Tumor Clones Research Articles

Reshaping the Pancreatic Cancer Microenvironment at Different Stages with Chemotherapy.

The dynamic tumor microenvironment, especially the immune microenvironment, during the natural progression and/or chemotherapy treatment is a critical frontier in understanding the effects of chemotherapy on pancreatic cancer. Non-stratified pancreatic cancer patients always receive chemotherapeutic strategies, including neoadjuvant chemotherapy and adjuvant chemotherapy, predominantly according to their physical conditions and different disease stages. An increasing number of studies demonstrate that the pancreatic cancer tumor microenvironment could be reshaped by chemotherapy, an outcome caused by immunogenic cell death, selection and/or education of preponderant tumor clones, adaptive gene mutations, and induction of cytokines/chemokines. These outcomes could in turn impact the efficacy of chemotherapy, making it range from synergetic to resistant and even tumor-promoting. Under chemotherapeutic impact, the metastatic micro-structures in the primary tumor may be built to leak tumor cells into the lymph or blood vasculature, and micro-metastatic/recurrent niches rich in immunosuppressive cells may be recruited by cytokines and chemokines, which provide housing conditions for these circling tumor cells. An in-depth understanding of how chemotherapy reshapes the tumor microenvironment may lead to new therapeutic strategies to block its adverse tumor-promoting effects and prolong survival. In this review, reshaped pancreatic cancer tumor microenvironments due to chemotherapy were reflected mainly in immune cells, pancreatic cancer cells, and cancer-associated fibroblast cells, quantitatively, functionally, and spatially. Additionally, small molecule kinases and immune checkpoints participating in this remodeling process caused by chemotherapy are suggested to be blocked reasonably to synergize with chemotherapy.

M2 macrophages drive leukemic transformation by imposing resistance to phagocytosis and improving mitochondrial metabolism

It is increasingly becoming clear that cancers are a symbiosis of diverse cell types and tumor clones. Combined single-cell RNA sequencing, flow cytometry, and immunohistochemistry studies of the innate immune compartment in the bone marrow of patients with acute myeloid leukemia (AML) reveal a shift toward a tumor-supportive M2-polarized macrophage landscape with an altered transcriptional program, with enhanced fatty acid oxidation and NAD+ generation. Functionally, these AML-associated macrophages display decreased phagocytic activity and intra-bone marrow coinjection of M2 macrophages together with leukemic blasts strongly enhances in vivo transformation potential. A 2-day in vitro exposure to M2 macrophages results in the accumulation of CALRlow leukemic blast cells, which are now protected against phagocytosis. Moreover, M2-exposed "trained" leukemic blasts display increased mitochondrial metabolism, in part mediated via mitochondrial transfer. Our study provides insight into the mechanisms by which the immune landscape contributes to aggressive leukemia development and provides alternatives for targeting strategies aimed at the tumor microenvironment.

Abstract 4277: Measuring proliferation rates of distinct tumour clones using single-cell DNA sequencing

Abstract Background: Tumour proliferation rate is a key phenotypic feature of cancer, with higher rates linked to poorer clinical outcomes. Thus far, proliferation rates have been measured using pathological or experimental techniques on bulk tumour samples. However, while tumours are heterogeneous compositions of distinct clones with varying levels of fitness, measuring the proliferation of individual clones has not been possible to date. We hypothesise that enabling the identification of the most proliferative clones would reveal genomic hallmarks of aggressive clones, or the prediction of their potential phenotype, e.g., metastatic potential. Methods: We have developed SPRINTER (Single-cell Proliferation Rate Inference in Neoplasms Through Evolutionary Routes), a novel computational method to measure proliferation rates in individual tumour clones using single-cell whole-genome DNA sequencing. To assess the accuracy and power of SPRINTER, we have also developed an experimental approach to DNA sequence >18,000 single cells, accurately separated in different DNA-replication phases. We have sequenced and applied SPRINTER to >10,000 non-small cell lung cancer cells from longitudinal and metastatic tumour samples within the TRACERx study and PEACE autopsy programme. We have further analysed published data from >10,000 breast cancer cells. Results: We demonstrate that SPRINTER can accurately identify subpopulations of cells with different proliferation rates using relatively small numbers of cells, in contrast to previous preliminary approaches. While our estimates are concordant with previous bulk experimental studies (5-40%), we importantly have identified clonal heterogeneity in proliferation rates. Using bulk analysis, we have identified patterns of dissemination of tumour clones in non-small cell lung cancer. Integrating this with single-cell data, our results indicate that more widely disseminating tumour clones have higher proliferation rates, suggesting a link between proliferation and dissemination potential. We additionally find that clones are more proliferative in the metastatic versus the primary setting. Furthermore, we have identified high proliferation clones that may have a selective advantage in a breast tumour, and have inferred that they likely arose recently in cancer evolution. Conclusions: We have developed a novel method that enables accurate identification of proliferation rates of individual tumour clones using single-cell DNA sequencing data, allowing the investigation of genomic hallmarks in highly proliferative clones that might lead to higher fitness. Citation Format: Olivia Lucas, Sophie Ward, Rija Zaidi, Mark Hill, Emilia Lim, Haoran Zhai, Abigail Bunkum, Sonya Hessey, Michelle Dietzen, Andrew Rowan, Cristina Naceur-Lombardelli, Nnenna Kanu, Mariam Jamal-Hanjani, Charles Swanton, Simone Zaccaria. Measuring proliferation rates of distinct tumour clones using single-cell DNA sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4277.

