Degree Of Clonal Expansion Research Articles

Abstract LB310: Single-cell transcriptomics and TCR clonality reveal a role for immunotherapy in pediatric brain tumors

Abstract Central nervous system tumors are the most common solid tumors and the leading cause of cancer-related deaths in children. Outcomes remain dismal for a significant proportion of patients despite aggressive multimodal therapy and survivors suffer from treatment-related morbidity and neurological deficits. Despite their remarkable success in adult cancers, immunotherapies are not approved for pediatric brain tumors. Several factors like the relatively smaller patient population, a tumor location that makes them difficult to access through biopsies and poorly-studied tumor immune microenvironment (TME) have posed challenges in advancing immunotherapeutic options for these patients. Immune checkpoint blockers (ICB) tested in unstratified cohorts of children with brain tumors have not shown promising results; therefore, the role of immunotherapy remains unclear. Because the magnitude and nature of pre-existing T cell responses in the tumor has been shown to correlate with response to ICB, we asked if pediatric brain tumors show evidence of such favorable T cell responses. In the current study, we address this question using an unbiased approach to analyze the clonal relationship and transcriptional status of T cells present in tumor tissue of children with brain tumors using paired single-cell RNA-sequencing and TCR-sequencing. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with potential to recognize tumor antigens. Such clonally-expanded T cells display states linked to anti-tumor immunity, express higher levels of transcripts encoding for molecules linked to T cell activation, effector functions, immune cell recruitment, tissue-residency, immune checkpoints, and importantly, show significant enrichment of signatures linked to neoantigen-specific T cells and immunotherapy response. Notably, we identify several neoantigens in pediatric brain tumors, and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes in high-grade glioma. We further show that PD1-expressing CD8+ T cells in pediatric brain tumors are indeed functional as evidenced by their capacity for cytotoxicity, cytokine production and proliferation. Among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and expression of transcripts encoding immune checkpoints in tumor-infiltrating T cells. Overall, our findings suggest that accurate characterization of intra-tumoral T cell responses for features linked to immune check point therapy (ICB) response may enable selection of patients where immunotherapy is likely to be beneficial, an approach that requires prospective validation in future clinical trials. Citation Format: Anusha Preethi Ganesan, Aditi Upadhye, Kevin Meza Landeros, Ciro Ramírez-Suástegui1, Benjamin Schmiedel, Edwin Woo, Serena J. Chee, Denise Malicki, Nicole Coufal, David Gonda, Michael Levy, Jason Greenbaum, Gregory Seumois, John Crawford, William Roberts, Stephen Schoenberger, Hilde Cheroutre, Christian Ottensmeier, Pandurangan Vijayanand. Single-cell transcriptomics and TCR clonality reveal a role for immunotherapy in pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB310.

Intra-tumoral T cells in pediatric brain tumors display clonal expansion and effector properties.

Brain tumors in children are a devastating disease in a high proportion of patients. Owing to inconsistent results in clinical trials in unstratified patients, the role of immunotherapy remains unclear. We performed an in-depth survey of the single-cell transcriptomes and clonal relationship of intra-tumoral T cells from children with brain tumors. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with the potential to recognize tumor antigens. Such clonally expanded T cells display enrichment of transcripts linked to effector function, tissue residency, immune checkpoints and signatures of neoantigen-specific T cells and immunotherapy response. We identify neoantigens in pediatric brain tumors and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes. Notably, among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and magnitude of T cell response. Our findings suggest that characterization of intra-tumoral T cell responses may enable selection of patients for immunotherapy, an approach that requires prospective validation in clinical trials.

Prognostication of Large Granular Lymphocytic Leukemia in Clinical Practice: Three New Cases and Literature Review

