Inflammatory Rhabdomyoblastic Tumor: Clinicopathologic and Molecular Analysis of 13 Cases.

Inflammatory rhabdomyoblastic tumors (IRMTs) are newly recognized skeletal muscle tumors with uncertain malignant potential. We investigated 13 IRMTs using clinicopathologic, genetic, and epigenetic methods. The cohort included 7 men and 6 women, aged 23 to 80 years (median, 50 years), of whom 2 had neurofibromatosis type 1. Most tumors occurred in the deep soft tissues of the lower limbs, head/neck, trunk wall, and retroperitoneum/pelvis. Two tumors involved the hypopharyngeal submucosa as polypoid masses. Eight tumors showed conventional histology of predominantly spindled cells with nuclear atypia, low mitotic activity, and massive inflammatory infiltrates. Three tumors showed atypical histology, including uniform epithelioid or plump cells and mitotically active histiocytes. The remaining 2 tumors demonstrated malignant progression to rhabdomyosarcoma; one had additional IRMT histology and the other was a pure sarcoma. All 11 IRMTs without malignant progression exhibited indolent behavior at a median follow-up of 43 months. One of the 2 patients with IRMTs with malignant progression died of lung metastases. All IRMTs were positive for desmin and PAX7, whereas myogenin and MyoD1 were expressed in a subset of cases. Targeted next-generation sequencing identified pathogenic mutations in NF1 (5/8) and TP53 (4/8). All TP53 mutations co-occurred with NF1 mutations. TP53 variant allele frequency was much lower than that of NF1 in 2 cases. These tumors showed geographic (subclonal) strong p53 immunoreactivity, suggesting the secondary emergence of a TP53-mutant clone. DNA methylation-based copy number analysis conducted in 11 tumors revealed characteristic flat patterns with relative gains, including chromosomes 5, 18, 20, 21, and/or 22 in most cases. Widespread loss of heterozygosity with retained biparental copies of these chromosomes was confirmed in 4 tumors analyzed via allele-specific profiling. Based on unsupervised DNA methylation analysis, none of the 11 tumors tested clustered with existing reference entities but formed a coherent group, although its specificity warrants further study.

The development of a custom RNA-sequencing panel for the identification of predictive and diagnostic biomarkers in glioma.

PURPOSE: Various molecular profiles are needed to classify malignant brain tumors, including gliomas, based on the latest classification criteria of the World Health Organization, and their poor prognosis necessitates new therapeutic targets. The Todai OncoPanel 2 RNA Panel (TOP2-RNA) is a custom-target RNA-sequencing (RNA-seq) using the junction capture method to maximize the sensitivity of detecting 455 fusion gene transcripts and analyze the expression profiles of 1,390 genes. This study aimed to classify gliomas and identify their molecular targets using TOP2-RNA. METHODS: A total of 124 frozen samples of malignant gliomas were subjected to TOP2-RNA for classification based on their molecular profiles and the identification of molecular targets. RESULTS: Among 55 glioblastoma cases, gene fusions were detected in 11 cases (20%), including novel MET fusions. Seven tyrosine kinase genes were found to be overexpressed in 15 cases (27.3%). In contrast to isocitrate dehydrogenase (IDH) wild-type glioblastoma, IDH-mutant tumors, including astrocytomas and oligodendrogliomas, barely harbor fusion genes or gene overexpression. Of the 34 overexpressed tyrosine kinase genes, MDM2 and CDK4 in glioblastoma, 22 copy number amplifications (64.7%) were observed. When comparing astrocytomas and oligodendrogliomas in gene set enrichment analysis, the gene sets related to 1p36 and 19q were highly enriched in astrocytomas, suggesting that regional genomic DNA copy number alterations can be evaluated by gene expression analysis. CONCLUSIONS: TOP2-RNA is a highly sensitive assay for detecting fusion genes, exon skipping, and aberrant gene expression. Alterations in targetable driver genes were identified in more than 50% of glioblastoma. Molecular profiling by TOP2-RNA provides ample predictive, prognostic, and diagnostic biomarkers that may not be identified by conventional assays and, therefore, is expected to increase treatment options for individual patients with glioma.

