Clonal Evolution Research Articles

Decoding ribosome complexity: role of ribosomal proteins in cancer and disease.

The ribosome is a remarkably complex machinery, at the interface with diverse cellular functions and processes. Evolutionarily conserved, yet intricately regulated, ribosomes play pivotal roles in decoding genetic information into the synthesis of proteins and in the generation of biomass critical for cellular physiological functions. Recent insights have revealed the existence of ribosome heterogeneity at multiple levels. Such heterogeneity extends to cancer, where aberrant ribosome biogenesis and function contribute to oncogenesis. This led to the emergence of the concept of 'onco-ribosomes', specific ribosomal variants with altered structural dynamics, contributing to cancer initiation and progression. Ribosomal proteins (RPs) are involved in many of these alterations, acting as critical factors for the translational reprogramming of cancer cells. In this review article, we highlight the roles of RPs in ribosome biogenesis, how mutations in RPs and their paralogues reshape the translational landscape, driving clonal evolution and therapeutic resistance. Furthermore, we present recent evidence providing new insights into post-translational modifications of RPs, such as ubiquitylation, UFMylation and phosphorylation, and how they regulate ribosome recycling, translational fidelity and cellular stress responses. Understanding the intricate interplay between ribosome complexity, heterogeneity and RP-mediated regulatory mechanisms in pathology offers profound insights into cancer biology and unveils novel therapeutic avenues targeting the translational machinery in cancer.

Evolutionary history of adenomas to colorectal cancer in FAP families.

Familial adenomatous polyposis (FAP) is a genetic syndrome characterized by multiple polyps at various evolutionary stages, which, if left untreated, inevitably progress to colorectal cancer (CRC). In this study, we present a comprehensive analysis of the evolutionary history of FAP-CRC from precancerous adenoma to carcinoma. Tissues were collected from gastrointestinal endoscopy or surgical resection. Exome sequencing was performed on multiple regions of adenocarcinoma (n = 8), villous adenoma (n = 10), tubular adenoma (n = 9) and blood samples were obtained from 9 patients belonging to 7 Chinese FAP families. Phylogenetic trees were reconstructed, and evolutionary analysis was conducted to reveal the temporal sequence of events leading to CRC. Inherited germline mutation sites in APC gene were identified in FAP01 (p.S1281*, COSM19212), FAP03 (p.S384Tfs*19), FAP04 (p.E1538*, COSM6041693), FAP05 (p.Q1062*, COSM3696862), and FAP07-FAP09 (p.V677Sfs*3). Notably, p.V677Sfs*3 mutation was recognized as a novel germline mutation in APC, supported by evidence of genotype-phenotype correlation in pedigree analysis. Adenomas exhibited lower mutational rates than FAP-CRC and displayed recurrent alterations in well-known chromosomal instability (CIN) genes (APC, RAS, SMAD4 and TP53) and DNA damage repair genes (SUZ12, KMT2C, BCLAF1, RUNX1, and ARID1B), suggesting the presence of genomic instability. Furthermore, a progressive increase in the HRD score (a measure of "genomic scars") was observed from tubular adenomas to villous adenomas and ultimately to carcinomas. TP53 emerged as the primary driver gene for adenoma-carcinoma transition, with driver mutations consistently appearing simultaneously rather than sequentially acquired from adenomas to carcinomas. Clonal evolution demonstrated that liver metastases can originate from the same cancer-primed cell present in a primary cancerous lesion. We identified a novel pathogenic variant in APC, namely, p.V677Sfs*3. The process of carcinogenesis in FAP-CRC supports the classical cancerization model, where an initial APC mutation leads to the activation of the WNT signaling pathway and CIN. Subsequently, additional mutations occur in other putative CIN genes (e.g., DNA repair, chromatin remodeling), ultimately leading to the development of microsatellite stable (MSS) tumors. Our study provides a comprehensive understanding of the genomic landscapes that underlie the transition from adenoma to carcinoma.

Autocrine glutamate signaling drives cell competition in Drosophila.