Abstract 2037: Tracking the emergence of immunotherapy resistance in lung cancer metastases

Abstract Metastatic non-small cell lung cancer (NSCLC) results from a complex evolutionary process in which cancer cells migrate from a primary tumour to a new anatomical site. Immunotherapy is frequently used to treat NSCLC, but drug resistance is frequent (>50%) and metastasis remains the most common cause of death. Recent studies of primary and metastatic tumours suggest that anti-cancer treatments can impact cancer evolutionary dynamics by applying selective pressures that promote the development of treatment resistance in genetically distinct subpopulations of cancer cells, or tumour clones. However, the relationship between metastatic progression and the development of resistance to immunotherapy has not been fully explored. In particular, whether metastases become resistant to immunotherapy as a result of the migration of resistant cells or whether distinct metastatic sites converge in parallel toward an immunotherapy resistance phenotype irrespective of cell migration, is not known. In this work, we investigate the role of metastatic cell migration in the development of immunotherapy resistance using DNA sequencing data from primary and metastatic tumour samples from twelve immunotherapy-treated patients with NSCLC co-recruited to the national TRACERx and PEACE studies. Using computational approaches, we identified distinct tumour clones in primary and metastatic tumours and used these to reconstruct tumour phylogenies for each patient. By evaluating tumour clonal dynamics in relation to treatment, we classified tumour clones as either sensitive or resistant to immunotherapy. We reconstructed the metastatic migration histories of these clones using existing computational methods based on somatic single nucleotide variants. Our results suggest that heterogeneous mechanisms of immune evasion develop in parallel at distinct metastatic sites. The ongoing analysis of the genetic changes that distinguish resistant from sensitive clones, and migrating from non-migrating clones, will provide further detail regarding the impact of metastatic migrations on immunotherapy resistance. Using this approach to map the evolutionary history of metastatic NSCLC, we provide insight into the mechanism by which immunotherapy resistance develops at distinct metastatic sites. Citation Format: Sonya Hessey, Ariana Huebner, Oriol Pich, Abi Bunkum, Wing Liu, David A. Moore, Cristina Naceur-Lombardelli, Selvaraju Veeriah, Sophie Ward, Roberto Salgado, Nicholas McGranahan, Simone Zaccaria, Charles Swanton, Mariam Jamal-Hanjani. Tracking the emergence of immunotherapy resistance in lung cancer metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2037.

Abstract 5934: Tracking T cell clonal dynamics across time and space in metastatic colorectal cancer

