Progress of single-cell sequencing in lymphoma / 白血病·淋巴瘤

Single-cell sequencing (SCS) sequences the genetic information of a single cell to better understand the differences amongst cells and reveal the unique changes of each cell type. The specific analysis of cell subsets at the single-cell level can accurately evaluate tumor cells and microenvironment cells to reveal the complexity of molecular components and the difference from the corresponding components in non-malignant tissues. Lymphoma is highly heterogeneous, some have unknown pathological types, etiology and poor prognosis. SCS is helpful to clarify the molecular mechanisms of lymphomagenesis and pathological staging, and guide clinical practice. This article reviews SCS and its application in lymphoma.

Poly-G for tumor matched samples chronicles the evolution of human colorectal cancer / 中华肿瘤杂志

Objective: To analyze poly-guanine (poly-G) genotypes and construct the phylogenetic tree of colorectal cancer (CRC) and provide an efficient and convenient method for the study of intra-tumor heterogeneity and tumor metastasis pathway. Methods: The clinicopathological information of patients with primary colorectal cancer resection with regional lymph node metastases were retrospectively collected in the Department of General Surgery, General Hospital of Tianjin Medical University from January 2017 to December 2017. The paraffin sections of the paired tumor samples were performed consecutively, and multi-region microdissection was performed after histogene staining. The phenol-chloroform extraction and ethanol precipitation scheme was used to obtain DNA, and Poly-G multiplex PCR amplification and capillary electrophoresis detection were performed. The correlation between Poly-G mutation frequency and clinicopathological parameters was analyzed. Based on the difference of Poly-G genotypes between paired samples, the distance matrix was calculated, and the phylogenetic tree was constructed to clarify the tumor metastasis pathway. Results: A total of 237 paired samples were collected from 20 patients including 134 primary lesions, 66 lymph node metastases, 37 normal tissues, and Poly-G mutation was detected in 20 patients (100%). The mutation frequency of Poly-G in low and undifferentiated patients was (74.10±23.11)%, higher than that in high and medium differentiated patients [(31.36±12.04)%, P<0.001]. In microsatellite instability patients, the mutation frequency of Poly-G was (68.19±24.80)%, which was higher than that in microsatellite stable patients [(32.40±14.90)%, P=0.003]. The Poly-G mutation frequency was not correlated with age, gender, and pathological staging (all P>0.05). Based on Poly-G genotype difference of the paired samples, the phylogenetic trees of 20 patients were constructed, showing the evolution process of the tumor, especially the subclonal origins of lymph node metastasis. Conclusion: Poly-G mutations accumulate in the occurrence and development of CRC, and can be used as genetic markers to generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.

Does focal heterogeneity affect survival in postoperative ipsilateral multifocal and multicentric breast cancers?

SUMMARY OBJECTIVE: In multicentric/multifocal breast tumors, there may be immunological and histological differences between foci that may affect survival and treatment choice. We aimed to evaluate the effect of focal heterogeneity seen in multicentric/multifocal breast tumors on survival. METHODS: We retrospectively collected and analyzed the clinicopathological data of 89 female patients with multifocal/multicentric breast cancer, whose surgical and medical treatment was completed and who were followed up for 5 years. RESULTS: Of all patients, 29.2% (26/89) were heterogeneous. Heterogeneity of these foci was as follows: histologic heterogeneity of index foci (mix type): 15.7% (14/89), histologic heterogeneity of inter-foci: 7.9% (7/89), and immunohistochemical heterogeneity of inter-foci: 10.1% (9/89). When additional foci were evaluated, oncological therapy was changed for 3 (3.3%) of 89 patients. Heterogeneity does not have a significant (p>0.05) effect on recurrence and survival in multicentric/multifocal breast cancers. Pathological N stage is an independent risk factor for disease-free survival (hazard ratio=2.29, 95% confidence interval=1.39-3.76, p=0.001). CONCLUSIONS: In multifocal/multicentric breast cancers, less than 4% of patients may experience heterogeneity requiring change in the therapeutic decision. However, heterogeneity does not have a significant effect on recurrence and survival in multifocal/multicentric breast cancers. The pathological N stage is an independent risk factor for disease-free survival.