Cell competition is an evolutionarily conserved quality control process that eliminates suboptimal or potentially dangerous cells. Although differential metabolic states act as direct drivers of competition, how these are measured across tissues is not understood. Here, we demonstrate that vesicular glutamate transporter (VGlut) and autocrine glutamate signaling are required for cell competition and Myc-driven super-competition in the Drosophila epithelia. We find that the loss of glutamate-stimulated VGlut>NMDAR>CaMKII>CrebB signaling triggers loser status and cell death under competitive settings via the autocrine induction of TNF. This in turn drives TNFR>JNK activation, triggering loser cell elimination and PDK/LDH-dependent metabolic reprogramming. Inhibiting caspases or preventing loser cells from transferring lactate to their neighbors nullifies cell competition. Further, in a Drosophila model for premalignancy, Myc-overexpressing clones co-opt this signaling circuit to acquire super-competitor status. Targeting glutamate signaling converts Myc "super-competitor" clones into "losers," highlighting new therapeutic opportunities to restrict the evolution of fitter clones.

Clonal competition assays identify fitness signatures in cancer progression and resistance in multiple myeloma.

Multiple myeloma (MM) is a genetically heterogeneous disease and the management of relapses is one of the biggest clinical challenges. TP53 alterations are established high-risk markers and are included in the current disease staging criteria. KRAS is the most frequently mutated gene affecting around 20% of MM patients. Applying Clonal Competition Assays (CCA)byco-culturing color-labeled genetically modified cell models, we recently showed that mono- and biallelic alterations in TP53 transmit a fitness advantage to the cells. Here, we report a similar dynamic for two mutations in KRAS (G12A and A146T), providing a biological rationale for the high frequency of KRAS and TP53 alterations at MM relapse. Resistance mutations, on the other hand, did not endow MM cells with a general fitness advantage but rather presented a disadvantage compared to the wild-type. CUL4B KO and IKZF1 A152T transmit resistance against immunomodulatory agents, PSMB5 A20T to proteasome inhibition. However, MM cells harboring such lesions only outcompete the culture in the presence of the respective drug. To better prevent the selection of clones with the potential of inducing relapse, these results argue in favor of treatment-free breaks or a switch of the drug class given as maintenance therapy. In summary, the fitness benefit of TP53 and KRAS mutations was not treatment-related, unlike patient-derived drug resistance alterations that may only induce an advantage under treatment. CCAs are suitable models for the study of clonal evolution and competitive (dis)advantages conveyed by a specific genetic lesion of interest, and their dependence on external factors such as the treatment.

Transformation of t(14;18)-negative follicular lymphoma to plasmablastic lymphoma: a case report with analysis of genetic evolution

BackgroundFollicular lymphoma (FL) is characterized by t(14;18)(q32;q21) involving the IGH and BCL2 genes. However, 10–15% of FLs lack the BCL2 rearrangement. These BCL2-rearrangement−negative FLs are clinically, pathologically, and genetically heterogeneous. The biological behavior and histological transformation of such FLs are not adequately characterized. Here, we report the first case of t(14;18)-negative FL that rapidly progressed to plasmablastic lymphoma (PBL).Case presentationA previously healthy 51-year-old man presented with leg swelling. Computed tomography (CT) showed enlarged lymph nodes (LNs) throughout the body, including both inguinal areas. Needle biopsy of an inguinal LN suggested low-grade B-cell non-Hodgkin lymphoma. Excisional biopsy of a neck LN showed proliferation of centrocytic and centroblastic cells with follicular and diffuse growth patterns. Immunohistochemical analysis showed that the cells were positive for CD20, BCL6, CD10, and CD23. BCL2 staining was negative in the follicles and weak to moderately positive in the interfollicular areas. BCL2 fluorescence in situ hybridization result was negative. Targeted next-generation sequencing (NGS) revealed mutations in the TNFRSF14, CREBBP, STAT6, BCL6, CD79B, CD79A, and KLHL6 genes, without evidence of BCL2 or BCL6 rearrangement. The pathologic and genetic features were consistent with t(14;18)-negative FL. Two months after one cycle of bendamustine and rituximab chemotherapy, the patient developed left flank pain. Positron emission tomography/CT showed new development of a large hypermetabolic mass in the retroperitoneum. Needle biopsy of the retroperitoneal mass demonstrated diffuse proliferation of large plasmablastic cells, which were negative for the B-cell markers, BCL2, BCL6, and CD10; they were positive for MUM-1, CD138, CD38, and C-MYC. The pathologic findings were consistent with PBL. The clonal relationship between the initial FL and subsequent PBL was analyzed via targeted NGS. The tumors shared the same CREBBP, STAT6, BCL6, and CD79B mutations, strongly suggesting that the PBL had transformed from a FL clone. The PBL also harbored BRAF V600E mutation and IGH::MYC fusion in addition to IGH::IRF4 fusion.ConclusionsWe propose that transformation or divergent clonal evolution of FL into PBL can occur when relevant genetic mutations are present. This study broadens the spectrum of histological transformation of t(14;18)-negative FL and emphasizes its biological and clinical heterogeneity.

