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.

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.

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.

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.

Characterization of Philadelphia-like Pre-B Acute Lymphoblastic Leukemia: Experiences in Mexican Pediatric Patients.

Ph-like subtypes with CRLF2 abnormalities are frequent among Hispano-Latino children with pre-B ALL. Therefore, there is solid ground to suggest that this subtype is frequent in Mexican patients. The genomic complexity of Ph-like subtype constitutes a challenge for diagnosis, as it requires diverse genomic methodologies that are not widely available in diagnostic centers in Mexico. Here, we propose a diagnostic strategy for Ph-like ALL in accordance with our local capacity. Pre-B ALL patients without recurrent gene fusions (104) were classified using a gene-expression profile based on Ph-like signature genes analyzed by qRT-PCR. The expressions of the CRLF2 transcript and protein were determined by qRT-PCR and flow cytometry. The P2RY8::CRLF2, IGH::CRLF2, ABL1/2 rearrangements, and Ik6 isoform were screened using RT-PCR and FISH. Surrogate markers of Jak2-Stat5/Abl/Ras pathways were analyzed by phosphoflow. Mutations in relevant kinases/transcription factors genes in Ph-like were assessed by target-specific NGS. A total of 40 patients (38.5%) were classified as Ph-like; of these, 36 had abnormalities associated with Jak2-Stat5 and 4 had Abl. The rearrangements IGH::CRLF2,P2RY8::CRLF2, and iAMP21 were particularly frequent. We propose a strategy for the detection of Ph-like patients, by analyzing the overexpression/genetic lesions of CRLF2, the Abl phosphorylation of surrogate markers confirmed by gene rearrangements, and Sanger sequencing.

Integrating copy number data of 64 iAMP21 BCP-ALL patients narrows the common region of amplification to 1.57 Mb.

Background and purpose: Intrachromosomal amplification of chromosome 21 (iAMP21) is a rare subtype of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). It is unknown how iAMP21 contributes to leukaemia. The currently known commonly amplified region is 5.1 Mb. Methods: We aimed to narrow down the common region of amplification by using high resolution techniques. Array comparative genomic hybridization (aCGH) was used to determine copy number aberrations, Affymetrix U133 Plus2 expression arrays were used to determine gene expression. Genome-wide expression correlations were evaluated using Globaltest. Results: We narrowed down the common region of amplification by combining copy number data from 12 iAMP21 cases with 52 cases from literature. The combined common region of amplification was 1.57 Mb, located from 36.07 to 37.64 Mb (GRCh38). This region is located telomeric from, but not including, RUNX1, which is the locus commonly used to diagnose iAMP21. This narrow region, which falls inside the Down Syndrome critical region, includes 13 genes of which the expression of eight genes was significantly upregulated compared with 143 non-iAMP21 B-other cases. Among these, transcriptional repressor RIPPLY3 (also known as DSCR6) was the highest overexpressed gene (fold change = 4.2, FDR < 0.001) and most strongly correlated (R = 0.58) with iAMP21-related genome-wide expression changes. Discussion: The more precise definition of the common region of amplification could be beneficial in the diagnosis of iAMP21 based on copy number analysis from DNA sequencing or arrays as well as stimulate functional research into the role of the included genes in iAMP21 biology.

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.

Successful hematopoietic stem cell transplantation for two patients with relapse of intrachromosomal amplification of chromosome 21-positive B-cell precursor acute lymphoblastic leukemia.

In children with relapsed acute lymphoblastic leukemia (ALL), it is essential to identify patients in need of treatment intensification. Minimal residual disease (MRD)-based treatment stratification resulted in excellent survival in children with late relapsed B-cell precursor (BCP)-ALL. Chemotherapy alone produced a favorable outcome in patients with negative MRD after induction. The genetic abnormality also plays an important role in determining the prognosis and stratification for treatment. Intrachromosomal amplification of chromosome 21 (iAMP21) is associated with a poor outcome and a high risk for relapse, and there is no standard treatment after relapse. Herein, we present two patients with relapsed iAMP21-positive ALL who were successfully treated by cord blood transplantation (CBT). Although both patients had late bone marrow relapse and favorable MRD response, CBT was performed due to iAMP21 positive. Patients 1 and 2 have been in remission post-CBT for 15 and 45 months, respectively. Patients with relapsed iAMP21-positive ALL may be considered for stem cell transplantation even in late relapses and favorable MRD response.