Research progress on the heterogeneity of hepatocellular carcinoma / 中华肝胆外科杂志

Hepatocellular carcinoma cells interact with tumor microenvironment, and exhibits There are temporal and spatial heterogeneity of hepatocellular carcinoma which interacts with tumor microinvironment. In this paper, we summarized the characteristics of hepatocellular carcinoma heterogeneity, the mechanism of heterogeneity, and the impact of hepatocellular carcinoma heterogeneity on diagnosis and treatment, so as to provide a new thinking direction for the diagnosis and treatment of hepatocellular carcinoma.

Research progress of radiomics toward lung adenocarcinoma manifesting as solitary ground glass nodule / 国际肿瘤学杂志

The detection of early lung adenocarcinoma manifesting as ground glass nodule (GGN) is increasing. The promoting precision targeted therapy arises the need of radiomics (RM) and radiogenomics, a series of noninvasively radiological technology based on multiple modality, to assist the process including determination in diagnosis, treatment and follow-up strategy and release burden in clinical practice. RM, playing an important role in lung adenocarcinoma manifesting as GGN, can provide information towards the different components of the nodules, the analysis of peritumoral areas, the reduction of over diagnosis and treatment, the selection of targeted therapy and follow-up.

Preliminary research on breast cancer organoids as a radiobiological model / 中华放射肿瘤学杂志

Objective:To construct breast cancer organoid culture system and conduct histological characterization and preliminary radiobiological characteristics study.Methods:Different molecular types of breast cancer cell lines and patient-derived tumor cells were cultured in vitro to form breast cancer organoids and characterize their tissue structure. In addition, Ki-67, ER, PR and Her2 markers were evaluated by immunohistochemical staining. Breast cancer organoids were irradiated with 4 Gy and 8 Gy. The numbe and diameter changes of breast cancer organoids at 0, 24, 48 and 96 h after irradiation were observed to evaluate the irradiation-induced damage to the organoids. Results:Breast cancer cell lines and patient-derived tissues formed organoid structures at 6 d. HE staining showed the microstructures, and the expression profile of markers was spatially heterogeneous. The expression patterns of markers were similar between patient-derived organoids and original tumor tissues. Irradiation of MCF-7 breast cancer organoids led to growth arrested, and some of the formed organoids collapsed and the proliferating trend gradually recovered from 48 h to 96 h. MDA-MB-231 breast tumor organoids showed radioresistance, growth arrested, but the structures remained intact, the recovery trend was still not observed at 96 h. The tissue-derived organoids from triple-negative patients also showed radiation tolerance. After irradiation, the organoids continued to grow without significant structural changes, whereas the growth trend was significantly smaller than that in the non-irradiated group.Conclusions:Breast cancer organoids formed by in vitro culture of breast cancer cells from different sources and different molecular types have microstructure and heterogeneity, which can reflect the expression of source tissue markers and show different radioresistance. Organoids derived from triple-negative breast cancer are more resistant to irradiation.

Brain Tumor Heterogeneity

Tumor heterogeneity is the concept that different tumor cells provide distinct biomorphological lesions, gene expressions, proliferation, microenvironment and graduated capacity of metastatic lesions. Brain tumor heterogeneity has been recently discussed about the interesting interaction of chronic inflammation, microenvironment, epigenetics and glioma steam cells. Brain tumors remain a challenge with regards to medication and disease, due to the lack of treatment options and unsatisfactory results. These results might be the result of the brain tumor heterogeneity and its multiple resistance mechanisms to chemo and radiotherapy.

Current state and progression of advanced melanoma treatment under precision medicine / 中国肿瘤生物治疗杂志

@#[Abstract] In the past, surgery and chemotherapy were the main treatment strategies for malignant melanoma, but companied with poor prognosis. With the development of high-throughput gene sequencing technology and the deepening understanding of tumor molecular mechanisms, it has been found that tumor heterogeneity and the diversity of tumor microenvironment affect tumor formation, drug resistance and treatment selection, leading to different responses and benefits of melanoma patients to the same treatment. The emerge and progression of targeted therapy and immunotherapy have significantly increased the survival rates of patients with metastatic melanoma, promoting the individualization and precision of melanoma treatment and making precise treatment a research hotspot as well as a trend. This review mainly summarizes the research progress of systemic individualized treatment of advanced melanoma based on precise subtyping and molecular level, and to get a more comprehensive view of the survival status of melanoma patients in the era of precision medicine, as well as the prospect and necessity of developing various targeted therapy, immunotherapy or combination therapy.