Identification of Therapy-Induced Clonal Evolution and Resistance Pathways in Minimal Residual Clones in Multiple Myeloma through Single-Cell Sequencing.

In multiple myeloma (MM), therapy-induced clonal evolution is associated with treatment resistance and is one of the most important hindrances toward a cure for MM. To further understand the molecular mechanisms controlling the clonal evolution of MM, we applied single-cell RNA sequencing (scRNA-seq) to paired diagnostic and posttreatment bone marrow (BM) samples. scRNA-seq was performed on 38 BM samples from patients with monoclonal gammopathy of undetermined significance (n = 1), MM patients at diagnosis (n = 19), MM posttreatment (n = 17), and one healthy donor (HD). The single-cell transcriptome data of malignant plasma cells (PC) and the surrounding immune microenvironment were analyzed. Profiling by scRNA-seq data revealed three primary trajectories of transcriptional evolution after treatment: clonal elimination in patients with undetectable minimal residual disease (MRD-) and clonal stabilization and clonal selection in detectable MRD (MRD+) patients. We noted a metabolic shift toward fatty acid oxidation in cycling-resistant PCs, whereas selective PCs favored the NF-κB pathway. Intriguingly, when comparing the genetic and transcriptional dynamics, we found a significant correlation between genetic and nongenetic factors in driving the clonal evolution. Furthermore, we identified variations in cellular interactions between malignant PCs and the tumor microenvironment. Selective PCs showed the most robust cellular interactions with the tumor microenvironment. These data suggest that MM cells could rapidly adapt to induction treatment through transcriptional adaptation, metabolic adaptation, and specialized immune evasion. Targeting therapy-induced resistance mechanisms may help to avert refractory disease in MM.

Characterization of transcriptional heterogeneity and novel therapeutic targets using single cell RNA-sequencing of primary and circulating Ewing sarcoma cells.

Ewing sarcoma is the second most common bone cancer in children, accounting for 2% of pediatric cancer diagnoses. Patients who present with metastatic disease at the time of diagnosis have a dismal prognosis, compared to the >70% 5-year survival of those with localized disease. Here, we utilized single cell RNA-sequencing to characterize the transcriptional landscape of primary Ewing sarcoma tumors and surrounding tumor microenvironment (TME). Copy-number analysis identified subclonal evolution within patients prior to treatment. Primary tumor samples demonstrate a heterogenous transcriptional landscape with several conserved gene expression programs, including those composed of genes related to proliferation and EWS targets. Single cell RNA-sequencing and immunofluorescence of circulating tumor cells at the time of diagnosis identified TSPAN8 as a novel therapeutic target.

DIPG-86. INVESTIGATING THE TUMOR ENVIRONMENT AND SIGNALING DEPENDENCIES DRIVING DIFFUSE MIDLINE GLIOMA TUMOR INITIATION, PROGRESSION, AND RESISTANCE: A COMPREHENSIVE APPROACH LEVERAGING BULK MULTIOMICS DECONVOLUTION AND SINGLE-CELL OMICS VALIDATION

