Clonal dynamics in pediatric B-cell precursor acute lymphoblastic leukemia with very early relapse.

INTRODUCTION: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. METHODS: In this study, we performed a genomic analysis of diagnosis-relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. RESULTS: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. CONCLUSION: Comprehensive genomic characterization of diagnosis-relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

Multiclonal complexity of pediatric acute lymphoblastic leukemia and the prognostic relevance of subclonal mutations

Genomic studies of pediatric acute lymphoblastic leukemia (ALL) have shown remarkable heterogeneity in initial diagnosis, with multiple (sub)clones harboring lesions in relapse-associated genes. However, the clinical relevance of these subclonal alterations remains unclear. We assessed the clinical relevance and prognostic value of subclonal alterations in the relapse-associated genes IKZF1, CREBBP, KRAS, NRAS, PTPN11, TP53, NT5C2, and WHSC1 in 503 ALL cases. Using Molecular Inversion Probe sequencing and breakpoint-spanning PCR we reliably detected alterations below 1% allele frequency. We identified 660 genomic alterations in 285 diagnosis samples of which 495 (75%) were subclonal. RAS pathway mutations were common, particularly in minor subclones, and comparisons between RAS hotspot mutations revealed differences in their capacity to drive clonal expansion in ALL. We did not find an association of subclonal alterations with unfavorable outcome. Particularly for IKZF1, an established prognostic marker in ALL, all clonal but none of the subclonal alterations were preserved at relapse. We conclude that, for the genes tested, there is no basis to consider subclonal alterations detected at diagnosis for risk group stratification of ALL treatment.

Presence of Genetic Variants Among Young Men With Severe COVID-19.

Importance: Severe coronavirus disease 2019 (COVID-19) can occur in younger, predominantly male, patients without preexisting medical conditions. Some individuals may have primary immunodeficiencies that predispose to severe infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Objective: To explore the presence of genetic variants associated with primary immunodeficiencies among young patients with COVID-19. Design, Setting, and Participants: Case series of pairs of brothers without medical history meeting the selection criteria of young (age <35 years) brother pairs admitted to the intensive care unit (ICU) due to severe COVID-19. Four men from 2 unrelated families were admitted to the ICUs of 4 hospitals in the Netherlands between March 23 and April 12, 2020. The final date of follow-up was May 16, 2020. Available family members were included for genetic variant segregation analysis and as controls for functional experiments. Exposure: Severe COVID-19. Main Outcome and Measures: Results of rapid clinical whole-exome sequencing, performed to identify a potential monogenic cause. Subsequently, basic genetic and immunological tests were performed in primary immune cells isolated from the patients and family members to characterize any immune defects. Results: The 4 male patients had a mean age of 26 years (range, 21-32), with no history of major chronic disease. They were previously well before developing respiratory insufficiency due to severe COVID-19, requiring mechanical ventilation in the ICU. The mean duration of ventilatory support was 10 days (range, 9-11); the mean duration of ICU stay was 13 days (range, 10-16). One patient died. Rapid clinical whole-exome sequencing of the patients and segregation in available family members identified loss-of-function variants of the X-chromosomal TLR7. In members of family 1, a maternally inherited 4-nucleotide deletion was identified (c.2129_2132del; p.[Gln710Argfs*18]); the affected members of family 2 carried a missense variant (c.2383G>T; p.[Val795Phe]). In primary peripheral blood mononuclear cells from the patients, downstream type I interferon (IFN) signaling was transcriptionally downregulated, as measured by significantly decreased mRNA expression of IRF7, IFNB1, and ISG15 on stimulation with the TLR7 agonist imiquimod as compared with family members and controls. The production of IFN-γ, a type II IFN, was decreased in patients in response to stimulation with imiquimod. Conclusions and Relevance: In this case series of 4 young male patients with severe COVID-19, rare putative loss-of-function variants of X-chromosomal TLR7 were identified that were associated with impaired type I and II IFN responses. These preliminary findings provide insights into the pathogenesis of COVID-19.

TRIM28 haploinsufficiency predisposes to Wilms tumor.