Intratumor heterogeneity, microenvironment, and mechanisms of drug resistance in glioma recurrence and evolution / 医学前沿

Glioma is the most common lethal tumor of the human brain. The median survival of patients with primary World Health Organization grade IV glioma is only 14.6 months. The World Health Organization classification of tumors of the central nervous system categorized gliomas into lower-grade gliomas and glioblastomas. Unlike primary glioblastoma that usually develop de novo in the elderly, secondary glioblastoma enriched with an isocitrate dehydrogenase mutant typically progresses from lower-grade glioma within 5-10 years from the time of diagnosis. Based on various evolutional trajectories brought on by clonal and subclonal alterations, the evolution patterns of glioma vary according to different theories. Some important features distinguish the normal brain from other tissues, e.g., the composition of the microenvironment around the tumor cells, the presence of the blood-brain barrier, and others. The underlying mechanism of glioma recurrence and evolution patterns of glioma are different from those of other types of cancer. Several studies correlated tumor recurrence with tumor heterogeneity and the immune microenvironment. However, the detailed reasons for the progression and recurrence of glioma remain controversial. In this review, we introduce the different mechanisms involved in glioma progression, including tumor heterogeneity, the tumor microenvironment and drug resistance, and their pre-clinical implements in clinical trials. This review aimed to provide new insights into further clinical strategies for the treatment of patients with recurrent and secondary glioma.

Research Progress of Immune Checkpoint Inhibitors in Malignant Pleural Mesothelioma / 中国肺癌杂志

Malignant pleural mesothelioma (MPM) is a malignant tumor with strong invasiveness, low survival rate and lack of effective treatment options. As the only first-line treatment plan for the advanced MPM, combination of pemetrexed and cisplatin chemotherapy have been existing since the last 20 years. Immunotherapy has long been considered as a potential treatment plan for MPM, mainly including immune checkpoint inhibitors (ICIs), immunotoxin therapy, anti-cancer vaccine and adoptive T-cell therapy. This review focuses on summarizing the current research status of immune checkpoint inhibitors in MPM, discusses the effect of tumor heterogeneity on ICIs treatment, and describes that the biomarker-oriented immunotherapy is a new vision for the realization of individualized treatment of MPM.
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Research Progress of Single Cell Sequencing in Lung Cancer / 中国肺癌杂志

Lung cancer is the malignant tumor with the highest mortality rate in the world. Heterogeneity of lung cancer, usually studied by sequencing technology, is considered to have important clinical significance in current studies. However, general sequencing technology can only explain the differences between samples integrally and its resolution is not enough to describe the differences between the individual cells. Therefore, people urgently hope to understand the cell type, state, subgroup distribution in the tumor microenvironment and the communication behavior between cells in the single cell level. Single-cell sequencing technology solves this problem. Using this technique will contribute to further understanding the mechanism of the occurrence and development of lung cancer, discovering new diagnostic markers and therapeutic targets, and providing theoretical references for the precise treatment of lung cancer patients in the future. This article reviews the progress of single-cell sequencing technology and focuses on its research on lung cancer tumor heterogeneity, tumor microenvironment, invasion and metastasis, treatment response, and drug resistance.
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Application of Single-cell RNA Sequencing in Research on Tumor Immune Microenvironment / 中国医学科学院学报

Tumors are highly complex systems. Understanding the compositions and functions of the tumor immune microenvironment is a prerequisite for effective tumor immunotherapy. Single-cell RNA sequencing can detect the transcriptome of a cell at the resolution of single-cell level,describe its functional status,and thus deepen the understanding of the composition and function of different cell clusters in tumor immune microenvironment. This article reviews the application of single-cell RNA sequencing in research on tumor immune microenvironment.