Abstract BACKGROUND Diffuse Midline Glioma (DMG) is a incurable tumor affecting children. Recent genomic investigations have identified a recurrent H3K27M mutation which induces global alterations in histone methylation patterns and DNA methylation. These epigenetic modifications hint at pivotal roles in DMG pathogenesis, yet effective therapeutic strategies remain elusive, with median survival rates stagnant at approximately one year. This shortfall stems from two main factors: 1) Inadequate multiomics studies hindering our understanding of DMG evolution and tumor progression, and 2) Limited comprehension of the tumor microenvironment in DMG. METHODS To elucidate DMG’s clonal evolution, we adopted a comprehensive multi-region sampling approach, acquiring 33 specimens from seven patients. Whole-exome and transcriptome sequencing, alongside DNA methylation profiling, were performed. Additionally, we generated one of the most extensive single-cell multiomics datasets (>300,000 cells from 21 additional samples), with a focus on pre- and post-treatment, and dissemination effects in DMG. RESULTS Our analysis delineated a tumor-promoting microenvironment characterized by hypoxia and pro-inflammatory conditions, nurturing genomic alterations and specialized biological processes such as proliferation and epithelial-mesenchymal transition. Notably, a majority of immune cells exhibited M1-like polarization, bolstering pro-inflammatory programs within the tumor milieu. Subclones within the DMG cohort demonstrated dissemination potential, often exhibiting up-regulation of NOTCH, P53, and WNT beta-catenin signaling pathways. These findings suggest that DMG clones harboring dissemination capabilities outside the pons acquire additional phenotypic features, possibly mediated by epigenetic or transcriptional alterations, contributing to enhanced migratory and aggressive behaviors. CONCLUSIONS Our study unveils the parallel evolution of DMG at genetic, epigenetic, and transcriptional levels, unveiling novel subclonal phenotypes governing tumor behavior. Crucially, we identify critical environmental shifts such as hypoxia and inflammatory changes, coupled with specialized signaling programs, driving dissemination and resistance phenotypes. These insights may pave the way for generating accurate genetically mouse models and targeted therapeutic interventions to combat this challenging malignancy.

Neutral selection and clonal expansion during the development of colon cancer metastasis.

Intratumour heterogeneity has been shown to play a role in the malignant progression of cancer. The clonal evolution in primary cancer has been well studied, however, that in metastatic tumorigenesis is not fully understood. In this study, we established human colon cancer-derived organoids and investigated clonal dynamics during liver metastasis development by tracking barcode-labelled subclones. Long-term subclone co-cultures showed clonal drift, with a single subclone becoming dominant in the cell population. Interestingly, the selected subclones were not always the same, suggesting that clonal selection was not based on cell intrinsic properties. Furthermore, liver tumours developed by co-transplantation of organoid subclones into the immunodeficient mouse spleen showed a progressive drastic reduction in clonal diversity, and only one or two subclones predominated in the majority of large metastatic tumours. Importantly, selections were not limited to particular subclones but appeared to be random. A trend towards a reduction in clonal diversity was also found in liver metastases of multiple colour-labelled organoids of mouse intestinal tumours. Based on these results, we propose a novel mechanism of metastasis development, i.e. a subclone population of the disseminated tumour cells in the liver is selected by neutral selection during colonization and constitutes large metastatic tumours.

GCT-22. MEDICAL AND SURGICAL VARIABLES ASSOCIATED WITH RELAPSE IN CENTRAL NERVOUS SYSTEM (CNS) NONGERMINOMATOUS GERM CELL TUMORS (NGGCTS): SECONDARY ANALYSIS OF STRATUM 1 OF THE CHILDREN’S ONCOLOGY GROUP ACNS1123 PHASE 2 CLINICAL TRIAL

Abstract BACKGROUND ACNS1123 was a Phase 2 trial that evaluated the survival benefit of whole ventricular irradiation (WVI) instead of craniospinal irradiation in children with localized CNS NGGCTs who achieved a complete/partial radiographic response (CR/PR) to induction chemotherapy with negative tumor markers (beta-HCG/AFP) in serum and cerebrospinal fluid (CSF). Patients who did not achieve a CR/PR were considered inevaluable and removed from study therapy post-induction. METHODS In unplanned, exploratory analyses, we evaluated the association of age <15/>15-years at diagnosis, tumor location (pineal/suprasellar/bifocal/ventricular), tumor markers (beta-HCG/AFP) in serum and cerebrospinal fluid (CSF) at baseline (>/<1000), and rate of decline during induction in CR/PR and <CR/PR patients. Since most evaluable patients with relapse experienced distant failure in the spine, we assessed the impact of surgical variables, such as endoscopic third ventriculostomy (ETV)/biopsy/resection/combination, on spinal relapse in patients with or without disease progression. RESULTS 66 of 107 eligible patients achieved CR/PR with negative tumor markers while 41 patients did not. Bifocal tumors appeared to demonstrate a higher hazard for disease progression compared to suprasellar tumors (Hazard Ratio=5.4; p=0.0279). Otherwise, we did not find an association with progression-free-survival for any of the analyzed medical variables. Of 66 evaluable patients who received WVI, eight patients experienced relapse, with all patients demonstrating disease in the spine. No significant association was detected between any surgical variable (ETV/biopsy/resection) and spinal failure among evaluable patients who did or did not relapse; however, we had very limited power for these analyses due to very few events. CONCLUSION Current reliance on conventional parameters appears inadequate to identify biomarkers of response as well as relapse. Innovative approaches such as comprehensive genomic profiling of primary and recurrence tumor specimens could inform somatic clonal evolution and potential targeted therapies.