Somatic homozygous loss of SH2B3, and a non-Robertsonian translocation t(15;21)(q25.3;q22.1) with NTRK3 rearrangement, in an adolescent with progenitor B-cell acute lymphoblastic leukemia with the iAMP21.

Intrachromosomal amplification of chromosome 21 (iAMP21) occurs in ∼2% of B-cell acute lymphoblastic leukemia (ALL) and is considered to confer a poor prognosis. The relapse risk is associated with therapy intensity, suggesting that other somatic mutations may influence iAMP21-ALL prognosis. This abnormality is characterized by multiple copies of the RUNX1 gene in chromosome 21 and appears to arise through multiple breakage-fusion bridge cycles and chromothripsis. Rob(15;21) or a ring chromosome 21 have been associated with an increased risk for iAMP21-ALL, suggesting that constitutional genetic abnormalities may also drive leukemogenesis. Here we describe homozygous deletion of the SH2B3 gene, chromothripsis of chromosome 21, and a non-Robertsonian somatic t(15;21)(q25.3;q22.1) with NTRK3 gene rearrangement in an adolescent with iAMP21-B-ALL. Molecular cytogenetic studies detected iAMP21 with aCGH analysis revealing further genomic imbalances. The RT-qPCR analysis detected elevated expression levels of RUNX1 (68-fold) and reduced expression of CDK6 (0.057-fold). Studies with constitutive cells collected from mouth swabs showed that SH2B3 biallelic deletion was a somatic alteration occurring during clonal evolution. The identification of novel secondary genetic changes was valuable to discuss sporadic iAMP21 leukemogenic mechanisms. For the first time, we show a t(15;21)(q25.3;q22.1) with NTRK3 rearrangement in an adolescent with iAMP21-ALL.

Case Report: Precision Medicine Target Revealed by In Vitro Modeling of Relapsed, Refractory Acute Lymphoblastic Leukemia From a Child With Neurofibromatosis.

Children with neurofibromatosis have a higher risk of developing juvenile myelomonocytic leukemia and acute myeloid leukemia, but rarely develop B-cell acute lymphoblastic leukemia (B-ALL). Through in-vitro modeling, a novel NF1 p.L2467 frameshift (fs) mutation identified in a relapsed/refractory Ph-like B-ALL patient with neurofibromatosis demonstrated cytokine independence and increased RAS signaling, indicative of leukemic transformation. Furthermore, these cells were sensitive to the MEK inhibitors trametinib and mirdametinib. Bi-allelic NF1 loss of function may be a contributing factor to relapse and with sensitivity to MEK inhibitors, suggests a novel precision medicine target in the setting of neurofibromatosis patients with B-ALL.

Immunophenotypic challenges in diagnosis of CD79a negativity in a patient with B acute lymphoblastic leukemia harboring intrachromosomal amplification of chromosome 21: a case report.

BACKGROUND: Being expressed in all stages of B-cell development and having a significant value on the European Group for the Immunological Characterization of Acute Leukemias scoring system, CD79a is considered as an excellent pan-marker for lineage assignment of B cells by flow cytometry. Therefore, any lack or decrease in CD79a expression makes the diagnosis of B acute lymphoblastic leukemia cases very challenging, especially in developing country laboratories where flow cytometry analyses are not always available and, when they are, they are limited in the number of markers used for lineage assignment. Since this case is potentially interesting, we report a B acute lymphoblastic leukemia case with a lack of expression CD79a associated with intrachromosomal amplification of chromosome 21 genetic abnormality. We further discuss the practical challenges in the diagnosis of this case. CASE PRESENTATION: We present the case of an 8-year-old Caucasian boy from eastern Morocco who was initially hospitalized for a hemorrhagic syndrome. Peripheral blood smear examination showed a significant number of blasts suggesting acute leukemia. Bone marrow was studied for morphology, cytochemistry, immunophenotyping, and cytogenetics. Flow cytometry analyses showed expression of CD19, CD22, CD10, CD34, and HLA-DR markers by leukemic blasts. The expression of CD79a, which was checked with two different monoclonal antibodies, confirms that this marker was severely decreased in this case. Cytogenetic study performed by fluorescence in situ hybridization revealed the presence of intrachromosomal amplification of chromosome 21, a cytogenetic abnormality that is specific for B acute lymphoblastic leukemia. CONCLUSION: CD79a is one of the critical markers in the assignment of B acute lymphoblastic leukemia. In our case, we were lucky enough to be assisted by a few other markers of the B lineage that were positive in this case. Also, we mention the importance of proceeding to cytogenetic study, which in our case helped us to confirm the diagnosis made by flow cytometry by highlighting a cytogenetic abnormality that is specific to B acute lymphoblastic leukemia.