Two percent of patients with Wilms tumors have a positive family history. In many of these cases the genetic cause remains unresolved. By applying germline exome sequencing in two families with two affected individuals with Wilms tumors, we identified truncating mutations in TRIM28. Subsequent mutational screening of germline and tumor DNA of 269 children affected by Wilms tumor was performed, and revealed seven additional individuals with germline truncating mutations, and one individual with a somatic truncating mutation in TRIM28. TRIM28 encodes a complex scaffold protein involved in many different processes, including gene silencing, DNA repair and maintenance of genomic integrity. Expression studies on mRNA and protein level showed reduction of TRIM28, confirming a loss-of-function effect of the mutations identified. The tumors showed an epithelial-type histology that stained negative for TRIM28 by immunohistochemistry. The tumors were bilateral in six patients, and 10/11 tumors are accompanied by perilobar nephrogenic rests. Exome sequencing on eight tumor DNA samples from six individuals showed loss-of-heterozygosity (LOH) of the TRIM28-locus by mitotic recombination in seven tumors, suggesting that TRIM28 functions as a tumor suppressor gene in Wilms tumor development. Additionally, the tumors showed very few mutations in known Wilms tumor driver genes, suggesting that loss of TRIM28 is the main driver of tumorigenesis. In conclusion, we identified heterozygous germline truncating mutations in TRIM28 in 11 children with mainly epithelial-type Wilms tumors, which become homozygous in tumor tissue. These data establish TRIM28 as a novel Wilms tumor predisposition gene, acting as a tumor suppressor gene by LOH.

Independent prognostic value of BCR-ABL1-like signature and IKZF1 deletion, but not high CRLF2 expression, in children with B-cell precursor ALL.

Most relapses in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are not predicted using current prognostic features. Here, we determined the co-occurrence and independent prognostic relevance of 3 recently identified prognostic features: BCR-ABL1-like gene signature, deletions in IKZF1, and high CRLF2 messenger RNA expression (CRLF2-high). These features were determined in 4 trials representing 1128 children with ALL: DCOG ALL-8, ALL9, ALL10, and Cooperative ALL (COALL)-97/03. BCR-ABL1-like, IKZF1-deleted, and CRLF2-high cases constitute 33.7% of BCR-ABL1-negative, MLL wild-type BCP-ALL cases, of which BCR-ABL1-like and IKZF1 deletion (co)occurred most frequently. Higher cumulative incidence of relapse was found for BCR-ABL1-like and IKZF1-deleted, but not CRLF2-high, cases relative to remaining BCP-ALL cases, reflecting the observations in each of the cohorts analyzed separately. No relapses occurred among cases with CRLF2-high as single feature, whereas 62.9% of all relapses in BCR-ABL1-negative, MLL wild-type BCP-ALL occurred in cases with BCR-ABL1-like signature and/or IKZF1 deletion. Both the BCR-ABL1-like signature and IKZF1 deletions were prognostic features independent of conventional prognostic markers in a multivariate model, and both remained prognostic among cases with intermediate minimal residual disease. The BCR-ABL1-like signature and an IKZF1 deletion, but not CRLF2-high, are prognostic factors and are clinically of importance to identify high-risk patients who require more intensive and/or alternative therapies.

The origin and nature of tightly clustered BTG1 deletions in precursor B-cell acute lymphoblastic leukemia support a model of multiclonal evolution.

Recurrent submicroscopic deletions in genes affecting key cellular pathways are a hallmark of pediatric acute lymphoblastic leukemia (ALL). To gain more insight into the mechanism underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B-cell translocation gene 1 (BTG1), in a large cohort of pediatric ALL cases. BTG1 was found to be exclusively affected by genomic deletions, which were detected in 65 out of 722 B-cell precursor ALL (BCP-ALL) patient samples (9%), but not in 109 T-ALL cases. Eight different deletion sizes were identified, which all clustered at the telomeric site in a hotspot region within the second (and last) exon of the BTG1 gene, resulting in the expression of truncated BTG1 read-through transcripts. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests illegitimate RAG1/RAG2-mediated recombination as the responsible mechanism. Moreover, high levels of histone H3 lysine 4 trimethylation (H3K4me3), which is known to tether the RAG enzyme complex to DNA, were found within the BTG1 gene body in BCP-ALL cells, but not T-ALL cells. BTG1 deletions were rarely found in hyperdiploid BCP-ALLs, but were predominant in other cytogenetic subgroups, including the ETV6-RUNX1 and BCR-ABL1 positive BCP-ALL subgroups. Through sensitive PCR-based screening, we identified multiple additional BTG1 deletions at the subclonal level in BCP-ALL, with equal cytogenetic distribution which, in some cases, grew out into the major clone at relapse. Taken together, our results indicate that BTG1 deletions may act as "drivers" of leukemogenesis in specific BCP-ALL subgroups, in which they can arise independently in multiple subclones at sites that are prone to aberrant RAG1/RAG2-mediated recombination events. These findings provide further evidence for a complex and multiclonal evolution of ALL.

