Cell-free DNA from germline TP53 mutation carriers reflect cancer-like fragmentation patterns.

Germline pathogenic TP53 variants predispose individuals to a high lifetime risk of developing multiple cancers and are the hallmark feature of Li-Fraumeni syndrome (LFS). Our group has previously shown that LFS patients harbor shorter plasma cell-free DNA fragmentation; independent of cancer status. To understand the functional underpinning of cfDNA fragmentation in LFS, we conducted a fragmentomic analysis of 199 cfDNA samples from 82 TP53 mutation carriers and 30 healthy TP53-wildtype controls. We find that LFS individuals exhibit an increased prevalence of A/T nucleotides at fragment ends, dysregulated nucleosome positioning at p53 binding sites, and loci-specific changes in chromatin accessibility at development-associated transcription factor binding sites and at cancer-associated open chromatin regions. Machine learning classification resulted in robust differentiation between TP53 mutant versus wildtype cfDNA samples (AUC-ROC = 0.710-1.000) and intra-patient longitudinal analysis of ctDNA fragmentation signal enabled early cancer detection. These results suggest that cfDNA fragmentation may be a useful diagnostic tool in LFS patients and provides an important baseline for cancer early detection.

Spontaneous expression of the CIC::DUX4 fusion oncoprotein from a conditional allele potently drives sarcoma formation in genetically engineered mice.

CIC::DUX4 sarcoma (CDS) is a rare but highly aggressive undifferentiated small round cell sarcoma driven by a fusion between the tumor suppressor Capicua (CIC) and DUX4. Currently, there are no effective treatments and efforts to identify and translate better therapies are limited by the scarcity of patient tumor samples and cell lines. To address this limitation, we generated three genetically engineered mouse models of CDS (Ch7CDS, Ai9CDS, and TOPCDS). Remarkably, chimeric mice from all three conditional models developed spontaneous soft tissue tumors and disseminated disease in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic Cic function and the distance between adjacent loxP sites. Characterization of soft tissue and presumed metastatic tumors showed that they consistently expressed the CIC::DUX4 fusion protein and many downstream markers of the disease credentialing the models as CDS. In addition, tumor-derived cell lines were generated and ChIP-seq was preformed to map fusion-gene specific binding using an N-terminal HA epitope tag. These datasets, along with paired H3K27ac ChIP-sequencing maps, validate CIC::DUX4 as a neomorphic transcriptional activator. Moreover, they are consistent with a model where ETS family transcription factors are cooperative and redundant drivers of the core regulatory circuitry in CDS.

The BRD4-NUT Fusion Alone Drives Malignant Transformation of NUT Carcinoma.

NUT carcinoma (NC) is an aggressive squamous carcinoma defined by the BRD4-NUT fusion oncoprotein. Routinely effective systemic treatments are unavailable for most NC patients. The lack of an adequate animal model precludes identifying and leveraging cell-extrinsic factors therapeutically in NC. Here, we created a genetically engineered mouse model (GEMM) of NC that forms a Brd4::NUTM1 fusion gene upon tamoxifen induction of Sox2-driven Cre. The model displayed complete disease penetrance, with tumors arising from the squamous epithelium weeks after induction and all mice succumbing to the disease shortly thereafter. Closely resembling human NC (hNC), GEMM tumors (mNC) were poorly differentiated squamous carcinomas with high expression of MYC that metastasized to solid organs and regional lymph nodes. Two GEMM-derived cell lines were developed whose transcriptomic and epigenetic landscapes harbored key features of primary GEMM tumors. Importantly, GEMM tumor and cell line transcriptomes co-classified with those of human NC. BRD4-NUT also blocked differentiation and maintained the growth of mNC as in hNC. Mechanistically, GEMM primary tumors and cell lines formed large histone H3K27ac-enriched domains, termed megadomains, that were invariably associated with the expression of key NC-defining proto-oncogenes, Myc and Trp63. Small-molecule BET bromodomain inhibition (BETi) of mNC induced differentiation and growth arrest and prolonged survival of NC GEMMs, as it does in hNC models. Overall, tumor formation in the NC GEMM is definitive evidence that BRD4-NUT alone can potently drive the malignant transformation of squamous progenitor cells into NC. SIGNIFICANCE: The development of an immunocompetent model of NUT carcinoma that closely mimics the human disease provides a valuable global resource for mechanistic and preclinical studies to improve treatment of this incurable disease.

Myeloproliferative Neoplasm Driven by ETV6-ABL1 in an Adolescent with Recent History of Burkitt Leukemia.

