Frequency Of Clones Research Articles

Preliminary assessment of the diagnostic accuracy of cutaneous T-cell lymphoma through deep sequencing of T-cell receptor gamma gene.

The diagnostic challenges in early mycosis fungoides (MF) and other cutaneous T-cell lymphomas (CTCL) persist despite advancements in molecular methods. The study aims to provide a preliminary assessment of next-generation sequencing in analyzing T-cell receptor gamma (TRG) sequences for distinguishing CTCL from benign inflammatory disorders. Skin samples from CTCL and benign inflammatory skin disorders proven clinicopathologically were assessed for TRG by NGS. Our study analyzed skin samples from a total of 36 subjects, comprising 22 cases of CTCL, including 14 MF and 8 other CTCLs, alongside 14 cases of benign inflammatory skin disorders. According to the LymphoTrack criteria, monoclonality was detected in 75.0 % of the overall 24 CTCL patients. Specifically, in MF cases, 10 out of 14 were identified as monoclonality, with all four non-monoclonal cases being in the patch stage. For the other CTCL, 6 out of 8 cases displayed monoclonality. Among the overall 22 CTCL patients, 11 had multiple biopsies, with 9 displaying the same dominant clone across different sites. Among the 14 benign cases, only the case with erythrodermic psoriasis exhibited monoclonality. Our decision tree analysis suggests that a high frequency of the most abundant clone, its ratio to the third most abundant clone, and TRG V-I segment usage are effective markers aiding in diagnosing CTCL. Combination of the clone frequencies, and TRG V segment usage may enhance diagnosis for MF and other CTCLs, aiding in differentiating them from benign conditions. However, molecular diagnosis for patch-stage MF remains challenging.

Nanopore Sequencing for T-Cell Receptor Rearrangement Analysis in Cutaneous T-Cell Lymphoma.

Background: Analysis of T-cell receptor (TCR) clonality is a major diagnostic tool for lymphomas, particularly for cutaneous T-cell lymphomas (CTCL) like Mycosis fungoides and Sézary syndrome. However, a fast and cost-effective workflow is needed to enable widespread use of this method. Methods: We established a procedure for TCR rearrangement analysis via Oxford Nanopore Technology (ONT) sequencing. TCR receptor rearrangements (TCR-gamma and TCR-beta chains) were analyzed in samples from 45 patients with various diagnoses: Mycosis fungoides (37/45), Sézary Syndrome (2/45), folliculotropic CTCL (1/45), and non-CTCL diagnoses as polyclonal controls (5/45). Sample types included formalin-fixed paraffin-embedded (FFPE) samples (27/45), fresh frozen samples (9/45), and CD3-isolated cells (9/45). In addition, DNA of a Jurkat cell line was used as a monoclonal control. TCR amplicons were generated employing an optimized version of the protocol from the Euro Clonality consortium. Sequencing was conducted on the ONT GridION and Illumina MiSeq platforms, followed by similar bioinformatic analysis protocols. The tumor clone frequency (TCF), a crucial prognostic factor for CTCL patients, was used for method comparison. Results: The use of an optimized amplicon protocol and adapted bioinformatic tools demonstrated a strong correlation in TCF values between both sequencing methods across all sample types (range R: 0.992-0.996; range r2: 0.984-0.991). Conclusions: In summary, ONT sequencing was able to detect TCR clonality comparable to NGS, indicating its potential as a faster and more cost-effective option for routine diagnostic use.

TIRTL-seq: Deep, quantitative, and affordable paired TCR repertoire sequencing.

ɑ/β T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) ɑ and β chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies. Here, we present TIRTL-seq (Throughput-Intensive Rapid TCR Library sequencing), a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well. The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired αβTCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is ~$200. We benchmarked TIRTL-seq against state-of-the-art 5'RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. The sequences of both ɑ and β TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire.