Large Granular Lymphocytic Leukemia (LGLL) is a rare lymphoproliferative disorder of either CD3+ cytotoxic T-lymphocytes or CD3− natural killer cells with chronic and ill-defined clinical progression. Presentation at diagnosis can include asymptomatic or symptomatic cytopenias, recurrent infections, splenomegaly and rheumatoid arthritis. Multiple cell survival pathways are involved in the pathogenesis of LGLL, and it has been found to be specifically associated with aberrant STAT3 signaling. An increased number of clonal LGL cells in the blood establishes the diagnosis. Treatment regimens historically have included cyclophosphamide, methotrexate, steroids, cyclosporine, and splenectomy, but there are no available targeted therapies or definitive cure available for LGLL. Here we present three cases with varying treatment responses and outcomes that highlight the need for targeted therapy development. All three patients were confirmed to have T-LGLL by flow cytometry analysis. Patient one is a 64 year old female, presented for moderate recurrent symptoms of LGLL, initially diagnosed 17 years prior and treated with 6 months of cyclophosphamide. On the second presentation she was found to have STAT3 missense mutation and was treated for a second time with cyclophosphamide which improved symptoms. She additionally required subcutaneous immunoglobulin for treatment of recurrent infection. Patient two is a 72 year old male, presented for recurrent aphthous ulcers with severe leukopenia and neutropenia, found to have STAT3 negative LGLL. He received multiple courses of cyclophosphamide and was refractory to treatment with persistent symptoms despite improvement in leukopenia. Patient three is a 70 year old male, presented for symptomatic lymphocytosis. He was found on flow cytometry to have LGLL. He improved without further testing or treatment and has remained in remission for several years. These cases show the variation in presentation and response to therapy for patients with T-LGLL. Previous research has demonstrated the heterogeneity of LGLL and has additionally shown that STAT3 mutation was associated with decreased overall survival. This may be as yet simplifying the pathogenesis for targeting as we can still see heterogeneous clinical response even within STAT3 positive patients. One study showed that the size of the mutated clone correlated with degree of clonal expansion and that immunosuppressive therapy may not be able to reduce the clonal burden at high degrees of mutation. Our cases show that STAT3 positive status is not enough to determine prognosis and treatment course. The degree and types of mutation may need to be quantified as well as additional targets identified. While pursuing targeted treatment therapies for LGLL we must also pursue testing to assess disease burden.

Analysis of Gene Expression and TCR/B Cell Receptor Profiling of Immune Cells in Primary Sjögren's Syndrome by Single-Cell Sequencing.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease that is estimated to affect 35 million people worldwide and is characterized by lymphocytic infiltration, elevated circulating autoantibodies, and proinflammatory cytokines. The key immune cell subset changes and the TCR/BCR repertoire alterations in pSS patients remain unclear. In this study, we sought to comprehensively characterize the transcriptional changes in PBMCs of pSS patients by single-cell RNA sequencing and single-cell V(D)J sequencing. Naive CD8+ T cells and mucosal-associated invariant T cells were markedly decreased but regulatory T cells were increased in pSS patients. There were a large number of differentially expressed genes shared by multiple subpopulations of T cells and B cells. Abnormal signaling pathways, including Ag processing and presentation, the BCR signaling pathway, the TCR signaling pathway, and Epstein-Barr virus infection, were highly enriched in pSS patients. Moreover, there were obvious differences in the CD30, FLT3, IFN-II, IL-1, IL-2, IL-6, IL-10, RESISTIN, TGF-β, TNF, and VEGF signaling networks between pSS patients and healthy controls. Single-cell TCR and BCR repertoire analysis showed that there was a lower diversity of T cells in pSS patients than in healthy controls; however, there was no significant difference in the degree of clonal expansion, CDR3 length distribution, or degree of sequence sharing. Notably, our results further emphasize the functional importance of αβ pairing in determining Ag specificity. In conclusion, our analysis provides a comprehensive single-cell map of gene expression and TCR/BCR profiles in pSS patients for a better understanding of the pathogenesis, diagnosis, and treatment of pSS.

Abstract 6054: Genomic and transcriptomic remodeling of esophageal squamous cell carcinoma by neo-adjuvant radiochemotherapy