Construction of monoclonal cells derived from the human glioblastoma U87 cell line and study of their heterogeneity / 医学研究生学报

Objective The human glioblastoma (GBM) U87 cell line is employed as a model for studying the heterogeneity of GBM. This study was to examine the phenotypic profiles and genetic backgrounds of different monoclonal cells derived from the human GBM U87 cell line and explore the molecular mechanisms underlying the phenotypic difference. Methods Using the finite dilution method labeled with 5(6)-carboxyfluorescein diacetate N-hydroxy succinimidyl ester (CFSE), we constructed the monoclonal cell lines CF5 and G11 with typical morphological characteristics derived from the human GBM U87 cell line and identified them by short tandem repeat (STR). We detected the proliferation of the cells by CCK8 assay, EdU incorporation and colony-formation assay, their self-renewal capability by tumor sphere formation assay, their adhesion ability by immunofluorescence and CCK8 adhesion assay, their invasion ability with a 3D culture model, and their sensitivity to chemotherapeutic agents by Annexin V/PI double-staining flow cytometry. We performed transcriptome sequencing and bioinformatics analysis on the genetic profiles and determined the mRNA expressions of the representative differential genes in the enriched pathway by real-time quantitative PCR (qRT-PCR). Results The CF5 and G11 monoclonal cell lines morphologically typical of U87 were successfully constructed, the former small, short and thick, while the latter big, long and thin. Compared with the U87 and G11 cell lines, the CF5 cells showed a significantly higher proliferation ability (P < 0.01), though higher in the U87 than in the G11 cell line, a higher proportion of EdU-positive cells (0.35 ± 0.03 and 0.44 ± 0.03 vs 0.54 ± 0.05, P < 0.01), though higher in the U87 than in the G11 cell line, and a higher tumor-sphere formation ability (P < 0.01), though higher in the U87 than in the G11 cell line. In comparison with the U87 and CF5 cell lines, the G11 cells exhibited remarkably higher abilities of adhesion (P < 0.01) and invasion (P < 0.05), though both higher in the U87 than in the CF5 group. Totally, 159 genes were down-regulated and 303 up-regulated in the CF5 cells compared with those in the U87 and G11 cells, while 281 were down-regulated and 116 up-regulated in the G11 cells compared with those in the CF5 and U87 cells. The CF5 and G11 cells manifested the highest enrichment in the extracellular matrix-associated pathways, which were shown to be closely associated with the invasiveness and drug-resistance of the tumor. Conclusion We successfully constructed human GBM U87-derived monoclonal cell lines CF5 and G11 with different morphological features, phenotypic profiles and genetic backgrounds, which has paved the ground for further studies of the heterogeneity of GBM.

Application of single cell sequencing in breast cancer research / 中华内分泌外科杂志

Single cell sequencing developed in recent years,which studies genome and transcriptome at single cell level,is more suitable for solving problems on minor special sample studies,heterogenous population analysis and finding concurrent or mutually exclusive genomic events,compared to bulk sequencing.For breast cancer,which is highly heterogeneous,bulk sequencing data is not enough for solving many clinical problems,and single cell sequencing precisely plays an important role in it.This review,focusing on a minor special cell population in breast cancer (such as cancer stem cells,circulating tumor cells,et al),tumor heterogeneity and clonal evolution,and chemotherapy resistance,summarized application of single cell sequencing in breast cancer research in recent years,and discussed the future research directions.

Identificação e anotação funcional de RNAs longos não codificadores associados à subtipos moleculares em tumores pancreáticos / Identification of long non coding RNAs associated to molecular subtypes in pancreatic tumors

