Gene Panel Testing Research Articles

Comprehensive genomic profiling of Japanese patients with thoracic malignancies: A single-center retrospective study

BackgroundFew studies have been conducted on comprehensive genomic profiling (CGP) panels in Japanese patients with thoracic malignancies after completing standard treatment. Consequently, its value in clinical practice remains unclear. MethodsWe conducted a retrospective study of Japanese patients with thoracic malignancies who underwent CGP between June 2019 and November 2022 at our hospital. We evaluated the detection rate of actionable genetic alterations and percentage of patients who received genomically-matched therapy. Furthermore, we examined the value of the CGP panel in patients who underwent multiplex gene-panel testing prior to their initial treatment. This study was performed in accordance with the principles of the Declaration of Helsinki. ResultsThe study included 56 patients, of whom 47 (83.9%) had actionable genetic alterations and 8 (14.3%) received genomically-matched therapy. Of these, four patients were treated with approved drugs and three patients were treated with investigational agents. In addition, one patient was treated with approved drugs using the patient-directed care system. Of the 17 patients who had multiplex gene-panel testing performed at the start of their initial therapy, two (11.8%) were newly identified by the CGP panel and subsequently received genomically-matched therapy. EGFR L718Q and MET amplification were observed in two of the seven patients with epidermal growth factor receptor-tyrosine kinase inhibitor resistance. ConclusionsThe CGP panel could identify genetic alterations, thereby facilitating genomically-matched therapy, even in patients with thoracic malignancies who could not be identified using multiplex gene-panel testing.

DMD-Associated Dilated Cardiomyopathy: Genotypes, Phenotypes, and Phenocopies.

Variants in the DMD gene, that encodes the cytoskeletal protein, dystrophin, cause a severe form of dilated cardiomyopathy (DCM) associated with high rates of heart failure, heart transplantation, and ventricular arrhythmias. Improved early detection of individuals at risk is needed. Genetic testing of 40 male probands with a potential X-linked genetic cause of primary DCM was undertaken using multi-gene panel sequencing, multiplex polymerase chain reaction, and array comparative genomic hybridization. Variant location was assessed with respect to dystrophin isoform patterns and exon usage. Telomere length was evaluated as a marker of myocardial dysfunction in left ventricular tissue and blood. Four pathogenic/likely pathogenic DMD variants were found in 5 probands (5/40: 12.5%). Only one rare variant was identified by gene panel testing with 3 additional multi-exon deletion/duplications found following targeted assays for structural variants. All of the pathogenic/likely pathogenic DMD variants involved dystrophin exons that had percent spliced-in scores >90, indicating high levels of constitutive expression in the human adult heart. Fifteen DMD variant-negative probands (15/40: 37.5%) had variants in autosomal genes including TTN, BAG3, LMNA, and RBM20. Myocardial telomere length was reduced in patients with DCM irrespective of genotype. No differences in blood telomere length were observed between genotype-positive family members with/without DCM and controls. Primary genetic testing using multi-gene panels has a low yield and specific assays for structural variants are required if DMD-associated cardiomyopathy is suspected. Distinguishing X-linked causes of DCM from autosomal genes that show sex differences in clinical presentation is crucial for informed family management.

Genetic diagnosis of skeletal dysplasias causing short stature in children

Short stature may often be due to skeletal dysplasias affecting the limbs, spine, or both. A careful clinical evaluation will help in identifying the rhizomelic and mesomelic shortening of the limbs and scoliosis. The mutations in different genes involved in various pathways in skeletal development lead to phenotypes that present from infancy to childhood or adulthood. A systematic clinical evaluation with identification of the limb bowing or deformity, dysmorphic features, radiological findings from skull to toes, and a single gene or multi-gene panel testing will help in making an appropriate diagnosis. The clinical clues to skeletal dysplasia include skeletal disproportion, unexplained limb bowing, recurrent fractures, facial dysmorphism including flat facies and blue sclera in severe cases, and sometimes typical digital or cardiac abnormalities. The following review focuses on the postnatal presentation of skeletal dysplasias mostly referred for evaluation of short stature.

Comparison of the novel Franseen needle versus the fine-needle aspiration needle in endoscopic ultrasound-guided tissue acquisition for cancer gene panel testing: A propensity score-matching analysis.