Abstract Objective: To identify genetic and clinicopathological factors that affect the response of patients with esophageal squamous cell carcinoma (ESCC) to neoadjuvant chemoradiotherapy (nCRT), profile the genomic and transcriptomic alterations after nCRT and relate the alterations to patient’s prognosis. Methods: A total of 137 samples from 57 ESCC patients undergoing nCRT were collected. 82 samples including 54 pre-treatment samples and 28 post-treatment samples were subjected to whole-exome sequencing, and 55 samples including 26 pre-treatment samples and 29 post-treatment samples were subjected to RNA-seq analysis. Genetic and clinicopathological factors associated with patient’s response to nCRT were identified by comparing baseline features of patients achieving pathological response (pCR) and patients not achieving pCR. Genomic and transcriptomic profiles before and after nCRT were compared to identify features acquired during nCRT and the association of identified features with patient’s recurrence-free survival (RFS) and overall survival (OS) were investigated. Results: Co-deficiency of DDR and HIPPO pathway at baseline synergistically sensitized ESCC to nCRT. The arachidonic acid metabolism pathway was upregulated at baseline in patients with pCR. The proportion of small deletion and insertion (INDEL %) significantly increased in acquired mutations (p<0.001) and was positively correlated with focal chromosomal loss (Spearman's ρ=0.52, p=0.005) and negatively correlated with broad chromosomal gain (Spearman's ρ=-0.61, p<0.001) after nCRT. Acquired INDEL% was associated with tumor regression grade (TRG) (p=0.06) and predicted the worse RFS (p=0.16) and OS (p=0.067). The degree of clonal expansion during nCRT was associated with patient’s RFS (p=0.0087) and OS (p=0.023), and negative correlated with acquired INDEL % (Spearman's ρ=-0.45, p=0.02). The expression profile of tumor tissue was changed after nCRT and displayed upregulation of cell adhesion and stromal related pathways and downregulation of DNA replication and cell cycle pathways, and the alteration of expression profile was associated with patient’s prognosis. Conclusions: nCRT remodels the genome and transcriptome of ESCC. Small deletion/insertion and focal chromosomal loss may be characteristic genomic alterations imposed by radiation. Acquired INDEL proportion is a biomarker to indicate the efficacy of nCRT and predict patient’s prognosis. The degree of clonal expansion during nCRT reflects the treatment resistance and associated with patient’s prognosis. Citation Format: Yang Yang, TingTing Feng, Xiaojun Fan, Xia Zhou, Wu'an Bao, Danhong Zhang, Hua Bao, Junrong Yan, Xue Wu, Yang Shao, Guoqin Qiu, Dan Su, Qixun Chen. Genomic and transcriptomic remodeling of esophageal squamous cell carcinoma by neo-adjuvant radiochemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6054.

Dissecting Multicellular Ecosystems of HTLV-1 Infection and ATL By Multi-Omics Single Cell Analysis

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell malignancy, caused by human T-cell leukemia virus type-1 (HTLV-1) infection. To elucidate immune microenvironment and heterogeneity of HTLV-1-infected normal and leukemic cells, we performed multi-omics single cell analysis, evaluating whole-transcriptome, 101 surface marker proteins, and T/B-cell receptor repertoires in the same single cells. We analyzed 236,192 peripheral blood mononuclear cells (PBMCs) from 31 ATL patients (35 samples including 4 sequential samples), 11 HTLV-1-infected carriers, and 4 healthy donors. In our analysis, expression of HTLV-1-related genes, such as HBZ, clearly identified a distinct cluster of HTLV-1-infected cells within non-malignant CD4+ T cells. These cells are characterized by a CD45RO+CD62L-CD7-CCR4+CD25+CD73+ memory/effector phenotype. By contrast, malignant ATL cells were segregated into different clusters across patients, suggestive of inter-tumor heterogeneity. Transcriptome analysis of CD4+ T cells revealed up-regulation of interferon (IFN) responses and down-regulation of TNFa signaling in malignant ATL cells compared with HTLV-1-infected normal CD4+ T cells. Likewise, sequential sample analysis showed that progression from indolent to aggressive disease enhanced IFN responses, suggesting a pivotal role of this pathway in the ATL pathogenesis. Surface marker protein analysis demonstrated that HTLV-1 infection up-regulated the expression of stimulatory and inhibitory immune checkpoint molecules (such as OX40 and TIGIT, respectively), which was further augmented by ATL progression. Within malignant cells, we identified a fraction of cycling cells present across most ATL samples. This fraction showed an enhanced T-cell activation markers, such as CD25 and HLA-DR, and their frequency was increased in aggressive subtypes. On the other hand, in HTLV-1-infected carriers, HTLV-1-infected CD4+ T cells contained a small population of malignant-like cells showing clonal expansion. The degree of clonal expansion was significantly correlated with HTLV-1 viral load in PBMCs. These results clarify the heterogeneity within HTLV-1-infected cells and ATL malignant cells, pointing to its relevance during ATL initiation and progression. We also observed dynamic changes of the immune microenvironment in ATL. Although the relative frequencies of other cell types remained almost the same or reduced, only myeloid cells were increased in ATL patients compared with in HTLV-1-infected carriers. Re-clustering of myeloid cells identified a novel cluster of monocytes expressing FCGR1A, encoding CD64, a biomarker of IFN-stimulated gene levels. Transcriptome analysis revealed increased IFN signaling and decreased TNFa in myeloid cells from ATL patients compared with HTLV-1-infected carriers. Similar expression signatures changes were also observed in various immune cell types, such as B, CD8+ T, and NK cells, in ATL patients. In addition, substantial changes of surface marker proteins were also found in ATL patients. Particularly, T-cell activation markers, such as HLA-DR, and inhibitory immune checkpoint molecules, such as PD-1 and TIM-3, were up-regulated in CD8+ T cells from ATL patients. A co-culture experiment of ATL cell lines with PBMCs from healthy volunteers demonstrated that ATL cells induced immune-phenotypic changes of myeloid and CD8+ T cells, similar to those observed in ATL patient by our single-cell analysis, confirming the role of ATL cells in the modulation of the immune system. Taken together, the composition and function of immune microenvironment is dramatically altered in ATL patients, which may contribute to immunosuppression and disease progression in ATL. In summary, our multi-omics single-cell analysis comprehensively dissects the cellular and molecular architecture in HTLV-1-infected carriers and ATL patients. In particular, our approach clearly defines HTLV-1-infected cells by the expression of HTLV-1-related genes, leading to the detailed characterization of HTLV-1-infected cells and elucidation of their difference from ATL malignant cells. These findings will help to devise novel diagnostic and therapeutic strategies for HTLV-1-related disorders. Disclosures Kogure: Takeda Pharmaceutical Company Limited.: Honoraria. Shimoda:Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company: Honoraria; Novartis: Honoraria, Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Shire plc: Honoraria; Celgene: Honoraria; Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding. Kataoka:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Genomics: Current equity holder in private company.