Abstract Here we tracked T cell dynamics in 15 metastatic colorectal cancer (mCRC) patients who had multiple resections (median: 3, range: 2-7) over a period of >7 years (median: 10 years, range: 7-16 years), using a novel T cell receptor sequencing (TCRseq) method. These samples had been previously characterized for DNA and methylation alterations and gene expression, enabling an integrated analysis to determine potential cellular and molecular drivers and consequences of immune dynamics. This multi-region, multi-timepoint dataset presents a unique opportunity to study the co-evolution of mCRC and the T cell response over time, across metastatic sites and in response to therapy. T cell antigen specificity is defined by the TCR - a highly diverse sequence that enables tracking of specific T cell expansions across time and space. TCRseq quantifies the abundance of T cell clones and maps the dynamics of the TCR repertoire, however little is known about how these are altered in different metastatic sites, and post-chemotherapy. We developed and validated a new FFPE-compatible TCRseq method and sequenced 216 longitudinal samples representing the mCRC cohort described above. We detected a median of 348 unique TCRs per sample (range 69-9700), and revealed high levels of intra-tumor spatial heterogeneity. No significant difference was detected in the number of T cell clones or the TCR clonality between primary tumors (n=40), lung metastases (n=30) and liver metastases (n=50). Furthermore, these were not significantly different between tumor regions (n=139) and surrounding stroma (n=38). We found that compared to chemo-naïve tumors (n=36), those that had been exposed to recent chemotherapy (n=41) had a significant increase in both the number of unique T cell clones and Simpson’s evenness, likely reflecting a broad T cell response to DNA-damaging agents. After more than a year had passed without chemotherapy (n=64) these returned to pre-chemotherapy levels. In most cases the 10 most abundant TCR sequences in the primary tumor represented 15-40% of the total repertoire. We tracked these expanded clones through metastases, identifying ubiquitous clones which persisted across metastatic sites and through therapy. We found other expanded clones which were no longer present in metastases lesions, although some of these were found to expand again in later metastases. Finally, we examined correlations with tumor genomics, finding evidence in some patients that the TCR repertoire tracks closely with tumor clones even through metastasis and chemotherapy. In summary, this project represents the first comprehensive analysis of T cell dynamics through colorectal cancer metastasis, highlighting the changing T cell landscape post-chemotherapy and correlating the TCR repertoire with tumor genomics. Our results could have important clinical implications for mCRC immunotherapy and the design of personalized T cell-based therapy. Citation Format: Ann-Marie Baker, Alison Berner, Tahel Ronel, Nick Trahearn, Barbara Bravi, John Bridgewater, Benny Chain, Trevor A. Graham. Tracking T cell clonal dynamics across time and space in metastatic colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5934.

Abstract 258: Whole genome analysis of hereditary leiomyomatosis and renal cell cancer identifies role of localized hypermutational events

Abstract Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a rare hereditary condition affecting over 1 in 200,000 people worldwide. Individuals with HLRCC are also at risk of developing an aggressive form of renal cell carcinoma (RCC), as 15-20% of patients develop type 2 papillary RCC associated with poor prognosis. Although RCC risk and cancer penetrance of this condition has been studied, whole genome sequencing of associated tumors has yet to be examined, and the landscape of structural variants of HLRCC is largely unknown. We have therefore performed whole genome sequencing of tumor/normal pairs of HLRCC patients to study the tumor molecular architecture in this form of RCC. Through germline variant and somatic mutation calling, we have identified specific somatic mutational drivers, including those at the structural variant level for the first time in HLRCC. We have also quantified both chromothripsis and kataegis in these tumors, identifying the specific role of localized hypermutational events. Similarly, using tumour subclonal reconstruction techniques, we have inferred the clonality of HLRCC-associated RCC tumors and elucidate the relative timing of key oncogenic events and mutational processes. We also compare each of these mutational features to their equivalents in other RCC tumour subtypes. These studies shed key light on the specific mutational processes and drivers of this rare hereditary malignancy and on its lethality. Citation Format: Selina Wu, Paul C. Boutros. Whole genome analysis of hereditary leiomyomatosis and renal cell cancer identifies role of localized hypermutational events [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 258.

Abstract 4621: Distinct spatial and molecular maps in the tumour microenvironment of resistant melanoma patients treated with checkpoint inhibitor immunotherapy