Lymphangioleiomyomatosis as a potent lung cancer risk factor: Insights from a Japanese large cohort study.

BACKGROUND AND OBJECTIVE: Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease associated with the functional tumour suppressor genes TSC1 and TSC2 and causes structural destruction in the lungs, which could potentially increase the risk of lung cancer. However, this relationship remains unclear because of the rarity of the disease. METHODS: We investigated the relative risk of developing lung cancer among patients diagnosed with LAM between 2001 and 2022 at a single high-volume centre in Japan, using data from the Japanese Cancer Registry as the reference population. Next-generation sequencing (NGS) was performed in cases where tumour samples were available. RESULTS: Among 642 patients diagnosed with LAM (sporadic LAM, n = 557; tuberous sclerosis complex-LAM, n = 80; unclassified, n = 5), 13 (2.2%) were diagnosed with lung cancer during a median follow-up period of 5.13 years. All patients were female, 61.5% were never smokers, and the median age at lung cancer diagnosis was 53 years. Eight patients developed lung cancer after LAM diagnosis. The estimated incidence of lung cancer was 301.4 cases per 100,000 person-years, and the standardized incidence ratio was 13.6 (95% confidence interval, 6.2-21.0; p = 0.0008). Actionable genetic alterations were identified in 38.5% of the patients (EGFR: 3, ALK: 1 and ERBB2: 1). No findings suggested loss of TSC gene function in the two patients analysed by NGS. CONCLUSION: Our study revealed that patients diagnosed with LAM had a significantly increased risk of lung cancer. Further research is warranted to clarify the carcinogenesis of lung cancer in patients with LAM.

Detection of Novel Tyrosine Kinase Fusion Genes as Potential Therapeutic Targets in Bone and Soft Tissue Sarcomas Using DNA/RNA-based Clinical Sequencing.