Determination of the complexity and diversity of the TCR β-chain CDR3 repertoire in bladder cancer using high-throughput sequencing.

The present study aimed to investigate the complexity and diversity of the T lymphocyte immune repertoire in patients with bladder cancer. To do so, the immune state of patients was assessed. The study also aimed to elucidate the aetiology and pathogenesis of bladder cancer to provide a novel theoretical basis for disease prevention, diagnosis, treatment and prognosis monitoring. Cancerous and paracancerous (control) tissue samples were collected from five patients diagnosed with muscle-invasive bladder cancer. Multiplex polymerase chain reaction and Illumina high-throughput sequencing were used to determine the characteristics and clonal diversity of the T-cell receptor (TCR) β-chain complementarity-determining region 3 (CDR3) gene in the cancerous and paracancerous tissues of patients with bladder cancer. The degree of clonal expansion in malignant samples was significantly higher than in adjacent samples. Furthermore, ΤCRβ variable (TRBV), ΤCRβ diversity (TRBD) and ΤCRβ joining (TRBJ) repertoires were significantly different in cancerous samples compared with adjacent samples. In addition, 13 identified V-J pairs were highly expressed in cancerous samples whereas they had low expression in control samples. In conclusion, the degree of T-cell clonal expansion in bladder cancerous tissue was higher than in paracancerous tissue, whereas the immune diversity of the tissues of patients with bladder cancer was significantly lower. The DNA sequence and amino acid sequences, and V-J combination level may be used to comprehensively understand the diversity and characteristics of TCR CDR3 in bladder cancer and paracancerous tissues, and to evaluate the immune status of bladder cancer to develop therapeutic targets and biomarkers for prognosis monitoring.

Next generation sequencing reveals novel alterations in B-cell heavy chain receptor repertoires associated with acute-on-chronic liver failure.

Acute-on-chronic liver failure (ACLF) is a newly-defined serious syndrome with major features of acute decompensation (AD) of hepatic cirrhosis, liver failure and failure of multiple other organs. To date, the mechanism underlying the development and progression of ACLF remains to be fully elucidated. It has been noted that ACLF is associated with immune dysregulation. However, studies have mainly focused on T-cell responses. The present study aimed to determine the composition and alterations of B-cell receptor (BCR) heavy chain repertoires associated with ACLF using next generation sequencing (NGS). A total of six patients with hepatitis B virus (HBV)-related ACLF and six healthy control subjects were prospectively enrolled in the present study. The B-cell immunoglobulin heavy chain (IGH) repertoires in peripheral blood mononuclear cells (PBMCs) obtained from the patients with HBV-related ACLF and the control subjects were analyzed using NGS, coupled with multiplex polymerase chain reaction, were Illumina sequenced, and were further characterized using the international ImMunoGeneTics database. The distribution of the BCR complementarity-determining region 3 (CDR3) variable (V), diversity (D) and joining (J) and V-J gene segments were found to be comparable between the ACLF and control groups. Of note, the degree of clonal expansion in the ACLF group was significantly higher than that in the control group (P<0.05). Furthermore, a t-test of the distribution ratio of the V, D, J and V-J combinations in patients with ACLF and control subjects revealed differentially expressed genes. In total, six genes were upregulated and 19 genes were downregulated in response to ACLF. The difference between these two groups was statistically significant (P<0.05). The approach used in the present study was feasible and effective for analyzing peripheral B-cell repertoires in HBV-related ACLF. These results provide direct evidence that the BCR repertoire is important in immune responses, autoimmunity and alloreactivity, and that there is a link between the BCR repertoire and HBV-ACLF. Therefore, ACLF-specific BCR CDR3 sequences hold promise for therapeutic benefit to HBV-ACLF in the future.