Overexpression of chromatin remodeling and tyrosine kinase genes in iAMP21-positive acute lymphoblastic leukemia.

Intrachromosomal amplification of chromosome 21 (iAMP21) is a cytogenetic subtype associated with relapse and poor prognosis in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). The biology behind the high relapse risk is unknown and the aim of this study was to further characterize the genomic and transcriptional landscape of iAMP21. Using DNA arrays and sequencing, we could identify rearrangements and aberrations characteristic for iAMP21. RNA sequencing revealed that only half of the genes in the minimal region of amplification (20/45) were differentially expressed in iAMP21. Among them were the top overexpressed genes (p < 0.001) in iAMP21 vs. BCP ALL without iAMP21 and three candidate genes could be identified, the tyrosine kinase gene DYRK1A and chromatin remodeling genes CHAF1B and SON. While overexpression of DYRK1A and CHAF1B is associated with poor prognosis in malignant diseases including myeloid leukemia, this is the first study to show significant correlation with iAMP21-positive ALL.

MLPA as a complementary tool for diagnosis of chromosome 21 aberrations in childhood BCP-ALL.

Chromosome 21 abnormalities are the most frequent genetic findings in childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) cases. Majority of patients are effectively diagnosed with fluorescence in situ hybridization (FISH) and karyotyping; however, some cases may require additional tools to be used. Bone marrow samples of 373 childhood BCP-ALL patients were tested for chromosome 21 copy number variations (CNVs) with Multiplex Ligation-dependent Probe Amplification (MLPA) P327 array. Results from MLPA and cytogenetics were compared between groups according to the type of abnormality found on chromosome 21. Out the group of 235 patients, chromosome 21 multiplication was found by FISH assay in 56 cases (23.81%), ETV6-RUNX1 fusion in 34 (14.47%) and iAMP21 in 3 (1.28%) children, remaining 142 (60.43%) patients had no known chromosome 21 aberration. Median peak ratios of all tested probes in MLPA in aforementioned groups were 1.47 (IQR 1.28-1.77) vs. 1.00 (IQR 1.00-1.09) vs. 2.79 (IQR 1.97-2.83) vs. 1.00 (1.00-1.11), respectively. Aforementioned peak ratio of ETV6-RUNX1 fusion group was similar with patients of no known chromosome 21 aberration (p = 0.71). Interestingly, both groups differed from patients with chromosome 21 multiplication (p < 10-5) and with iAMP21 (p < 10-5). All cases of iAMP21 were correctly recognized by MLPA. MLPA seems to be good additional tool in the diagnostic process of chromosome 21 CNVs, especially in cases with iAMP21.

SH2B3 aberrations enriched in iAMP21 B lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) represents the most common childhood malignancy. Although survival for pediatric B-ALL has approached 90%, variability in outcome among and within cytogenetically defined subgroups persists. While G-banding and fluorescence in situ hybridization (FISH) have been used to characterize leukemic clones, there is added value of chromosomal microarray and next generation sequencing in screening genome-wide for copy number aberrations, copy neutral loss of heterozygosity and nucleotide variations. Evaluation of novel genetic aberrations can provide information about the biologic mechanisms of cytogenetically defined subgroups associated with poor prognosis, explain heterogeneity in patient outcome and identify novel targets for therapeutic intervention. The high risk B-ALL intrachromosomal amplification of chromosome 21, (iAMP21), subtype is characterized by amplification of a region of chromosome 21 that typically encompasses the RUNX1 gene and is associated with poor prognosis. Analysis of chromosomal microarray and FISH data revealed that deletions of SH2B3, encoding a negative regulator of multiple tyrosine kinase and cytokine signaling pathways, are enriched among leukemias harboring iAMP21. Enrichment of SH2B3 aberrations in the iAMP21 subtype may indicate that loss of SH2B3 contributes to disease progression and raises the possibility that these leukemias may be sensitive to tyrosine kinase inhibitors.