BACKGROUND: Approximately 1% of clinically treatable tyrosine kinase fusions, including anaplastic lymphoma kinase, neurotrophic tyrosine receptor kinase, RET proto-oncogene, and ROS proto-oncogene 1, have been identified in soft tissue sarcomas via comprehensive genome profiling based on DNA sequencing. Histologic tumor-specific fusion genes have been reported in approximately 20% of soft tissue sarcomas; however, unlike tyrosine kinase fusion genes, these fusions cannot be directly targeted in therapy. Approximately 80% of tumor-specific fusion-negative sarcomas, including myxofibrosarcoma and leiomyosarcoma, that are defined in complex karyotype sarcomas remain genetically uncharacterized; this mutually exclusive pattern of mutations suggests that other mutually exclusive driver oncogenes are yet to be discovered. Tumor-specific, fusion-negative sarcomas may be associated with unique translocations, and oncogenic fusion genes, including tyrosine kinase fusions, may have been overlooked in these sarcomas. QUESTIONS/PURPOSES: (1) Can DNA- or RNA-based analysis reveal any characteristic gene alterations in bone and soft tissue sarcomas? (2) Can useful and potential tyrosine kinase fusions in tumors from tumor-specific, fusion-negative sarcomas be detected using an RNA-based screening system? (3) Do the identified potential fusion tumors, especially in neurotrophic tyrosine receptor kinase gene fusions in bone sarcoma, transform cells and respond to targeted drug treatment in in vitro assays? (4) Can the identified tyrosine kinase fusion genes in sarcomas be useful therapeutic targets? METHODS: Between 2017 and 2020, we treated 100 patients for bone and soft tissue sarcomas at five institutions. Any biopsy or surgery from which a specimen could be obtained was included as potentially eligible. Ninety percent (90 patients) of patients were eligible; a further 8% (8 patients) were excluded because they were either lost to follow-up or their diagnosis was changed, leaving 82% (82 patients) for analysis here. To answer our first and second questions regarding gene alterations and potential tyrosine kinase fusions in eight bone and 74 soft tissue sarcomas, we used the TruSight Tumor 170 assay to detect mutations, copy number variations, and gene fusions in the samples. To answer our third question, we performed functional analyses involving in vitro assays to determine whether the identified tyrosine kinase fusions were associated with oncogenic abilities and drug responses. Finally, to determine usefulness as therapeutic targets, two pediatric patients harboring an NTRK fusion and an ALK fusion were treated with tyrosine kinase inhibitors in clinical trials. RESULTS: DNA/RNA-based analysis demonstrated characteristic alterations in bone and soft tissue sarcomas; DNA-based analyses detected TP53 and copy number alterations of MDM2 and CDK4 . These single-nucleotide variants and copy number variations were enriched in specific fusion-negative sarcomas. RNA-based screening detected fusion genes in 24% (20 of 82) of patients. Useful potential fusions were detected in 19% (11 of 58) of tumor-specific fusion-negative sarcomas, with nine of these patients harboring tyrosine kinase fusion genes; five of these patients had in-frame tyrosine kinase fusion genes ( STRN3-NTRK3, VWC2-EGFR, ICK-KDR, FOXP2-MET , and CEP290-MET ) with unknown pathologic significance. The functional analysis revealed that STRN3-NTRK3 rearrangement that was identified in bone had a strong transforming potential in 3T3 cells, and that STRN3-NTRK3 -positive cells were sensitive to larotrectinib in vitro. To confirm the usefulness of identified tyrosine kinase fusion genes as therapeutic targets, patients with well-characterized LMNA-NTRK1 and CLTC-ALK fusions were treated with tyrosine kinase inhibitors in clinical trials, and a complete response was achieved. CONCLUSION: We identified useful potential therapeutic targets for tyrosine kinase fusions in bone and soft tissue sarcomas using RNA-based analysis. We successfully identified STRN3-NTRK3 fusion in a patient with leiomyosarcoma of bone and determined the malignant potential of this fusion gene via functional analyses and drug effects. In light of these discoveries, comprehensive genome profiling should be considered even if the sarcoma is a bone sarcoma. There seem to be some limitations regarding current DNA-based comprehensive genome profiling tests, and it is important to use RNA testing for proper diagnosis and accurate identification of fusion genes. Studies on more patients, validation of results, and further functional analysis of unknown tyrosine kinase fusion genes are required to establish future treatments. CLINICAL RELEVANCE: DNA- and RNA-based screening systems may be useful for detecting tyrosine kinase fusion genes in specific fusion-negative sarcomas and identifying key therapeutic targets, leading to possible breakthroughs in the treatment of bone and soft tissue sarcomas. Given that current DNA sequencing misses fusion genes, RNA-based screening systems should be widely considered as a worldwide test for sarcoma. If standard treatments such as chemotherapy are not effective, or even if the sarcoma is of bone, RNA sequencing should be considered to identify as many therapeutic targets as possible.

Dramatic response to entrectinib in a patient with malignant peripheral nerve sheath tumor harboring novel SNRNP70-NTRK3 fusion gene.

Neurotropic tropomyosin receptor kinase (NTRK) gene rearrangements have been reported in limited cases of sarcomas; however, to date, there has been only one report of such rearrangements in malignant peripheral nerve sheath tumors (MPNSTs). Herein, we describe a 51-year-old male patient with a buttock tumor arising from the sciatic nerve, which was diagnosed as MPNST with positive S-100 staining, negative SOX10 staining, and loss of trimethylation at lysine 27 of histone H3 (H3K27me3) confirmed by immunohistochemistry. Soon after the resection of the primary tumor, the patient was found to have pulmonary and lymph node metastases. Chemotherapy with eribulin and trabectedin showed limited effects. However, the patient responded rapidly to pazopanib, but severe side effects caused discontinuation of the treatment. RNA panel testing revealed a novel fusion gene between Small Nuclear Ribonucleoprotein U1 Subunit 70 (SNRNP70) gene and NTRK3 gene. Furthermore, loss of NF1, SUZ12, and CDKN2A genes was confirmed by DNA panel testing, which is compatible with a histological diagnosis of MPNST. SNRNP70 possesses a coiled-coiled domain and seems to induce constitutive activation of NTRK3 through dimerization. In fact, immunohistochemistry revealed diffuse staining of pan-TRK within tumor cells. Treatment with entrectinib, which is an NTRK inhibitor, showed a quick and durable response for 10 months. Although NTRK rearrangements are very rare in MPNST, this case highlights the importance of genetic testing in MPNST, especially using an RNA panel for the detection of rare fusion genes.