Abstract Background: Checkpoint-based immunotherapies can achieve durable clinical responses in a variety of advanced cancers, including melanoma. However, clinical responses are not universal and there is still a substantial gap in understanding the mechanistic insights of immunotherapy resistance. This study aims to better understand patients’ resistance to immune checkpoint therapies by dissecting and tracing individual tumour and immune cell clones. Methods: Melanoma tumour dissociates and lesion matched formalin-fixed paraffin-embedded tissue samples were collected from patients with advanced melanoma receiving ipilimumab in combination with nivolumab; 2 patients were long-term responders, 2 had innate resistance and 1 acquired resistance. The molecular characteristics and spatial cellular relations associated with tumour and immune subsets in response to treatment were identified using single-cell indexing of transcriptomes and epitopes by sequencing and CODEX (CO-Detection by indEXing) multiplexed tissue imaging. Integration of spatial and molecular information was performed by combining reference-based phenotyping, Louvain clustering, and a mutual nearest neighbours-based transfer learning algorithm. Results: Pre-treatment melanoma biopsies from 5 patients (3 responding and 2 non-responding) along with a subsequent acquired resistance biopsy from an initially responding patient were analysed. Comparisons of cellular phenotypes identified higher proportions of gamma delta T cells (1.98% versus 0.2%) and enriched memory T cell profiles (CD3E, CD44, CD45RO, CD8) in responding compared to non-responding pre-treatment biopsies. Fibroblast from non-responding lesions overexpressed stromal related cytokine, IL1A (log2 fold change (FC) = 3.3) and CXCL1 (log2 FC = 1.4), which were enrichment at the tumour margin compared to responding lesions. Comparisons of a patient’s pre-treatment biopsy that initially responded to a subsequent progressing biopsy with acquired resistance had altered expression of transcript factor, ZFX (log2 FC = -1.59; adjusted p = 0.0119), and MBD2 (log2 FC = -1.5; adjusted p = 0.0225) within the patient’s memory T-cells. Conclusion: The combination of high dimensional single cell transcriptomics and spatial tissue imaging are effective tools to identify specific changes in cell phenotypes, intracellular signalling, and spatial location. This analysis identified reduced memory T cells, increased fibroblast, and altered cellular signalling were associated with resistance to immunotherapies. Citation Format: Camelia Quek, Aditya Pratapa, Ines Pires da Silva, Ghamdan Al-Eryani, Aaron Mayer, Nenad Bartonicek, Kate Harvey, Oliver Braubach, Robyn P.M. Saw, Jonathan Stretch, Kerwin F. Shannon, Alexander M. Menzies, Richard A. Scolyer, Georgina V. Long, Alexander Swarbrick, James S. Wilmott. Distinct spatial and molecular maps in the tumour microenvironment of resistant melanoma patients treated with checkpoint inhibitor immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4621.

Abstract 2789: Tumor suppressor genotype dramatically impacts lung cancer response to KRAS G12C inhibitors in vivo

Abstract Cancer is characterized by diverse genomic alterations that change cell state and can modify therapy responses. In some cases, the relationships between genotype and drug responses are obvious, such as the response of tumors with defined oncogenic mutations to inhibitors of those mutant proteins. However, covalent KRAS G12C inhibitors (G12Ci) induce clinical responses in less than half of patients with oncogenic KRAS G12C-driven lung cancer, illustrating that genomic alterations not obviously related to the drug mechanism can have a dramatic impact. We have developed and optimized an in vivo platform to identify pharmacogenomic interactions that dictate lung cancer responses to therapies. By coupling somatic genome editing, tumor barcoding, ultra-deep barcode sequencing, and robust statistical methods in genetically engineered mouse models of human lung cancer, we can quantify and compare candidate therapies across thousands of clonal tumors of diverse tumor suppressor genotypes. This platform provides a uniquely high-throughput and quantitative assessment of tumors that are initiated de novo within the natural immunocompetent tissue environment. To uncover genotypes that are particularly important in controlling the response of tumors to oncogenic KRAS inhibition, we applied this platform to quantify the impact of 59 tumor suppressor genes on KRASG12C-driven lung tumor responses to G12Ci. Treatment resulted in approximately three-quarters reduction in both average tumor sizes and overall tumor burden, with a clear drug and dose dependence. Approximately one-quarter of inactivated genes significantly and consistently altered tumor responses across numerous G12Ci and replicate studies. Interestingly, most of these genes are thought to be in pathways not directly related to RAS signaling, and they exhibit a striking pattern in which treatment sensitivity is positively correlated with strength of the tumor suppressor effect. This pharmacogenomic profile for G12Ci was categorically different than the profiles we have observed for chemotherapy and other therapies. Overall, these results provide direct causal evidence that certain tumor suppressor genotypes dramatically shift the effectiveness of G12Ci in vivo and generate hypotheses about patients likely to benefit from G12Ci relative to alternatives. Analysis of available human data provides early clinical support for some of these hypotheses. Ongoing efforts to define pharmacogenomic profiles of combination therapies could be of even greater importance. Platforms that can accurately predict how tumor genotype drives responses have the potential to transform precision cancer therapy, enabling more effective patient stratification and therapy combinations. Citation Format: Paul J. McMurdie, Ian P. Winters, Lily M. Blair, Lafia Sebastian, Vy Tran, Gabriel Grenot, Edwin A. Apilado, Edwin A. Apilado, Ian K. Lai, Gregory D. Wall, Dmitri A. Petrov, Monte M. Winslow, Michael J. Rosen, Joseph Juan. Tumor suppressor genotype dramatically impacts lung cancer response to KRAS G12C inhibitors in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2789.

