Tracing Oncogene Rearrangements in the Mutational History of Lung Adenocarcinoma.

Mutational processes giving rise to lung adenocarcinomas (LADCs) in non-smokers remain elusive. We analyzed 138 LADC whole genomes, including 83 cases with minimal contribution of smoking-associated mutational signature. Genomic rearrangements were not correlated with smoking-associated mutations and frequently served as driver events of smoking-signature-low LADCs. Complex genomic rearrangements, including chromothripsis and chromoplexy, generated 74% of known fusion oncogenes, including EML4-ALK, CD74-ROS1, and KIF5B-RET. Unlike other collateral rearrangements, these fusion-oncogene-associated rearrangements were frequently copy-number-balanced, representing a genomic signature of early oncogenesis. Analysis of mutation timing revealed that fusions and point mutations of canonical oncogenes were often acquired in the early decades of life. During a long latency, cancer-related genes were disrupted or amplified by complex rearrangements. The genomic landscape was different between subgroups-EGFR-mutant LADCs had frequent whole-genome duplications with p53 mutations, whereas fusion-oncogene-driven LADCs had frequent SETD2 mutations. Our study highlights LADC oncogenesis driven by endogenous mutational processes.

Widespread somatic L1 retrotransposition in normal colorectal epithelium.

Throughout an individual's lifetime, genomic alterations accumulate in somatic cells1-11. However, the mutational landscape induced by retrotransposition of long interspersed nuclear element-1 (L1), a widespread mobile element in the human genome12-14, is poorly understood in normal cells. Here we explored the whole-genome sequences of 899 single-cell clones established from three different cell types collected from 28 individuals. We identified 1,708 somatic L1 retrotransposition events that were enriched in colorectal epithelium and showed a positive relationship with age. Fingerprinting of source elements showed 34 retrotransposition-competent L1s. Multidimensional analysis demonstrated that (1) somatic L1 retrotranspositions occur from early embryogenesis at a substantial rate, (2) epigenetic on/off of a source element is preferentially determined in the early organogenesis stage, (3) retrotransposition-competent L1s with a lower population allele frequency have higher retrotransposition activity and (4) only a small fraction of L1 transcripts in the cytoplasm are finally retrotransposed in somatic cells. Analysis of matched cancers further suggested that somatic L1 retrotransposition rate is substantially increased during colorectal tumourigenesis. In summary, this study illustrates L1 retrotransposition-induced somatic mosaicism in normal cells and provides insights into the genomic and epigenomic regulation of transposable elements over the human lifetime.

Clonal dynamics in early human embryogenesis inferred from somatic mutation.

Cellular dynamics and fate decision in early human embryogenesis remain largely unknown owing to the challenges of performing studies in human embryos1. Here, we explored whole-genomes of 334 single-cell colonies and targeted deep sequences of 379 bulk tissues obtained from various anatomical locations of seven recently deceased adult human donors. Using somatic mutations as an intrinsic barcode, we reconstructed early cellular phylogenies that demonstrate (1) an endogenous mutational rate that is higher in the first cell division but decreases to approximately one per cell per cell division later in life; (2) universal unequal contribution of early cells to embryo proper, resulting from early cellular bottlenecks that stochastically set aside epiblast cells within the embryo; (3) examples of varying degrees of early clonal imbalances between tissues on the left and right sides of the body, different germ layers and specific anatomical parts and organs; (4) emergence of a few ancestral cells that will substantially contribute to adult cell pools in blood and liver; and (5) presence of mitochondrial DNA heteroplasmy in the fertilized egg. Our approach also provides insights into the age-related mutational processes and loss of sex chromosomes in normal somatic cells. In sum, this study provides a foundation for future studies to complete cellular phylogenies in human embryogenesis.

Effects of tertiary palliative care on the pattern of end-of-life care in patients with hematologic malignancies in Korea.