ETV6-ABL1 gene fusion is a rare genetic rearrangement in a variety of malignancies, including myeloproliferative neoplasms (MPN), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML). Here, we report the case of a 16-year-old male diagnosed with a MPN, 7 months post-completion of treatment for Burkitt leukaemia. RNA sequencing analysis confirmed the presence of an ETV6-ABL1 fusion transcript, with an intact, in-frame ABL tyrosine-kinase domain. Of note, secondary ETV6-ABL1-rearranged neoplastic diseases have not been reported to date. The patient was started on a tyrosine kinase inhibitor (TKI; imatinib) and, subsequently, underwent a 10/10 matched unrelated haematopoietic stem cell transplant. He is disease-free five years post-transplant. Definitive evidence of the prognostic influence of the ETV6-ABL1 fusion in haematological neoplasms is lacking; however, overall data suggest that it is a poor prognostic factor, particularly in patients with ALL and AML. The presence of this ETV6-ABL1 fusion should be more routinely investigated, especially in patients with a CML-like picture. More routine use of whole-genome and RNA sequencing analyses in clinical diagnostic care, in conjunction with conventional cytogenetics, will facilitate these investigations.

Genomic Microsatellite Signatures Identify Germline Mismatch Repair Deficiency and Risk of Cancer Onset.

PURPOSE: Diagnosis of Mismatch Repair Deficiency (MMRD) is crucial for tumor management and early detection in patients with the cancer predisposition syndrome constitutional mismatch repair deficiency (CMMRD). Current diagnostic tools are cumbersome and inconsistent both in childhood cancers and in determining germline MMRD. PATIENTS AND METHODS: We developed and analyzed a functional Low-pass Genomic Instability Characterization (LOGIC) assay to detect MMRD. The diagnostic performance of LOGIC was compared with that of current established assays including tumor mutational burden, immunohistochemistry, and the microsatellite instability panel. LOGIC was then applied to various normal tissues of patients with CMMRD with comprehensive clinical data including age of cancer presentation. RESULTS: Overall, LOGIC was 100% sensitive and specific in detecting MMRD in childhood cancers (N = 376). It was more sensitive than the microsatellite instability panel (14%, P = 4.3 × 10-12), immunohistochemistry (86%, P = 4.6 × 10-3), or tumor mutational burden (80%, P = 9.1 × 10-4). LOGIC was able to distinguish CMMRD from other cancer predisposition syndromes using blood and saliva DNA (P < .0001, n = 277). In normal cells, MMRDness scores differed between tissues (GI > blood > brain), increased over time in the same individual, and revealed genotype-phenotype associations within the mismatch repair genes. Importantly, increased MMRDness score was associated with younger age of first cancer presentation in individuals with CMMRD (P = 2.2 × 10-5). CONCLUSION: LOGIC was a robust tool for the diagnosis of MMRD in multiple cancer types and in normal tissues. LOGIC may inform therapeutic cancer decisions, provide rapid diagnosis of germline MMRD, and support tailored surveillance for individuals with CMMRD.

The clinical utility of integrative genomics in childhood cancer extends beyond targetable mutations.

We conducted integrative somatic-germline analyses by deeply sequencing 864 cancer-associated genes, complete genomes and transcriptomes for 300 mostly previously treated children and adolescents/young adults with cancer of poor prognosis or with rare tumors enrolled in the SickKids Cancer Sequencing (KiCS) program. Clinically actionable variants were identified in 56% of patients. Improved diagnostic accuracy led to modified management in a subset. Therapeutically targetable variants (54% of patients) were of unanticipated timing and type, with over 20% derived from the germline. Corroborating mutational signatures (SBS3/BRCAness) in patients with germline homologous recombination defects demonstrates the potential utility of PARP inhibitors. Mutational burden was significantly elevated in 9% of patients. Sequential sampling identified changes in therapeutically targetable drivers in over one-third of patients, suggesting benefit from rebiopsy for genomic analysis at the time of relapse. Comprehensive cancer genomic profiling is useful at multiple points in the care trajectory for children and adolescents/young adults with cancer, supporting its integration into early clinical management.

TMBur: a distributable tumor mutation burden approach for whole genome sequencing.