Abstract 3891: Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer

Abstract Prostate cancer (PCa) is the most diagnosed cancer among American men, which has traditionally been treated through hormone therapy. However, after hormone therapy many patients with PCa still develop a more aggressive stage of PCa, called metastasis castration resistant prostate cancer (mCRPC). The second- generation antiandrogens, such as enzalutamide or apalutamide, are used to competitively inhibit the androgen receptor (AR) signaling and achieved great clinical success. However, most of the mCRPC patients would develop resistance to the targeted therapy drugs within 6 months to 2 years after initial administration. Consequently, understanding the molecular mechanism of antiandrogen resistance, has become a critical endeavor to provide a greater benefit to patients. mCRPC is characterized by extensive heterogeneity of genomic copy number alterations, which may lead to antiandrogen resistance. TP53 and RB1 alterations have been reported to be dramatically enriched in mCRPC neuroendocrine cancer and we have previously found that inactivation of both TP53 and RB1 confers resistance to antiandrogen through lineage plasticity, where cancer cells can transdifferentiate from a luminal lineage to a mixture of basal and neuroendocrine lineages, which is no longer dependent on AR signaling. Despite these exciting discoveries, only 10% of patients have TP53 and RB1 loss and it is estimated that approximately 40% of patients develop antiandrogen resistance through an undiscovered mechanism.To identify more genomic alterations which confer antiandrogen resistance, we performed an in vivo library screening and identified some of the frequently depleted genes as top candidates mediating antiandrogen response, including the chromatin helicase DNA-binding factor (CHD1). Strikingly, the loss of CHD1 establishes an altered chromatin landscape and enables the activation of heterogenous resistant subclones to emerge, including clones with ectopic NR3C1/GR, POU3F2/BRN2, TBX2, and NR2F1. This work provided an innovative model to explain the dramatically increased tumor heterogeneity in therapy resistant prostate cancer and suggested potential druggable targets to overcome resistance. Building on those exciting results, we are now examining the underlying mechanism how the epigenetic rewiring in resistant tumor clones confer resistance and monitoring the evolution of those heterogenous resistant subclones. Furthermore, we are revealing the collaborative function of those four resistant driver genes in promoting metastasis to various distal organs through multidisplinary approaches including single cell transcriptomics and 3D organoid modeling. The completion of this study will not only add clarity to the mechanism of antiandrogen resistance but may also lead to the development of novel biomarker and therapeutic approaches to overcome resistance. Citation Format: Xiaong li, Su Deng, Julisa Gonzalez, Carla Rodriguez Tirado, Choushi Wang, Nickolas A. Johnson, Lauren Metang, Ping Mu. Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3891.

Nonrandom X Chromosome Inactivation Detection

X chromosome inactivation patterns may be clinically useful in assessing tumor clonality, determining carrier status for certain X-linked disorders, and evaluating the pathogenicity of a genetic variant identified in an X-linked gene. The protocols in this article utilize the highly polymorphic trinucleotide repeat within the first exon of the human androgen receptor gene (AR) and the methylation-sensitive restriction enzyme HpaII to distinguish between the maternal and paternal alleles and simultaneously determine their methylation status. The data obtained from these protocols can be used to calculate the ratio of inactivation between the two alleles that ultimately reflects whether a female has a random or nonrandom pattern of X chromosome inactivation. © 2023 Wiley Periodicals LLC. Basic Protocol 1: X chromosome inactivation assay Basic Protocol 2: PCR amplification and labeling of digested and undigested DNA templates.