Reports have indicated that a surface area of 4 mm2 or more of collected tissue sections could provide the recommended total DNA for the OncoGuide NCC Oncopanel system, which is a cancer gene panel test developed in Japan. We wished to compare the percentage of tissue sections collected by endoscopic ultrasound-assisted tissue acquisition (EUS-TA) with surface areas of ≥4 mm2 between a conventional needle, namely the EZ Shot 3 Plus (Olympus Medical Japan, Tokyo, Japan) (EZ3), and the recent SonoTip TopGain (MediGlobe, Rohrdorf, Germany) (TopGain). From April 2010 to December 2021, among 693 EUS-TA cases, EZ3 was used in 390 cases and TopGain in 45. The EZ3 and TopGain groups were matched in a 1:1 ratio with a tolerance of 0.2, with 35 patients each matched using propensity score analysis. The TopGain group had a significantly higher percentage of cases with a tissue area of ≥4 mm2 than the EZ3 group (42.9% vs 68.6%, P = 0.030). Multivariate analysis revealed an association between TopGain and tissue areas of ≥4 mm2 (odds ratio 2.996, 95% confidence interval 1.068-8.403, P = 0.037). EUS-TA using TopGain significantly collected more ≥4 mm2 tissue area compared with EZ3, suggesting its usefulness for cancer gene panel testing.

Pathogenic Germline Mutational Landscape in Patients With Renal Cell Carcinoma and Associated Clinicopathologic Features.

A subset of renal cell carcinoma (RCC) cases occur because of a hereditary predisposition. However, the prevalence and profiling of germline alterations in RCC have not been fully characterized. Additionally, clinicopathologic factors associated with pathogenic or likely pathogenic (P/LP) germline variants in patients with RCC remain poorly understood. A retrospective analysis of patients with RCC who underwent genetic evaluation was performed. The frequency of P/LP germline variants and genes was evaluated in this cohort. The association between genetic testing outcomes and clinicopathologic features was also assessed. A total of 321 patients with RCC who had germline testing were identified. Within this cohort, 42 patients (13.1%) had P/LP variants. Genes with the most frequent germline mutations were FLCN (n = 10, 3.1%), SDHB (n = 4, 1.2%), VHL (n = 4, 1.2%), MLH1 (n = 3, 0.9%), and CHEK2 (n = 4, 1.2%). Among patients with P/LP variants, 19 (45.2%) had a potentially targetable mutation. The presence of bilateral or multifocal tumors was associated with P/LP variants (P = .0012 and P = .0098, respectively). Patients who had targeted gene testing had higher rates of P/LP variants compared with multigene panel testing (P = .015). Age and family history of cancers (RCC and non-RCC) did not have any statistically significant association with germline testing outcomes. Among patients with RCC, unselected for a known familial predisposition, 13.4% had P/LP variants. Almost half of patients with P/LP variants had a potentially targetable mutation. Targeted gene panel testing is a feasible option for patients, particularly if syndromic features are present. Age and family history were not associated with P/LP variants. Future studies are needed to optimize current genetic evaluation criteria to expand the detection of patients with RCC who may have germline mutations.

Organization of the epileptogenic zone and signal analysis at seizure onset in patients with drug-resistant epilepsy due to focal cortical dysplasia with mTOR pathway gene mutations-An SEEG study.

Epilepsy surgery in genetic drug-resistant epilepsy is a debated subject as more histological and molecular data are available. We retrospectively collected data from focal drug-resistant epilepsy patients that underwent stereoelectroencephalography (SEEG) invasive recordings. Patients with nonlesional brain imaging or in whom a first epilepsy surgery failed to control seizures were selected. We computed and displayed the intracranial ictal onset activity pattern on structural imaging. Patients underwent epilepsy gene panel testing, next generation sequencing-NGS. Of 113 patients, 13 underwent genetic testing, and in 6 patients, a mechanistic target of rapamycin pathway gene germline mutation (mTOR) was identified. Brain imaging was nonlesional except for one patient in whom two abnormalities suggestive of focal cortical dysplasia (FCD) were found. Patients underwent tailored brain surgery based on SEEG data, tissue analysis revealed FCD and postsurgical outcome was favorable. Our findings are similar to previous case series suggesting that epilepsy surgery can be a treatment option in patients with mTOR pathway mutation. In patients with mTOR pathway mutation, the postsurgical outcome is favorable if complete resection of the epileptogenic zone is performed. Electrophysiological seizure onset patterns in FCDs associated with mTOR pathway mutations display low-voltage fast activity as previously described.