Extensive analysis of 59 sarcoma-related fusion genes identified pazopanib as a potential inhibitor to COL1A1-PDGFB fusion gene.

Sarcomas are malignant mesenchymal tumors that are extremely rare and divergent. Fusion genes are involved in approximately 30% of sarcomas as driver oncogenes; however, their detailed functions are not fully understood. In this study, we determined the functional significance of 59 sarcoma-related fusion genes. The transforming potential and drug sensitivities of these fusion genes were evaluated using a focus formation assay (FFA) and the mixed-all-nominated-in-one (MANO) method, respectively. The transcriptome was also examined using RNA sequencing of 3T3 cells transduced with each fusion gene. Approximately half (28/59, 47%) of the fusion genes exhibited transformation in the FFA assay, which was classified into five types based on the resulting phenotype. The sensitivity to 12 drugs including multityrosine kinase inhibitors was assessed using the MANO method and pazopanib was found to be more effective against cells expressing the COL1A1-PDGFB fusion gene compared with the others. The downstream MAPK/AKT pathway was suppressed at the protein level following pazopanib treatment. The fusion genes were classified into four subgroups by cluster analysis of the gene expression data and gene set enrichment analysis. In summary, the oncogenicity and drug sensitivity of 59 fusion genes were simultaneously evaluated using a high-throughput strategy. Pazopanib was selected as a candidate drug for sarcomas harboring the COL1A1-PDGFB fusion gene. This assessment could be useful as a screening platform and provides a database to evaluate customized therapy for fusion gene-associated sarcomas.

The pathogenic role of the BRCA2 c.7847C>T (p.Ser2616Phe) variant in breast and ovarian cancer predisposition.

Substantial numbers of variants of unknown significance (VUSs) have been identified in BRCA1/2 through genetic testing, which poses a significant clinical challenge because the contribution of these VUSs to cancer predisposition has not yet been determined. Here, we report 10 Japanese patients from seven families with breast or ovarian cancer harboring the BRCA2 c.7847C>T (p.Ser2616Phe) variant that was interpreted as a VUS. This variant recurs only in families from Japan and has not been reported in the global general population databases. A Japanese patient with Fanconi anemia with compound heterozygous variants c.7847C>T (p.Ser2616Phe) and c.475+1G>A in BRCA2 was reported. In silico predictions and quantitative cosegregation analysis suggest a high probability of pathogenicity. The clinical features of the variant carriers were not specific to, but were consistent with, those of patients with hereditary breast and ovarian cancer. A validated functional assay, called the mixed-all-nominated-in-one-BRCA (MANO-B) method and the accurate BRCA companion diagnostic (ABCD) test, demonstrated the deleterious effects of the variant. Altogether, following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, this variant satisfied the "PS3," "PM2," "PM3," and "PP3" criteria. We thus conclude that the BRCA2 c.7847C>T (p.Ser2616Phe) variant is a "likely pathogenic" variant that is specifically observed in the Japanese population, leading to a breast and ovarian cancer predisposition.

Molecular pathology quality control in Southeast Asia: Results of a multiregional quality assurance study from MASTER KEY Asia.

Tissue specimen quality assurance is a major issue of precision medicine for rare cancers. However, the laboratory standards and quality of pathological specimens prepared in Asian hospitals remain unknown. To understand the methods in Southeast Asian oncology hospitals and to clarify how pre-analytics affect the quality of formalin-fixed paraffin-embedded (FFPE) specimens, a questionnaire surveying pre-analytical procedures (Part I) was administered, quality assessment of immunohistochemistry (IHC) staining and DNA/RNA extracted from the representative FFPE specimens from each hospital (Part II) was conducted, and the quality of DNA/RNA extracted from FFPE of rare-cancer patients for genomic sequencing (Part III) was examined. Quality measurements for DNA/RNA included ΔΔCt, DV200, and cDNA yield. Six major cancer hospitals from Malaysia, Philippines, and Vietnam participated. One hospital showed unacceptable quality for the DNA/RNA assessment, but improved by revising laboratory procedures. Only 57% (n = 73) of the 128 rare-cancer patients' specimens met both DNA and RNA quality criteria for next-generation sequencing. Median DV200 was 80.7% and 64.3% for qualified and failed RNA, respectively. Median ΔΔCt was 1.25 for qualified and 4.89 for failed DNA. Longer storage period was significantly associated with poor DNA (fail to qualify ratio = 1579:321 days, p < 0.001) and RNA (fail to qualify ratio = 1070:280 days, p < 0.001). After improvement of pre-analytical factors, the qualification rate increased for hospitals A and E from 41.5% to 70.5% and 62.5% to 86%, respectively. This is the first report to elucidate the pre-analytical laboratory procedures of main Southeast Asian oncology hospitals. An external quality assessment program may improve factors associated with tumor FFPE specimen quality.