ARF suppression by MYC but not MYCN confers increased malignancy of aggressive pediatric brain tumors

Medulloblastoma, the most common malignant pediatric brain tumor, often harbors MYC amplifications. Compared to high-grade gliomas, MYC-amplified medulloblastomas often show increased photoreceptor activity and arise in the presence of a functional ARF/p53 suppressor pathway. Here, we generate an immunocompetent transgenic mouse model with regulatable MYC that develop clonal tumors that molecularly resemble photoreceptor-positive Group 3 medulloblastoma. Compared to MYCN-expressing brain tumors driven from the same promoter, pronounced ARF silencing is present in our MYC-expressing model and in human medulloblastoma. While partial Arf suppression causes increased malignancy in MYCN-expressing tumors, complete Arf depletion promotes photoreceptor-negative high-grade glioma formation. Computational models and clinical data further identify drugs targeting MYC-driven tumors with a suppressed but functional ARF pathway. We show that the HSP90 inhibitor, Onalespib, significantly targets MYC-driven but not MYCN-driven tumors in an ARF-dependent manner. The treatment increases cell death in synergy with cisplatin and demonstrates potential for targeting MYC-driven medulloblastoma.

A Clustering Method Unifying Cell-Type Recognition and Subtype Identification for Tumor Heterogeneity Analysis.

The rapid development of single-cell technology has opened up a whole new perspective for identifying cell types in multicellular organisms and understanding the relationships between them. Distinguishing different cell types and subtypes can identify the components of different immune cells and different tumor clones in the tumor microenvironment, which is the basic work of tumor heterogeneity analysis and can help researchers understand the mechanism of tumor immune escape. Existing algorithms treat both cell types and subtypes as populations of cells with specific gene expression patterns, which is not conducive to accurate cell typing. For that, we proposed a cell similarity metric that unifies cell type recognition and subtype identification (UCRSI), with the assumption that selectively expressed genes represent differences in underlying cell type with on/off manner, while differences in expression level represent different cell subtype with more/less manner. Our method calculates these two kinds of differences separately, and then combines them using a consensus adjacency matrix, and finally cell typing is completed using spectral clustering algorithm. The results show that UCRSI can reconstruct expert annotation of single-cell RNA sequencing datasets more robustly than existing methods. And, UCRSI is useful for analyzing tumor heterogeneity and improving visualization of large-scale cell clustering.

Anemia in Neoplastic Disease: Current Treatment Options

Anemia is one of the most common complications of cancer. The severity of anemia depends on the severity of the disease and the course of its development. The review of the literature presents the causes and mechanisms of anemia in patients with malignant neoplasms, discussing its negative impact not only on the quality of life, but also on the effectiveness of chemotherapy, existing clinical recommendations for the treatment of anemia with the use of erythropoiesis stimulating agents. The attention is paid to the effectiveness of modern intravenous iron preparations. A brief overview of the literature data on the problem of use of intravenous iron preparations in clinical practice is presented. The review includes mainly the data of controlled randomized clinical studies, which are considered to be most informative in evidence. Recently, effective and safe intravenous iron preparations have appeared, the ways of their introduction, doses and formulas have been developed to calculate the overall deficit of iron in the organism. The data on the anemia importance, the incidence of which in tumor diseases, increases constantly, were found. In numerous randomized studies that have taken place over the open protocol, the value of hemoglobin (Hb) level for the quality of life of cancer patients receiving chemotherapy is demonstrated. Before recombinant human erythropoietins (rHuEPO) the transfusion of erythrocyte mass was used in cancer patients with anemia. However, hemotransfusion could be accompanied by the development of complications, transmission of transmissible infections, post-transfusion reactions, aloimmunization and the effect on the tumor clone with the possibility of stimulating tumor growth. The safety of iron parenteral preparations has increased because of the creation of a new generation of iron drugs.