Patient-Derived Ex Vivo Cultures and Endpoint Assays with Surrogate Biomarkers in Functional Testing for Prediction of Therapeutic Response.

Prediction of therapeutic outcomes is important for cancer patients in order to reduce side effects and improve the efficacy of anti-cancer drugs. Currently, the most widely accepted method for predicting the efficacy of anti-cancer drugs is gene panel testing based on next-generation sequencing. However, gene panel testing has several limitations. For example, only 10% of cancer patients are estimated to have druggable mutations, even if whole-exome sequencing is applied. Additionally, even if optimal drugs are selected, a significant proportion of patients derive no benefit from the indicated drug treatment. Furthermore, most of the anti-cancer drugs selected by gene panel testing are molecularly targeted drugs, and the efficacies of cytotoxic drugs remain difficult to predict. Apart from gene panel testing, attempts to predict chemotherapeutic efficacy using ex vivo cultures from cancer patients have been increasing. Several groups have retrospectively demonstrated correlations between ex vivo drug sensitivity and clinical outcome. For ex vivo culture, surgically resected tumor tissue is the most abundant source. However, patients with recurrent or metastatic tumors do not usually undergo surgery, and chemotherapy may be the only option for those with inoperable tumors. Therefore, predictive methods using small amounts of cancer tissue from diagnostic materials such as endoscopic, fine-needle aspirates, needle cores and liquid biopsies are needed. To achieve this, various types of ex vivo culture and endpoint assays using effective surrogate biomarkers of drug sensitivity have recently been developed. Here, we review the variety of ex vivo cultures and endpoint assays currently available.

Clinical availability and characteristics of multigene panel testing for recurrent/advanced gynecologic cancer.

Japan's health insurance covers multigene panel testing. This study aimed to determine the potential availability and utility of gene panel testing clinically in gynecologic oncology. We analyzed the characteristics of patients with gynecologic cancer who underwent gene panel testing using FoundationOne® CDx or OncoGuide™ NCC Oncopanel between November 2019 and October 2022. Out of 102 patients analyzed, 32, 18, 43, 8, and 1 had cervical, endometrial, ovarian cancers, sarcoma, and vaginal cancer, respectively. Druggable gene alteration was found in 70 patients (68.6%; 21 with cervical cancer, 15 with endometrial cancer, 28 with ovarian cancer, 5 with sarcoma, and 1 with other). The most common druggable gene alteration was PIK3CA mutation (n = 21), followed by PTEN mutation (n = 12) and high tumor mutation burden (TMB-H) (n = 11). TMB-H was detected in 5 patients with cervical cancer, 5 with endometrial cancer, and 1 with endometrial stromal sarcoma. Eleven patients (10.8%) received molecularly targeted therapy according to their gene aberrations. Gene panel testing was mostly performed when the second-line treatment was ineffective. Of all 102 patients, 60 did not have recommended treatment, and 15 died or had worsened conditions before obtaining the test results. Through multigene panel testing, although many patients had druggable gene alterations, 10.8% of them received the recommended treatment. TMB-H was mainly observed in cervical/endometrial cancer, suggesting its potential as a therapeutic biomarker of immune checkpoint inhibitors. Furthermore, patients' prognosis and performance status should be considered before performing the test.

Confirmation of Insertion, Deletion, and Deletion-Insertion Variants Detected by Next-Generation Sequencing.

Despite clinically demonstrated accuracy in next generation sequencing (NGS) data, many clinical laboratories continue to confirm variants with Sanger sequencing, which increases cost of testing and turnaround time. Several studies have assessed the accuracy of NGS in detecting single nucleotide variants; however, less has been reported about insertion, deletion, and deletion-insertion variants (indels). We performed a retrospective analysis from 2015-2022 of indel results from a subset of NGS targeted gene panel tests offered through the Mayo Clinic Genomics Laboratories. We compared results from NGS and Sanger sequencing of indels observed in clinical runs and during the intra-assay validation of the tests. Results demonstrated 100% concordance between NGS and Sanger sequencing for over 490 indels (217 unique), ranging in size from 1 to 68 basepairs (bp). The majority of indels were deletions (77%) and 1 to 5 bp in length (90%). Variant frequencies ranged from 11.4% to 67.4% and 85.1% to 100% for heterozygous and homozygous variants, respectively, with a median depth of coverage of 2562×. A subset of indels (7%) were located in complex regions of the genome, and these were accurately detected by NGS. We also demonstrated 100% reproducibility of indel detection (n = 179) during intra-assay validation. Together this data demonstrates that reportable indel variants up to 68 bp can be accurately assessed using NGS, even when they occur in complex regions. Depending on the complexity of the region or variant, Sanger sequence confirmation of indels is usually not necessary if the variants meet appropriate coverage and allele frequency thresholds.