INTRODUCTION: Patients with hematologic malignancies (HMs) often face challenges in accessing palliative care (PC) and receiving quality end-of-life (EOL) care. We examined factors associated with referrals to tertiary PC and the effects of tertiary PC on EOL care in patients with HMs. METHOD: We included patients with HMs who were admitted to a university-affiliated hospital and died during hospitalization between January 2018 and December 2021. We investigated the receipt of PC consultations, patient characteristics, and EOL care indicators. RESULTS: Overall, 487 patients were included in the analysis, with 156 (32%) undergoing PC consultation. Sex, residence, disease status, and admission purpose were factors associated with the likelihood of PC consultation, and there has been an increasing trend in the frequency of consultations in recent cases. A higher proportion of patients who received PC completed advance statements and life-sustaining treatment documents. Patients who received PC had lower rates of aggressive EOL care, including chemotherapy and intensive care unit admission, than those who did not receive PC. Notably, PC reduced the number of blood transfusions. CONCLUSION: Tertiary PC aims to reduce aggressive EOL care through patient-centered goal-of-care discussions. Therefore, there is an imperative need for concerted efforts toward seamless integration of PC.

Eltrombopag for Post-Transplant Poor Graft Function in East Asian Patients.

Poor graft function (PGF) is a serious, potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation. Eltrombopag has shown multilineage responses in patients with refractory severe aplastic anemia, supporting the idea that it may improve cytopenia in patients with PGF. This retrospective, single center analysis included 8 Korean patients receiving eltrombopag for PGF. Median interval between transplant and eltrombopag treatment was 73 days, and the median duration treatment was 3.5 weeks. With median maximum daily dose of 50 mg, the time to best response was 93 days. Median hemoglobin increased from 8.2 g/dL to 10.9 g/dL, platelet from 18.5 × 109/L to 54 × 109/L, and absolute neutrophil count from 1.25 × 109/L to 3.32 × 109/L. In conclusion, eltrombopag is a good option for PGF in Korean patients, even at a lower dose compared to western patients.

The genome-wide landscape of C:G > T:A polymorphism at the CpG contexts in the human population.

BACKGROUND: The C:G > T:A substitution at the CpG dinucleotide contexts is the most frequent substitution type in genome evolution. The mutational process is obviously ongoing in the human germline; however, its impact on common and rare genomic polymorphisms has not been comprehensively investigated yet. Here we observed the landscape and dynamics of C:G > T:A substitutions from population-scale human genome sequencing datasets including ~ 4300 whole-genomes from the 1000 Genomes and the pan-cancer analysis of whole genomes (PCAWG) Project and ~ 60,000 whole-exomes from the Exome Aggregation Consortium (ExAC) database. RESULTS: Of the 28,084,558 CpG sites in the human reference genome, 26.0% show C:G > T:A substitution in the dataset. Remarkably, CpGs in CpG islands (CGIs) have a much lower frequency of such mutations (5.6%). Interestingly, the mutation frequency of CGIs is not uniform with a significantly higher C:G > T:A substitution rate for intragenic CGIs compared to other types. For non-CGI CpGs, the mutation rate was positively correlated with the distance from the nearest CGI up to 2 kb. Finally, we found the impact of negative selection for coding CpG mutations resulting in amino acid change. CONCLUSIONS: This study provides the first unbiased rate of C:G > T:A substitution at the CpG dinucleotide contexts, using population-scale human genome sequencing data. Our findings provide insights into the dynamics of the mutation acquisition in the human genome.

Immune recurrence score using 7 immunoregulatory protein expressions can predict recurrence in stage I-III breast cancer patients.

BACKGROUND: Immune cells in the tumour microenvironment play an essential role in tumorigenesis. This study aimed to evaluate the immunoregulatory protein expression of breast cancer and reveal their prognostic role. METHODS: Expression of 10 immune markers (PD-1/PD-L1/PD-L2/IDO/TIM-3/OX40/OX40L/B7-H2/ B7-H3/B7-H4) with known/possible clinical relevance was identified in stromal tumour-infiltrating lymphocytes or tumour tissue of stage I-III breast cancer patients. RESULTS: A total of 392 patients, including 271(69.1%) luminal A, 36(9.2%) luminal B, 32(8.2%) HER2-positive and 53(13.5%) triple negative disease, were included. Expression of PD-1 and PD-L1 was higher in HER2-positive and triple negative disease. By contrast, expression of TIM-3, OX40 and OX40L were higher in luminal disease. We devised an immune recurrence score (IRS) using seven markers with prognostic value (B7-H2/B7-H3/B7-H4/OX40/OX40L/PD-L1/PD-L2). Patients were classified as high-risk (7.9%), intermediate-risk (67.6%), or low-risk (24.5%). In the multivariate analysis, IRS low-risk (adjusted HR 0.14, p = 0.001) and intermediate-risk (adjusted HR 0.32, p = 0.002) had significantly lower risk of recurrence compared with high-risk. The prognostic role of IRS was maintained in both luminal A and non-luminal A patients. CONCLUSIONS: This study identified immunoregulatory protein expression of breast cancer patients using 10 immune markers. In addition, we devised an IRS which may predict recurrence in stage I-III breast cancer patients.

