Profiling of hepatocellular carcinoma neoantigens reveals immune microenvironment and clonal evolution related patterns

Objective: Neoantigens derived from tumor-specific genomic alterations have demonstrated great potential for immunotherapeutic interventions in cancers. However, the comprehensive profile of hepatocellular carcinoma(HCC) neoantigens and their complex interplay with immune microenvironment and tumor evolution have not been fully addressed.Methods: Here we integrated whole exome sequencing data, transcriptome sequencing data and clinical information of 72 primary HCC patients to characterize the HCC neoantigen profile, and systematically explored its interactions with tumor clonal evolution, driver mutations and immune microenvironments.Results: We observed that higher somatic mutation/neoantigen load was associated with better clinical outcomes and HCC patients could be further divided into two subgroups with distinct prognosis based on their neoantigen expression patterns. HCC subgroup with neoantigen expression probability high(NEP-H) showed more aggressive pathologic features including increased incidence of tumor thrombus(P=0.038), higher recurrence rate(P=0.029),more inclined to lack tumor capsule(P=0.026) and with more microsatellite instability sites(P=0.006). In addition,NEP-H subgroup was also characterized by higher chance to be involved in tumor clonal evolution [odds ratio(OR)=46.7, P<0.001]. Gene set enrichment analysis revealed that upregulation of MYC and its targets could suppress immune responses, leading to elevated neoantigen expression proportion in tumor cells. Furthermore, we discovered an immune escape mechanism that tumors could become more inconspicuous by evolving subclones with less immunogenicity. We observed that smaller clonal mutation clusters with higher immunogenicity in tumor were more likely to involve in clonal evolution. Based on identified neoantigen profiles, we also discovered series of neoantigenic hotspot genes, which could serve as potential actionable targets in future.Conclusions: Our results revealed the landscape of HCC neoantigens and discovered two clinically relevant subgroups with distinct neoantigen expression patterns, suggesting the neoantigen expression should be fully considered in future immunotherapeutic interventions.

scDPN for High-throughput Single-cell CNV Detection to Uncover Clonal Evolution During HCC Recurrence

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution.Unfortunately,classical DNA amplification-based methods have low throughput and introduce coverage bias during sample preamplification.We developed a single-cell DNA library preparation method without preamplification in nanolitre scale(scDPN)to address these issues.The method achieved a throughput of up to 1800 cells per run for copy number variation(CNV)detection.Also,our approach demonstrated a lower level of amplification bias and noise than the multiple displacement amplification(MDA)method and showed high sensitivity and accuracy for cell line and tumor tissue evaluation.We used this approach to profile the tumor clones in paired primary and relapsed tumor samples of hepatocellular carcinoma(HCC).We identified three clonal subpopulations with a multitude of aneuploid alterations across the genome.Furthermore,we observed that a minor clone of the primary tumor containing additional alterations in chromosomes 1q,10q,and 14q developed into the dominant clone in the recurrent tumor,indicating clonal selection during recurrence in HCC.Overall,this approach provides a comprehensive and scalable solution to understand genome heterogeneity and evolution.

Unmasking tumor heterogeneity and clonal evolution by single-cell analysis

The intratumoral heterogeneity orchestrated by the tumor intrinsic and extrinsic mechanisms enable cancers to persist and spread notwithstanding the use of aggressive interventional therapies.The heterogeneity is revealed at multiple levels-at the level of individual tumor cells,in the cellular composition of tumor infiltrates and in the chemical microenvironment in which the cells reside.Deconvoluting the complex nature of the cell types present in the tumor,along with the homo and heterotypic interactions between different cell types can produce novel insights of biological and clinical relevance.However,most techniques analyze tumors at a gross level missing key inter-cell-type genotypic and phenotypic differences.The advent of single-cell sequencing has given an unprecedented opportunity to analyze the tumor at a resolution that not only captures the diversity of the cellular composition of a tumor but also provides information on the genetic,epigenetic and functional states of different cell types.In this review,we summarize the genesis of tumor heterogeneity,its impact on tumor growth and progression and their clinical consequences.We present an overview of the currently available platforms for isolation and sequencing of single tumor cells and provide evidence of its utility in precision medicine and personalized therapy.

Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue

AIM To investigate genotype variation among induced pluripotent stem cell(iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing. METHODS Human iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer(OCT3/4, SOX2, and KLF4). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using nextgeneration sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants(SNVs)(missense, nonsense,and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbS NP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer. RESULTS In eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2, TTN, ULK4, TSSK1 B, FLT4, STK19, STK31, TRRAP, WNK1, PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer(stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the noncancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer(stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells.CONCLUSION The genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from.

