This disease features protocol-dependent outcomes. For example, in pediatric patients, iAMP21-ALL presents poor outcomes when standard therapy is used. Although, when children receive intensive treatment, they usually show lower relapse risk and improved survival. Thus, the early detection of iAMP21 is crucial to guide therapeutic strategies. In this regard, FISH probes may provide a rapid and effective screening, being a valuable ally for banding cytogenetics. In this work, we describe the clinical and molecular data of four pediatric patients with BCP-ALL iAMP21, showing the importance of RUNX1-RUNX1T1 and ETV6-RUNX1 FISH probes in screening this high-risk entity. This collaborative study included four children with BCP-ALL iAMP21. The peripheral blood and Bone Marrow (BM) samples were referred to the cytogenetics laboratory at the Brazilian National Cancer Institute between 2013 and 2021. We performed G-banding and FISH experiments in interphase nuclei and metaphase spreads. We used commercial Locus-Specific (LSI) ETV6-RUNX1, RUNX1-RUNX1T1, and centromeric 13/21 FISH probes. Using the FISH technique with LSI ETV6-RUNX1 and RUNX1-RUNX1T1 probes, we identified the derivative chromosome 21 and the precise number of RUNX1 signals for each patient. It also helped disclose whether the RUNX1 amplifications were intra- and or extrachromosomal. The intrachromosomal amplification of chromosome 21 is an uncommon high-risk chromosomal aberration in pediatric B-ALL. iAMP21 can be elusive, sometimes escaping detection by banding cytogenetics. Thus, confirmation by molecular techniques is necessary. Besides, recent studies suggest a relapse risk associated with therapy intensity, emphasizing the importance of a rapid and early detection of iAMP21 for appropriate risk stratification and therapeutic intervention. The FISH approach used through our experimental design has proved efficient in the early detection of iAMP21 in all patients. Importantly, it helped determine the diagnosis of iAMP21 48 hours after initial diagnosis. Our approach can also be valuable in monitoring patients for Minimal Residual Disease (MRD). According to the literature, those who continuously present MRD should be eligible for BM transplantation. In summary, the flexibility of FISH probes enabled the rapid detection of this high-risk neoplasm. Besides, the precise characterization of iAMP21, including the aberrant chromosomes 21 and extra RUNX1 signals, was important to guide treatment strategies and the monitoring of patients. In addition, we reinforce the importance of investigating the heterogeneity of iAMP21 marker chromosomes, their molecular complexity, and genomic association with disease progression. Novel genetic findings may provide valuable information about this entity, which could open perspectives regarding novel therapeutic approaches for children with BCP-ALL iAMP21.

The genomic landscape of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Here, using whole-genome, exome and transcriptome sequencing of 2,754 childhood patients with ALL, we find that, despite a generally low mutation burden, ALL cases harbor a median of four putative somatic driver alterations per sample, with 376 putative driver genes identified varying in prevalence across ALL subtypes. Most samples harbor at least one rare gene alteration, including 70 putative cancer driver genes associated with ubiquitination, SUMOylation, noncoding transcripts and other functions. In hyperdiploid B-ALL, chromosomal gains are acquired early and synchronously before ultraviolet-induced mutation. By contrast, ultraviolet-induced mutations precede chromosomal gains in B-ALL cases with intrachromosomal amplification of chromosome 21. We also demonstrate the prognostic significance of genetic alterations within subtypes. Intriguingly, DUX4- and KMT2A-rearranged subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of the ALL genomic landscape and associated outcomes.

Clinicopathologic and genetic evaluation of B-lymphoblastic leukemia with intrachromosomal amplification of chromosome 21 (iAMP21) in adult patients

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a rare provisional entity of B-cell acute lymphoblastic leukemia (B-ALL) in the current WHO classification and has been described as specific for pediatric patients with a median age at diagnosis of 9–10 years. We report two adult cases of B-ALL with iAMP21, one 31-year-old woman and one 40-year-old man, identified by karyotyping and next generation sequencing (NGS), with fluorescence in situ hybridization (FISH) pattern meeting diagnostic criteria for iAMP21. Both patients were treated on high-risk chemotherapeutic regimen followed by stem cell transplant. In contrast to reported high relapse rate within the first three years in pediatric population, our adult patients are alive in remission, with the interval from diagnosis to last follow up of 2.95 and 3.96 years. Our cases illustrate the importance of screening for iAMP21 in adult population when ETV6-RUNX1 FISH testing is not routinely performed for adult patients.

Characterization of unusual iAMP21 B-lymphoblastic leukemia (iAMP21-ALL) from the Mayo Clinic and Children's Oncology Group.

Acute lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21-ALL) represents a recurrent high-risk cytogenetic abnormality and accurate identification is critical for appropriate clinical management. Identification of iAMP21-ALL has historically relied on fluorescence in situ hybridization (FISH) using a RUNX1 probe. Current classification requires ≥ five copies of RUNX1 per cell and ≥ three additional copies of RUNX1 on a single abnormal iAMP21-chromosome. We sought to evaluate the performance of the RUNX1 probe in the identification of iAMP21-ALL. This study was a retrospective evaluation of iAMP21-ALL in the Mayo Clinic and Children's Oncology Group cohorts. Of 207 cases of iAMP21-ALL, 188 (91%) were classified as "typical" iAMP21-ALL, while 19 (9%) cases were classified as "unusual" iAMP21-ALL. The "unusual" iAMP21 cases did not meet the current definition of iAMP21 by FISH but were confirmed to have iAMP21 by chromosomal microarray. Half of the "unusual" iAMP21-ALL cases had less than five RUNX1 signals, while the remainder had ≥ five RUNX1 signals with some located apart from the abnormal iAMP21-chromosome. Nine percent of iAMP21-ALL cases fail to meet the FISH definition of iAMP21-ALL demonstrating that laboratories are at risk of misidentification of iAMP21-ALL when relying only on the RUNX1 FISH probe. Incorporation of chromosomal microarray testing circumvents these risks.