Micafungin prophylaxis for acute leukemia patients undergoing induction chemotherapy.

BACKGROUND: Micafungin is a well-tolerated and effective prophylactic antifungal agent used in hematologic diseases. In this prospective trial, we evaluated the efficacy and safety of prophylactic micafungin during first induction chemotherapy in patients with acute leukemia. We also compared outcomes of prophylactic micafungin with those of prophylactic posaconazole in acute myeloid leukemia (AML). METHODS: Medically fit patients with newly diagnosed acute leukemia received 50 mg micafungin intravenously once daily from the initiation of first induction chemotherapy to recovery of neutrophil count, suspected fungal infection, or unacceptable drug-related toxicity ( Clinicaltrials.gov number, NCT02440178). The primary end point was incidence of invasive fungal infection, and the secondary end points were adverse events of prophylactic micafungin and mortality during induction therapy. RESULTS: The 65 patients (median age = 51 years, male:female = 34:31) enrolled in this study had diagnoses of AML (33, 50.8%), acute lymphoblastic leukemia (31, 47.7%), and acute biphenotypic leukemia (1, 1.5%). Median duration of micafungin treatment was 24 days (range 1-68), with proven invasive fungal disease in one patient (1.5%) and possible fungal infection in two patients (3.1%). Three of the patients (4.6%) experienced the following adverse events, but all events were tolerable: liver function abnormality (Grade 2, n = 1; Grade 3, n = 1) and allergic reaction (Grade 2, n = 1). Three patients died during induction therapy, and invasive aspergillosis pneumonia was the cause of death for one of those patients. Overall, 19 patients (29.2%) discontinued prophylactic micafungin, and 18 (27.7%) patients switched to another antifungal agent. We observed no fungal infections caused by amphotericin B-resistant organisms. In AML patients, outcomes of prophylactic micafungin during induction chemotherapy did not differ significantly with those of prophylactic posaconazole with regard to incidence of fungal infections, rate of discontinuation, or safety. CONCLUSIONS: Our study demonstrates that prophylactic micafungin is safe and effective in patients with acute leukemia undergoing induction chemotherapy. Outcomes in patients with AML were similar to those of prophylactic posaconazole, indicating the usefulness of micafungin as a prophylactic antifungal agent during induction chemotherapy for AML. TRIAL REGISTRATION: Clinicaltrials.gov NCT02440178, registered May 12th 2015.

Utility of allogeneic hematopoietic stem cell transplantation using international donors in a homogenous ethnic population: question in the era of various alternative donors.

The advent of various alternative donors in allogeneic hematopoietic stem cell transplantation (HSCT) raises the question of using international donors, especially in ethnically homogenous populations. We analyzed the clinical outcome and medical expense of human leukocyte antigen (HLA)-matched HSCT using domestic and international donors. We analyzed the patients who received allogeneic HSCT at five medical centers in Korea in the last 10 years. Using propensity-score matching, we compared overall survival (OS), relapse-free survival (RFS), and transplantation-related complications. Medical expense was analyzed based on National Health Insurance Service (NHIS) data. A total of 269 patients were analyzed after 3:1 (domestic/international) matching. There was no difference in OS (p = 0.395) and RFS (p = 0.604) between the domestic and international donor groups (5-year OS rate 42.9 and 37.8%, 5-year RFS rate 37.6 and 33.5% for domestic and international groups, respectively). No difference in chronic graft-versus-host disease (GVHD) incidence was observed (34.2% in domestic and 35.9% in international group, p = 0.804). Early infection was more frequent in the domestic group (55.0 vs. 35.8%, p = 0.007), whereas infection after 30 days was more frequent in the international group (28.7 vs. 49.3%, p = 0.001). Mean medical expense was far higher in the international group, by US $51,944 in the entire follow-up period (p < 0.001). We would expect similar outcomes for international and domestic donors in terms of survival and treatment-related complications with HLA-matched HSCT in other ethnically homogenous populations. These findings should be considered together with the high cost of using international donors in the era of various alternative donors.