BACKGROUND: Tumor mutation burden (TMB) is a key characteristic used in a tumor-type agnostic context to inform the use of immune checkpoint inhibitors (ICI). Accurate and consistent measurement of TMB is crucial as it can significantly impact patient selection for therapy and clinical trials, with a threshold of 10 mutations/Mb commonly used as an inclusion criterion. Studies have shown that the most significant contributor to variability in mutation counts in whole genome sequence (WGS) data is differences in analysis methods, even more than differences in extraction or library construction methods. Therefore, tools for improving consistency in whole genome TMB estimation are of clinical importance. METHODS: We developed a distributable TMB analysis suite, TMBur, to address the need for genomic TMB estimate consistency in projects that span jurisdictions. TMBur is implemented in Nextflow and performs all analysis steps to generate TMB estimates directly from fastq files, incorporating somatic variant calling with Manta, Strelka2, and Mutect2, and microsatellite instability profiling with MSISensor. These tools are provided in a Singularity container downloaded by the workflow at runtime, allowing the entire workflow to be run identically on most computing platforms. To test the reproducibility of TMBur TMB estimates, we performed replicate runs on WGS data derived from the COLO829 and COLO829BL cell lines at multiple research centres. The clinical value of derived TMB estimates was then evaluated using a cohort of 90 patients with advanced, metastatic cancer that received ICIs following WGS analysis. Patients were split into groups based on a threshold of 10/Mb, and time to progression from initiation of ICIs was examined using Kaplan-Meier and cox-proportional hazards analyses. RESULTS: TMBur produced identical TMB estimates across replicates and at multiple analysis centres. The clinical utility of TMBur-derived TMB estimates were validated, with a genomic TMB ≥ 10/Mb demonstrating improved time to progression, even after correcting for differences in tumor type (HR = 0.39, p = 0.012). CONCLUSIONS: TMBur, a shareable workflow, generates consistent whole genome derived TMB estimates predictive of response to ICIs across multiple analysis centres. Reproducible TMB estimates from this approach can improve collaboration and ensure equitable treatment and clinical trial access spanning jurisdictions.

Understanding barriers to diabetes eye screening in a large rural general practice: an audit of patients not reached by screening services.

Introduction Diabetes is a leading cause of blindness in New Zealanders, yet a significant proportion of patients with diabetes are not reached by diabetes eye screening. Aim Our study audited patients with diabetes in a large New Zealand rural general practice, to identify addressable barriers preventing patients from attending diabetes eye screening. Methods All patients who had missed a diabetes eye screening appointment at the Dargaville Hospital Eye Screening Clinic between 2018 and 2021 were identified. Demographic information, laboratory and disease status data were recorded and analysed. Semi-structured telephone interviews were undertaken with 66 patients exploring barriers to diabetes eye screening. Descriptive statistical analysis was performed on quantitative data and a thematic analysis on qualitative results. Results One-hundred and fifty-four (27%) of 571 patients invited to screening missed at least one appointment; of these, 66 (43%) were interviewed. Quantitative analysis suggested Maori patients were less likely to be reached, with a 20% higher number of missed appointments than European patients and a higher glycated haemoglobin (HbA1c). Maori patients reported greater barriers to attending eye screening. Common barriers identified by participants were transport, work and family commitments, financial, health and lack of appointment reminders. Discussion Increased barriers for Maori patients could explain the reduced ability of the screening service to reach Maori patients. In order to address inequity and increase overall screening rates, diabetes eye screening and primary care services need to improve the booking system, facilitate transport to screening, engage patients and their whanau and build trust.

Diverse Oncogenic Fusions and Distinct Gene Expression Patterns Define the Genomic Landscape of Pediatric Papillary Thyroid Carcinoma.

Pediatric papillary thyroid carcinoma (PPTC) is clinically distinct from adult-onset disease. Although there are higher rates of metastasis and recurrence in PPTC, prognosis remains highly favorable. Molecular characterization of PPTC has been lacking. Historically, only 40% to 50% of childhood papillary thyroid carcinoma (PTC) were known to be driven by genomic variants common to adult PTC; oncogenic drivers in the remainder were unknown. This contrasts with approximately 90% of adult PTC driven by a discrete number of variants. In this study, 52 PPTCs underwent candidate gene testing, followed in a subset by whole-exome and transcriptome sequencing. Within these samples, candidate gene testing identified variants in 31 (60%) tumors, while exome and transcriptome sequencing identified oncogenic variants in 19 of 21 (90%) remaining tumors. The latter were enriched for oncogenic fusions, with 11 nonrecurrent fusion transcripts, including two previously undescribed fusions, STRN-RET and TG-PBF. Most fusions were associated with 3' receptor tyrosine kinase (RTK) moieties: RET, MET, ALK, and NTRK3. For advanced (distally metastatic) tumors, a driver variant was described in 91%. Gene expression analysis defined three clusters that demonstrated distinct expression of genes involved in thyroid differentiation and MAPK signaling. Among RET-CCDC6-driven tumors, gene expression in pediatric tumors was distinguishable from that in adults. Collectively, these results show that the genomic landscape of pediatric PTC is different from adult PTC. Moreover, they identify genomic drivers in 98% of PPTCs, predominantly oncogenic fusion transcripts involving RTKs, with a pronounced impact on gene expression. Notably, most advanced tumors were driven by a variant for which targeted systemic therapy exists. SIGNIFICANCE: This study highlights important distinctions between the genomes and transcriptomes of pediatric and adult papillary thyroid carcinoma, with implications for understanding the biology, diagnosis, and treatment of advanced disease in children.