Intrachromosomal amplification of chromosome 21 in Korean pediatric patients with B-cell precursor acute lymphoblastic leukemia in a single institution.

BACKGROUND: Intrachromosomal amplification of chromosome 21 (iAMP21), defined as the presence of three or more RUNX1 signals on one marker chromosome, is a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that is known to have a poor prognosis when treated with standard therapy. The aim of this study was to evaluate the clinical characteristics of Korean children with iAMP21. METHODS: The cytogenetic data from BCP-ALL children were reviewed. The ETV6/RUNX1 ES Dual Color Probe was used for fluorescence in situ hybridization (FISH). RESULTS: In total, 295 children were included. Of these, 10 patients (3.4%) had iAMP21. The median age of iAMP21 patients was 9 years, and the median value of white blood cell count was 5.09×109/L. Slow early treatment response was observed more in iAMP21 patients. Patients with iAMP21 had a higher incidence of relapse and worse survival rates. In patients with iAMP21, the estimated 10-year cumulative incidence of relapse was 53.3%. The estimated 10-year event-free survival and overall survival rate were 46.7% and 64.8%, respectively. Most cases of leukemic relapse developed in the late period (median, 43 mo). In multivariate analysis, high risk group was the only factor that had a significant impact on death. CONCLUSION: The existence of iAMP21 was related to delayed treatment response and was likely to affect increased relapse and death in the late period. Further studies are needed to reveal its effect on BCP-ALL treatment outcomes and its role as an independent prognostic factor.

Is intrachromosomal amplification of chromosome 21 (iAMP21) always intrachromosomal?

Recurrent chromosomal abnormalities in childhood B-cell acute lymphoblastic leukemia (B-ALL) provide prognostic information that is useful in determining treatment stratification. iAMP21 is a more recently recognized cytogenetic entity of B-ALL that was originally described as multiple copies of the RUNX1 gene on a structurally abnormal chromosome 21. Subsequent studies elucidated a common region of highest-level amplification that includes RUNX1. Fluorescence in situ hybridization (FISH) is the most common method used to identify iAMP21, which is defined as the presence of five or more total copies of RUNX1, with three or more extra RUNX1 signals on a single abnormal chromosome 21. More recently, chromosomal microarray (CMA) and next generation sequencing have uncovered a characteristic chromosome 21 copy number profile in cases of iAMP21. We present a case of iAMP21 that does not fit the formal FISH definition. However, CMA uncovered the characteristic chromosome 21 copy number profile that is seen in iAMP21, demonstrating that CMA is helpful for the detection of this entity when FISH results are ambiguous. Furthermore, CMA showed that the highest level of amplification in this case did not include the RUNX1 gene, consistent with current evidence that RUNX1 is not the primary target of amplification.

Frequency and Clinical Characteristics of Intrachromosomal Amplification of Chromosome 21 in Korean Childhood B-lineage Acute Lymphoblastic Leukemia.

BACKGROUND: Intrachromosomal amplification of chromosome 21 (iAMP21) is known to be associated with poor prognosis in B-cell ALL (B-ALL). To determine the frequency and clinical characteristics of iAMP21 in Korean B-ALL patients, we performed FISH and multiplex ligation-dependent probe amplification (MLPA) analyses. METHODS: A total of 102 childhood B-ALL patients were screened with ETV6-RUNX1 FISH probes (Abbott Molecular, USA). The presence of an iAMP21 was confirmed by using MLPA P327 iAMP21-ERG probemix (MRC Holland, The Netherlands). RESULTS: iAMP21 was detected in one of the screened B-ALL patients (1/102 patients, 1.0%) who presented the ALL immunophenotype and complex karyotype at initial diagnosis. The patient relapsed twice after bone marrow transplantation. MLPA showed 12.5-Mb and 4.28-Mb regions of amplification and deletion, respectively. CONCLUSIONS: The frequency of iAMP21 is considerable in Korean pediatric patients. Our report suggests that iAMP21 in childhood B-ALL has very unfavorable impact on patient's prognosis. Additional methods such as MLPA analysis is essential to rule out patients with equivocal interphase FISH results.