Germline whole genome sequencing in adults with multiple primary tumors.

Multiple primary tumors (MPTs) are a harbinger of hereditary cancer syndromes. Affected individuals often fit genetic testing criteria for a number of hereditary cancer genes and undergo multigene panel testing. Other genomictesting options, such as whole exome (WES) and whole genome sequencing (WGS) are available, but the utility of these genomic approaches as a second-tier test for those with uninformative multigene panel testing has not been explored.Here, we report our germline sequencing results from WGS in 9 patients with MPTs who had non-informative multigene panel testing. Following germline WGS, sequence (agnostic or 735 selected genes) and copy number variant (CNV) analysis was performed according to the American College of Medical Genetics (ACMG) standards and guidelines for interpreting sequence variants and reporting CNVs.In this cohort, WGS, as a second-tier test, did not identify additional pathogenic or likely pathogenic variants in cancer predisposition genes. Although we identified aCHEK2likely pathogenic variant and a MUTYHpathogenic variant, both were previously identified in the multigene panels and were not explanatory for the presented type of tumors. CNV analysis also failed to identify any pathogenic or likely pathogenic variants in cancer predisposition genes.In summary, after multigene panel testing, WGS did not reveal any additional pathogenic variants in patients with MPTs. Our study, based on a small cohort of patients with MPT, suggests that germline gene panel testing may be sufficient to investigate these cases. Future studies with larger sample sizes may further elucidate the additional utility of WGS in MPTs.

Comprehensive Gene Panel Testing for Hearing Loss in Children: Understanding Factors Influencing Diagnostic Yield

To evaluate factors influencing the diagnostic yield of comprehensive gene panel testing (CGPT) for hearing loss (HL) in children and to understand the characteristics of undiagnosed probands. This was a retrospective cohort study of 474 probands with childhood-onset HL who underwent CGPT between 2016 and 2020 at a single center. Main outcomes and measures included the association between clinical variables and diagnostic yield and the genetic and clinical characteristics of undiagnosed probands. The overall diagnostic yield was 44% (209/474) with causative variants involving 41 genes. While the diagnostic yield was high in the probands with congenital, bilateral, and severe HL, it was low in those with unilateral, noncongenital, or mild HL; cochlear nerve deficiency; preterm birth; neonatal intensive care unit admittance; certain ancestry; and developmental delay. Follow-up studies on 49 probands with initially inconclusive CGPT results changed the diagnostic status to likely positive or negative outcomes in 39 of them (80%). Reflex to exome sequencing on 128 undiagnosed probands by CGPT revealed diagnostic findings in 8 individuals, 5 of whom had developmental delays. The remaining 255 probands were undiagnosed, with 173 (173/255) having only a single variant in the gene(s) associated with autosomal recessive HL and 28% (48/173) having a matched phenotype. CGPT efficiently identifies the genetic etiologies of HL in children. CGPT-undiagnosed probands may benefit from follow-up studies or expanded testing.

EML4-ALK Gene Mutation Detected with New NGS Lung Cancer Panel CDx Using Sputum Cytology in a Case of Advanced NSCLC.

The detection of driver gene mutations has become essential for lung cancer; however, insufficient sample sizes make gene panel tests difficult to use. We previously reported that the lung cancer compact panel TM (LCCP) could detect EGFR and MET gene mutations with sputum cytology. To date, the detection of gene mutation using RNA from sputum samples is considered practically difficult. We report a case in which the EML4-ALK fusion gene was successfully detected from a sputum sample using the LCCP that was just released in Japan as a new next-generation sequencing lung cancer panel, CDx.

Early onset ataxia with comorbid myoclonus and epilepsy: A disease spectrum with shared molecular pathways and cortico-thalamo-cerebellar network involvement.