Clinical utility of Todai OncoPanel in the setting of approved comprehensive cancer genomic profiling tests in Japan.

Comprehensive cancer genome profiling (CGP) has been nationally reimbursed in Japan since June 2019. Less than 10% of the patients have been reported to undergo recommended treatment. Todai OncoPanel (TOP) is a dual DNA-RNA panel as well as a paired tumor-normal matched test. Two hundred patients underwent TOP as part of Advanced Medical Care B with approval from the Ministry of Health, Labour and Welfare between September 2018 and December 2019. Tests were carried out in patients with cancers without standard treatment or when patients had already undergone standard treatment. Data from DNA and RNA panels were analyzed in 198 and 191 patients, respectively. The percentage of patients who were given therapeutic or diagnostic recommendations was 61% (120/198). One hundred and four samples (53%) harbored gene alterations that were detected with the DNA panel and had potential treatment implications, and 14 samples (7%) had a high tumor mutational burden. Twenty-two samples (11.1%) harbored 30 fusion transcripts or MET exon 14 skipping that were detected by the RNA panel. Of those 30 transcripts, 6 had treatment implications and 4 had diagnostic implications. Thirteen patients (7%) were found to have pathogenic or likely pathogenic germline variants and genetic counseling was recommended. Overall, 12 patients (6%) received recommended treatment. In summary, patients benefited from both TOP DNA and RNA panels while following the same indication as the approved CGP tests. (UMIN000033647).

Subclonal accumulation of immune escape mechanisms in microsatellite instability-high colorectal cancers.

BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.

Expression of paired box 9 defines an aggressive subset of lung adenocarcinoma preferentially occurring in smokers.

AIMS: A distinct subset of lung adenocarcinomas (LADs), arising from a series of peripheral lung cells defined as the terminal respiratory unit (TRU), is characterised by thyroid transcription factor 1 (TTF-1) expression. The clinical relevance of transcription factors (TFs) other than TTF-1 remains unknown in LAD and was explored in the present study. METHODS AND RESULTS: Seventy-one LAD samples were subjected to high-throughput transcriptome screening of LAD using cap analysis gene expression (CAGE) sequencing data; CAGE provides genome-wide expression levels of the transcription start sites (TSSs). In total, 1083 invasive LAD samples were subjected to immunohistochemical examination for paired box 9 (PAX9) and TTF-1 expression levels. PAX9 is an endoderm development-associated TF that most strongly and inversely correlates with the expression of TTF-1 TSS subsets. Immunohistochemically, PAX9 expression was restricted to the nuclei of ciliated epithelial and basal cells in the bronchi and bronchioles and the nuclei of epithelial cells of the bronchial glands; moreover, PAX9 expression was observed in 304 LADs (28%). PAX9-positive LADs were significantly associated with heavy smoking, non-lepidic subtype, EGFR wild-type tumours and PD-L1 expression (all P < 0.0001). All these characteristics were opposite to those of TRU-type LADs with TTF-1 expression. PAX9 expression was an independent prognostic factor for decreased overall survival (P = 0.022). CONCLUSIONS: Our results revealed that PAX9 expression defines an aggressive subset of LADs preferentially occurring in smokers that may arise from bronchial or bronchiolar cells.

Chronological improvement in precision oncology implementation in Japan.

