Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity.

BACKGROUND: Cancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms. RESULTS: In sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms. CONCLUSION: This work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

Germline PTCH1: c.361_362insAlu alteration identified by comprehensive exome and RNA sequencing in a patient with Gorlin syndrome.

Gorlin syndrome can be caused by pathogenic/likely pathogenic (P/LP) variants in the tumor suppressor gene PTCH1 (9q22.1-q31), which encodes the receptor for the sonic hedgehog (SHH) ligand. We present a 12-month-old boy clinically diagnosed with Gorlin syndrome who was found to have significantly delayed development, palmar pitting, palmar and plantar keratosis, short hands, frontal bossing, coarse face, hypertelorism, a bifid rib, misaligned and missing teeth, and SHH-activated medulloblastoma. Genetic testing, including a pediatric cancer panel and genome sequencing with peripheral blood, failed to identify any P/LP variants in PTCH1. Paired tumor/normal exome sequencing was performed, which identified a germline NM_000264.5 (PTCH1): c.361_362ins? alteration through manual review of sequencing reads. Clinical RNA sequencing further demonstrated an Alu insertion at this region (PTCH1: c.361_362insAlu), providing molecular confirmation of Gorlin syndrome. This finding exemplifies a unique mechanism for PTCH1 disruption in the germline and highlights the importance of comprehensive analysis, including manual review of DNA sequencing reads and the utility of RNA analysis to detect variant types which may not be identified by routine genetic screening techniques.

EGFR internal tandem duplications in fusion-negative congenital and neonatal spindle cell tumors.

Next-generation sequencing (NGS) assays can sensitively detect somatic variation, and increasingly can enable the identification of complex structural rearrangements. A subset of infantile spindle cell sarcomas, particularly congenital mesoblastic nephromas with classic or mixed histology, have structural rearrangement in the form of internal tandem duplications (ITD) involving EGFR. We performed prospective analysis to identify EGFR ITD through clinical or research studies, as well as retrospective analysis to quantify the frequency of EGFR ITD in pediatric sarcomas. Within our institution, three tumors with EGFR ITD were prospectively identified, all occurring in patients less than 1 year of age at diagnosis, including two renal tumors and one mediastinal soft tissue tumor. These three cases exhibited both cellular and mixed cellular and classic histology. All patients had no evidence of disease progression off therapy, despite incomplete resection. To extend our analysis and quantify the frequency of EGFR ITD in pediatric sarcomas, we retrospectively analyzed a cohort of tumors (n = 90) that were previously negative for clinical RT-PCR-based fusion testing. We identified EGFR ITD in three analyzed cases, all in patients less than 1 year of age (n = 18; 3/18, 17%). Here we expand the spectrum of tumors with EGFR ITD to congenital soft tissue tumors and report an unusual example of an EGFR ITD in a tumor with cellular congenital mesoblastic nephroma histology. We also highlight the importance of appropriate test selection and bioinformatic analysis for identification of this genomic alteration that is unexpectedly common in congenital and infantile spindle cell tumors.

Infantile metastatic ependymoma with a novel molecular profile and favorable outcome to intensive chemotherapy without irradiation: Case-based review.

Ependymal tumors are the third most common brain tumor under 14 years old. Even though metastatic disease is a rare event, it affects mostly young children and carries an adverse prognosis. The factors associated with dissemination and the best treatment approach have not yet been established and there is limited published data on how to manage metastatic disease, especially in patients under 3 years of age. We provide a review of the literature on clinical characteristics and radiation-sparing treatments for metastatic ependymoma in children under 3 years of age treated. The majority (73%) of the identified cases were above 12 months old and had the PF as the primary site at diagnosis. Chemotherapy-based approaches, in different regimens, were used with radiation reserved for progression or relapse. The prognosis varied among the studies, with an average of 50%-58% overall survival. This study also describes the case of a 7-month-old boy with metastatic posterior fossa (PF) ependymoma, for whom we identified a novel SPECC1L-RAF1 gene fusion using a patient-centric comprehensive molecular profiling protocol. The patient was successfully treated with intensive induction chemotherapy followed by high-dose chemotherapy and autologous hematopoietic progenitor cell rescue (AuHSCR). Currently, the patient is in continuous remission 5 years after his diagnosis, without radiation therapy. The understanding of the available therapeutic approaches may assist physicians in their management of such patients. This report also opens the perspective of newly identified molecular alterations in metastatic ependymomas that might drive more chemo-sensitive tumors.

