Variant detection from long-read genome sequencing (lrGS) has proven to be considerably more accurate and comprehensive than variant detection from short-read genome sequencing (srGS). However, the rate at which lrGS can increase molecular diagnostic yield for rare disease is not yet precisely characterized. We performed lrGS using Pacific Biosciences \"HiFi\" technology on 96 short-read-negative probands with rare disease that were suspected to be genetic. We generated hg38-aligned variants and de novo phased genome assemblies, and subsequently annotated, filtered, and curated variants using clinical standards. New disease-relevant or potentially relevant genetic findings were identified in 16/96 (16.7%) probands, eight of which (8/96, 8.33%) harbored pathogenic or likely pathogenic variants. Newly identified variants were visible in both srGS and lrGS in nine probands (~9.4%) and resulted from changes to interpretation mostly from recent gene-disease association discoveries. Seven cases included variants that were only interpretable in lrGS, including copy-number variants, an inversion, a mobile element insertion, two low-complexity repeat expansions, and a 1 bp deletion. While evidence for each of these variants is, in retrospect, visible in srGS, they were either: not called within srGS data, were represented by calls with incorrect sizes or structures, or failed quality-control and filtration. Thus, while reanalysis of older data clearly increases diagnostic yield, we find that lrGS allows for substantial additional yield (7/96, 7.3%) beyond srGS. We anticipate that as lrGS analysis improves, and as lrGS datasets grow allowing for better variant frequency annotation, the additional lrGS-only rare disease yield will grow over time.

Using next generation sequencing (NGS), The Cancer Genome Atlas (TCGA) found that endometrial carcinomas (ECs) fall under one of four molecular subtypes, and a POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)-based surrogate has been developed. We sought to retrospectively classify and characterize a large series of unselected ECs that were prospectively subjected to clinical sequencing by utilizing clinical molecular and IHC data.
All patients with EC with clinical tumor-normal MSK-IMPACT NGS from 2014 to 2020 (n = 2115) were classified by integrating molecular data (i.e., POLE mutation, TP53 mutation, MSIsensor score) and MMR and p53 IHC results. Survival analysis was performed for primary EC patients with upfront surgery at our institution.
Utilizing our integrated approach, significantly more ECs were molecularly classified (1834/2115, 87%) as compared to the surrogate (1387/2115, 66%, p < 0.001), with an almost perfect agreement for classifiable cases (Kappa 0.962, 95% CI 0.949-0.975). Discrepancies were primarily due to TP53 mutations in p53-IHC-normal ECs. Of the 1834 ECs, most were of copy number (CN)-high molecular subtype (40%), followed by CN-low (32%), MSI-high (23%) and POLE (5%). Histologic and genomic variability was present amongst all molecular subtypes. Molecular classification was prognostic in early- and advanced-stage disease, including early-stage endometrioid EC.
The integration of clinical NGS and IHC data allows for an algorithmic approach to molecularly classifying newly diagnosed EC, while overcoming issues of IHC-based genetic alteration detection. Such integrated approach will be important moving forward given the prognostic and potentially predictive information afforded by this classification.

With the advent of new molecular diagnostic techniques, retrieving DNA from the formalin-fixed paraffin-embedded (FFPE) tissues has become an essential yet challenging step for efficient downstream processes. Owing to low quality and quantity of DNA retrieved from the FFPE sections, the process is often impractical and needs significant improvements. Here, we established an efficient method for the purification of DNA from FFPE specimens by optimizing incubation temperature, incubation time, and the concentration of a formalin scavenger tris(hydroxymethyl)aminomethane (Tris) for reverse-crosslinking. The optimized method, named \"Highly concentrated Tris-mediated DNA extraction\" (HiTE), yielded three times the DNA yield per tissue slice compared with a representative DNA extraction kit. Moreover, the use of HiTE-extracted DNA increased the yield of the sequencing library three times and accordingly yielded a log higher and more reproducible sequencing library compared with that obtained using the commonly used commercial kit. The sequencing library prepared from HiTE-extracted FFPE-DNA had longer inserts and produced reads that evenly covered the reference genome. Successful application of HiTE-extracted FFPE-DNA for whole-genome and targeted gene panel sequencing indicates its practical usability.