Early onset ataxia (EOA) concerns a heterogeneous disease group, often presenting with other comorbid phenotypes such as myoclonus and epilepsy. Due to genetic and phenotypic heterogeneity, it can be difficult to identify the underlying gene defect from the clinical symptoms. The pathological mechanisms underlying comorbid EOA phenotypes remain largely unknown. The aim of this study is to investigate the key pathological mechanisms in EOA with myoclonus and/or epilepsy. For 154 EOA-genes we investigated (1) the associated phenotype (2) reported anatomical neuroimaging abnormalities, and (3) functionally enriched biological pathways through in silico analysis. We assessed the validity of our in silico results by outcome comparison to a clinical EOA-cohort (80 patients, 31 genes). EOA associated gene mutations cause a spectrum of disorders, including myoclonic and epileptic phenotypes. Cerebellar imaging abnormalities were observed in 73-86% (cohort and in silico respectively) of EOA-genes independently of phenotypic comorbidity. EOA phenotypes with comorbid myoclonus and myoclonus/epilepsy were specifically associated with abnormalities in the cerebello-thalamo-cortical network. EOA, myoclonus and epilepsy genes shared enriched pathways involved in neurotransmission and neurodevelopment both in the in silico and clinical genes. EOA gene subgroups with myoclonus and epilepsy showed specific enrichment for lysosomal and lipid processes. The investigated EOA phenotypes revealed predominantly cerebellar abnormalities, with thalamo-cortical abnormalities in the mixed phenotypes, suggesting anatomical network involvement in EOA pathogenesis. The studied phenotypes exhibit a shared biomolecular pathogenesis, with some specific phenotype-dependent pathways. Mutations in EOA, epilepsy and myoclonus associated genes can all cause heterogeneous ataxia phenotypes, which supports exome sequencing with a movement disorder panel over conventional single gene panel testing in the clinical setting.

010 Enhanced outcome prediction in cutaneous squamous cell carcinoma using deep-learning and computational histopathology (cSCCnet)

Abstract Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer and leads to a significant disease burden. Although metastases occur in < 5% of patients, effective treatments are limited and 5-year survival is poor, with mortality rates equivalent to those of melanoma in some regions of the world. Despite recent advances in understanding the molecular pathogenesis of cSCC, patient risk stratification is limited and based on clinicopathological staging, with few validated prognostic biomarkers available to guide management [de Jong E, Lammerts MUPA, Genders RE, Bowes Bavinck JN. Update of advanced cutaneous squamous cell carcinoma. J Eur Acad Dermatol Venereol 2022; 36(Suppl. 1):6–10]. In this study, we aimed to use artificial intelligence technology to develop a deep learning model using digitized whole-slide images of cSCC to quantify the risk of metastasis. Routine haematoxylin and eosin-stained slides of 256 primary cSCCs from immunocompetent patients at four collaborating centres were scanned. The area of interest encompassing tumour and peritumoral stroma was annotated, and 512 × 512 pixel tiles were generated from each slide. High-quality tiles (containing sufficient tumour content) were pooled together, with patient-level labels inherited by tiles. We trained multiple models using an 80:10:10 split for training, validation and testing, and the model achieving the highest tile- and slide-level validation accuracy was identified (ResNet50). The final model, namely, cSCCnet, achieved 88% sensitivity, 73% specificity, 64% positive predictive value and 92% negative predictive values on the independent test set. Blinded review of the top predictive tiles by three histopathologists suggested that the predictive features identified by cSCCnet were distinct from histopathological features commonly used by clinicians to estimate metastatic risk. We have demonstrated that it is possible to train a deep learning model using digital pathology to predict the risk of metastasis. cSCCnet surpassed the published performance of currently used staging systems, as well as commercially available gene panel testing (de Jong et al.; Wysong A, Newman JG, Covington KR et al. Validation of a 40-gene expression profile test to predict metastatic risk in localized high-risk cutaneous squamous cell carcinoma. J Am Acad Dermatol 2021; 84:361–9). The morphological features and patterns identified by cSCCnet are different from established histopathological high-risk criteria, indicating that this technology can enhance current practice. Although further validation in a prospective cohort is pending, this is an important step toward creating a risk stratification tool that can be incorporated into routine pathology workflows, significantly improving patient outcomes for this common cancer.