Detection of brain somatic variation in epilepsy-associated developmental lesions.

OBJECTIVE: Epilepsy-associated developmental lesions, including malformations of cortical development and low-grade developmental tumors, represent a major cause of drug-resistant seizures requiring surgical intervention in children. Brain-restricted somatic mosaicism has been implicated in the genetic etiology of these lesions; however, many contributory genes remain unidentified. METHODS: We enrolled 50 children who were undergoing epilepsy surgery into a translational research study. Resected tissue was divided for clinical neuropathologic evaluation and genomic analysis. We performed exome and RNA sequencing to identify somatic variation and we confirmed our findings using high-depth targeted DNA sequencing. RESULTS: We uncovered candidate disease-causing somatic variation affecting 28 patients (56%), as well as candidate germline variants affecting 4 patients (8%). In agreement with previous studies, we identified somatic variation affecting solute carrier family 35 member A2 (SLC35A2) and mechanistic target of rapamycin kinase (MTOR) pathway genes in patients with focal cortical dysplasia. Somatic gains of chromosome 1q were detected in 30% (3 of 10) of patients with Type I focal cortical dysplasia (FCD)s. Somatic variation in mitogen-activated protein kinase (MAPK) pathway genes (i.e., fibroblast growth factor receptor 1 [FGFR1], FGFR2, B-raf proto-oncogene, serine/threonine kinase [BRAF], and KRAS proto-oncogene, GTPase [KRAS]) was associated with low-grade epilepsy-associated developmental tumors. RNA sequencing enabled the detection of somatic structural variation that would have otherwise been missed, and which accounted for more than one-half of epilepsy-associated tumor diagnoses. Sampling across multiple anatomic regions revealed that somatic variant allele fractions vary widely within epileptogenic tissue. Finally, we identified putative disease-causing variants in genes not yet associated with focal cortical dysplasia. SIGNIFICANCE: These results further elucidate the genetic basis of structural brain abnormalities leading to focal epilepsy in children and point to new candidate disease genes.

Novel morphologic findings in PLAG1-rearranged soft tissue tumors.

Oncogenesis in PLAG1-rearranged tumors often results from PLAG1 transcription factor overexpression driven by promoter-swapping between constitutively expressed fusion partners. PLAG1-rearranged tumors demonstrate diverse morphologies. This study adds to this morphologic heterogeneity by introducing two tumors with PLAG1 rearrangements that display distinct histologic features. The first arose in the inguinal region of a 3-year-old, appeared well-circumscribed with a multinodular pattern, and harbored two fusions: ZFHX4-PLAG1 and CHCHD7-PLAG1. The second arose in the pelvic cavity of a 15-year-old girl, was extensively infiltrative and vascularized with an adipocytic component, and demonstrated a COL3A1-PLAG1 fusion. Both showed low-grade cytomorphology, scarce mitoses, no necrosis, and expression of CD34 and desmin. The ZFHX4-/CHCHD7-PLAG1-rearranged tumor showed no evidence of recurrence after 5 months. By contrast, the COL3A1-PLAG1-rearranged tumor quickly recurred following primary excision with positive margins; subsequent re-excision with adjuvant chemotherapy resulted in no evidence of recurrence after 2 years. While both tumors show overlap with benign and malignant fibroblastic and fibrovascular neoplasms, they also display divergent features. These cases highlight the importance of appropriate characterization in soft tissue tumors with unusual clinical and histologic characteristics.

Gastroblastoma with a novel EWSR1-CTBP1 fusion presenting in adolescence.

Gastroblastomas are rare tumors with a biphasic epithelioid/spindle cell morphology that typically present in early adulthood and have recurrent MALAT1-GLI1 fusions. We describe an adolescent patient with Wiskott-Aldrich syndrome who presented with a large submucosal gastric tumor with biphasic morphology. Despite histologic features consistent with gastroblastoma, a MALAT1-GLI1 fusion was not found in this patient's tumor; instead, comprehensive molecular profiling identified a novel EWSR1-CTBP1 fusion and no other significant genetic alterations. The tumor also overexpressed NOTCH and FGFR by RNA profiling. The novel fusion and expression profile suggest a role for epithelial-mesenchymal transition in this tumor, with potential implications for the pathogenesis of biphasic gastric tumors such as gastroblastoma.

Discovery of clinically relevant fusions in pediatric cancer.