Ethical and Analytic Challenges With Genomic Sequencing of Relapsed Hematologic Malignancies Following Allogeneic Hematopoietic Stem-Cell Transplantation.

The implementation of precision medicine and next-generation sequencing technologies in the field of oncology is a novel approach being more widely studied and used in cases of high-risk primary and recurrent malignancies. Leukemias are the second most common cause of cancer-related mortality in children and the sixth most in adults. Relapsed leukemia represents a major component of the population that may benefit from genomic sequencing. However, ethical and analytic challenges arise when considering sequencing of biologic samples obtained from patients with relapsed leukemia following allogeneic hematopoietic stem-cell transplantation. Blood from the recipient after transplantation would include donor-derived cells and thus, genomic sequencing of recipient blood will interrogate the donor germline in addition to the somatic genetic profile of the leukemia cells and the recipient germline. This is a situation for which the donor will not have typically provided consent and may be particularly problematic if actionable secondary or incidental findings related to the donor are uncovered. We present the challenges raised in this scenario and provide strategies to mitigate this risk.

DNA Polymerase and Mismatch Repair Exert Distinct Microsatellite Instability Signatures in Normal and Malignant Human Cells.

Although replication repair deficiency, either by mismatch repair deficiency (MMRD) and/or loss of DNA polymerase proofreading, can cause hypermutation in cancer, microsatellite instability (MSI) is considered a hallmark of MMRD alone. By genome-wide analysis of tumors with germline and somatic deficiencies in replication repair, we reveal a novel association between loss of polymerase proofreading and MSI, especially when both components are lost. Analysis of indels in microsatellites (MS-indels) identified five distinct signatures (MS-sigs). MMRD MS-sigs are dominated by multibase losses, whereas mutant-polymerase MS-sigs contain primarily single-base gains. MS deletions in MMRD tumors depend on the original size of the MS and converge to a preferred length, providing mechanistic insight. Finally, we demonstrate that MS-sigs can be a powerful clinical tool for managing individuals with germline MMRD and replication repair-deficient cancers, as they can detect the replication repair deficiency in normal cells and predict their response to immunotherapy. SIGNIFICANCE: Exome- and genome-wide MSI analysis reveals novel signatures that are uniquely attributed to mismatch repair and DNA polymerase. This provides new mechanistic insight into MS maintenance and can be applied clinically for diagnosis of replication repair deficiency and immunotherapy response prediction.This article is highlighted in the In This Issue feature, p. 995.

First report of t(5;11) KMT2A-MAML1 fusion in de novo infant acute lymphoblastic leukemia.

Infant acute lymphoblastic leukemia (ALL) comprises 2.5%-5% of pediatric ALL with inferior survival compared to older children. A majority of infants (80%) with ALL harbor KMT2A gene rearrangement, which portends a poor prognosis. Approximately 94 different partner genes have been identified to date. The common rearrangements include t(4;11)(q21;q23)KMT2A-AFF1,t(11;19) (q23;p13.3)KMT2A-MLLT1 and t(9;11)(p22;q23)KMT2A-MLLT3. We report a novel translocation t(5;11)(q35;q23)KMT2A-MAML1 in newly diagnosed infant precursor B-ALL. Long-term follow-up and a larger number of patients are needed to better understand its prognostic significance.

A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo.

In many repeat diseases, such as Huntington's disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSß. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats.

MYCN Amplified Relapse Following Resolution of MYCN Nonamplified 4S Neuroblastoma With Placental Involvement: A Case Report and Review of the Literature.