Tumor heterogeneity has been known to cause inter-assay discordance among next-generation sequencing (NGS) results. However, whether preclinical factors such as sample type, sample quality and analytical features of gene panel can affect the concordance between two different assays remains largely unexplored.
Replicate sets of DNA samples extracted from formalin-fixed paraffin-embedded tissues (FFPE) (n = 20) and fresh frozen (FF) tissues (n = 10) were herein analyzed using a tumor-only (TO) and paired tumor-normal (TN) gene panel in laboratories certified by the Clinical Laboratory Improvement Amendment. Reported variants from the TO and TN panels were then compared. Furthermore, additional FFPE samples were sequentially sliced from the same FFPE block and submitted to another TN panel assay.
Substantial discordance (71.8%) was observed between the results of the two panels despite using identical DNA samples, with the discordance rate being significantly higher for FFPE samples (p < 0.05). Among the 99 variants reported only in the TO panel, 32.3% were consistent with germline variants, which were excluded in the TN panel, while 30.3% had an allele frequency of less than 5%, some of which were highly likely to be artificial calls. The comparison of two independent TN panel assay results from the same FFPE block also showed substantial discordance rate (55.3%).
In the context of clinical settings, our comparative analysis revealed that inter-NGS assay discordance commonly occurred due to sample types and the different analytical features of each panel.

Metastatic progression is the main cause of death in cancer patients, whereas the underlying genomic mechanisms driving metastasis remain largely unknown. Here, we assembled MSK-MET, a pan-cancer cohort of over 25,000 patients with metastatic diseases. By analyzing genomic and clinical data from this cohort, we identified associations between genomic alterations and patterns of metastatic dissemination across 50 tumor types. We found that chromosomal instability is strongly correlated with metastatic burden in some tumor types, including prostate adenocarcinoma, lung adenocarcinoma, and HR+/HER2+ breast ductal carcinoma, but not in others, including colorectal cancer and high-grade serous ovarian cancer, where copy-number alteration patterns may be established early in tumor development. We also identified somatic alterations associated with metastatic burden and specific target organs. Our data offer a valuable resource for the investigation of the biological basis for metastatic spread and highlight the complex role of chromosomal instability in cancer progression.

During the past decade, next-generation sequencing (NGS) technologies have become widely adopted in cancer research and clinical care. Common applications within the clinical setting include patient stratification into relevant molecular subtypes, identification of biomarkers of response and resistance to targeted and systemic therapies, assessment of heritable cancer risk based on known pathogenic variants, and longitudinal monitoring of treatment response. The need for efficient downstream processing and reliable interpretation of sequencing data has led to the development of novel algorithms and computational pipelines, as well as structured knowledge bases that link genomic alterations to currently available drugs and ongoing clinical trials. Cancer centers around the world use different types of targeted solid-tissue and blood based NGS assays to analyze the genomic and transcriptomic profile of patients as part of their routine clinical care. Recently, cross-institutional collaborations have led to the creation of large pooled datasets that can offer valuable insights into the genomics of rare cancers.

Next-generation sequencing-based comprehensive genome profiling (CGP) testing, OncoGuide NCC Oncopanel System, and FoundationOne CDx Cancer Genomic Profile have been covered by the Japanese national health insurance system since June 2019. Because CGP was initially developed to enroll patients into an early-phase clinical trial for solid tumors, its approved indications have been limited to patients who have completed the standard chemotherapy treatment. Approximately 14,000 cases have been registered with the Center for Cancer Genomics and Advanced Therapeutics as of March 2021. Measuring the drug access rate is not enough due to patients\' deteriorating condition during CGP analysis and due to the limited number of ongoing clinical trials available, although tumor-agnostic therapies, such as the use of pembrolizumab in high microsatellite-instable solid tumors and in conditions with a high tumor mutational burden (≥10 mut/Mb) as well as the use of entrectinib and larotrectinib in NTRK fusion-positive tumors have been approved in Japan. Moreover, since this analysis is performed using DNA derived from tumor tissue, it is difficult to perform CGP in cases in which an insufficient amount of tissue exists. Thus, noninvasive blood-based assays have been developed, and CGP panels using circulating tumor DNA from blood were approved in March 2021. However, cost, timing, and the number of tests allowed by the health system have not yet been determined. Therefore, in this review, we outline the current status and issues of CGP testing using tumor tissues as well as the expectations and limitations of liquid biopsy for use in Japanese clinical practice.