DIPG-33. COMBINING CLONAL TRACKING WITH SINGLE CELL RNA SEQUENCING REVEALS TEMPORAL SHIFTS IN CELL STATES ASSOCIATED WITH TRAMETINIB RESISTANCE IN MAPK ALTERED DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND H3K27-altered diffuse midline glioma (DMG) is an aggressive and incurable paediatric high-grade glioma with median overall survival of 9-12 months. A distinct subset of DMG harbour alterations in the MAPK pathway, and we have previously shown that DMG-MAPK may exhibit sensitivity to MEK inhibitors but develop resistance under long-term exposure to drug. Elucidating these resistance mechanisms could spearhead better treatment strategies for these patients. METHODS To study clonal evolution under trametinib, we used CloneSifter lineage tracking technology to uniquely barcode 300,000 DMG-MAPK cells which were then treated with a GI80 dose of trametinib for 11 weeks. We extracted DNA to quantify barcode enrichment at endpoint across six technical replicate experiments and leveraged CloneSifter’s compatibility with single cell RNA sequencing (scRNA-seq) to couple clonal identity with transcriptional states in ~90,000 trametinib treated cells at varying timepoints. RESULTS DNA sequencing revealed 0.5% (1500/300,000) of the initial barcodes were enriched across trametinib replicates and were also detectable by scRNA-seq. Conducting cNMF analysis on the single cell dataset showed populations of trametinib treated cells associated with each other in a temporal manner, whereby early and late-stage resistant populations were defined by neuronal and stem cell like markers, respectively. A time-dependent enrichment of CloneSifter barcodes was also observed, suggesting dynamic expansion of clones over the treatment window. Notably, week 11 trametinib treated cells showed higher expression of resistance driver genes related to EMT, stemness and p38/MAPK upregulation. CONCLUSIONS Utilizing the sequence-specific reporter construct built in to the CloneSifter system allows for retrieval of resistant clones marked by enriched barcodes under treatment prior to drug exposure, enabling further characterization of monoclonal populations of trametinib-resistant cells. In summary, we have employed novel tools to study resistance development in DMG-MAPK that could be exploited to devise rational treatment strategies for these patients in an evolutionary-guided manner.

Synchronous Endometrial and Ovarian Endometrioid Carcinoma With MUTYH Germline Mutation: A Case Report With Genetic Analysis.

Synchronous endometrial and ovarian endometrioid carcinoma, which simultaneously involves the endometrium and ovary, is a relatively rare entity among gynecological cancers. Precise diagnosis and risk stratification are crucial for disease management. We present a unique case of a 40-year-old woman diagnosed with synchronous endometrial and ovarian endometrioid carcinoma carrying a monoallelic pathogenic MUTYH germline variant. Despite the histological morphology of the right ovarian tumor exhibiting some differences compared to the uterine tumor, we identified three identical somatic mutations shared between the uterine tumor and right ovarian tumor, along with four additional mutations exclusive to the uterine tumor, through the utilization of massively parallel sequencing of a 196-gene panel. These findings enabled us to elucidate the clonal relatedness and potential clonal evolution of the tumor across the two anatomical sites. Furthermore, in accordance with the 2023 FIGO staging system, the patient was diagnosed with Stage IIIB2 uterine cancer, and consequently, adjuvant radiation and chemotherapy were administered after surgery. She is being followed periodically and is normal 15 months after surgery. To the best of our knowledge, this study presents the first case of a patient with synchronous endometrial and ovarian endometrioid carcinoma harboring a monoallelic pathogenic MUTYH germline variant.