Accumulation of STR-Loci Aberrations in Subclones of Jurkat Cell Line as a Model of Tumor Clonal Evolution

Many genetic markers are known to distinguish tumor cells from normal. Genetic lesions found at disease onset often belong to a predominant tumor clone, and further observation makes it possible to assess the fate of this clone during therapy. However, minor clones escape monitoring and become unidentified, leading to relapses. Here we report the results of in vitro study of clonal evolution in cultured tumor cell line (Jurkat) compared to the cell line of non-tumor origin (WIL2-S). Cell lines were cultured and cloned by limiting dilutions. Subclones were tested by short tandem repeats (STR) profiling. Spontaneous STR aberrations in cells of non-tumor origin occur in less than 1 of 100 cultured cells. While in the cells of tumor origin, new aberrations appear in 1 or even more of 3 cultured cells. At the same time, a significant relationship was found between the accumulation of aberrations in the pool of subclones and the rate of cell growth. One can speculate that this approach could be applied for the analysis of primary patient tumor cell culture to obtain information concerning the evolutionary potential of the tumor cells that may be useful for the selection of a therapy approach.

N6-methyadenosine modified SUV39H2 regulates homologous recombination through epigenetic repression of DUSP6 in gastric cancer

Despite many advances in treatment over the past few years, the poor 5-year survival rate and high recurrence rate of gastric cancer (GC) remain unsatisfactory. As the most abundant epigenetic modification in the eukaryotic mRNA, N6-methyladenosine (m6A) methylation participates in tumor progression and tissue development. During tumor progression, DNA damage repair mechanisms can be reprogrammed to give new growth advantages on tumor clones whose genomic integrity is disturbed. Here we detected the elevated SUV39H2 expression in GC tissues and cell lines. Functionally, SUV39H2 promoted GC proliferation and inhibited apoptosis in vitro and in vivo. Mechanistically, METTL3-mediated m6A modification promotes mRNA stability of SUV39H2 in an IGF2BP2 dependent manner, resulting in upregulated mRNA expression of SUV39H2. As a histone methyltransferase, SUV39H2 was verified to increase the phosphorylation level of ATM through transcriptional repression of DUSP6, thereby promoting HRR and ultimately inhibiting GC chemosensitivity to cisplatin. Collectively, these results indicate the specific mechanism of m6A-modified SUV39H2 as a histone methyltransferase promoting HRR to inhibit the chemosensitivity of GC. SUV39H2 is expected to become a key target in the precision targeted therapy of GC.

The use of inotuzumab ozogamicin in children with relapsed/refractory B-lineage acute lymphoblastic leukemia

Today, treatment results for acute lymphoblastic leukemia (ALL) look encouraging, yet 10–15% patients still end up relapsing. The success of relapse treatment is directly dependent on whether or not a tumor clone has been completely eradicated before hematopoietic stem cell transplantation (HSCT). Immunotherapy made it possible to achieve minimal residual disease (MRD) – negative remission even in refractory patients. One example of such immunotherapeutic agents is inotuzumab ozogamicin (InO), an anti-CD22 monoclonal antibody conjugated to the cytotoxic agent calicheamicin. We included 17 patients under the age of 18 with relapsed or refractory precursor B-cell ALL (pre-B ALL) who had been treated with InO at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of Russia from 01.10.2016 to 01.09.2022. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The efficacy of the therapy was assessed based on the patients’ morphological response, MRD negativity and overall survival. Treatment toxicity was assessed according to CTCAE 5.0 (Common Terminology Criteria for Adverse Events). Statistical analysis was performed using the XLSTAT 2016 software. The majority of the patients (75%) responded to the therapy. MRD negativity was achieved in 41.2% of the study patients. The one-year overall survival rate was 40.3% (95% confidence interval 14.8–65.7). The treatment was well tolerated but 33% of the patients treated with standard-dose InO and subsequent HSCT developed veno-occlusive disease/sinusoidal obstruction syndrome. In our study, we demonstrated the high efficacy of InO both when used as a rescue therapy in patients with relapsed/refractory pre-B ALL and as a bridging therapy in patients before HSCT.

Langerhans cell histiocytosis as a clonal disease of mononuclear phagocyte system

Langerhans cell histiocytosis (LCH) belongs to histiocytic proliferative diseases, which are rare in clinical practice; however they pose significant challenges both for their diagnosis and choice of therapeutic strategies. Histiocytic proliferative diseases are the scope of oncology; nevertheless, at the diagnostic stage the patients are referred to pediatricians or dermatologists. That is why the interdisciplinary interaction of various specialties and common approaches to their classification, diagnosis and treatment are important for the management of patients with histiocytic proliferative disorders.
 Accumulation of the studies on the LCH pathophysiology has promoted the development of new diagnostic algorithms and treatment methods. After the fact of MAPK signal pathway activation had been established, the potential target for therapy was identified. Neoplastic nature of LCH has been hypothesized. If confirmed, we can expect actual diagnostic algorithms being elaborated, in particular, the potential to predict the disease depending on the tumor clone mutation type. The unique characteristics of LCH including proliferate clonality (presumable of neoplastic nature), the disease course with spontaneous regression and frequent relapses and tropism to certain tissues (target organs) make up the grounds for further in-depth studies of the disease.