In Japan, comprehensive genomic profiling (CGP) tests for refractory cancer patients have been approved since June 2019, under the requirement that all cases undergoing CGP tests are annotated by the molecular tumor board (MTB) at each government-designated hospital. To investigate improvement in precision oncology, we evaluated and compared the proportion of cases receiving matched treatments according to CGP results and those recommended to receive genetic counseling at all core hospitals between the first period (11 hospitals, June 2019 to January 2020) and second period (12 hospitals, February 2020 to January 2021). A total of 754 and 2294 cases underwent CGP tests at core hospitals in the first and second periods, respectively; 28 (3.7%) and 176 (7.7%) patients received matched treatments (p < 0.001). Additionally, 25 (3.3%) and 237 (10.3%) cases were recommended to receive genetic counseling in the first and second periods, respectively (p < 0.001). The proportion was associated with the type of CGP test: tumor-only (N = 2391) vs. tumor-normal paired (N = 657) analysis (10.0% vs. 3.5%). These results suggest that recommendations regarding available clinical trials in networked MTBs might contribute to increasing the numbers of matched treatments, and that tumor-normal paired rather than tumor-only tests can increase the efficiency of patient referrals for genetic counseling.

Recommendations related to the analytical equivalence assessment of gene panel testing.

Advances in cancer genome care over the past few years have included the development of gene panel testing for various biomarkers. This article summarizes issues and provides recommendations related to analytical performance evaluations for new oncology gene panels. The scope of these recommendations includes comprehensive genomic profiling assays related to gene panel testing that uses histological or serum specimens to detect gene mutations. As a research project of the Japan Agency for Medical Research and Development Research on Regulatory Science of Pharmaceuticals and Medical Devices, we convened the working group committee that consisted of more than 30 experts from academia, industry, and government. We have discussed the points that should be considered to allow maximal simplification of assessments using clinical specimens in evaluating accuracy and limit of detection in equivalence and analytical performance for 3 years. We provide recommendations specific to each type of gene mutation as well as to reference standards or specimens used for evaluations. In addition, in order to facilitate the discussion on the analytical performance of gene panel tests by multidisciplinary tumor boards of hospitals, the present recommendations also describe the items that companies are expected to provide information on in their packaging inserts and reports, and the items that are expected to be discussed by multidisciplinary tumor boards. Our working group document will be important for participants in multidisciplinary tumor boards, including medical oncologists and genome scientists, and developers of gene panels not only in Japan but also in other countries.

Expert panel consensus recommendations on the use of circulating tumor DNA assays for patients with advanced solid tumors.

Comprehensive genomic profiling is increasingly used to facilitate precision oncology based on molecular stratification. In addition to conventional tissue comprehensive genomic profiling, comprehensive genomic profiling of circulating tumor DNA has become widely utilized in cancer care owing on its advantages, including less invasiveness, rapid turnaround time, and capturing heterogeneity. However, circulating tumor DNA comprehensive genomic profiling has some limitations, mainly false negatives due to low levels of plasma circulating tumor deoxyribonucleic acid and false positives caused by clonal hematopoiesis. Nevertheless, no guidelines and recommendations fully address these issues. Here, an expert panel committee involving representatives from 12 Designated Core Hospitals for Cancer Genomic Medicine in Japan was organized to develop expert consensus recommendations for the use of circulating tumor deoxyribonucleic acid-based comprehensive genomic profiling. The aim was to generate guidelines for clinicians and allied healthcare professionals on the optimal use of the circulating tumor DNA assays in advanced solid tumors and to aid the design of future clinical trials that utilize and develop circulating tumor DNA assays to refine precision oncology. Fourteen clinical questions regarding circulating tumor deoxyribonucleic acid comprehensive genomic profiling including the timing of testing and considerations for interpreting results were established by searching and curating associated literatures, and corresponding recommendations were prepared based on the literature for each clinical question. Final consensus recommendations were developed by voting to determine the level of each recommendation by the Committee members.

Feasibility and clinical utility of comprehensive genomic profiling of hematological malignancies.