Nonequilibrium Antigen Recognition during Infections and Vaccinations

The initial immune response to an acute primary infection is a coupled process of antigen proliferation, molecular recognition by naive B cells, and their subsequent clonal expansion. This process contains a fundamental problem: the recognition of an exponentially time-dependent antigen signal. Here, we show that an efficient immune response must be stringently constrained to B-cell lineages with high antigen binding affinity. We propose a tuned proofreading mechanism for primary recognition of new antigens, where the molecular recognition machinery is adapted to the complexity of the immune repertoire. We show that this process produces potent, specific, and fast recognition of antigens, maintaining a spectrum of genetically distinct B-cell lineages as input for affinity maturation. Our analysis maps the proliferation-recognition dynamics of a primary infection to a generalized Luria-Delbrück process, akin to the dynamics of the classic fluctuation experiment. This map establishes a link between signal recognition dynamics and evolution. We derive the resulting statistics of the activated immune repertoire: Antigen binding affinity, expected size, and frequency of active B-cell clones are related by power laws, which define the class of generalized Luria-Delbrück processes. Their exponents depend on the antigen and B-cell proliferation rate, the number of proofreading steps, and the lineage density of the naive repertoire. We extend the model to include spatiotemporal processes, including the diffusion-recognition dynamics of a vaccination. Empirical data of activated mouse immune repertoires are found to be consistent with activation involving about three proofreading steps. The model predicts key clinical characteristics of acute infections and vaccinations, including the emergence of elite neutralizers and the effects of immune aging. More broadly, our results establish infections and vaccinations as a new probe into the global architecture and functional principles of immune repertoires. Published by the American Physical Society 2024

Light chain only variant of proliferative glomerulonephritis with monoclonal immunoglobulin deposits: a clinicopathological study of three cases

AimsTo explore the clinical and pathological features of light chain only variant of proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID-LC).MethodsFrom January 2010 to December 2022, patients who were diagnosed with...

Rapid evolution of consumptive and non-consumptive predator effects on prey population densities, bioenergetics and stoichiometry.

Predators can strongly influence prey populations not only through consumptive effects (CE) but also through non-consumptive effects (NCE) imposed by predation risk. Yet, the impact of NCE on bioenergetic and stoichiometric body contents of prey, traits that are shaping life histories, population and food web dynamics, is largely unknown. Moreover, the degree to which NCE can evolve and can drive evolution in prey populations is rarely studied. A 6-week outdoor mesocosm experiment with Caged-Fish (NCE) and Free-Ranging-Fish (CE and NCE) treatments was conducted to quantify and compare the effects of CE and NCE on population densities, bioenergetic and stoichiometric body contents of Daphnia magna, a keystone species in freshwater ecosystems. We tested for evolution of CE and NCE by using experimental populations consisting of D. magna clones from two periods of a resurrected natural pond population: a pre-fish period without fish and a high-fish period with high predation pressure. Both Caged-Fish and Free-Ranging-Fish treatments decreased the body size and population densities, especially in Daphnia from the high-fish period. Only the Free-Ranging-Fish treatment affected bioenergetic variables, while both the Caged-Fish and Free-Ranging-Fish treatments shaped body stoichiometry. The effects of CE and NCE were different between both periods indicating their rapid evolution in the natural resurrected population. Both the Caged-Fish and Free-Ranging-Fish treatments changed the clonal frequencies of the experimental Daphnia populations of the pre-fish as well as the high-fish period, indicating that not only CE but also NCE induced clonal sorting, hence rapid evolution during the mesocosm experiment in both periods. Our results demonstrate that CE as well as NCE have the potential to change not only the body size and population density but also the bioenergetic and stoichiometric characteristics of prey populations. Moreover, we show that these responses not only evolved in the studied resurrected population, but that CE and NCE also caused differential rapid evolution in a time frame of 6 weeks (ca. four to six generations). As NCE can evolve as well as can drive evolution, they may play an important role in shaping eco-evolutionary dynamics in predator-prey interactions.

Range of trait variation in prey determines evolutionary contributions to predator growth rates.