A Core Genome Multilocus Sequence Typing Scheme for Proteus mirabilis

Objective A core genome multilocus sequence typing(cgMLST)scheme to genotype and identify potential risk clonal groups(CGs)in Proteus mirabilis.Methods In this work,we propose a publicly available cgMLST scheme for P.mirabilis using chew BBACA.In total 72 complete P.mirabilis genomes,representing the diversity of this species,were used to set up a cgMLST scheme targeting 1,842 genes,635 unfinished(contig,chromosome,and scaffold)genomes were used for its validation.Results We identified a total of 205 CGs from 695 P.mirabilis strains with regional distribution characteristics.Of these,159 unique CGs were distributed in 16 countries.CG20 and CG3 carried large numbers of shared and unique antibiotic resistance genes.Nine virulence genes(papC,papD,papE,papF,papG,papH,papI,papJ,and papK)related to the P fimbrial operon that cause severe urinary tract infections were only found in CG20.These CGs require attention due to potential risks.Conclusion This research innovatively performs high-resolution molecular typing of P.mirabilis using whole-genome sequencing technology combined with a bioinformatics pipeline(chewBBACA).We found that the CGs of P.mirabilis showed regional distribution differences.We expect that our research will contribute to the establishment of cgMLST for P.mirabilis.

Clinical characteristics and prognostic significance of immunoglobulin isotype switch in patients with multiple myeloma

Immunoglobulin(Ig)isotype switching in multiple myeloma(MM)is a rare form of clonal evolution.The aim of this study was to investigate the clinical features and prognostic significance of Ig isotype switching by observing Ig transformation in patients with relapse.A retrospective analysis was performed on 506 patients with newly diagnosed MM who were treated at our hospital from February 2005 to February 2020.The patients who experienced relapse were divided into the following four groups according to Ig phenotype:original paraprotein,complete isotype switching,light chain escape(LCE),and non-secretory clinical relapse.For comparative purposes with the original paraprotein group,the last three groups were pooled as the transformation group.Among the 506 included patients,376(74.3%)relapsed.Among them,13/376(3.5%)patients exhibited Ig isotype switching,including 3 with complete isotype switching,3 with LCE,and 7 with non-secretory clinical relapse.Eleven remained sensitive to therapy,exhibiting at least a partial response.Seven patients survived for at least 20 months after relapse.The median overall survival time of the LCE,clinical relapse,and complete isotype switching groups were 6,20,and 76 months,respectively,after recurrence.The clinical manifestations and Ig phenotypes of MM recurrence were different from those at the initial diagnosis in the 13 patients exhibiting Ig isotype switching.These differences vividly conveyed the heterogeneity of the clonal populations and provides direct clinical evidence for MM clonal evolution.

Whole-exome mutational landscape of metastasis in patient-derived hepatocellular carcinoma cells

In order to explore the genomic basis for liver cancer metastasis,whole-exome sequencing(WES)was performed on patient-derived hepatocellular carcinoma(HCC)cell lines with differential metastatic potentials and analyzed their clonal evolution relationships.An evolutionary tree based on genomic single nucleotide polymorphism(SNP)was constructed in MegaX software.The WES data showed that the average percentage of heterogeneous mutations in each HCC cell lines was 16.55%(range,15.38%e18.17%).C:G>T:A and T:A>C:G somatic transitions were the two most frequent substitutions.In these metastatic HCC cell lines,non-silent gene mutations were found in 21.88%of known driver genes and 10 classical signaling pathways.The protein interaction network was constructed by STRING,and hub genes were found in the shared trunk mutation genes and the heterogeneous branch mutations respectively.In cBioPortal database,some of the selected hub genes were found to be associated with poor overall survival(OS)of HCC patients.Among the mutated HCC driver genes,a novel KEAP1 mutation with a homozygous frameshift truncation at the c-terminal Nrf2 binding region was detected and verified in MHCC97-H and HCC97LM3 cells.In conclusion,WES data demonstrate that HCC cell lines from tumor biopsy specimens of the same patient have obtained different metastatic potentials through repeated selection in rodents in vivo,and they do indeed have a genetic relationship at the genomic level.

BCR-ABL1 is a secondary event after JAK2V617F in a patient with essential thrombocythemia who develop chronic myeloid leukemia

Several cases such as myeloproliferative neoplasms(MPN)with the coexistence of JAK2 and BCR-ABL have been reported.However,cases of transformation of essential thrombocythemia(ET)into chronic myeloid leukemia(CML)during the disease progression were rarely reported.Here,we report the case of a patient with JAK2 V617F-positive ET who subsequently acquired BCR–ABL1,which transformed the disease into CML after 10 years from the initial diagnosis.In this study,we dynamically monitored JAK2 V617F and BCR-ABL and observed multiple gene mutations,including IDH2,IDH1,ASXL1,KRAS,and RUNX1.It is important to be aware of this potentially clone evolution in disease progression.

Preleukemic stem cells:leave it or not?

Acute myeloid leukemia(AML)has been shown to undergo multiple acquired mutations in hematopoietic cell lineages over years before becoming clinically apparent.The early stage of AML(before it becomes clinically recognizable)may be characterized by acquisition of some,but not all,leukemia-related somatic mutations in hematopoietic stem cells(HSCs).The physiological roles of these mutations remain puzzling.These HSCs have been termed as preleukemic HSCs.However,those frequent acquired somatic mutations are also found in healthy aging adults,namely,“age-related clonal hematopoiesis.”Multiple studies have demonstrated that the preleukemic HSCs survive through chemotherapy and then contribute to the relapse and the development of de novo AML.Whether preleukemic HSCs should be targeted or whether a preventive therapy should be considered for those individuals remains to be determined.This article aims to shed light on this special subject and to discuss the roles of preleukemic HSCs in leukemogenesis.