Congenital neuroblastoma with placental involvement is exceptionally rare, but mortality is high. Detailed examination of placenta including MYCN amplification and segmental chromosomal aberrations should be performed in all suspected cases, as it is noninvasive and readily available. Maternal dissemination has not been reported. In this manuscript, we describe an infant with placental diagnosis of MYCN nonamplified congenital neuroblastoma. This is the first report of a recurrence of congenital 4S neuroblastoma following resolution in which MYCN amplification is only detected in the recurrence. Germline sequencing using a large comprehensive cancer panel did not reveal variants in candidate cancer predisposition genes.

mTOR Inhibitors as a New Therapeutic Strategy in Treatment Resistant Epilepsy in Hemimegalencephaly: A Case Report.

Hemimegalencephaly is a hamartomatous malformation of one hemisphere. Functional hemispherectomy, the definitive treatment, is associated with significant morbidity and mortality in early infancy. Dysregulation of the mTOR pathway can result in malformations of cortical development, and mTOR inhibitors can effectively reduce seizures in tuberous sclerosis complex. We report a 6-day-old female with hemimegalencephaly and frequent seizures despite 9 antiseizure medications. At 3 months of age, while awaiting hemispherectomy, an mTOR inhibitor, rapamycin, was initiated by the neurologist. After 1 week of treatment, there was >50% reduction in seizures and total seizure burden, and after 2 weeks, development improved, resulting in deferral of surgery by 2.5 months with an increased body weight. Pathology demonstrated cortical dysplasia with upregulation of the mTOR pathway. Deep-sequencing of brain tissue demonstrated 16% mosaicism for a pathogenic de novo MTOR gene mutation. This case exemplifies how mTOR inhibitors could be considered for seizure reduction in patients with hemimegalencephaly while awaiting surgery.

Rearrangement bursts generate canonical gene fusions in bone and soft tissue tumors.

Sarcomas are cancers of the bone and soft tissue often defined by gene fusions. Ewing sarcoma involves fusions between EWSR1, a gene encoding an RNA binding protein, and E26 transformation-specific (ETS) transcription factors. We explored how and when EWSR1-ETS fusions arise by studying the whole genomes of Ewing sarcomas. In 52 of 124 (42%) of tumors, the fusion gene arises by a sudden burst of complex, loop-like rearrangements, a process called chromoplexy, rather than by simple reciprocal translocations. These loops always contained the disease-defining fusion at the center, but they disrupted multiple additional genes. The loops occurred preferentially in early replicating and transcriptionally active genomic regions. Similar loops forming canonical fusions were found in three other sarcoma types. Chromoplexy-generated fusions appear to be associated with an aggressive form of Ewing sarcoma. These loops arise early, giving rise to both primary and relapse Ewing sarcoma tumors, which can continue to evolve in parallel.

Response to Immune Checkpoint Inhibition in Two Patients with Alveolar Soft-Part Sarcoma.

Alveolar soft-part sarcoma (ASPS) is a morphologically distinctive mesenchymal tumor characterized by a canonical ASPL-TFE3 fusion product. In the metastatic setting, standard cytotoxic chemotherapies are typically ineffective. Studies have suggested modest clinical response to multitargeted receptor tyrosine kinase inhibitors. Here, we report sustained partial responses in two patients with immune checkpoint inhibition treated with either durvalumab (anti-PD-L1) alone or in combination with tremelimumab (anti-CTLA-4), which appeared unrelated to tumor immune infiltrates or mutational burden. Genomic analysis of these patients, and other cases of ASPS, demonstrated molecular mismatch-repair deficiency signatures. These findings suggest that immune checkpoint blockade may be a useful therapeutic strategy for ASPS. Cancer Immunol Res; 6(9); 1001-7. ©2018 AACR.

Aggressive embryonal rhabdomyosarcoma in a 3-month-old boy: A clinical and molecular analysis.

Rhabdomyosarcoma (RMS) represents the most common soft tissue sarcoma in the pediatric age group. While RMS has been traditionally classified on the basis of its histological appearance (with embryonal and alveolar being most common), it is now clear that the PAX-FOXO1 fusion product drives prognosis. We report here a case of pelvic embryonal RMS in a 3-month-old male who was subsequently found to have developed brain metastases during the course of chemotherapy. Cytogenetic analysis of the brain metastases at the time of autopsy as well as next-generation sequencing analysis revealed a reciprocal translocation involving the SH3 domain containing ring finger 3 gene (SH3RF3, on chromosome 2q13) and the Lipase C gene (LIPC, on chromosome 15q21.3). Due to the poor quality of the pretreatment and postresection samples, cytogenetics and NGS analysis looking for the presence of this balanced translocation in these specimens could not be performed. To the authors' knowledge, this translocation has never been described in RMS. Further studies are needed to determine the biological and clinical implications of this novel translocation.