BACKGROUND: Pediatric cancers typically have a distinct genomic landscape when compared to adult cancers and frequently carry somatic gene fusion events that alter gene expression and drive tumorigenesis. Sensitive and specific detection of gene fusions through the analysis of next-generation-based RNA sequencing (RNA-Seq) data is computationally challenging and may be confounded by low tumor cellularity or underlying genomic complexity. Furthermore, numerous computational tools are available to identify fusions from supporting RNA-Seq reads, yet each algorithm demonstrates unique variability in sensitivity and precision, and no clearly superior approach currently exists. To overcome these challenges, we have developed an ensemble fusion calling approach to increase the accuracy of identifying fusions. RESULTS: Our Ensemble Fusion (EnFusion) approach utilizes seven fusion calling algorithms: Arriba, CICERO, FusionMap, FusionCatcher, JAFFA, MapSplice, and STAR-Fusion, which are packaged as a fully automated pipeline using Docker and Amazon Web Services (AWS) serverless technology. This method uses paired end RNA-Seq sequence reads as input, and the output from each algorithm is examined to identify fusions detected by a consensus of at least three algorithms. These consensus fusion results are filtered by comparison to an internal database to remove likely artifactual fusions occurring at high frequencies in our internal cohort, while a "known fusion list" prevents failure to report known pathogenic events. We have employed the EnFusion pipeline on RNA-Seq data from 229 patients with pediatric cancer or blood disorders studied under an IRB-approved protocol. The samples consist of 138 central nervous system tumors, 73 solid tumors, and 18 hematologic malignancies or disorders. The combination of an ensemble fusion-calling pipeline and a knowledge-based filtering strategy identified 67 clinically relevant fusions among our cohort (diagnostic yield of 29.3%), including RBPMS-MET, BCAN-NTRK1, and TRIM22-BRAF fusions. Following clinical confirmation and reporting in the patient's medical record, both known and novel fusions provided medically meaningful information. CONCLUSIONS: The EnFusion pipeline offers a streamlined approach to discover fusions in cancer, at higher levels of sensitivity and accuracy than single algorithm methods. Furthermore, this method accurately identifies driver fusions in pediatric cancer, providing clinical impact by contributing evidence to diagnosis and, when appropriate, indicating targeted therapies.

Targeted Therapy in a Young Adult With a Novel Epithelioid Tumor Driven by a PRRC2B-ALK Fusion.

This case report describes an 18-year-old woman with an unusual epithelioid tumor of the omentum with a novel PRRC2B-ALK fusion. Although the atypical pathologic features raised significant diagnostic challenges, expression of CD30 on tumor cells and detection of an ALK rearrangement provided critical information for selecting targeted therapy in a patient not suitable for surgical resection. Despite an initially promising therapeutic response, the patient died. The efficacy of treatment was confirmed by the lack of viable tumor cells at autopsy. This case highlights the role of timely targeted therapy in patients with rare tumors and novel actionable molecular targets.

CD163L1+CXCL10+ Macrophages are Enriched Within Colonic Lamina Propria of Diverticulitis Patients.

BACKGROUND: Inflammation of diverticula, which are outpouchings of the colonic bowl wall, causes diverticulitis. Severe cases of diverticulitis require surgical intervention. Through RNA-seq analysis of intestinal tissues, we previously found that the innate immune response was deregulated in surgical diverticulitis patients. In that study, pro-inflammatory and macrophage markers were differentially expressed in the colons of diverticulitis versus control patients. Here we investigate CD163L1+ macrophages and the pro-inflammatory chemokine, CXCL10, in diverticulitis. MATERIALS AND METHODS: We assessed tissue from an uninvolved area adjacent to a region of the sigmoid colon chronically affected by diverticulitis and performed Spearman's correlation on transcripts associated with macrophage signaling. We identified altered CD163L1 and CXCL10 gene expression levels that we confirmed by RT-qPCR analysis on an independent cohort of diverticulitis patients and controls. We used immunofluorescence microscopy to localize CD163L1+ macrophages and CXCL10 levels in intestinal tissue and ELISA to measure CXCL10 levels in patient serum. RESULTS: We found a positive correlation between intestinal CD163L1 and CXCL10 gene expression and an increased number of CD163L1+ macrophages in the sigmoid colons of diverticulitis patients relative to controls (P = 0.036). Macrophages at the apices of colonic crypts expressed the chemokine CXCL10. Correspondingly, these diverticulitis patients also displayed heightened CXCL10 levels in their serum (P = 0.007). CONCLUSIONS: We identified a novel population of CD163L1+CXCL10+ macrophages in the colonic crypts of diverticulitis patients and demonstrated increased expression of serum CXCL10 in these patients. CXCL10 may serve as a prognostic biomarker to aid in clinical decision making for diverticulitis patients.