Comparison of cladribine- and fludarabine-based induction chemotherapy in relapsed or refractory acute myeloid leukaemia.

Induction regimens integrating cladribine or fludarabine have shown promising outcomes in relapsed or refractory (R/R) acute myeloid leukaemia (AML). We compared the outcome of a cladribine- versus a fludarabine-based regimen as induction chemotherapy for R/R-AML. We included patients with R/R-AML who were treated with a cladribine- or fludarabine-based chemotherapy between 2006 and 2015. We analysed 120 patients, 65 treated with cladribine and 55 treated with fludarabine. The CR rates were 62.7 and 61.4 % for the cladribine group and fludarabine group, respectively (p = 0.890). Poor prognostic factors included older age, secondary AML, poor cytogenetic risk group, prior induction failure, and short first CR duration. No significant overall survival (OS) or relapse-free survival (RFS) differences were found between the groups (OS, p = 0.213; RFS, p = 0.143). However, in a certain subset, survival outcomes were better with cladribine than with fludarabine, including de novo AML, CR at first induction therapy, and not-poor cytogenetic risk group inclusion without overt chemotherapy-refractoriness. By contrast, secondary AML patients had improved survival outcomes when treated with the fludarabine regimen. After CR, better outcomes were observed when allogeneic stem cell transplantation (SCT) was given as consolidation. In R/R-AML, cladribine- and fludarabine-based combination induction chemotherapy had differential survival outcomes according to disease characteristics. Allogeneic SCT after CR with a purine analogue-based regimen improved long-term outcome in these patients.

Analytical and Clinical Validation of a Highly Sensitive NGS-Based ctDNA Assay with Real-World Concordance in NSCLC.

Purpose: There have been needs to improve the sensitivity of liquid biopsy. This report aims to report the analytical and clinical validation of a next generation sequencing (NGS)-based circulating tumor DNA (ctDNA) assay. Materials and Methods: Analytical validation was conducted in vitro by evaluating the limit of detection (LOD), precision, and specificity for various genomic aberrations. The real-world performance in non-small cell lung cancer (NSCLC) was assessed by comparing the results of AlphaLiquid®100 to the tissue-based results. Results: The LODs with 30 ng input DNA were 0.11%, 0.11%, 0.06%, 0.21%, and 2.13 copies for detecting SNVs, insertions, deletions, fusions, and copy number alterations (CNA), respectively. Quantitatively, SNV/INDELs, fusions, and CNAs showed a good correlation (R2=0.91, 0.40, and 0.65; y=0.95, 1.06, and 1.19) to the manufacturer's values, and per-base specificities for all types of variants were near 100%. In real-world NSCLC (n=122), key actionable mutations in NSCLC were detected in 60.7% (74/122) with the ctDNA assay. Comparative analysis against the NGS-based tissue results for all key mutations showed positive percent agreement (PPA) of 85.3%. For individual genes, the PPA was as high as 95.7% for EGFR mutations and 83.3% for ALK translocations. AlphaLiquid 100 detected drug-sensitive EGFR mutation at a variant allele frequency as low as 0.02% and also identified an EGFR mutation in a case where tissue sample missed. Blood samples collected post-targeted therapies revealed additional acquired mutations. Conclusion: The AlphaLiquid®100 ctDNA assay demonstrates robust analytical validity, offering clinically important information for NSCLC patients.

Quantitative and qualitative mutational impact of ionizing radiation on normal cells.

The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.

Subset of retinoblastoma tumours is associated with BRCA1/2 mutations.