Diagnostic Gene Panel Testing in (Non)-Syndromic Patients with Cleft Lip, Alveolus and/or Palate in the Netherlands

Objectives: Clefts of the lip, alveolus and/or palate (CLA/P) are the most common craniofacial congenital malformations in humans. These oral clefts can be divided into non-syndromic (isolated) and syndromic forms. Many cleft-related syndromes are clinically variable and genetically heterogeneous, making it challenging to distinguish syndromic from non-syndromic cases. Recognition of syndromic/genetic causes is important for personalized tailored care, identification of (unrecognized) comorbidities, and accurate genetic counseling. Therefore, next generation sequencing (NGS)-based targeted gene panel testing is increasingly implemented in diagnostics of CLA/P patients. In this retrospective study, we assess the yield of NGS gene panel testing in a cohort of CLA/P cases. Methods: Whole exome sequencing (WES) followed by variant detection and interpretation in an a priori selected set of genes associated with CLA/P phenotypes was performed in 212 unrelated CLA/P patients after genetic counseling between 2015 and 2020. Medical records including family history and results of additional genetic tests were evaluated. Results: In 24 CLA/P cases (11.3%), a pathogenic genetic variant was identified. Twenty out of these 24 had a genetic syndrome requiring specific monitoring and follow-up. Six of these 24 cases (25%) were presumed to be isolated CLA/P cases prior to testing, corresponding to 2.8% of the total cohort. In eight CLA/P cases (3.8%) without a diagnosis after NGS-based gene panel testing, a molecular diagnosis was established by additional genetic analyses (e.g., SNP array, single gene testing, trio WES). Conclusion: This study illustrates NGS-based gene panel testing is a powerful diagnostic tool in the diagnostic workup of CLA/P patients. Also, in apparently isolated cases and non-familial cases, a genetic diagnosis can be identified. Early diagnosis facilitates personalized care for patients and accurate genetic counseling of their families.

Real life use of biomarkers of homologous recombination deficiency (HRD) status beyond BRCA to predict the effectiveness of PARP inhibitors in ovarian cancer patients.

10592 Background: Testing for BRCA mutations (BRCAm) and genomic instability can identify epithelial ovarian cancer (OC) patients most likely to benefit from PARP-inhibitors (PARPi). However, current biomarkers of non- BRCA Homologous Recombination Repair (HRR) mutations are insufficient for guiding use of PARPi in the clinic. Despite non- BRCA HRR pathway gene mutations are rare, these patients may benefit from PARPi. Furthermore, recent preclinical findings showed that sensitivity to PARPi could be associated also with mutations in mismatch repair (MMR) genes, although sensitivity in the clinic is not proven. Methods: We report our real-life experience to assess the HRD status beyond BRCAm in newly-diagnosed OC patients, with the use of HRR gene panel and HRD genomic instability tests. After primary diagnosis, tumor and germline BRCA (g BRCA) status were assessed and, if BRCA WT, tumor HRR deficiency status were centrally determined by myChoiceCDx (Myriad) assay. NGS panel evaluating 20 MMR and HRR-genes beyond BRCA1/2 was proposed to patients with significant personal and/or family history of cancer, resulted negative to g BRCA testing. Results: From January 2017 to January 2023, 540 unselected epithelial OC patients, aging 27 to 81, were tested for tumor and g BRCA status; 109 were carriers of germline pathogenic or likely pathogenic variants (PVs) in BRCA1/2 genes (20.2%): 72 in BRCA1 (66.1%) and 37 in BRCA2 gene (33.9%). Additional 19 patients showed somatic PVs confined to tumor samples (3.5%).In the population of 70 BRCA WT patients tested by multigene panel testing, 14 germline PVs in HRR-associated (7.1%) or MMR-associated genes (12.8%) were found, including 5 in MUTYH (35.7%), 2 in ATM (14.3%), 2 in MLH1 (14.3%), 2 in PMS2 (14.3%), 1 in RAD51C (7.14%), 1 in RAD51D (7.14%), and 1 in CHEK2 gene (7.14%).Out of 72 samples analyzed by HRD genomic instability test, 23 cases were identified as HRD positive (31.9%), with a median GIS score of 65.5 (44-83). Median GIS were 35 (1-72) in the 6 cases of non- BRCA mutated tumors (8.3%), 40 (4-82) in the 17 tumor with non- BRCA variant of uncertain significance (VUS) (23.6%), and 27 (2-83) in the WT samples. Median GIS were significantly higher in tumors with PVs/VUS of RAD51 than BRIP1 (55.5 vs 35). Conclusions: HRRm gene panel and HRD genomic instability tests are not interchangeable to study HRR deficiency beyond BRCAm. HRD genomic instability test is effective to identify patients most likely to gain benefit from PARPi, but not for predicting familial risk of cancer. In our population, NGS panel evaluating HRR genes, beyond BRCA1/2, improved the detection rate of HRRm by 7%, with additional finding of MMR germline mutations, and important clinical implications for family members. Prospective data are expected to evaluate the effectiveness of PARPi in these population.