Evolutionary and ecological dynamics can occur on similar timescales and thus influence each other. While it has been shown that the relative contribution of ecological and evolutionary change to population dynamics can vary, it still remains unknown what influences these differences. Here, we test whether prey populations with increased variation in their defense and competitiveness traits will have a stronger impact of evolution for predator growth rates. We controlled trait variation by pairing distinct clonal lineages of the green alga Chlamydomonas reinhardtii with known traits as prey with the rotifer Brachionus calyciforus as predator and compared those results with a mechanistic model matching the empirical system. We measured the impact of evolution (shift in prey clonal frequency) and ecology (shift in prey population density) for predator growth rate and its dependency on trait variation using an approach based on a two-way ANOVA. Our experimental results indicated that higher trait variation, i.e., a greater distance in trait space, increased the relative contribution of prey evolution to predator growth rate over 3-4 predator generations, which was also observed in model simulations spanning longer time periods. In our model, we also observed clone-specific results, where a more competitive undefended prey resulted in a higher evolutionary contribution, independent of the trait distance. Our results suggest that trait combinations and total prey trait variation combine to influence the contribution of evolution to predator population dynamics, and that trait variation can be used to identify and better predict the role of eco-evolutionary dynamics in predator-prey systems.

Profiling antigen-reactive T cells using high throughput single-cell T cell receptor sequencing.

Abstract Recent advances in single-cell sequencing allow for simultaneous profiling of T cell receptors (TCRs) and transcriptomes leading to the characterization of key T cell subsets with antigen recognition and killing capabilities. Despite their success, these methods rely on microfluidic devices or plate-based protocols with limited sensitivity and throughput (1,000s-10,000s of cells, 1-2 samples) making the study of disease-relevant T cells time consuming and costly. To overcome these limitations, we developed a split pool combinatorial barcoding technology to simultaneously characterize the TCRs alongside the full transcriptomes of up to 1 million T cells from up to 96 unique samples. Here, we profiled hundreds of thousands of human T cells stimulated with antigens from a variety of diseases. Applying our method, we performed deep characterization of antigen-reactive TCR clonotypes and assessed their sequence similarities. In addition, we performed transcriptome-based clustering analyses and identified highly proliferative and cytotoxic T cell subsets. Finally, we correlated T cell activation and cytotoxicity states to clonal expansion frequencies in order to identify TCRs with increased killing potential. In summary, we present a highly flexible and scalable combinatorial barcoding technology to allow researchers to profile up to 1 million T cells in one experiment and investigate their functional responses during infection, cancer, autoimmunity, or therapeutic interventions.

Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing

BackgroundUnderstanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing.MethodsA data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach.ResultsA 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92–0.99] and 0.92 [95% CI: 0.83–0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28–0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35–3.68].ConclusionsA region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

Abstract 2334: Computational methods for optimizing marker selection, clonal lineage reconstruction, and longitudinal tracking of clonal dynamics via circulating tumor DNA (ctDNA)

Abstract Background: Technologies for blood-based profiling of tumor DNA (“liquid biopsy”) have offered great prospects for non-invasive early cancer diagnosis, treatment monitoring, and clinical guidance, but further advances in computational models and data analysis to optimize monitoring protocols and develop liquid biopsy as a robust quantitative assay of tumor clonal evolution. Methods: We propose new computational methods to improve our ability to characterize tumor clonal dynamics from circulating tumor DNA (ctDNA). Our methods address how to apply ctDNA assays most effectively to two distinct questions in profiling tumor clonal dynamics: 1) How to apply longitudinal ctDNA data to refine phylogenetic tree models of clonal evolution, and 2) how to most effectively characterize changes in clonal frequencies that may be indicative of treatment response or tumor progression. We pose these questions computationally in terms of a probabilistic framework for optimally identifying maximum likelihood markers for the preceding tasks and applying measured marker concentrations to their solution. We apply this framework by first evaluating the distribution of plausible clonal lineage models using bootstrap samples over pre-treatment tissue-based genetic variation data, then refining these lineage models and the clonal frequencies we can infer from them over successive longitudinal samples of selected markers. Results: We tested our method on synthetic data under various model assumptions and showed the method to be effective at refining distribution tree models and clonal frequencies over longitudinal samples so as to minimize measures of tree distance relative to the ground truth. We further apply the methods to a real ovarian cancer case and show their ability to develop and refine a clonal lineage model and assess clonal frequencies. Conclusion: Our methods show the power of ctDNA assays in conjunction with computational optimization to lead to improved marker selection, clonal lineage reconstruction, and tracking of clonal dynamics, with potential for more precise and quantitative profiling of tumor progression enabling improved clinical decision-making. Citation Format: Xuecong Fu, Yueqian Deng, Zhicheng Luo, William Laframboise, David Bartlett, Russell Schwartz. Computational methods for optimizing marker selection, clonal lineage reconstruction, and longitudinal tracking of clonal dynamics via circulating tumor DNA (ctDNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2334.