Elevated enhancer-oncogene contacts and higher oncogene expression levels by recurrent CTCF inactivating mutations in acute T cell leukemia.

Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene expression. In patients with T cell acute lymphoblastic leukemia (T-ALL), we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5; 14) (q35; q32.2) rearranged patients, especially when CTCF-binding sites are preserved in between the BCL11B enhancer and the TLX3 oncogene. These solitary intervening sites insulate TLX3 from the enhancer by inducing competitive looping to multiple binding sites near the TLX3 promoter. Reduced CTCF levels or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3 promoter to BCL11B enhancer looping, which elevates oncogene expression levels and leukemia burden.

BTG1 regulates glucocorticoid receptor autoinduction in acute lymphoblastic leukemia.

Resistance to glucocorticoids (GCs) is a major clinical problem in the treatment of acute lymphoblastic leukemia (ALL), but the underlying mechanisms are not well understood. Although mutations in the glucocorticoid receptor (GR) gene can give rise to therapy resistance in vitro, acquired somatic mutations in the GR are rarely encountered in patients. Here we report that the protein encoded by the BTG1 gene, which is frequently deleted in (pediatric) ALL, is a key determinant of GC responsiveness. Using RNA interference, we show that loss of BTG1 expression causes GC resistance both by decimating GR expression and by controlling GR-mediated transcription. Conversely, reexpression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression, a phenomenon known as GR autoinduction. In addition, the arginine methyltransferase PRMT1, a BTG1-binding partner and transcriptional coactivator, is recruited to the GR gene promoter in a BTG1-dependent manner. These results implicate the BTG1/PRMT1 complex in GR-mediated gene expression and reveal that deregulation of a nuclear receptor coactivator complex can give rise to GC resistance. Further characterization of this complex as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC-based therapies in ALL and other hematologic malignancies.

Prevalence of (Epi)genetic Predisposing Factors in a 5-Year Unselected National Wilms Tumor Cohort: A Comprehensive Clinical and Genomic Characterization.

PURPOSE: Wilms tumor (WT) is associated with (epi)genetic predisposing factors affecting a growing number of WT predisposing genes and loci, including those causing Beckwith-Wiedemann spectrum (BWSp) or WT1-related syndromes. To guide genetic counseling and testing, we need insight into the prevalence of WT predisposing (epi)genetic factors. PATIENTS AND METHODS: All children diagnosed with WT in the Netherlands between 2015 and 2020 were referred to a clinical geneticist. Phenotypic data, disease characteristics, and diagnostic test results were collected. If no genetic predisposition was identified by targeted diagnostic testing, germline (trio-)whole-exome sequencing and BWSp testing on normal kidney-derived DNA were offered. RESULTS: A total of 126 cases were analyzed of 128 identified patients. (Epi)genetic predisposing factors were present in 42 of 126 patients (33.3%) on the basis of a molecular diagnosis in blood-derived DNA (n = 26), normal kidney-derived DNA (n = 12), or solely a clinical diagnosis of BWSp (n = 4). Constitutional, heterozygous DIS3L2 variants were identified as a recurrent predisposing factor in five patients (4%), with a second somatic hit in 4 of 5 tumors. Twenty patients (16%) were diagnosed with BWSp while four additional patients without BWSp features harbored chromosome 11p15 methylation defects in normal kidney tissue. Remaining findings included WT1-related syndromes (n = 10), Fanconi anemia (n = 1), neurofibromatosis type 1 (n = 1), and a pathogenic REST variant (n = 1). In addition, (likely) pathogenic variants in adult-onset cancer predisposition genes (BRCA2, PMS2, CHEK2, and MUTYH) were identified in 5 of 56 (8.9%) patients with available whole-exome sequencing data. Several candidate WT predisposition genes were identified, which require further validation. CONCLUSION: (Epi)genetic WT predisposing factors, including mosaic aberrations and recurrent heterozygous DIS3L2 variants, were present in at least 33.3% of patients with WT. On the basis of these results, we encourage standard genetic testing after counseling by a clinical geneticist.