Clonal Dynamics and Relapse Risk Revealed by High-Sensitivity FLT3-Internal Tandem Duplication Detection in Acute Myeloid Leukemia

The ability to detect low-level disease is key to our understanding of clonal heterogeneity in acute myeloid leukemia (AML) and residual disease that elude conventional assays and seed relapse. We developed a high-sensitivity next-generation sequencing (HS-NGS) clinical assay, able to reliably detect low levels (1 × 10−5) of FLT3-ITD, a frequent, therapeutically targetable and prognostically relevant mutation in AML. By applying this assay to 289 longitudinal samples from 62 patients at initial diagnosis and/or clinical follow-up (mean follow-up of 22 months), we reveal the frequent occurrence of FLT3-ITD subclones at diagnosis and demonstrate a significantly decreased relapse risk when FLT3-ITD is cleared after induction or thereafter. We perform pairwise sequencing of diagnosis and relapse samples from 23 patients to uncover more detailed patterns of FLT3-ITD clonal evolution at relapse than is detectable by less-sensitive assays. Finally, we show that rising ITD level during consecutive biopsies is a harbinger of impending relapse. Our findings corroborate the emerging clinical utility of high-sensitivity FLT3-ITD testing and expands our understanding of clonal dynamics in FLT3-ITD–positive AML.

Current Novel Targeted Therapeutic Strategies in Multiple Myeloma.

Multiple myeloma (MM) is a hematologic malignancy caused by the clonal expansion of immunoglobulin-producing plasma cells in the bone marrow and/or extramedullary sites. Common manifestations of MM include anemia, renal dysfunction, infection, bone pain, hypercalcemia, and fatigue. Despite numerous recent advancements in the MM treatment paradigm, current therapies demonstrate limited long-term effectiveness and eventual disease relapse remains exceedingly common. Myeloma cells often develop drug resistance through clonal evolution and alterations of cellular signaling pathways. Therefore, continued research of new targets in MM is crucial to circumvent cumulative drug resistance, overcome treatment-limiting toxicities, and improve outcomes in this incurable disease. This article provides a comprehensive overview of the landscape of novel treatments and emerging therapies for MM grouped by molecular target. Molecular targets outlined include BCMA, GPRC5D, FcRH5, CD38, SLAMF7, BCL-2, kinesin spindle protein, protein disulfide isomerase 1, peptidylprolyl isomerase A, Sec61 translocon, and cyclin-dependent kinase 6. Immunomodulatory drugs, NK cell therapy, and proteolysis-targeting chimera are described as well.

Genomic alterations and transcriptional phenotypes in circulating tumor DNA and matched metastatic tumor.

Profiling circulating cell-free DNA (cfDNA) has become a fundamental practice in cancer medicine, but the effectiveness of cfDNA at elucidating tumor-derived molecular features has not been systematically compared to standard single-lesion tumor biopsies in prospective cohorts of patients. The use of plasma instead of tissue to guide therapy is particularly attractive for patients with small cell lung cancer (SCLC), a cancer whose aggressive clinical course making it exceedingly challenging to obtain tumor biopsies. Here, a prospective cohort of 49 plasma samples obtained before, during, and after treatment from 20 patients with recurrent SCLC, we study cfDNA low pass whole genome (0.1X coverage) and exome (130X) sequencing in comparison with time-point matched tumor, characterized using exome and transcriptome sequencing. Direct comparison of cfDNA versus tumor biopsy reveals that cfDNA not only mirrors the mutation and copy number landscape of the corresponding tumor but also identifies clinically relevant resistance mechanisms and cancer driver alterations not found in matched tumor biopsies. Longitudinal cfDNA analysis reliably tracks tumor response, progression, and clonal evolution. Genomic sequencing coverage of plasma DNA fragments around transcription start sites shows distinct treatment-related changes and captures the expression of key transcription factors such as NEUROD1 and REST in the corresponding SCLC tumors, allowing prediction of SCLC neuroendocrine phenotypes and treatment responses. These findings have important implications for non-invasive stratification and subtype-specific therapies for patients with SCLC, now treated as a single disease.

ScSNViz: a user-friendly toolset for visualization and analysis of Cell-Specific Expressed SNVs.

Understanding genetic variation at the single-cell level is crucial for insights into cellular heterogeneity, clonal evolution, and gene expression regulation, but there is a scarcity of tools for visualizing and analyzing cell-level genetic variants. We introduce scSNViz, a comprehensive R-based toolset for visualization and analysis of cell-specific expressed Single Nucleotide Variants (sceSNVs) within cell-barcoded single-cell RNA-sequencing (scRNA-seq) data. ScSNViz offers 3D sceSNV visualization capabilities for dimensionally reduced scRNA-seq gene expression data, compatibility with popular scRNA-seq processing tools like Seurat, cell-type classification tools such as SingleR and scType, and trajectory inference computation using Slingshot. Furthermore, scSNViz conducts estimation, summary, and graphical representation of statistical metrics pertaining to sceSNVs distribution and expression across individual cells. It also provides support for the analysis of individual sceSNVs as well as sets comprising multiple expressed sceSNVs of interest. ScSNViz is implemented as user-friendly R-scripts, freely available on https://horvathlab.github.io/NGS/scSNViz , supported by help utilities, and requiring no specialized bioinformatics skills for use.