Abstract A004: TP53 mutational landscape in non-cancerous endometrium during the lifespan of Black and White individuals

Abstract Background TP53 somatic mutations accumulate and clonally expand in histologically normal tissues with aging, however, little is known about this process and how it influences cancer development. Within endometrial carcinoma, TP53 mutations tend to be found in aggressive, non-endometrioid histologies associated with poor survival outcomes, more commonly affecting Black women. We seek to understand the TP53 mutational burden in benign endometrium by characterizing mutations acquired throughout a lifespan with the integration of social determinants of health in Black and White women. Methods Endometrial tissue was collected at autopsy or hysterectomy, confirmed with H&amp;E staining, and macrodissected. Ultra-deep duplex sequencing (~10,000x) of isolated DNA was used to sequence TP53somatic mutations with high resolution. Each individual’s TP53 mutation frequency was calculated as the number of unique TP53 variants divided by the total duplex nucleotides sequenced. Mutations were classified as pathogenic if insertion-deletion, splice, or nonsense in addition to all pathogenic substitutions identified by the Seshat TP53 database. Socioeconomic metrics were acquired using zip codes and the 2021 US Census Bureau American Community Survey. Individuals with a history of endometriosis, prior malignancy, or prior chemotherapy were excluded. Results Benign endometrial tissue was collected from 69 individuals (24 Black, 45 White), including 22 aged 0-19 years, 25 aged 20-39 years, 17 aged 40-59 years, and 5 aged over 60 years. 174 TP53 variants were identified. Variant types included 66.7% missense (n=116), 13.2% insertion-deletion (n=23), 12.1% synonymous (n=21), 4.6% nonsense (n=8), and 3.4% splice (n=6). 102 TP53 mutations (58.6%) were pathogenic. TP53 mutation frequency increased linearly with age (Spearman correlation p&amp;lt;0.01). 12.6% of mutations were identified in more than one duplex mutant read (large clone) beginning at age 29 through age 81. The frequency of large TP53 mutant clones increased exponentially with age. In this limited dataset, there was no statistical association between socioeconomic metrics and the mutational burden of Black and White individuals. Conclusions The number and size of TP53 mutant clones in benign endometrium are associated with older age, indicating clonal evolution through life. Most somatic mutations identified were pathogenic substitutions or loss-of-function variants, confirming positive selection of cancer-like mutations. Additional data are needed to determine the role of socioeconomic factors in TP53 genomic burden. Citation Format: Eric Rios-Doria, Elizabeth U. Parker, Brendan F. Kohrn, Elena Latorre-Estevez, Jeanne Fredrickson, Elizabeth M. Swisher, Kemi M. Doll, Rosana Risques. TP53 mutational landscape in non-cancerous endometrium during the lifespan of Black and White individuals [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A004.

Prevalence of Paroxysmal Nocturnal Haemoglobinuria Clone in Aplastic Anaemia: A Single Centre Study

Background: Flow cytometry assay for PNH clone is a compulsory routine test for all aplastic anaemia patients. Objective: To estimate the frequency of PNH clone in aplastic anaemia. Method: Twenty-two known cases of aplastic anaemia patients were enrolled for the study. Flow cytometric quantitation of glycosyl phosphatidyl-inositol (GPI)-anchored proteins deficiency using markers CD14, CD24, CD45, CD59, Fluorescent Aerolysin (FLAER), CD235a (6 markers) were performed. Result: PNH clone was identified in 8 (36.4%) of the study population. Among PNH clone positive patients 7 (87.5%) were suffering from non-severe aplastic anaemia. Conclusion: PNH clone is significantly associated with aplastic anaemia and PNH clone assay should be regularly assayed in aplastic anaemia patients for specific management.