Trio-based whole exome sequencing in patients with suspected sporadic inborn errors of immunity: A retrospective cohort study.

Background: De novo variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) analysis in patients with suspected inborn errors of immunity (IEI). Methods: This study explored the potential added value of systematic assessment of DNVs in a retrospective cohort of 123 patients with a suspected sporadic IEI that underwent patient-parent trio-based WES. Results: A (likely) molecular diagnosis for (part) of the immunological phenotype was achieved in 12 patients with the diagnostic in silico IEI WES gene panel. Systematic evaluation of rare, non-synonymous DNVs in coding or splice site regions led to the identification of 14 candidate DNVs in genes with an annotated immune function. DNVs were found in IEI genes (NLRP3 and RELA) and in potentially novel candidate genes, including PSMB10, DDX1, KMT2C, and FBXW11. The FBXW11 canonical splice site DNV was shown to lead to defective RNA splicing, increased NF-κB p65 signalling, and elevated IL-1ß production in primary immune cells extracted from the patient with autoinflammatory disease. Conclusions: Our findings in this retrospective cohort study advocate the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI. Furthermore, we provide functional evidence supporting a causal role for FBXW11 loss-of-function mutations in autoinflammatory disease. Funding: This research was supported by grants from the European Union, ZonMW and the Radboud Institute for Molecular Life Sciences.

Genetic Screening for TLR7 Variants in Young and Previously Healthy Men With Severe COVID-19.

Introduction: Loss-of-function TLR7 variants have been recently reported in a small number of males to underlie strong predisposition to severe COVID-19. We aimed to determine the presence of these rare variants in young men with severe COVID-19. Methods: We prospectively studied males between 18 and 50 years-old without predisposing comorbidities that required at least high-flow nasal oxygen to treat COVID-19. The coding region of TLR7 was sequenced to assess the presence of potentially deleterious variants. Results: TLR7 missense variants were identified in two out of 14 patients (14.3%). Overall, the median age was 38 (IQR 30-45) years. Both variants were not previously reported in population control databases and were predicted to be damaging by in silico predictors. In a 30-year-old patient a maternally inherited variant [c.644A>G; p.(Asn215Ser)] was identified, co-segregating in his 27-year-old brother who also contracted severe COVID-19. A second variant [c.2797T>C; p.(Trp933Arg)] was found in a 28-year-old patient, co-segregating in his 24-year-old brother who developed mild COVID-19. Functional testing of this variant revealed decreased type I and II interferon responses in peripheral mononuclear blood cells upon stimulation with the TLR7 agonist imiquimod, confirming a loss-of-function effect. Conclusions: This study supports a rationale for the genetic screening for TLR7 variants in young men with severe COVID-19 in the absence of other relevant risk factors. A diagnosis of TLR7 deficiency could not only inform on treatment options for the patient, but also enables pre-symptomatic testing of at-risk male relatives with the possibility of instituting early preventive and therapeutic interventions.

Upfront Treatment Influences the Composition of Genetic Alterations in Relapsed Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

Genomic alterations in relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) may provide insight into the role of specific genomic events in relapse development. Along this line, comparisons between the spectrum of alterations in relapses that arise in different upfront treatment protocols may provide valuable information on the association between the tumor genome, protocol components and outcome. Here, we performed a comprehensive characterization of relapsed BCP-ALL cases that developed in the context of 3 completed Dutch upfront studies, ALL8, ALL9, and ALL10. In total, 123 pediatric BCP-ALL relapses and 77 paired samples from primary diagnosis were analyzed for alterations in 22 recurrently affected genes. We found pronounced differences in relapse alterations between the 3 studies. Specifically, CREBBP mutations were observed predominantly in relapses after treatment with ALL8 and ALL10 which, in the latter group, were all detected in medium risk-treated patients. IKZF1 alterations were enriched 2.2-fold (p = 0.01) and 2.9-fold (p < 0.001) in ALL8 and ALL9 relapses compared to diagnosis, respectively, whereas no significant enrichment was found for relapses that were observed after treatment with ALL10. Furthermore, IKZF1 deletions were more frequently preserved from a major clone at diagnosis in relapses after ALL9 compared to relapses after ALL8 and ALL10 (p = 0.03). These data are in line with previous studies showing that the prognostic value of IKZF1 deletions differs between upfront protocols and is particularly strong in the ALL9 regimen. In conclusion, our data reveal a correlation between upfront treatment and the genetic composition of relapsed BCP-ALL.