Immune Repertoire Sequencing Using Molecular Identifiers Enables Accurate Clonality Discovery and Clone Size Quantification.

Unique molecular identifiers (MIDs) have been demonstrated to effectively improve immune repertoire sequencing (IR-seq) accuracy, especially to identify somatic hypermutations in antibody repertoire sequencing. However, evaluating the sensitivity to detect rare T cells and the degree of clonal expansion in IR-seq has been difficult due to the lack of knowledge of T cell receptor (TCR) RNA molecule copy number and a generalized approach to estimate T cell clone size from TCR RNA molecule quantification. This limited the application of TCR repertoire sequencing (TCR-seq) in clinical settings, such as detecting minimal residual disease in lymphoid malignancies after treatment, evaluating effectiveness of vaccination and assessing degree of infection. Here, we describe using an MID Clustering-based IR-Seq (MIDCIRS) method to quantitatively study TCR RNA molecule copy number and clonality in T cells. First, we demonstrated the necessity of performing MID sub-clustering to eliminate erroneous sequences. Further, we showed that MIDCIRS enables a sensitive detection of a single cell in as many as one million naïve T cells and an accurate estimation of the degree of T cell clonal expression. The demonstrated accuracy, sensitivity, and wide dynamic range of MIDCIRS TCR-seq provide foundations for future applications in both basic research and clinical settings.

Single-cell analysis of glandular T cell receptors in Sjögren's syndrome.

CD4+ T cells predominate in salivary gland (SG) inflammatory lesions in Sjögren's syndrome (SS). However, their antigen specificity, degree of clonal expansion, and relationship to clinical disease features remain unknown. We used multiplex reverse-transcriptase PCR to amplify paired T cell receptor α (TCRα) and β transcripts of single CD4+CD45RA- T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype. TCRα and β sequences were obtained from a median of 91 SG and 107 PB cells per subject. The degree of clonal expansion and frequency of cells expressing two productively rearranged α genes were increased in SG versus PB. Expanded clones from SG exhibited complementary-determining region 3 (CDR3) sequence similarity both within and among subjects, suggesting antigenic selection and shared antigen recognition. CDR3 similarities were shared among expanded clones from individuals discordant for canonical Ro and La autoantibodies, suggesting recognition of alternative SG antigen(s). The extent of SG clonal expansion correlated with reduced saliva production and increased SG fibrosis, linking expanded SG T cells with glandular dysfunction. Knowledge of paired TCRα and β sequences enables further work toward identification of target antigens and development of novel therapies.

Deep sequencing of the TCRβ repertoire reveals T cell clonal expansion is associated with HTLV-1 proviral load in HAM/TSP patients

HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) is associated with a chronic inflammatory central nervous system in which the host immune response against HTLV-1 infection is considered immunopathogenic. We have applied a new unbiased deep sequencing method of TCR repertoire to accurately measure the diversity and degree of clonal expansion of peripheral circulating T-cells in patients with HAM/TSP compared to age, gender, and ethnicity matched healthy controls. TCR libraries were generated using 5'rapid amplification of cDNA ends to amplify the V-D-J genes of unsorted T-cells obtained from peripheral blood. Over 100 million reads were analyzed using MiGec software (Shugay, et al., Nat Methods. 2014 Jun;11(6):653-5), that align and match the human TCR β-chain nucleotide sequences through IMGT database. T-cell clonotypes with high-quality CDR3 sequence were selected by a molecular identifier PCR strategy, which allowed for filtering PCR errors and correct for artificial sequences generated on a HiSeq 2000 Illumina system platform. By calculating the coefficient of variation of our dataset we considered clonal expansion as those unique TCR clonotypes showing reads >8. While we did not observe any significant statistical differences of the number of single clones (clonotypes with 1 read, P=0.62) between HAM/TSP patients and healthy controls groups, by contrast, a higher clonal expansion of the T-cell repertoire was observed in HAM/TSP patients when compared to healthy controls (unpaired parametric T-test with P=0.026). Interestingly, this clonal expansion of the HAM/TSP TCR repertoire correlated with the HTLV-I provirus load (as defined by digital PCR quantification of the HTLV-I tax gene) (r=0.64, P=0.008). Our findings strongly suggest that in this chronic neurological disorder, a strong dysregulated T-cell response may be driven by HTLV-I and that strategies to decrease virus may be of clinical benefit.