BACKGROUND: The advancement of cancer genomics has allowed for multiplex gene assays using next-generation sequencing (NGS) to be practically implemented, however, a clinical practice system remains to be established.
OBJECTIVE: We evaluated the feasibility of clinical sequencing using NGS-based multiplex gene assays between cooperating medical institutions in patients with advanced cancers.
METHODS: In this observational study, DNA and RNA samples prepared from existing tumor tissues were subjected to comprehensive genomic profiling using targeted sequencing.
RESULTS: From January 2017 to March 2019, 36 samples from 33 patients were assessed. Of all patients, 27 (82%) had lung cancer, with the median age of 50 years (range 38-83). Multiplex gene panel tests were successfully carried out on 35/36 (97%) samples. Potentially actionable gene alterations were identified in 10/30 (33%) samples (3 HER2, 2 KRAS, 2 ALK, 1 PIK3CA, 1 RET, and 1 CDKN2A). In the 6 samples examined for resistant mechanisms, ALK I1171N mutation and MET copy number gain were detected in 2 patients with ALK rearrangement-positive lung cancer.
CONCLUSIONS: Clinical sequencing using NGS-based multiplex gene assays between collaborating domestic medical institutions was feasible, with a success rate of > 97%. Overall, clinical sequencing benefits therapeutic decision-making in patients with advanced cancer.

The Tohoku Medical Megabank (TMM) project was established to provide creative reconstruction of the Tohoku area that suffered from a huge earthquake and ensuing tsunami (the Great East Japan Earthquake, GEJE). TMM aims to establish two large-scale genome cohorts and an integrated biobank managing biospecimen and related information. It supports community medicine by establishing next-generation medical systems through a combination of the prospective genome cohort studies with a total of 150,000 participants and genomic medicine. The strategies for genome analyses in TMM are to develop an elaborate genome reference panel by means of high-fidelity Japanese whole-genome sequence, to design custom single nucleotide polymorphism (SNP) arrays based on the reference panel, and to obtain genotype data for all the TMM cohort participants subsequently. Disease-associated genomic information and omics data, including metabolomics and microbiome analysis, provide an essential platform for precision medicine and personalized healthcare (PHC). Ethical, legal, and social issues (ELSI) and education are important for implementing genomic medicine. The major considerations of ELSI regarding each participant of the cohort studies are the respect for the autonomy and the protection of privacies. Moreover, developing and provide human resources not only for the TMM project but also for the social implementation of precision medicine and PHC is required. We started a pilot study of the return of genomic results for familial hypercholesterolemia (FH) as a target disease. TMM aims to establish solid platforms that support precision medicine and PHC based on the genomic and omics information and environmental and lifestyle factors of the individuals, which is one of the most advanced medical care beyond the evidenced-based medicine in the near future.

In debates about genetic testing of children, as well as about disclosing unsolicited findings (UFs) of pediatric exome sequencing, respect for future autonomy should be regarded as a prima facie consideration for not taking steps that would entail denying the future adult the opportunity to decide for herself about what to know about her own genome. While the argument can be overridden when other, morally more weighty considerations are at stake, whether this is the case can only be determined in concrete cases. Importantly, when children grow into adolescents, respect for future autonomy will have to give way to respecting their emerging autonomy. When pediatric exome sequencing is done for complex conditions not involving developmental delay, respect for the child\'s future or emerging autonomy should be a primary consideration for those charged with deciding on behalf of the child. Building on what Emanuel and Emanuel have termed the \'deliberative model\' of shared decision making, we argue that if parents fail to give these considerations their due, professionals should actively invite them to do so. Taking a directive stance may be needed in order to make sure that the future or emerging autonomy of the child are duly considered in the decision-making process, but also to help the parents and themselves to shape their respective roles as responsible care-givers.