COLQ and ARHGAP15 are Associated with Diverticular Disease and are Expressed in the Colon.

BACKGROUND: Diverticular disease is a common but poorly understood disease of the gastrointestinal tract. Recent studies have identified several single nucleotide polymorphisms (SNPs) that are associated with diverticular disease. MATERIALS AND METHODS: The genotypes of three SNPs (rs4662344 in ARHGAP15, rs7609897 in COLQ, and rs67153654 in FAM155A) were identified by Taqman assay in 204 patients with diverticular disease. Clinical characteristics were obtained from the medical record to study association with genotype. To evaluate gene expression in colon tissue, qPCR was performed on 24 patients with diverticulitis, and COLQ was localized using immunohistochemistry. RESULTS: The ARHGAP15 and COLQ SNPs were significantly associated with both diverticular disease and specifically diverticulitis, while the FAM155A was not associated with either. No association was found with clinical disease characteristics. Heterozygous genotypes at the ARHGAP15 SNP was associated with lower ARHGAP15 expression in colon tissues. COLQ protein localized to the myenteric plexus in the colon. CONCLUSIONS: This study confirmed association of the ARHGAP15 and COLQ SNPs with diverticular disease in our patients but could not confirm FAM155A SNP association. Neither of these SNPs appeared to associate with more severe disease, but genotype at the ARHGAP15 SNP did impact expression of ARHGAP15 in the colon. Additionally, this study is the first to localize COLQ in the colon. Its presence in the myenteric nervous system suggests COLQ SNP variants may contribute to diverticular disease by altering motility.

Multifocal Versus Conventional Unifocal Diverticulitis: A Comparison of Clinical and Transcriptomic Characteristics.

BACKGROUND: The management of diverticulitis is compromised by difficulty in identifying patients who require surgery for recurrent or persistent disease. Here, we introduce the concept of multifocal diverticulitis (MFD), characterized by multiple episodes of diverticulitis occurring at different locations within the colon. AIMS: To compare clinical characteristics, success of surgical management, and colonic transcriptomes of MFD patients to patients with conventional unifocal diverticulitis (UFD). METHODS: This retrospective study included 404 patients with CT-confirmed diverticulitis episodes. Patients with diverticulitis seen in at least two different colonic locations were classified as the MFD group and compared to the UFD group based on number of episodes, sites of disease, family history, surgeries performed, and postoperative recurrence. RNA-seq was conducted on full-thickness colonic tissues of ten MFD and 11 UFD patients. RESULTS: Twenty-eight patients (6.9%) with MFD were identified. MFD patients had more diverticulitis episodes and were more likely to have positive family history, have right-sided disease, require surgery, and have recurrence after surgery. All MFD patients treated with segmental resection had recurrence, while recurrence was less common in patients undergoing more extensive surgery (P < 0.001). Using RNA-seq, we identified 69 genes that were differentially expressed between MFD and UFD patients. Significantly down-regulated genes were associated with immune response pathways. CONCLUSIONS: MFD appears to be a more severe subset of diverticulitis with a possible genetic component. Transcriptomic data suggest that MFD may be associated with alteration of the immune response.

RNA-seq implicates deregulation of the immune system in the pathogenesis of diverticulitis.

Individuals with diverticula or outpouchings of the colonic mucosa and submucosa through the colonic wall have diverticulosis, which is usually asymptomatic. In 10-25% of individuals, the diverticula become inflamed, resulting in diverticulitis. Very little is known about the pathophysiology or gene regulatory pathways involved in the development of diverticulitis. To identify these pathways, we deep sequenced RNAs isolated from full-thickness sections of sigmoid colon from diverticulitis patients and control individuals. Specifically for diverticulitis cases, we analyzed tissue adjacent to areas affected by chronic disease. Since the tissue was collected during elective sigmoid resection, the disease was in a quiescent state. A comparison of differentially expressed genes found that gene ontology (GO) pathways associated with the immune response were upregulated in diverticulitis patients compared with nondiverticulosis controls. Next, weighted gene coexpression network analysis was performed to identify the interaction among coexpressed genes. This analysis revealed RASAL3, SASH3, PTPRC, and INPP5D as hub genes within the brown module eigengene, which highly correlated (r = 0.67, P = 0.0004) with diverticulitis. Additionally, we identified elevated expression of downstream interacting genes. In summary, transcripts associated with the immune response were upregulated in adjacent tissue from the sigmoid colons of chronic, recurrent diverticulitis patients. Further elucidating the genetic or epigenetic mechanisms associated with these alterations can help identify those at risk for chronic disease and may assist in clinical decision management.NEW & NOTEWORTHY By using an unbiased approach to analyze transcripts expressed in unaffected colonic tissues adjacent to those affected by chronic diverticulitis, our study implicates that a defect in the immune response may be involved in the development of the disease. This finding expands on the current data that suggest the pathophysiology of diverticulitis is mediated by dietary, age, and obesity-related factors. Further characterizing the immunologic differences in diverticulitis may better inform clinical decision-making.