Identification of genetic alterations through next-generation sequencing (NGS) can guide treatment decision-making by providing information on diagnosis, therapy selection, and prognostic stratification in patients with hematological malignancies. Although the utility of NGS-based genomic profiling assays was investigated in hematological malignancies, no assays sufficiently cover driver mutations, including recently discovered ones, as well as fusions and/or pathogenic germline variants. To address these issues, here we have devised an integrated DNA/RNA profiling assay to detect various types of somatic alterations and germline variants at once. Particularly, our assay can successfully identify copy number alterations and structural variations, including immunoglobulin heavy chain translocations, IKZF1 intragenic deletions, and rare fusions. Using this assay, we conducted a prospective study to investigate the feasibility and clinical usefulness of comprehensive genomic profiling for 452 recurrently altered genes in hematological malignancies. In total, 176 patients (with 188 specimens) were analyzed, in which at least one alteration was detected in 171 (97%) patients, with a median number of total alterations of 7 (0-55). Among them, 145 (82%), 86 (49%), and 102 (58%) patients harbored at least one clinically relevant alteration for diagnosis, treatment, and prognosis, respectively. The proportion of patients with clinically relevant alterations was the highest in acute myeloid leukemia, whereas this assay was less informative in T/natural killer-cell lymphoma. These results suggest the clinical utility of NGS-based genomic profiling, particularly for their diagnosis and prognostic prediction, thereby highlighting the promise of precision medicine in hematological malignancies.

A multicenter investigator-initiated Phase 2 trial of E7090 in patients with advanced or recurrent solid tumor with fibroblast growth factor receptor (FGFR) gene alteration: FORTUNE trial.

BACKGROUND: Aberrant fibroblast growth factor receptor (FGFR) signaling can substantially influence oncogenicity. Despite that FGFR gene abnormality is often detected by cancer genome profiling tests, there is no tumor-agnostic approval yet for these aberrations. E7090 (tasurgratinib) is an orally available selective tyrosine kinase inhibitor of FGFR1-3. Specific FGFR alterations were previously reported to be highly sensitive to E7090 based on a high-throughput functional evaluation method, called mixed-all-nominated-mutants-in-one (MANO) method, narrowing down the most promising targets. This trial was focused on the alterations identified by the MANO method and was performed under the nationwide large registry network for rare cancers in Japan (MASTER KEY Project). METHODS/DESIGN: This single-arm Phase 2 trial was designed to evaluate the safety and efficacy of E7090 in patients with advanced or recurrent solid tumors harboring FGFR alterations. Three cohorts were set based on the type of FGFR alterations and the results of MANO method. A maximum of 45 patients will be enrolled from 5 institutions over 2.5 years. E7090 will be administered once daily as an oral single agent in 28-day cycles. The primary endpoint is the objective overall response rate; whereas, the secondary endpoints include progression-free survival, overall survival, disease control rate, safety, duration of response, and time to response. Ethics approval was granted by the National Cancer Center Hospital Certified Review Board. Patient enrollment began in June 2021. DISCUSSION: A unique investigator-initiated multicenter Phase 2 trial was designed based on the results of preclinical investigation aiming to acquire the approval of E7090 for solid tumors harboring FGFR gene alterations. The findings may serve as a novel model for the development of tumor-agnostic molecular targeted therapies against rare genetic abnormalities. TRIAL REGISTRATION: Japan Registry of Clinical Trial: jRCT2031210043 (registered April 20, 2021) ClinicalTrials.gov: NCT04962867 (registered July 15, 2021).

Tumor mutational burden measurement using comprehensive genomic profiling assay.