Abstract A027: Detecting and modeling frequency-dependent evolution in serially passaged primary triple negative breast cancer patient derived xenografts

Abstract Tumor evolution is a contributing factor to most failed cancer treatments. The intratumor heterogeneity of cellular phenotypes found in the tumor microenvironment acts as an evolutionary substate for cancer cells during tumorigenesis, subsequent growth, and after the patient is in the clinic. Selective forces imposed by certain medications can cause the emergence of a tumor with a resistant phenotype. Chemotherapies that target rapidly dividing cells will select for slower dividing ones which are less sensitive to such agents. However, many selective pressures acting on the tumor microenvironment are yet to be elucidated. For example, the effect one cancer cell has on another’s growth through competitive (i.e., the struggle for resources) or cooperative (e.g., exchange of growth factors) interactions remains poorly understood. Our central hypothesis is that cancer cells with distinct karyotypes will have a varying growth rate influenced by the karyotype landscape of the entire tumor. That is, we expect the fitness of a cancer cell to be largely determined by its karyotype and the karyotypes of its neighbors. Thus, we predict that frequency-dependent selection has a significant impact on clonal evolution in cancer. To test this, we have developed two novel methods for respectively identifying likely instances of frequency-dependent selection and modeling the long-term evolutionary trajectory in such cases. To detect frequency-dependent effects, we investigate the relationship between the frequency of one clone and the growth rate of all others (including itself). If a statistically significant correlation can be found, we proceed to modeling clonal evolution. To do this, we use a penalized least squares method and find the best-fitting parameterization of the replicator equation to the clonally resolved frequency data. This provides us with a payoff matrix describing the intensity of competition between each pair of clones. Furthermore, it allows us to model the long-term evolutionary dynamics of the tumor and to make predictions regarding co-existence of clones or clonal sweeps. Recently, investigators serially passaged primary triple negative breast cancer (TNBC) patient derived xenograft (PDX) tumors in NRG mice for over a year, inferring clonal frequencies at multiple timepoints using single cell whole genome (scWGS) and RNA sequencing (scRNA-seq). From this data, we were able to detect statistically significant frequency-dependent effects in the majority of serially passaged PDX tumors. Of these, most are predicted to result in a clonal sweep. Three of the PDX tumors were treated with cisplatin which revealed clone-specific drug response. This presents an opportunity for adaptive therapy scheduling which takes advantage of fitness tradeoffs under different contexts. A greater understanding of the effects frequency-dependent selection has on tumor evolution will contribute to the design of therapies intended to preclude or delay the emergence of resistance to treatment. Citation Format: Thomas A. Veith, Richard Beck, Noemi Andor. Detecting and modeling frequency-dependent evolution in serially passaged primary triple negative breast cancer patient derived xenografts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A027.