Development of a total colectomy and ileorectal anastomosis rat model to evaluate colonic metaplasia.

BACKGROUND: Ulcerative colitis is an idiopathic inflammatory condition of the colon that may require surgical intervention including proctocolectomy and either ileal pouch-anal anastomosis or in the pediatric population, low ileorectal anastomosis (IRA). Often, subsequent physiologic alteration (or colonic metaplasia) occurs in the anastomosed small bowel that includes changes in mucin content, villous blunting, and increased expression of WNT5A, a marker of colonic crypt regeneration. We developed a rat low IRA model to assess and study the development of colonic metaplasia. MATERIALS AND METHODS: We subjected male Sprague-Dawley rats (n = 17) to total colectomy and low IRA surgery and evaluated healing periodically by endoscopic evaluation. The ileum upstream of the anastomosis was assessed by hematoxylin and eosin staining, and the mucin content was measured by high iron diamine-Alcian blue staining. Wnt5a transcripts were quantified by reverse transcription and quantitative polymerase chain reaction at the 8-wk study end point. RESULTS: Although no gross endoscopic evidence of inflammation was seen throughout the course of the study, colonic metaplasia in the small bowel was detected in 7 out of 10 (70%) rats at the study end point. In rats with colonic metaplasia, enhanced expression of Wnt5a was evident at the study end point compared to levels in the terminal ileum at the time of surgery. CONCLUSIONS: Within 4-8 wk, the majority of rats subjected to IRA developed colonic metaplasia defined by villous blunting, changes in mucin content, and increased expression of Wnt5a. This model provides a method to study small bowel colonic metaplasia.

Clinical Cytogenetics: Current Practices and Beyond.

BACKGROUND: Throughout history, the field of cytogenetics has witnessed significant changes due to the constant evolution of technologies used to assess chromosome number and structure. Similar to the evolution of single nucleotide variant detection from Sanger sequencing to next-generation sequencing, the identification of chromosome alterations has progressed from banding to fluorescence in situ hybridization (FISH) to chromosomal microarrays. More recently, emerging technologies such as optical genome mapping and genome sequencing have made noteworthy contributions to clinical laboratory testing in the field of cytogenetics. CONTENT: In this review, we journey through some of the most pivotal discoveries that have shaped the development of clinical cytogenetics testing. We also explore the current test offerings, their uses and limitations, and future directions in technology advancements. SUMMARY: Cytogenetics methods, including banding and targeted assessments like FISH, continue to hold crucial roles in cytogenetic testing. These methods offer a rapid turnaround time, especially for conditions with a known etiology involving recognized cytogenetic aberrations. Additionally, laboratories have the flexibility to now employ higher-throughput methodologies to enhance resolution for cases with greater complexity.

A LINE-1 mediated deletion resulting in germline retinoblastoma predisposition.

Retinoblastoma is an ocular cancer associated with genomic variation in the RB1 gene. In individuals with bilateral retinoblastoma, a germline variant in RB1 is identified in virtually all cases. We describe herein an individual with bilateral retinoblastoma for whom multiple clinical lab assays performed by outside commercial laboratories failed to identify a germline RB1 variant. Paired tumor/normal exome sequencing, long-read whole genome sequencing, and long-read isoform sequencing was performed on a translational research basis ultimately identified a germline likely de novo Long Interspersed Nuclear Element (LINE)-1 mediated deletion resulting in a premature stop of translation of RB1 as the underlying genetic cause of retinoblastoma in this individual. Based on these research findings, the LINE-1 mediated deletion was confirmed via Sanger sequencing in our clinical laboratory, and results were reported in the patient's medical record to allow for appropriate genetic counseling.