Abstract 3671: Association between residence in a Hispanic enclave and end-induction minimal residual disease among children with acute lymphoblastic leukemia in Texas

Abstract Background: Acute lymphoblastic leukemia (ALL) remains a leading cause of death in children and adolescents. We have demonstrated that Hispanic enclaves - neighborhoods with high proportions of Spanish-speaking residents, recent immigrants, and ethnic-specific businesses - are associated with inferior overall survival in children with ALL. However, associations of enclaves with other outcomes remain poorly understood. In the present study, our objective was to determine whether residence in an enclave was associated with end-induction minimal residual disease (MRD), a strong predictor of ALL mortality. Methods: This was a retrospective study of N=142 children aged 0-18 years, treated at Texas Children’s Hospital (Houston, TX) from 2007-2018. MRD was defined as ≥0.01% leukemic blasts in bone marrow at day 29 of therapy. We used the 2010 census tract geography and a modified version of the Hispanic enclave index to identify enclaves. We assigned an enclave index score to each census tract, computed quartiles based on the statewide distribution, and mapped children to quartiles based on address at diagnosis (Q1: least ethnically distinct neighborhoods; Q4: most ethnically distinct neighborhoods). Data on sex, self-reported race/ethnicity, National Cancer Institute (NCI) risk group, primary language, and ALL cytogenetics were abstracted from electronic health records. Cytogenetics were considered either unfavorable (BCR-ABL1 fusion, KMT2A rearrangement, hypodiploidy, or intrachromosomal amplification of chromosome 21) or favorable (ETV6-RUNX1 fusion, double trisomies of chromosomes 4 and 10)/neutral (neither favorable nor unfavorable). We estimated the odds ratio (OR) and 95% confidence interval (95% CI) of MRD according to enclave index score using logistic regression. Results: Neither individual ethnicity nor primary language varied by MRD status, but a greater proportion of MRD-positive children lived in the most ethnically distinct neighborhoods (53% vs. 36%, p=0.04). Enclave index score was associated with MRD after adjusting for sex, NCI risk group, and cytogenetics (OR 1.57 per quartile increment, 95% CI 1.09-2.33). We also observed non-significantly increased odds of MRD among children in Q3 (OR 1.53, 95% CI 0.41-6.55) and Q4 (OR 2.84, 0.83-11.58) relative to Q1. Conclusions: We found that residence in a Hispanic enclave was associated with MRD in children with ALL. Neither ethnicity nor primary language differed by MRD status, suggesting that these did not confound the association with enclave index score. Neighborhood factors may influence early treatment outcomes in ALL, and children living in Hispanic enclaves may constitute a high-risk population. Citation Format: Joshua P. Muniz, John P. Woodhouse, Amy E. Hughes, Sandi L. Pruitt, Karen R. Rabin, Michael E. Scheurer, Philip J. Lupo, Jeremy M. Schraw. Association between residence in a Hispanic enclave and end-induction minimal residual disease among children with acute lymphoblastic leukemia in Texas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3671.

B‐cell acute lymphoblastic leukemia with iAMP21 in a patient with Down syndrome due to a constitutional isodicentric chromosome 21

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is associated with various specific cytogenetic and molecular markers that have significant influence on treatment and prognosis. A subset of children has a much higher risk of developing B-ALL due to constitutional genetic alterations such as trisomy 21 (Down's syndrome). In these patients, B-ALL is often associated with specific genomic profiles leading to leukemic transformation. In rare cases, constitutional structural chromosomal abnormalities involving chromosome 21, such as the der(15;21) Robertsonian translocation and a ring 21 chromosome, have been associated with intrachromosomal amplification of chromosome 21 (iAMP21) B-ALL. Here, we report the development of B-ALL in a child with Down's syndrome who carries a constitutional isodicentric chromosome 21 [idic(21)], described previously by Putra et al., 2017. This idic(21) appeared to be unstable during mitosis, leading to somatic rearrangements consistent with iAMP21 amplification, resulting in the development of leukemia. In this case, a single constitutional structural chromosome 21 rearrangement resulted in a B-ALL with Down syndrome-associated genomic lesions as well as genomic lesions not common to the Down syndrome subtype of B-ALL. Our findings highlight the need for counseling of individuals with constitutional structural chromosome 21 rearrangements regarding their risks of developing a B-ALL.

ERG amplification is a secondary recurrent driver event in myeloid malignancy with complex karyotype and TP53 mutations.

ERG is a transcription factor encoded on chromosome 21q22.2 with important roles in hematopoiesis and oncogenesis of prostate cancer. ERG amplification has been identified as one of the most common recurrent events in acute myeloid leukemia with complex karyotype (AML-CK). In this study, we uncover three different modes of ERG amplification in AML-CK. Importantly, we present evidence to show that ERG amplification is distinct from intrachromosomal amplification of chromosome 21 (iAMP21), a hallmark segmental amplification frequently encompassing RUNX1 and ERG in a subset of high-risk B-lymphoblastic leukemia. We also characterize the association with TP53 aberrations and other chromosomal aberrations, including chromothripsis. Lastly, we show that ERG amplification can initially emerge as subclonal events in low-grade myeloid neoplasms. These findings demonstrate that ERG amplification is a recurrent secondary driver event in AML and raise the tantalizing possibility of ERG as a therapeutic target.

A case of childhood acute lymphoblastic leukemia suggesting intrachromosomal amplification of chromosome 21 (iAMP21)

A case of childhood acute lymphoblastic leukemia suggesting intrachromosomal amplification of chromosome 21 (iAMP21)