Distinct Immune and Molecular Profiles of Hepatitis-Associated Aplastic Anemia

Background: Hepatitis-associated aplastic anemia (HAAA) is characterized by the development of hematopoietic failure within months following an acute episode of non-infectious hepatitis. Although disease presentation with pancytopenia and a hypocellular marrow in HAAA is similar to non-hepatitis immune AA (non-HAAA), the immune and molecular profile in HAAA has not been completely elucidated. Here, we aimed to assess HAAA patients for cytokines and presence of clonal events linked to AA, the presence of paroxysmal nocturnal hemoglobinuria (PNH) clones and somatic mutations in peripheral blood mononuclear cells (PBMC) subpopulations, and compared to a non-HAAA cohort. Methods: A cohort of HAAA patients (n=30), age and sex-matched non-HAAA patients (n=24) and healthy controls (n=23) seen at the National Institutes of Health since 2003 were retrospectively screened for a panel of inflammatory cytokines and growth factors by Luminex, and somatic mutations in sorted CD3-, CD3+CD4+, and CD3+CD8+ PBMC fractions by error-corrected DNA sequencing. A customized panel with 42 myeloid-related and 49 autoimmunity-related genes at minimum limited of detection of 0.1% were used. All samples were before any immunosuppressive treatment (IST). Cytokine levels and frequencies of PNH clones and clonal hematopoiesis (CH) in different fractions were compared among groups. Results: HAAA patients were in average young (median age=23); 37% were at age &amp;lt; 18 (Table 1). Although disease severity and blood counts were similar to the non-HAAA cohort, immune and molecular markers of immune AA were distinct among cohorts. The frequency of PNH clones was significantly lower in HAAA compared to the non-HAAA cohort (15% v 58%, p=0.0008). The median size of PNH clones in HAAA and non-HAAA was 1.4% and 7.4%, respectively (p =0.03). The cytokine profiles performed in the sera of 22 HAAA before IST were marked by significantly higher levels of IL-1ra (p=0.0008), CXCL-10 (p=0.0038), and VEGF (p=0.0023) in comparison to 31 non-HAAA assessed patients; IL-6 (p=0.0002) and CD40L (p=0.0001) levels were significantly lower than in non-HAAA. As reported in immune AA, HAAA patients had higher levels of TPO (p=0.0001), G-CSF (p=0.0001), and EPO (p=0.0001) than controls; lower levels of CCL-5 (p=0.0001) and CCL-11 (p=0.0025) were seen in the HAAA. FLT3-L levels were markedly elevated in both AA cohorts when compared to HC. The clonal landscape was also strikingly different between HAAA and non-HAAA in all fractions evaluated (Figure 1). In HAAA, 3 out of 21 assessed patients (14%) had CH in the CD3- fraction at maximum variant allele frequency (VAF) of 1.8%; one patient had two RUNX1 mutations while a JAK2 and DNMT3A mutation were found in the other two patients. No mutations were found in CD4+ and CD8+ compartments in subjects with HAAA. In contrast, 13 of 24 non-HAAA patients (54%) had CH in the CD3- populations, which was dominated by PIGA, BCOR, and DNMT3A clones, a pattern consistent with the known clonal landscape of immune AA. The frequency of non-HAAA with CH in the CD4+ and CD8+ compartments was 7/24 (29%) and 8/24 (30%) patients, respectively; CH in these fractions was similar to those found in CD3- dominated by mutations in PIGA, BCOR, and DNMT3A at VAFs ranging from 0.2% to 15% (Figure 1). CH in lymphoid-related genes was only found in a single patient with a STAT3 p.S614Rmutation at VAF of 3% exclusive to the CD8+ fraction. Conclusion: HAAA has a distinct immunologic and molecular profile than non-HAAA patients. PNH clones were less frequent in HAAA and patterns of cytokines were significantly different than the non-HAAA cohort. CH in PIGA and BCOR, highly associated with AA, were not present in our small HAAA cohort. Our results indicate a distinct underlying pathophysiology that initiates the immune destruction resulting in marrow failure in HAAA patients. Confirming these findings in a larger cohort and correlating with clinical outcomes is ongoing.

Unraveling Clonal Evolution and Mechanisms of Treatment Resistance in Murine KMT2A-Rearranged Leukemia with Subclonal FLT3 N676K