Advances in the pathogenesis of multiple myeloma bone disease.

Multiple myeloma (MM) is a clonal proliferative malignant tumor of plasma cells in bone marrow. With the aging of population in China, the incidence of MM is on the rise. Multiple myeloma bone disease (MBD) is one of the common clinical manifestations of MM, and 80%-90% of MM patients are accompanied by osteolytic lesions at the time of their first visit to the clinic. MBD not only increases the disability rate of patients, but also severely reduces the physical function of patients due to skeletal lesions and bone-related events. Currently available drugs for treating of MBD are ineffective and associated with side effects. Therefore, it is important to find new therapeutic approaches for the treatment of MBD. It is generally believed that the increased osteoclast activity and suppressed osteoblast function are the main pathologic mechanisms for MBD. However, more and more studies have suggested that soluble molecules in the bone marrow microenvironment, including cytokines, extracellular bodies, and metabolites, play an important role in the development of MBD. Therefore, exploring the occurrence and potential molecular mechanisms for MBD from multiple perspectives, and identifying the predictive biomarkers and potential therapeutic targets are of significance for the clinical treatment of MBD.

Loss of Heterozygosity in the Circulating Tumor DNA and CD138+ Bone Marrow Cells in Multiple Myeloma.

Multiple myeloma (MM) is characterized by heterogeneity of tumor cells. The study of tumor cells from blood, bone marrow, plasmacytoma, etc., allows us to identify similarities and differences in tumor lesions of various anatomical localizations. The aim of this study was to compare the loss of heterozygosity (LOH) by tumor cells by assessing STR profiles of different MM lesions. We examined paired samples of plasma circulating tumor DNA (ctDNA) and CD138+ bone marrow cells in MM patients. For patients with plasmacytomas (66% of 38 patients included), the STR profile of plasmacytomas was also studied when biopsy samples were available. Diverse patterns of LOH were found in lesions of different localization for most patients. LOH in plasma ctDNA, bone marrow, and plasmacytoma samples was found for 55%, 71%, and 100% of patients, respectively. One could expect a greater variety of STR profiles in aberrant loci for patients with plasmacytomas. This hypothesis was not confirmed-no difference in the frequency of LOH in MM patients with or without plasmacytomas was found. This indicates the genetic diversity of tumor clones in MM, regardless of the presence of extramedullar lesions. Therefore, we conclude that risk stratification based on molecular tests performed solely on bone marrow samples may not be sufficient for all MM patients, including those without plasmacytomas. Due to genetic heterogeneity of MM tumor cells from various lesions, the high diagnostic value of liquid biopsy approaches becomes obvious.

Pseudo-temporal dynamics of chemoresistant triple negative breast cancer cells reveal EGFR/HER2 inhibition as synthetic lethal during mid-neoadjuvant chemotherapy

In the absence of targetable hormonal axes, chemoresistance for triple-negative breast cancer (TNBC) often compromises patient outcomes. To investigate the underlying tumor dynamics, we performed trajectory analysis on the single-nuclei RNA-seq (snRNA-seq) of chemoresistant tumor clones during neoadjuvant chemotherapy (NAC). It revealed a common tumor trajectory across multiple patients with HER2-like expansions during NAC. Genome-wide CRISPR-Cas9 knock-out on mammary epithelial cells revealed chemosensitivity-promoting knock-outs were up-regulated along the tumor trajectory. Furthermore, we derived a consensus gene signature of TNBC chemoresistance by comparing the trajectory transcriptome with chemoresistant transcriptomes from TNBC cell lines and poor prognosis patient samples to predict FDA-approved drugs, including afatinib (pan-HER inhibitor), targeting the consensus signature. We validated the synergistic efficacy of afatinib and paclitaxel in chemoresistant TNBC cells and confirmed pharmacological suppression of the consensus signature. The study provides a dynamic model of chemoresistant tumor transcriptome, and computational framework for pharmacological intervention.