BACKGROUND: We investigated the potential association between pathogenic BRCA1/2 variants and retinoblastoma pathogenicity. METHODS: In this single-centre, retrospective case series, we performed hereditary cancer panel tests using blood samples for patients with retinoblastoma diagnosed between March 2017 and October 2021. Bioinformatics prediction tools were then used to conduct in silico pathogenicity assessments for patients with BRCA1/2 family variants, in addition to the American College of Medical Genetics and Genomics (ACMG) variant classification. One patient with a germline BRCA1 variant was analysed with whole-genome sequencing (WGS), mutational signature analysis and methylation analysis for RB1 and BRCA using the patient's tumour and blood samples. RESULTS: Of 30 retinoblastoma patients who underwent panel sequencing, six (20%) were found to carry germline variants in the BRCA1/2 or BRIP1 genes. Among these six patients, two had pathogenic or likely pathogenic variants as per the ACMG variant classification. Additionally, three patients showed potential pathogenic BRCA1/2 family variants through further analysis with alternative bioinformatics prediction tools. In the WGS analysis of a tumour from a patient with a germline likely pathogenic BRCA1 variant in one allele, we observed the loss of one RB1 allele due to a large deletion. No somatic non-synonymous mutations or frameshift indels were detected in the RB1 locus of the remaining allele. This sample also showed BRCA1 gene promoter hypermethylation in the tumour, indicating additional epigenetic silencing. CONCLUSION: This study demonstrated that some retinoblastoma patients harboured germline BRCA1/2 family variants, which may be associated with the development of retinoblastoma along with RB1 mutations.

Prognostic utility of body composition parameters based on computed tomography analysis of advanced non-small cell lung cancer treated with immune checkpoint inhibitors.

OBJECTIVE: The purpose of this study was to evaluate the prognostic impact of body composition parameters based on computed tomography (CT) in patients with non-small cell lung cancer (NSCLC) who received ICI treatment. METHODS: This retrospective study analyzed the data from advanced NSCLC patients treated with ICI therapy between 2013 and 2019. We included patients with NSCLC who underwent baseline CT scans. The exclusion criteria included patients who received three or more lines of chemotherapy, those with insufficient clinical information, or those without treatment response evaluation. RESULTS: A total of 136 patients were enrolled. Among the volumetric body composition parameters, patients in the highest quartiles (Q2-4) of the visceral fat index (VFI) exhibited a higher response rate to ICI therapy than those in the lowest quartile (Q1) of VFI (Q1 vs. Q2-4: 18.2% vs. 43.1%, p = 0.012). Patients with a VFI in Q2-4 had significantly prolonged progression-free survival (PFS) and overall survival (OS) (PFS, Q1 vs. Q2-4: 3.0 months vs. 6.4 months, p = 0.043; OS, Q1 vs. Q2-4: 5.6 months vs. 16.3 months, p = 0.004). Kaplan-Meier analysis based on the VFI and visceral fat Hounsfield unit (HU) revealed that patients with VFI in Q1 and HU in Q2-4 had the worst prognosis. CONCLUSIONS: Visceral fat volume is significantly associated with treatment outcomes in ICI-treated patients with NSCLC. Moreover, fat quality may impact the treatment outcomes. This finding underscores the potential significance of both fat compartments and fat quality as prognostic indicators. CRITICAL RELEVANCE STATEMENT: Visceral fat volume is significantly associated with treatment outcomes in ICI-treated patients with non-small cell lung cancer. Moreover, fat quality may impact the treatment outcomes. This finding underscores the potential significance of both fat compartments and fat quality as prognostic indicators. KEY POINTS: • We found that visceral fat volume positively correlated with treatment response and survival in patients with non-small cell lung cancer receiving immune checkpoint inhibitors. • Additionally, a trend toward a negative correlation between visceral fat attenuation and survival was observed. • The findings highlight the prognostic utility of fat compartments and fat quality.

Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes as a biomarker in locally advanced unresectable thymic epithelial neoplasm: A single-center, retrospective, longitudinal cohort study.