Our understanding of how individual mutations, whether present in all or just a subset of the leukemia cells, affect cellular response to therapy is limited. Herein, we investigate the impact of different therapies on survival, evolution, and resistance patterns in an acute myeloid leukemia (AML) mouse model driven by KMT2A::MLLT3 and subclonal FLT3 N676K. Leukemic bone marrow expressing KMT2A::MLLT3-mCherry in all cells and FLT3 N676K-GFP in 40% of cells was re-transplanted into sublethally irradiated recipients. Upon engraftment, recipients were subjected to different treatments, including chemotherapy (cytarabine for 5 days+doxorubicin for 3 days), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Targeted treatment was administered for 28 days, while the control group received vehicle ( Fig 1a). Each treatment prolonged survival with a median latency of 30 days for chemotherapy 37.5 days for AC220, 42 days for chemotherapy+AC220, and 45 days for AC220+Trametenib, and 25.5 days for control mice. At sacrifice, all mice displayed splenomegaly and leukocytosis. The treatment impacted the evolution of the KMT2A:: MLLT3+ FLT3 N676K cells and they expanded to clonal dominance in control and chemotherapy-treated mice. However, three distinct patterns of evolution were detected in AC220-treated mice with the FLT3 N676K-containing cells either expanding to clonal dominance (&amp;gt;80% of KMT2A:: MLLT3+ FLT3 N676K), the FLT3 N676K-containing cells being eradicated (&amp;gt;80% of KMT2A::MLLT3-alone), or dual similar sized clones expressing KMT2A:: MLLT3-alone or KMT2A::MLLT3+ FLT3 N676K(Fig1 b).Eradication of the FLT3 N676K-cells was rare, predominantly observed upon AC220+Trametinib treatment and the frequency of dual clones increased when chemotherapy was followed by AC220, in line with treatment affecting the evolution of genetically distinct clones. RNA-sequencing was used to determine altered gene expression patterns upon resistance and unveiled three major clusters including 1) mice expressing KMT2A:: MLLT3-alone, 2) control and chemotherapy-treated mice, and 3) AC220-treated mice. Notably, a subset of AC220-treated mice clustered close to the control and chemotherapy-treated mice suggesting potential resistance mechanisms through activation of the MEK/ERK-pathway. All leukemias had a myeloid Cd11b + phenotype. However, the control and chemotherapy-treated leukemias also displayed an aberrant expression of the lymphoid marker B220 and showed enrichment of gene sets correlating with stemness and oxidative phosphorylation, that suggested a switch in cellular phenotype and metabolic state. In contrast, AC220-treated leukemias lacked or had a low fraction of B220 + cells and showed enrichment of gene sets correlating with granulocyte-monocyte progenitors (GMP) and immune regulatory pathways (Fig 1 c). Acquired AC220-resistance mutations was rare (n=2), suggesting activation of alternative pathways upon resistance. Re-treatment of established cell lines from treated animals with AC220 showed that the cells were resistant. To uncover mechanisms of resistance at the cellular level, single cell RNA-sequencing was performed on four mice and revealed the emergence of distinct cellular subpopulations in AC220-treated mice, with a strong GMP signature and upregulation of the homeobox gene Six1(Fig 1d).Overexpression of Six1 in all AC220-treated leukemias was further confirmed by bulk RNA-sequencing. Interestingly, Six1 has previously been described as a regulator for leukemia stem cell maintenance in AML and under Wnt/ß-catenin signalling control. Subsequent drug assays showed AC220-treated cells exhibited sensitivity to Six1 pharmacological inhibition, suggesting a dependency on Six1, as well as sensitivity to Wnt/ß-catenin inhibition. Taken together, the treatment given affected survival and impacted evolution of the FLT3 N676K-leukemia cells. The general absence of acquired mutations in most mice suggests that target-independent mechanisms may underline acquired resistance in most mice, and we propose the Six1/Wnt/ß-catenin axis as a potential vulnerability upon AC220-resistance.

Genetic Engineering of Hematopoietic Progenitor Stem Cells for Targeted IFN-α Immunotherapy Reprogramming the Solid Tumor Microenvironment: A First-in-Man Study in Glioblastoma Multiforme (NCT03866109)