Frequency and spectrum of clinically significant molecular alterations detected by tumor gene panel testing for pediatric cancer patients in the Texas KidsCanSeq study.

10018 Background: Tumor sequencing has the potential to inform diagnosis, prognosis, and treatment of childhood cancer patients. Prospective data are needed to evaluate the clinical utility of these tests. Methods: Patients less than 18 years of age with extracranial solid tumors (ST), central nervous system tumors (CNS), lymphomas and histiocytic disorders (L/H) were enrolled in the Texas KidsCanSeq clinical genomics study at six Texas institutions. Available tumor samples from a subset of patients designated as high risk (based on poor prognosis at diagnosis or tumor recurrence) were sequenced using pediatric cancer-focused targeted DNA (initially 124 genes; expanded to 169 genes) and RNA (81 genes) panels developed at Texas Children’s Hospital. DNA variants (single nucleotide variants, insertion/deletions, splice site variants, copy number alterations) and fusion genes detected by RNA testing were analyzed. Tumor alterations were classified by molecular pathologists and oncologists as carrying strong (Tier 1) or potential (Tier 2) clinical significance following consensus guidelines from the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. Results: A total of 626 patients were enrolled between 2018 and 2021, of whom 380 were designated as high risk. Tumor samples adequate for molecular testing were available from 342 of the 380 (90%) high risk patients (196 ST [57%], 132 CNS [39%] and 14 L/H [4%]). Clinically significant alterations were identified in 246 of 342 (72%) tumors: 165 (48%) from DNA testing only, 34 (10%) from RNA testing only, and 47 (14%) from both modalities. The frequency of clinically significant alterations was similar across tumor cohorts (74% ST, 68% CNS, 71% L/H). Alterations relevant to therapy-choice were reported in 127 of 342 (37%) tumors (35% ST, 26% CNS, 57% L/H); 87 of these 127 (69%) were not known from prior clinical testing. The most frequently altered therapeutic gene alterations (Table) involved BRAF (n=25), CDKN2A/B (n=20), SMARCB1 (n=17), FGFR1/2/3/4 (n=14), NF1 (n=13), PIK3CA (n=11), ALK (n=10), CDK4 (n=9), and RET (n=7). One hypermutated tumor was identified: a high-grade glioma (tumor mutation burden >190 mut/Mb) in a patient with Lynch syndrome due to a germline MSH6 variant. Conclusions: Tumor alterations of clinical significance according to consensus guidelines were reported in a large proportion of children with diverse high risk or recurrent cancer diagnoses, the majority of which were not detected by other standard pathologic analysis. These data further support broader adoption of tumor molecular profiling for these patients. [Table: see text]

Comprehensive clinical tumor RNA sequencing for children with cancer.