BACKGROUND: Thymic epithelial tumors (TET) are rare malignancies and lack well-defined biomarkers for neoadjuvant therapy. This study aimed to evaluate the clinical utility of artificial intelligence (AI)-powered tumor-infiltrating lymphocyte (TIL) analysis in TET. METHODS: Patients initially diagnosed with unresectable thymoma or thymic carcinoma who underwent neoadjuvant therapy between January 2004 and December 2021 formed our study population. Hematoxylin and eosin-stained sections from the initial biopsy and surgery were analyzed using an AI-powered spatial TIL analyzer. Intratumoral TIL (iTIL) and stromal TIL (sTIL) were quantified and their immune phenotype (IP) was identified. RESULTS: Thirty-five patients were included in this study. The proportion of patients with partial response to neoadjuvant therapy was higher in the group with nondesert IP in preneoadjuvant biopsy (63.6% vs. 17.6%, p = 0.038). A significant increase in both iTIL (median 22.18/mm2 vs. 340.69/mm2 , p < 0.001) and sTIL (median 175.19/mm2 vs. 531.02/mm2 , p = 0.004) was observed after neoadjuvant therapy. Patients with higher iTIL (>147/mm2 ) exhibited longer disease-free survival (median, 29 months vs. 12 months, p = 0.009) and overall survival (OS) (median, 62 months vs. 45 months, p = 0.002). Patients with higher sTIL (>232.1/mm2 ) exhibited longer OS (median 62 months vs. 30 months, p = 0.021). CONCLUSIONS: Nondesert IP in initial biopsy was associated with a better response to neoadjuvant therapy. Increased infiltration of both iTIL and sTIL in surgical specimens were associated with longer OS in patients with TET who underwent resection followed by neoadjuvant therapy.

Treated chronic hepatitis B is a good prognostic factor of diffuse large B-cell lymphoma.

BACKGROUND/AIMS: Chronic hepatitis B (CHB) is a risk factor for non-Hodgkin lymphoma (NHL). Our recent study suggested that antiviral treatment may reduce the incidence of NHL in CHB patients. This study compared the prognoses of hepatitis B virus (HBV)-associated diffuse large B-cell lymphoma (DLBCL) patients receiving antiviral treatment and HBV-unassociated DLBCL patients. METHODS: This study comprised 928 DLBCL patients who were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) at two referral centers in Korea. All patients with CHB received antiviral treatment. Time-to-progression (TTP) and overall survival (OS) were the primary and secondary endpoints, respectively. RESULTS: Among the 928 patients in this study, 82 were hepatitis B surface antigen (HBsAg)-positive (the CHB group) and 846 were HBsAg-negative (the non-CHB group). The median follow-up time was 50.5 months (interquartile range [IQR]=25.6-69.7 months). Multivariable analyses showed longer TTP in the CHB group than the non-CHB group both before inverse probability of treatment weighting (IPTW; adjusted hazard ratio [aHR]=0.49, 95% confidence interval [CI]=0.29-0.82, p=0.007) and after IPTW (aHR=0.42, 95% CI=0.26-0.70, p<0.001). The CHB group also had a longer OS than the non-CHB group both before IPTW (HR=0.55, 95% CI=0.33-0.92, log-rank p=0.02) and after IPTW (HR=0.53, 95% CI=0.32-0.99, log-rank p=0.02). Although liver-related deaths did not occur in the non-CHB group, two deaths occurred in the CHB group due to hepatocellular carcinoma and acute liver failure, respectively. CONCLUSION: Our findings indicate that HBV-associated DLBCL patients receiving antiviral treatment have significantly longer TTP and OS after R-CHOP treatment than HBV-unassociated DLBCL patients.

Patient-derived organoids as a preclinical platform for precision medicine in colorectal cancer.

Patient-derived organoids are being considered as models that can help guide personalized therapy through in vitro anticancer drug response evaluation. However, attempts to quantify in vitro drug responses in organoids and compare them with responses in matched patients remain inadequate. In this study, we investigated whether drug responses of organoids correlate with clinical responses of matched patients and disease progression of patients. Organoids were established from 54 patients with colorectal cancer who (except for one patient) did not receive any form of therapy before, and tumor organoids were assessed through whole-exome sequencing. For comparisons of in vitro drug responses in matched patients, we developed an 'organoid score' based on the variable anticancer treatment responses observed in organoids. Very interestingly, a higher organoid score was significantly correlated with a lower tumor regression rate after the standard-of-care treatment in matched patients. Additionally, we confirmed that patients with a higher organoid score (≥ 2.5) had poorer progression-free survival compared with those with a lower organoid score (< 2.5). Furthermore, to assess potential drug repurposing using an FDA-approved drug library, ten tumor organoids derived from patients with disease progression were applied to a simulation platform. Taken together, organoids and organoid scores can facilitate the prediction of anticancer therapy efficacy, and they can be used as a simulation model to determine the next therapeutic options through drug screening. Organoids will be an attractive platform to enable the implementation of personalized therapy for colorectal cancer patients.