Accurate detection of low-level mosaic mutations in pediatric acute lymphoblastic leukemia using single molecule tagging and deep-sequencing.

Pathogenic mutations in relapse-associated genes in pediatric acute lymphoblastic leukemia may improve risk stratification when detected at subclonal levels at primary diagnosis. However, to detect subclonal mutations upfront, a deep-sequencing approach with high specificity and sensitivity is required. Here, we performed a proof-of-principle study to detect low-level mosaic RAS pathway mutations by deep sequencing using random tagging-based single molecule Molecular Inversion Probes (smMIPs). The smMIP-based approach could sensitively detect variants with allele frequency as low as 0.4%, which could all be confirmed by other techniques. In comparison, with standard deep-sequencing techniques we reached a detection threshold of only 2.5%, which hampered detection of seven low-level mosaic mutations representing 24% of all detected mutations. We conclude that smMIP-based deep-sequencing outperforms standard deep-sequencing techniques by showing lower background noise and high specificity, and is the preferred technology for detecting mutations upfront, particularly in genes in which mutations show limited clustering in hotspots.

Validation study of existing gene expression signatures for anti-TNF treatment in patients with rheumatoid arthritis.

So far, there are no means of identifying rheumatoid arthritis (RA) patients who will fail to respond to tumour necrosis factor blocking agents (anti-TNF), prior to treatment. We set out to validate eight previously reported gene expression signatures predicting therapy outcome. Genome-wide expression profiling using Affymetrix GeneChip Exon 1.0 ST arrays was performed on RNA isolated from whole blood of 42 RA patients starting treatment with infliximab or adalimumab. Clinical response according to EULAR criteria was determined at week 14 of therapy. Genes that have been reported to be associated with anti-TNF treatment were extracted from our dataset. K-means partition clustering was performed to assess the predictive value of the gene-sets. We performed a hypothesis-driven analysis of the dataset using eight existing gene sets predictive of anti-TNF treatment outcome. The set that performed best reached a sensitivity of 71% and a specificity of 61%, for classifying the patients in the current study. We successfully validated one of eight previously reported predictive expression profile. This replicated expression signature is a good starting point for developing a prediction model for anti-TNF treatment outcome that can be used in a daily clinical setting. Our results confirm that gene expression profiling prior to treatment is a useful tool to predict anti-TNF (non) response.

High-resolution genomic profiling of pediatric lymphoblastic lymphomas reveals subtle differences with pediatric acute lymphoblastic leukemias in the B-lineage.

Lymphoblastic lymphoma (LBL) is one of the most frequent occurring pediatric non-Hodgkin lymphomas. In the WHO classification scheme, pediatric LBL is considered to be the same disease entity as pediatric acute lymphoblastic leukemia (ALL). However, it is unclear whether the genetic basis of pediatric LBL development is similar to that of pediatric ALL. We performed genome-wide analyses of copy number aberrations in 12 T-LBL and 7 precursor B-cell LBL pediatric cases using high-resolution SNP-based array CGH. Similar to what previously has been found in T-ALL, T-LBL exhibited recurrent deletions of the CDKN2A locus, occurring in 92% of the cases. Additionally, we detected deletions of RB1 (16%), duplications of MYB (16%), and an amplification of ABL1 in one case. These results show that, similar to T-ALL, the genomic alterations in T-LBL predominantly target genes involved in cell cycle progression. The majority of precursor B-cell LBL was characterized by high-hyperdiploidy (71%), and showed high resemblance with high-hyperdiploid precursor B-cell ALL. Taken together, our data suggest that pediatric LBL and ALL exhibit similar genomic abnormalities within confined immunophenotypic and cytogenetic subgroups, but that the representations of these subgroups differs between the two entities.