Composition and variation analysis of the TCR β-chain CDR3 repertoire in systemic lupus erythematosus using high-throughput sequencing.

The ability of T lymphocytes to mount an immune response against a diverse array of pathogens is primarily conveyed by the amino acid (aa) sequence of the hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (TCR). In this study, we used a combination of multiplex-PCR, Illumina sequencing and IMGT/HighV-QUEST for a standardized analysis of the characteristics and polymorphisms of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy donors (NC). We found the distributions of CDR3, VD indel, and DJ indel lengths to be comparable between the SLE and NC groups. The degree of clonal expansion in the SLE group was significantly greater than in the NC group, and the expression levels of 10 TRβV segments and 6 TRβJ segments were also significantly different in the SLE group. Regarding public T cell responses, 3CDR3 DNA sequences and 4 aa sequences were shared by all SLE patients and may serve as biomarkers for SLE disease risk, diagnosis and/or prognosis.

Spatial and temporal heterogeneity in high-grade serous ovarian cancer: a phylogenetic analysis.

BackgroundThe major clinical challenge in the treatment of high-grade serous ovarian cancer (HGSOC) is the development of progressive resistance to platinum-based chemotherapy. The objective of this study was to determine whether intra-tumour genetic heterogeneity resulting from clonal evolution and the emergence of subclonal tumour populations in HGSOC was associated with the development of resistant disease.Methods and FindingsEvolutionary inference and phylogenetic quantification of heterogeneity was performed using the MEDICC algorithm on high-resolution whole genome copy number profiles and selected genome-wide sequencing of 135 spatially and temporally separated samples from 14 patients with HGSOC who received platinum-based chemotherapy. Samples were obtained from the clinical CTCR-OV03/04 studies, and patients were enrolled between 20 July 2007 and 22 October 2009. Median follow-up of the cohort was 31 mo (interquartile range 22–46 mo), censored after 26 October 2013. Outcome measures were overall survival (OS) and progression-free survival (PFS). There were marked differences in the degree of clonal expansion (CE) between patients (median 0.74, interquartile range 0.66–1.15), and dichotimization by median CE showed worse survival in CE-high cases (PFS 12.7 versus 10.1 mo, p = 0.009; OS 42.6 versus 23.5 mo, p = 0.003). Bootstrap analysis with resampling showed that the 95% confidence intervals for the hazard ratios for PFS and OS in the CE-high group were greater than 1.0. These data support a relationship between heterogeneity and survival but do not precisely determine its effect size. Relapsed tissue was available for two patients in the CE-high group, and phylogenetic analysis showed that the prevalent clonal population at clinical recurrence arose from early divergence events. A subclonal population marked by a NF1 deletion showed a progressive increase in tumour allele fraction during chemotherapy.ConclusionsThis study demonstrates that quantitative measures of intra-tumour heterogeneity may have predictive value for survival after chemotherapy treatment in HGSOC. Subclonal tumour populations are present in pre-treatment biopsies in HGSOC and can undergo expansion during chemotherapy, causing clinical relapse.