Flow cytometric profiles with CD7 and CADM1 in CD4+ T cells are promising indicators for prognosis of aggressive ATL

AbstractAdult T-cell leukemia/lymphoma (ATL) is a poor prognosis hematological malignancy originating from human T-cell leukemia virus 1 (HTLV-1)–infected CD4+ T cells. Flow cytometric plots of CADM1 and CD7 in CD4+ T cells are useful for separating HTLV-1–uninfected T cells and ATL cells. They are indicators of clonal evolution of HTLV-1–infected cells and disease progression of asymptomatic carriers or indolent ATL. However, the impacts of the plots on the clinical course or prognosis of ATL, especially in aggressive ATL, remain unclear. We focused on the N fraction (CD4+ CADM1+ CD7–) reflecting ATL cells and analyzed the flow cytometric profiles and clinical course of 497 samples from 92 HTLV-1–infected patients who were mainly aggressive ATL. The parameters based on N fractions showed significant correlations with known indicators of ATL disease status (soluble interleukin-2 receptor, lactate dehydrogenase, abnormal lymphocytes, etc.) and sensitively reflected the treatment response of aggressive ATL. The parameters based on N fractions significantly stratified the prognosis of aggressive ATL at 4 different time points: before treatment, after 1 course of chemotherapy, at the best response after chemotherapy, and before allogeneic hematopoietic cell transplantation. Even after mogamulizumab administration, which shows potent effects for peripheral blood lesions, the N fraction was still a useful indicator for prognostic estimation. In summary, this report shows that CADM1 vs CD7 plots in CD4+ T cells are useful indicators of the clinical course and prognosis of aggressive ATL. Therefore, this CADM1 and CD7 profile is suggested to be a useful prognostic indicator consistently from HTLV-1 carriers to aggressive ATL.

A bibliometric study of the intellectual base and global research hotspots for single-cell sequencing [2009–2022] in breast cancer

BackgroundBreast cancer is the most widespread malignant tumor worldwide. Single-cell sequencing technology offers novel insights and methods to understand the onset, progression, and treatment of tumors. Nevertheless, there is currently an absence of a thorough and unbiased report on the comprehensive research status of single-cell sequencing in breast cancer. This study seeks to summarize and quantify the dynamics and trends of research on breast cancer single-cell sequencing by bibliometric analysis. MethodsResearch articles and reviews related to breast cancer single-cell sequencing were selected from the WoSCC database. Visualization of data regarding countries, institutions, authors, references, and keywords was performed by CiteSpace and VOSviewer software. Results583 articles and reviews were analyzed in this study. The quantity of publications related to breast cancer single-cell sequencing has been increasing annually. These studies originate from 302 institutions in 46 countries, with YMAX S WICHA producing the most publications and WANG Y being the most cited author. Nature Communications is the most researched journal, while Nature holds the highest number of citations. These journals predominantly cover topics in the molecular/biological/immunological fields. Moreover, an analysis of reference and keyword bursts revealed that current research trends in this area are primarily centered on “clonal evolution,” “tumor microenvironment,” and “immunotherapy.” ConclusionBreast cancer single-cell sequencing is a rapidly growing area of scientific interest. Future research requires more frequent and in-depth collaborations among countries, institutions, and authors. Furthermore, “clonal evolution,” “tumor microenvironment,” and “immunotherapy” are likely to become major focal points in upcoming research on breast cancer single-cell sequencing.