BACKGROUND: Tumors with a high number of mutations in the genome, or tumor mutational burden, are presumed to be more likely to respond to immune checkpoint inhibitors. However, the optimal method to calculate tumor mutational burden using comprehensive genomic profiling assays is unknown. METHODS: Todai OncoPanel is a dual panel of a deoxyribonucleic acid panel and a ribonucleic acid panel. Todai OncoPanel deoxyribonucleic acid panel version 6 is an improvement over version 3 with increased number of targeted genes and limited targeting of intronic regions. We calculated tumor mutational burden measured by Todai OncoPanel deoxyribonucleic acid panel versions 3 and 6 using three different calculation methods: all mutations within the targeted region (target tumor mutational burden), all mutations within the coding region (all coding tumor mutational burden) and non-synonymous mutations (non-synonymous coding tumor mutational burden). We then compared them with whole exosome sequencing tumor mutational burden. In addition, 16 lung cancer patients whose samples were analyzed using Todai OncoPanel deoxyribonucleic acid version 3 were treated with anti-PD-1 or PD-L1 antibody monotherapy. RESULTS: When compared with whole exosome sequencing tumor mutational burden as the standard, tumor mutational burden measured by Todai OncoPanel deoxyribonucleic acid version 3 resulted in accuracy of 71% for all three calculation methods. In version 6, accuracy was 96% for target tumor mutational burden and all coding tumor mutational burden and 91% for non-synonymous coding tumor mutational burden. Patients with either partial response or stable disease had higher non-synonymous coding tumor mutational burden (6.7/Mb vs. 1.6/Mb, P = 0.02) and higher PD-L1 expression (40% vs. 3%, P = 0.01) and a trend toward higher target tumor mutational burden (9.2/Mb vs. 2.4/Mb, P = 0.09) compared with patients with progressive disease. CONCLUSIONS: Increase in targeted gene number and limiting intronic regions improved tumor mutational burden measurement by Todai OncoPanel when compared with whole exosome sequencing tumor mutational burden. Target tumor mutational burden may be the method of choice to measure tumor mutational burden.

Development of an optimal protocol for molecular profiling of tumor cells in pleural effusions at single-cell level.

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single-cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic-activated cell sorting system, and CD45-negative and cytokeratin-positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole-genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4-ALK fusion (17/20 cells, 85%) with an alectinib-resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA-associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single-cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA-associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.

Appropriate use of cancer comprehensive genome profiling assay using circulating tumor DNA.

Comprehensive genomic profiling (CGP) is being increasingly used for the routine clinical management of solid cancers. In July 2018, the use of tumor tissue-based CGP assays became available for all solid cancers under the universal health insurance system in Japan. Several restrictions presently exist, such as patient eligibility and limitations on the opportunities to perform such assays. The clinical implementation of CGP based on plasma circulating tumor DNA (ctDNA) is also expected to raise issues regarding the selection and use of tissue DNA and ctDNA CGP. A Joint Task Force for the Promotion of Cancer Genome Medicine comprised of three Japanese cancer-related societies has formulated a policy proposal for the appropriate use of plasma CGP (in Japanese), available at https://www.jca.gr.jp/researcher/topics/2021/files/20210120.pdf, http://www.jsco.or.jp/jpn/user_data/upload/File/20210120.pdf, and https://www.jsmo.or.jp/file/dl/newsj/2765.pdf. Based on these recommendations, the working group has summarized the respective advantages and cautions regarding the use of tissue DNA CGP and ctDNA CGP with reference to the advice of a multidisciplinary expert panel, the preferred use of plasma specimens over tissue, and multiple ctDNA testing. These recommendations have been prepared to maximize the benefits of performing CGP assays and might be applicable in other countries and regions.

Highly sensitive fusion detection using plasma cell-free RNA in non-small-cell lung cancers.

ALK, ROS1, and RET kinase fusions are important predictive biomarkers of tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC). Analysis of cell-free DNA (cfDNA) provides a noninvasive method to identify gene changes in tumor cells. The present study sought to use cfRNA and cfDNA for identifying fusion genes. A reliable protocol was established to detect fusion genes using cfRNA and assessed the analytical validity and clinical usefulness in 30 samples from 20 cases of fusion-positive NSCLC. The results of cfRNA-based assays were compared with tissue biopsy and cfDNA-based liquid biopsy (Guardant360 plasma next-generation sequencing [NGS] assay). The overall sensitivity of the cfRNA-based assay was 26.7% (8/30) and that of cfDNA-based assay was 16.7% (3/18). When analysis was limited to the samples collected at chemo-naïve or progressive disease status and available for both assays, the sensitivity of the cfRNA-based assay was 77.8% (7/9) and that of cfDNA-based assay was 33.3% (3/9). Fusion gene identification in cfRNA was correlated with treatment response. These results suggest that the proposed cfRNA assay is a useful diagnostic test for patients with insufficient tissues to facilitate effective administration of first-line treatment and is a useful tool to monitor the progression of NSCLC for consideration of second-line treatments.