The structure and genomic integration site of the HTLV-1 provirus determine selective clonal expansion and transformation to adult T cell leukaemia/lymphoma

Following infection and integration into the genome of host CD4+ T-lymphocytes, human T-lymphotropic virus type-1 (HTLV-1) persists by driving proliferation of infected T-cell clones which are counter-selected by HTLV-1-specific cytotoxic T-lymphocytes (CTLs). HTLV-1 Tax protein is the dominant target antigen recognized by these CTLs, and Tax is thought to cause the infected T-cell proliferation. In 40% of cases of adult T-cell leukaemia/lymphoma (ATLL) Tax protein is not expressed, suggesting that the provirus has down-regulated Tax to allow immune escape and viral persistence. Here, we tested the hypothesis that the genomic site of proviral integration determines the degree of clonal expansion and therefore determines the risk of ATLL. Proviral integration sites in 242 ATLL cases and 96 asymptomatic carriers were mapped and quantified using our recently described high-throughput technique (Gillet et al 2011, Blood, Cook et al 2012, Blood). In the ATLL cases, we identified promoter deletions predicted to cause tax gene silencing, we sequenced the tax gene and quantified promoter methylation. The results showed that 65% cases of ATLL could silence Tax by promoter deletion, tax gene nonsense mutations or tax promoter hypermethylation. High-throughput sequencing data showed that ATLL cases were not invariably monoclonal and that the provirus is typically integrated into a transcriptionally active region of the host genome. The results also suggest that transcriptional activity of the flanking host genome contributes to tax gene silencing, allowing CTL escape and clonal expansion.

Natural history of pancreatic mucinous cystic neoplasms.

e21115 Background: Pancreatic cysts (PC) are increasingly being detected; proper management requires a balance between aggressive intervention and surveillance. Mucinous cysts may develop adenocarcinoma; however, only a minority will do so. To better understand the evolution of mucinous cystic neoplasms, we studied the natural history of PC by integrating molecular analyses of cyst fluid with other clinical parameters in a continuous series of patients with extended follow-up. Methods: A continuous series of 299 patients with pancreatic cysts yielded 43 mucinous cysts with one or more ASGE criteria and/or risk factors for cancer formation. Patients with overt pancreatic cancer were excluded. Molecular analyses measured 1) DNA quantity, 2) DNA quality, 3) Kras point mutation, 4) allelic imbalance (loss of heterozygosity [LOH]) for 16 markers, and 5) degree of clonal expansion of DNA alterations (RedPath PathFinderTG). Follow up ranged from 25 to 45 months. Results: Of 43 patients predicted to be non-malign...

High Resolution Karyotyping of Large Granular Lymphocyte Leukemia by SNP Arrays Reveals Clonal Defects Leading to LOH at Loci Integral to Lymphocyte Function.

Large granular lymphocyte leukemia (LGL leukemia) is a semiautonomous clonal lymphoproliferation of cytotoxic T cells associated with various immune cytopenias. Within the spectrum of acquired immune-mediated bone marrow failure states, LGL leukemia can serve as monoclonal model of usually polyclonal T cell-mediated pathology. Mechanisms of unopposed clonal expansion of LGL cells in many aspects resemble true lymphoma and are not well understood. In addition to its reactive character, intrinsic clonal defects may be present in some patient with LGL, in particular those with more pronounced lymphoproliferative features. However, unlike in B-cell lymphomas, recurrent chromosomal abnormalities have not been frequently identified in LGL leukemia using traditional metaphase karyotyping techniques. We have applied Affymetrix 250K single nucleotide polymorphism-arrays (SNP-A) and 6.0 SNP-A in 28 patients with LGL leukemia, to elicit a far higher resolution of chromosomal content through genomic mapping of individual SNP and respective copy number analysis in LGL leukemia. SNP-A based cytogenetics have been applied successfully to MDS patients and has increased prognostic reliability. Blood mononuclear cells containing high proportion of clonal cells as determined by TCR Vβ flow cytometry were used as a source of DNA. For comparison, a large number of control blood and marrow specimens (N=119 for 6.0 and 124 for 250K SNP arrays) were analyzed. Data were processed using Genotyping Console v2.1 software (Affymetrix, Santa Clara, CA). After exclusion of known copy number variants (CNV) referenced in public databases and our own set of 178 normal controls, we found distinct chromosomal changes in 16/28 (57%) of LGL leukemia patients. Consensus regions of deletion/gain or uniparental disomy (UPD) were identified. The most common abnormalities included either UPD or copy number loss of chromosome 3q21.2–q21.31, which was identified in 6 (21%) patients. This region harbors CD86, the gene encoding the B7.2 protein responsible for T cell activation and regulation through costimulatory mechanisms. Copy number loss/UPD was also identified at chromosome 1p31.1–p32.3 in 4 (18%) patients. Interestingly, copy number gain in this same region was identified in 2 (9%) patients suggesting that this region may correspond to either a new, infrequent germ line encoded CNV or represents a somatic microdeletion. Recent studies indicated a role for SIL/SCL, which resides at this locus, in V(D)J recombination, lymphocyte development, and maturation. Additional conserved chromosomal abnormalities included copy number gains in 14q (11%) and copy number loss at 11p15 (14%), all possibly representing germ line or somatic CNV physiologically acquired during lymphocyte ontogenesis. We compared chromosomal lesions identified in our LGL cohort with clinical features including age, presence of neutropenia, anemia, thrombocytopenia, splenomegaly, immunophenotype, and degree of clonal expansion. No difference in clinical course was found between patients with and without cytogenetic abnormalities with regard to type and severity of cytopenias or size of the LGL clone. However, patients with 1p31.1–p32.3 deletions were found to have lesser degree of neutropenia compared to patients without 1p31.1–p32.3 deletions (p<0.001). While these data may reflect chromosomal variants and random somatic abnormalities, the recurrent loss of heterozygosity and gains within several loci with known regulatory function for lymphocyte biology suggests the involvement of these defects in the mechanism of clonal evolution.