O Adenocarcinoma Pancreático Ductal (Pancreatic Ductal Adenocarcinoma - PDAC) é a sé- tima causa de mortes por câncer no mundo, com uma taxa de sobrevida de apenas 6%. Embora alguns genes estejam recorrentemente mutados em grande parte dos tumores e sejam críticos para a oncogênese, a heterogeneidade das alterações moleculares tanto no tumor quanto em componentes do microambiente tumoral se reflete em diferentes características fenotípicas com comportamentos clínicos distintos e que têm sido associados a diferentes subtipos moleculares através da análise computacional de dados de alterações somáticas e transcricionais no PDAC. RNAs não codificadores longos (lncRNAs) têm sido reconhecidos como importantes reguladores da expressão gênica em doenças proliferativas mas sua associação com subtipos em PDAC e sua contribuição para o estabelecimento de diferentes fenótipos moleculares e clínicos da doença não foi explorada até o momento. Neste trabalho, foi implementada uma abordagem computacional com o objetivo de identificar e anotar funcionalmente lncRNAs associados a subtipos moleculares de PDAC. Inicialmente, a classificação não supervisionada por Fatoração Matricial Não Negativa (Non-Negative Matrix Factorization - NMF) de dados de expressão gê- nica global de amostras clínicas disponíveis publicamente (The Cancer Genome Atlas - TCGA) resultou na identificação de quatro subgrupos distintos de PDAC, que recapitulam os fenótipos Exócrino/Endócrino, Imunogênico, Escamoso e Progenitor descritos na literatura. Uma análise de expressão diferencial permitiu a identificação de assinaturas de expressão gênica características que incluem lncRNAs associados a cada subgrupo. Através da construção de redes de coexpressão de mRNAs e lncRNAs e a identificação de módulos da rede significativamente enriquecidos em genes que participam em vias moleculares conhecidas foi possível inferir possíveis funções biológicas à lncRNAs associados aos diferentes subtipos moleculares, tais como funções exócrinas/neuroendócrinas, imunogênicas, reparo de DNA/progressão do ciclo celular e progenitoras/morfogênicas. Entre ele, o subgrupo 3, enriquecido para fenótipo Escamoso e associado a hiper-expressão do supressor tumoral TP63, possui dois lncRNAS hiper-expressos neste subgrupo em relação aos outros subgrupos, sendo que o lncRNA antissenso FAM83A-AS1 tem a predição de interagir com as proteínas FGFR2, AXIN1, PTEN, BRAF, SMAD4, TGFBR2, TP53 e CDKN2A, que exercem funções importantes na transdução de sinal e supressão tumoral no câncer incluindo o de pâncreas. Entre os lncRNAs hipo-regulados no subgrupo 3 em relação ao outros subgrupos, alguns, como FLJ42875, LOC338651, C20orf56 e LOC38838 tem predição de interação com alta afinidade à proteína BRCA2, que está envolvida no reparo de DNA e participa de processos de resistência à quimioterápicos. As informações trazidas por este estudo permitem gerar hipóteses sobre a contribuição de lncRNAs para a definição de subtipos moleculares de PDAC e priorizar candidatos e experimentos para estudos funcionais de modo a contribuir para um melhor entendimento sobre os mecanismos de ação de lncRNAs na tumorigênese e agressividade do câncer de pâncreas

Pancreatic Ductal Adenocarcinoma (PDAC) is the seventh cause of worldwide cancer related deaths, with an overall survival rate of only 6%. Some genes might be recurrently mutated in a large number of tumors, and be critical for oncogenesis, molecular alteration heterogeneity both in the tumor as all as in the tumor microenvironment is reflected in diverse phenotypic features with distinct clinical outcomes, and this distinction in multiple molecular subtypes has been drawn through transcriptional and somatic alteration computational analysis within PDAC. Long Non Coding RNAs (lncRNAs) have been recognized as important gene expression regulators in proliferative diseases, but its association to molecular subtypes in PDAC and its contribution in the establishment of diverse molecular and clinical phenotypes hasnt been explored at length until the present. This work focused on the implementation of a computational approach with the objective of lncRNA identification and functional annotation associated to distinct molecular subtypes in PDAC. Initially, Non-negative Matrix Factorization (NMF), an unsupervised classification method, applied to global gene expression data from publicly available clinical samples (The Cancer Genome Atlas - TCGA) resulted in the identification of four distinct PDAC molecular subgroups reminiscent of Exocrine/Endocrine, Immunogenic, Squamous and Progenitor phenotypes. Differential expression analysis allowed a characteristic gene expression signature identification, including distinct molecular subtype associated lncRNAs. mRNA and lncRNA containing gene co-expression modules significantly enriched annotated pathways containing the molecular subtype associated lncRNAs allowed to designate possible molecular functions of the distinct molecular subtype associated lncRNAs, such as exocrine/neuroendocrine, immunogenic, DNA repair/cell cycle progression and progenitor/morphogenic functions. Subgroup 3, enriched with a Squamous phenotype and associated to TP63 over-expression contains two lncRNAs over-expressed compared to other subgroups; furthermore, the antisense lncRNA FAM83A-AS1 yielded a predicted lncRNA-protein interaction to FGFR2, AXIN1, PTEN, BRAF, SMAD4, TGFBR2, TP53 and CDKN2A, proteins that play important signal transduction and tumor suppressor roles in several cancer types, including pancreas. Among under-expressed lncRNAs in subgroup 3 compared to the other subgroups, some, such as FLJ42875, LOC338651, C20orf56 and LOC38838 yilded a high protein interaction prediction score with BRCA2, a protein involved in DNA repair and processes resuling in chemotherapy resistance. The information brought by this study allowed to generate hypothesis on lncRNA contribution to define PDAC molecular subtypes, helping prioritize candidates and experiments for functional studies, thus contributing to a better understanding on lncRNA mechanisms related to tumor progression and aggressiveness in pancreatic cancer