e15067 Background: Cancer is the number one cause of death by disease for children in the United States. The underlying cause for most childhood cancers is unknown and unlike most adult cancers, childhood cancers harbor fewer coding mutations compared to adult cancers and are often driven by epigenetic deregulation. The functional impact of both genetic and epigenetic abnormalities can be detected from the analysis of tumor RNA. While tumor RNA profiling has entered several clinical trials, it is not utilized comprehensively and there is limited availability of clinical-grade RNA-Seq assays. We have published preliminary evidence of the utility of using research tumor RNA information in the pediatric oncology clinic. The next step is to develop an RNA-Seq assay that identifies gene fusions, expressed mutations, and important gene expression abnormalities in pediatric tumors. Methods: To validate our RNA-Seq assay pipeline, we have started with fusion detection and compared our results with a CLIA-validated DNA-based assay. We used RNA-Seq data generated from three fusion-positive cell lines to define the minimum of Mapped, Exonic, Non-Duplicate (MEND) reads necessary to accurately identify known fusions. Results: We found that the fusion, which has moderate expression (above 3 log2(TPM+1)), was consistently detectable in subsets containing at least 1 million MEND reads (Table). We next evaluated the precision of this finding. The fusion was detected in each of 21 additional subsets of 2 million MEND reads with replacement, but in only 71% of 21 subsets of 1 million MEND reads. In RNA-Seq data generated from tumor samples preserved with FFPE, we compared our RNA-based fusion findings with structural variants detected with the DNA-based gene panel test UCSF 500. Seven positive results were concordant, and one negative result was concordant. Two negative findings were positive by UCSF 500. Evaluation of these discordant results is ongoing, but the expression data indicates that the RNA result for one, a TMPRSS-ERG fusion, is accurate. To incorporate gene expression into our assay, we assessed the precision of gene expression measurements. In a preliminary dataset of three technical replicates, we found that the 95% confidence interval of expression values greater than 3 log2(TPM+1) is 4.5% of the measured value. Comprehensive clinical validation of our protocol will involve additional previously-characterized cancer samples. Conclusions: In summary, these data establish and validate key analytical parameters necessary for successful implementation of RNA-Seq analysis in the pediatric oncology clinic and advance our ultimate goal of bringing the most innovative molecular diagnostic methods to every child with cancer. [Table: see text]

Clinical Characteristics and Genetic Analyses of Patients with Idiopathic Hypogonadotropic Hypogonadism

Idiopathic hypogonadotropic hypogonadism (IHH) is classified into two groups-Kalman syndrome and normosmic IHH (nIHH). Half of all cases can be explained by mutations in >50 genes. Targeted gene panel testing with nexrt generation sequencing (NGS) is required for patients without typical phenotypic findings. The aim was to determine the genetic etiologies of patients with IHH using NGS, including 54 IHH-associated genes, and to present protein homology modeling and protein stability analyzes of the detected variations. Clinical and demographic data of 16 patients (eight female), aged between 11.6-17.8 years, from different families were assessed. All patients were followed up for a diagnosis of nIHH, had normal cranial imaging, were without anterior pituitary hormone deficiency other than gonadotropins, had no sex chromosome anomaly, had no additional disease, and underwent genetic analysis with NGS between the years 2008-2021. Rare variants were classified according to the variant interpretation framework of the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology. Changes in protein structure caused by variations were modeled using RoseTTAFold and changes in protein stability resulting from variation were analyzed. Half of the 16 had no detectable variation. Three (18.75%) had a homozygous (pathogenic) variant in the GNRHR gene, one (6.25%) had a compound heterozygous [likely pathogenic-variants of uncertain significance (VUS)] variant in PROK2 and four (25%) each had a heterozygous (VUS) variant in HESX1, FGF8, FLRT3 and DMXL2. Protein models showed that variants interpreted as VUS according to ACMG could account for the clinical IHH. The frequency of variation detection was similar to the literature. Modelling showed that the variant in five different genes, interpreted as VUS according to ACMG, could explain the clinical IHH.

Possible False Results With cobas® EGFR Mutation Test v2 and Oncomine Dx Target Test for EGFR Mutation.

Disparities in the results of next-generation sequencing-based multiplex gene panel tests and those of single-gene tests when detecting epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) have been reported. However, the possible underlying causes have not been investigated. The aim of this study was to explore the possibilities and causes of false results obtained using cobas® EGFR Mutation Test v2 (cobas® EGFR) and Oncomine Dx Target Test (ODxTT). The data of patients with NSCLC who underwent gene assessment using both cobas® EGFR and ODxTT between April 2021 and May 2022 were retrospectively reviewed. Disparate results of EGFR mutation analyses were then reviewed. One hundred and sixteen patients were included in the analysis. The results of six samples were inconsistent. In four samples, exon 20 insertion mutations were detected using cobas® EGFR, but not identified using ODxTT. A fragment analysis was performed on three of the four samples, and all showed negative results for exon 20 insertion. Furthermore, one false negative result was obtained in the ODxTT for both exon 19 deletion and L858R mutations. For exon 19 deletion mutation, a single nucleotide variant from adenine to thymine was identified close to the mutation site. False positives for exon 20 insertion may occur when using cobas® EGFR, and false negatives for exon 19 deletion and L858R mutations may occur when using ODxTT.