Fate of Premalignant Clones during the Asymptomatic Phase Preceding Lymphoid Malignancy

Almost all cancers are preceded by a prolonged period of clinical latency during which a combination of cellular events helps move carcinogen-exposed cells towards a malignant phenotype. Hitherto, investigating the fate of premalignant cells in vivo remained strongly hampered by the fact that these cells are usually indistinguishable from their normal counterparts. Here, for the first time, we have designed a strategy able to reconstitute the replicative history of the bona fide premalignant clone in an animal model, the sheep experimentally infected with the lymphotropic bovine leukemia virus. We have shown that premalignant clones are early and clearly distinguished from other virus-exposed cells on the basis of their degree of clonal expansion and genetic instability. Detectable as early as 0.5 month after the beginning of virus exposure, premalignant cells displayed a two-step pattern of extensive clonal expansion together with a mutation load approximately 6 times higher than that of other virus-exposed cells that remained untransformed during the life span of investigated animals. There was no fixation of somatic mutations over time, suggesting that they regularly lead to cellular death, partly contributing to maintain a normal lymphocyte count during the prolonged premalignant stage. This equilibrium was finally broken after a period of 18.5 to 60 months of clinical latency, when a dramatic decrease in the genetic instability of premalignant cells coincided with a rapid increase in lymphocyte count and lymphoma onset.

Induced drug resistance inhibits selection of initiated cells and cancer development.

Compounds exerting a mitoinhibitory effect on normal hepatocytes are potent promoters in the resistant hepatocyte model of chemical carcinogenesis in combination with stimulation of regenerative growth by partial hepatectomy or treatment with carbon tetrachloride. 2-Acetylaminofluorene (2-AAF) almost completely inhibits liver cell regeneration after partial hepatectomy, allowing only resistant cells to participate in regenerative growth. After initiation by diethylnitrosamine and promotion with 2-AAF and partial hepatectomy (PH), focal growth of initiated cells generates liver lesions which occupy 40% of the hepatic volume three weeks after PH. In this work the mechanism for the anti promoting effects of phenobarbital and 3-methylcholantrene were investigated as well as their effects on the development of malignant hepatocellular carcinoma in the resistant hepatocyte model. Treatment with phenobarbital or, especially, 3-methylcholanthrene rendered normal rat hepatocytes resistant to the mitoinhibitory effect of 2-AAF. In combination with 2-AAF/PH, 3-methylcholanthrene shortened the regenerative growth period to less than one week. In the Solt-Farber protocol for experimental hepatocarcinogenesis, treatment with phenobarbital or 3-methylcholanthrene during promotion with 2-AAF/PH permitted hepatocytes surrounding the focal lesions to respond with regenerative growth. The foci and surrounding liver grew until the liver/body mass index reached the control value. With phenobarbital treatment the total focal volume was 20% of the liver volume three weeks after PH, whereas the corresponding value in the case of 3-methylcholanthrene was only 1%. Labelling index data supported the conclusion that growth of the liver lesions in the resistant hepatocyte model was dependent on differential inhibition of normal hepatocyte growth by the promoter and that the size of the foci obtained was related to the length of time after PH required to complete liver regeneration. 3-methylcholanthrene induced 2-AAF resistance prevented the development of large persistent nodules and hepatocellular carcinoma while phenobarbital delayed cancer development with several month. The data thus supports the idea that the degree of clonal expansion during promotion determines the size of the population at risk for malignant transformation, as well as the final frequency of carcinomas.