Analysis of intra-tumor heterogeneity in breast cancer precision medicine / 国际肿瘤学杂志

As a breakthrough of precision medicine,intra-tumor heterogeneity is a research hotspot and is correlated to tumorigenesis,metastasis and resistance to therapies.In breast cancer,evidence of intra-tumor heterogeneity has been documented by numerous studies and it is the main obstacle to find ideal tumor markers and personalized medicine.Further analysis including the feature and generation mechanism of intra-tumor heterogeneity and the methods to assessment intra-tumor heterogeneity in the clinic are the key to cancer precision medicine.

Analysis of intra-tumor heterogeneity in breast cancer precision medicine / 国际肿瘤学杂志

As a breakthrough of precision medicine,intra-tumor heterogeneity is a research hotspot and is correlated to tumorigenesis,metastasis and resistance to therapies.In breast cancer,evidence of intra-tumor heterogeneity has been documented by numerous studies and it is the main obstacle to find ideal tumor markers and personalized medicine.Further analysis including the feature and generation mechanism of intra-tumor heterogeneity and the methods to assessment intra-tumor heterogeneity in the clinic are the key to cancer precision medicine.

Tumor heterogeneity and drug resistance of targeted antitumor agents / 药学学报

The popular application of targeted antitumor agents has greatly improved the efficacies of tumor therapy. However, some patients develop drug resistance after the administration with them for six to twelve months, leading to the failure of treatment. The cause of it is mainly due to tumor heterogeneity. The genesis of tumor heterogeneity is closely associated with tumor stem cells, genetic instability, cell competition and stochastic events. There are a lot of mechanisms involved in the drug resistance to the targeted agents. Tumor heterogeneity and drug resistance are great challenges for precision oncology and are taken account for the process of research and development of new antitumor agents.

Expressional Subpopulation of Cancers Determined by G64, a Co-regulated Module

Studies of cancer heterogeneity have received considerable attention recently, because the presence or absence of resistant sub-clones may determine whether or not certain therapeutic treatments are effective. Previously, we have reported G64, a co-regulated gene module composed of 64 different genes, can differentiate tumor intra- or inter-subpopulations in lung adenocarcinomas (LADCs). Here, we investigated whether the G64 module genes were also expressed distinctively in different subpopulations of other cancers. RNA sequencing-based transcriptome data derived from 22 cancers, except LADC, were downloaded from The Cancer Genome Atlas (TCGA). Interestingly, the 22 cancers also expressed the G64 genes in a correlated manner, as observed previously in an LADC study. Considering that gene expression levels were continuous among different tumor samples, tumor subpopulations were investigated using extreme expressional ranges of G64-i.e., tumor subpopulation with the lowest 15% of G64 expression, tumor subpopulation with the highest 15% of G64 expression, and tumor subpopulation with intermediate expression. In each of the 22 cancers, we examined whether patient survival was different among the three different subgroups and found that G64 could differentiate tumor subpopulations in six other cancers, including sarcoma, kidney, brain, liver, and esophageal cancers.