Indeterminate measurable residual disease by multiparameter flow cytometry is associated with an intermediate risk of clinical relapse in adult patients with acute leukaemia

Measurable residual disease (MRD) is useful for prognostication and for monitoring response to treatment in patients with acute leukaemia. MRD by multiparametric flow cytometry (MFC-MRD) utilises the leukaemia-associated immunophenotype (LAIP) and difference from normal (DfN) strategies to identify the leukaemic clone. Difficulties arise when the LAIP overlaps with normal regeneration, there is clonal evolution, or when the abnormal clone population is exceptionally small e.g., <0.01% of CD45+ cells. Such cases are reported as 'indeterminate'; however, there is little international consensus on this reporting. The relationship between clinical outcomes and indeterminate MFC-MRD is unknown. Here we determine the rate of indeterminate MFC-MRD reporting, its relationship to concurrent molecular MRD results when available, and to clinical outcomes to 12 months. We performed an internal audit of all adult testing for MFC-MRD between January and December 2021. A total of 153 consecutive patients with a diagnosis of acute leukaemia were included. Successive MFC-MRD results and clinical outcomes were recorded over a 12-month period from time of inclusion into the study. In total, 460 MFC-MRD tests from 153 patients were reviewed and 73 (16%) MFC-MRD tests from 54 (35%) patients were reported as indeterminate. The majority (70%) were at low levels between 0.01-0.1% of CD45+ cells. Compared to patients with a negative result, acute myeloid leukaemia (AML) was more frequent in patients who had an indeterminate MFC-MRD (70% vs 36%), and B-cell acute lymphoblastic leukaemia was less common (20% vs 55%). In patients with indeterminate MFC-MRD results, one-third had received either chemotherapy or allogeneic haemopoietic stem cell transplant (aHSCT) within the preceding 3 months. Agreement between MFC and molecular MRD testing was low. Patients with indeterminate MFC-MRD had leukaemia relapse rates below patients with a positive MFC-MRD, but greater than those with negative MFC-MRD (positive 33% vs indeterminate 21% vs negative 8%, p=0.038). Overall, these findings indicate that indeterminate MFC-MRD results are more common in adults with AML and also in those who have received chemotherapy or aHSCT within the previous 3 months. We report for the first time that indeterminate MFC-MRD is a finding of potential clinical significance, which associates with a numerically higher median relapse rate within 12 months when compared to a negative MFC-MRD result.

Understanding clonal heterogeneity and mechanisms of methotrexate resistance in two osteosarcoma PDX models.

e23512 Background: Osteosarcoma is a primary malignant bone tumor that is known to have significant intratumoral heterogeneity with branched cancer evolution. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % children and young adults. It has been revealed that the metastatic ability and resistance of cancers including osteosarcoma might be due to the presence of a subset of cells within the intra-tumorally heterogeneous population. We investigated the emergence of resistant clonal subpopulations following targeted methotrexate treatment in two patient derived xenograft (PDX) models in vitro and in vivo. Our aim was to not only track the subclonal population of cells that establish in the lungs but also to identify clonal drivers and the mutational signature of the genes and pathways involved in MTX resistance. Methods: We previously established 20 barcoded osteosarcoma PDX models and demonstrated that the application of methotrexate (MTX-IC95) treatment as a selective pressure in seven models produces clonal subpopulations that were more resistant to MTX. In the current work we used barcoded next generation and RNA sequencing techniques to map the clonal and transcriptomic architecture of MTX resistant single clones in M36 and OS252 PDX models. We next injected the barcoded PDX cells intratibially in twenty severe combined immunodeficiency disease (SCID) mice to track the clonal population/subpopulation of cells that metastasize to the lungs. Following primary tumor growth, limb amputation was carried out and mice were treated with methotrexate or vehicle for two weeks. Primary tumor, blood and lung samples were collected after death from metastasis or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -800C. DNA and RNA was extracted and analyzed by PCR, NGS or WES to map clonality, mutational and evolutionary profiles. Results: MTX treated single clones showed diversity and barcode abundance when compared to vehicle; barcode abundance reduced in the resistant clonal subpopulations when compared to the vehicles. Ten unique subclones were shared in both vehicle and MTX resistant clones in M36 while OS252 had only one shared subclone respectively. More than 50% of the injected mice developed primary tumors and matched lung micrometastases. We are currently analyzing both targeted and RNA sequencing results to further understand clonality, the mutational signature of the genes (DHFR, MTR, FPGS, GGH) and pathways involved in MTX resistance. Conclusions: We investigated mechanism of methotrexate resistance in two barcoded PDX models in vitro and in vivo using cytotoxicity, next generation and RNA sequencing techniques. Targeted amplicon sequencing of key genes involved in antifolate pathway will yield new information on clonal evolution and mechanisms of resistance.