Clinically significant findings in a decade-long retrospective study of prenatal chromosomal microarray testing.

BACKGROUND: Chromosomal microarray (CMA) is commonly utilized in the obstetrics setting. CMA is recommended when one or more fetal structural abnormalities is identified. CMA is also commonly used to determine genetic etiologies for miscarriages, fetal demise, and confirming positive prenatal cell-free DNA screening results. METHODS: In this study, we retrospectively examined 523 prenatal and 319 products-of-conception (POC) CMA cases tested at Nationwide Children's Hospital from 2011 to 2020. We reviewed the referral indications, the diagnostic yield, and the reported copy number variants (CNV) findings. RESULTS: In our cohort, the diagnostic yield of clinically significant CNV findings for prenatal testing was 7.8% (n = 41/523) compared to POC testing (16.3%, n = 52/319). Abnormal ultrasound findings were the most common indication present in 81% of prenatal samples. Intrauterine fetal demise was the common indication identified in POC samples. The most common pathogenic finding observed in all samples was isolated trisomy 21, detected in seven samples. CONCLUSION: Our CMA study supports the clinical utility of prenatal CMA for clinical management and identifying genetic etiology in POC arrays. In addition, it provides insight to the spectrum of prenatal and POC CMA results as detected in an academic hospital clinical laboratory setting that serves as a reference laboratory.

CHEK2 Alterations in Pediatric Malignancy: A Single-Institution Experience.

BACKGROUND: Approximately 10% of pediatric malignancies are secondary to germline alterations in cancer-predisposing genes. Checkpoint kinase 2 (CHEK2) germline loss-of-function variants have been reported in pediatric cancer patients, but clinical phenotypes and outcomes are poorly described. We present our single-institution experience of pediatric oncology patients with CHEK2 germline alterations, including clinical presentations and outcomes. METHODS: Pediatric oncology patients with CHEK2 germline alterations were identified among those assessed by clinical or translational research at the Institute for Genomic Medicine at Nationwide Children's Hospital. A chart review of disease course was conducted on identified patients. RESULTS: We identified 6 patients with germline CHEK2 variants from a cohort of 300 individuals, including 1 patient with concurrent presentation of Burkitt lymphoma and neuroblastoma, 3 patients with brain tumors, 1 patient with Ewing sarcoma, and 1 patient with myelodysplastic syndrome. Three patients had a family history of malignancies. Four patients were in remission; one was undergoing treatment; one patient had developed treatment-related meningiomas. We review prior data regarding CHEK2 variants in this population, challenges associated with variant interpretation, and genetic counseling for individuals with CHEK2 variants. CONCLUSIONS: CHEK2 germline loss-of-function alterations occur in patients with a variety of pediatric tumors. Larger multicenter studies will improve our understanding of the incidence, phenotype, and molecular biology of CHEK2 germline variants in pediatric cancers.

Expanding the Clinical Utility of Targeted RNA Sequencing Panels beyond Gene Fusions to Complex, Intragenic Structural Rearrangements.

Gene fusions are a form of structural rearrangement well established as driver events in pediatric and adult cancers. The identification of such events holds clinical significance in the refinement, prognostication, and provision of treatment in cancer. Structural rearrangements also extend beyond fusions to include intragenic rearrangements, such as internal tandem duplications (ITDs) or exon-level deletions. These intragenic events have been increasingly implicated as cancer-promoting events. However, the detection of intragenic rearrangements may be challenging to resolve bioinformatically with short-read sequencing technologies and therefore may not be routinely assessed in panel-based testing. Within an academic clinical laboratory, over three years, a total of 608 disease-involved samples (522 hematologic malignancy, 86 solid tumors) underwent clinical testing using Anchored Multiplex PCR (AMP)-based RNA sequencing. Hematologic malignancies were evaluated using a custom Pan-Heme 154 gene panel, while solid tumors were assessed using a custom Pan-Solid 115 gene panel. Gene fusions, ITDs, and intragenic deletions were assessed for diagnostic, prognostic, or therapeutic significance. When considering gene fusions alone, we report an overall diagnostic yield of 36% (37% hematologic malignancy, 41% solid tumors). When including intragenic structural rearrangements, the overall diagnostic yield increased to 48% (48% hematologic malignancy, 45% solid tumor). We demonstrate the clinical utility of reporting structural rearrangements, including gene fusions and intragenic structural rearrangements, using an AMP-based RNA sequencing panel.