Gallbladder adenocarcinomas undergo subclonal diversification and selection from precancerous lesions to metastatic tumors.

We aimed to elucidate the evolutionary trajectories of gallbladder adenocarcinoma (GBAC) using multi-regional and longitudinal tumor samples. Using whole-exome sequencing data, we constructed phylogenetic trees in each patient and analyzed mutational signatures. A total of 11 patients including 2 rapid autopsy cases were enrolled. The most frequently altered gene in primary tumors was ERBB2 and TP53 (54.5%), followed by FBXW7 (27.3%). Most mutations in frequently altered genes in primary tumors were detectable in concurrent precancerous lesions (biliary intraepithelial neoplasia [BilIN]), but a substantial proportion was subclonal. Subclonal diversity was common in BilIN (n=4). However, among subclones in BilIN, a certain subclone commonly shrank in concurrent primary tumors. In addition, selected subclones underwent linear and branching evolution, maintaining subclonal diversity. Combined analysis with metastatic tumors (n=11) identified branching evolution in nine patients (81.8%). Of these, eight patients (88.9%) had a total of 11 subclones expanded at least sevenfold during metastasis. These subclones harbored putative metastasis-driving mutations in cancer-related genes such as SMAD4, ROBO1, and DICER1. In mutational signature analysis, six mutational signatures were identified: 1, 3, 7, 13, 22, and 24 (cosine similarity >0.9). Signatures 1 (age) and 13 (APOBEC) decreased during metastasis while signatures 22 (aristolochic acid) and 24 (aflatoxin) were relatively highlighted. Subclonal diversity arose early in precancerous lesions and clonal selection was a common event during malignant transformation in GBAC. However, selected cancer clones continued to evolve and thus maintained subclonal diversity in metastatic tumors.

Dissecting single-cell genomes through the clonal organoid technique.

The revolution in genome sequencing technologies has enabled the comprehensive detection of genomic variations in human cells, including inherited germline polymorphisms, de novo mutations, and postzygotic mutations. When these technologies are combined with techniques for isolating and expanding single-cell DNA, the landscape of somatic mosaicism in an individual body can be systematically revealed at a single-cell resolution. Here, we summarize three strategies (whole-genome amplification, microdissection of clonal patches in the tissue, and in vitro clonal expansion of single cells) that are currently applied for single-cell mutational analyses. Among these approaches, in vitro clonal expansion, particularly via adult stem cell-derived organoid culture technologies, yields the most sensitive and precise catalog of somatic mutations in single cells. Moreover, because it produces living mutant cells, downstream validation experiments and multiomics profiling are possible. Through the synergistic combination of organoid culture and genome sequencing, researchers can track genome changes at a single-cell resolution, which will lead to new discoveries that were previously impossible.

Mutational spectrum of SARS-CoV-2 during the global pandemic.

Viruses accumulate mutations under the influence of natural selection and host-virus interactions. Through a systematic comparison of 351,525 full viral genome sequences collected during the recent COVID-19 pandemic, we reveal the spectrum of SARS-CoV-2 mutations. Unlike those of other viruses, the mutational spectrum of SARS-CoV-2 exhibits extreme asymmetry, with a much higher rate of C>U than U>C substitutions, as well as a higher rate of G>U than U>G substitutions. This suggests directional genome sequence evolution during transmission. The substantial asymmetry and directionality of the mutational spectrum enable pseudotemporal tracing of SARS-CoV-2 without prior information about the root sequence, collection time, and sampling region. This shows that the viral genome sequences collected in Asia are similar to the original genome sequence. Adjusted estimation of the dN/dS ratio accounting for the asymmetrical mutational spectrum also shows evidence of negative selection on viral genes, consistent with previous reports. Our findings provide deep insights into the mutational processes in SARS-CoV-2 viral infection and advance the understanding of the history and future evolution of the virus.