Background and methods . TEM-GBM is a Phase 1/2a dose-escalating non-randomized open label study, involving a single intravenous injection of Temferon. Temferon is constituted by autologous CD34+ HSPCs genetically modified with a lentiviral vector encoding for human interferon-a2 and has been designed to deliver IFNa in the tumor microenvironment (TME) in a targeted manner via Tie-2 expressing monocytes (TEMs). The patient population is newly diagnosed GBM patients with unmethylated MGMT gene promoter. The study aims to evaluate the short-term (up to 90 Days) and long-term (up to 2 years) tolerability and safety of five escalating doses of Temferon in up to 27 glioblastoma patients. The secondary objective is to evaluate biological activity and efficacy when used following SOC first line therapeutic approaches. Autologous CD34+ HSPC are mobilized with lenograstim and plerixafor, collected by apheresis, purified and ex vivo modified with a lentiviral vector. So far, up to 3 million Temferon cells/kg have been co-administered with a fixed dose of non-manipulated CD34+ supporter cells following a sub-myeloablative conditioning regimen (Thiotepa + BCNU or Busulfan or Busulfan alone). Results. As of 30th June 2023, twenty-one patients have been enrolled across 7 cohorts evaluating 4 incremental doses (0.5-3.0x10 6/kg) of Temferon. To date, no DLTs have been identified. Median Overall Survival after 1st surgery is 17 months (5-40 months). In all patients, rapid engraftment of gene modified progenitors and fast recovery from sub-myeloablative conditioning regimens have been observed (median engraftment across all the cohorts: Neutrophils D+12, Platelets D+13,5 following Temferon infusion). The percentage of transduced cells found in the BM obtained for the highest dose reached 50% at 1 month and persisted at detectable level in the long-term. Very low median concentrations of IFNa were detected in the plasma, indicating tight regulation of vector expression. Notably, the concentration of IFNa in the CSF increased concomitantly with disease progression suggesting active recruitment of TEMs into the tumor and subsequent release of IFNa. 57% of the treated patients underwent a 2nd-line treatment (either pharmacological or surgical) with an interim survival rate at 2-years of 28% (5 of 18 patients; 3 patients excluded as follow-up is below 12 months), which is higher than what is reported in literature (15%). One out of the surviving patients was enrolled in a long-term follow-up study and survived up to 3 years after surgery without any 2nd-line therapy. Genetically engineered cells were detected within the tumor lesion at levels &amp;gt;3% in 3/6 patients at second surgery. Single cell RNA seq analysis unveiled reprogramming of the myeloid TME, with an increase in pro-inflammatory (M1-like) and a decrease in hypoxic (M2-like) macrophages following Temferon exposure. Of note, this population shift was particularly evident when comparing a stable with a progressive tumor lesion biopsied contemporaneously in one patient, reproducing preclinical findings from a GBM mouse model (PMID: 35857642). Analysis of the T cell compartment highlighted an overall increase of CD8+ T cells (mainly effector T cell subsets) and a decrease in CD4+ T cells in Temferon patients. By mapping published signatures of antitumor neoantigen-reactive T cells (NeoTCR; PMID: 35113651) onto the GBM T cell landscape and integrating this data with clonal frequency metrics from concurrent scTCRseq data, we observed an approximately 3-fold increase of predicted tumor-reactive CD8+ T cells bearing expanded clonotypes (&amp;gt;1% within each patient) in Temferon vs. control patients. Strong up-regulation of IFNa and inflammatory responses in most of the clusters from myeloid and T cells, but also CD45- tumor and stromal cell compartments from Temferon patients suggested diffuse IFNa payload delivery into the GBM TME. Conclusion: These data show that Temferon is safe and biologically active at the tumor site and favors anti-tumor immunity. The results provide initial evidence of Temferon's potential to modulate the TME of GBM patients and to counteract disease progression and improve the survival of uMGMT GBM patients.

Clonal Hematopoiesis Is Common in Long-Term Survivors of Pediatric Hematopoietic Cell Transplantation, Including Umbilical Cord Blood Transplantation

Background 1. Approximately 20.000 to 200.000 hematopoietic stem and progenitor cells (HSPCs) contribute to steady-state adult hematopoiesis. During ageing, the overall diversity of the HSPC pool decreases, which is in part driven by the selective expansion of HSPC clones with driver mutations, a process called clonal hematopoiesis (CH). CH is an age-related condition that predisposes to hematologic malignancy, cardiovascular disease, and all-cause mortality. Hematopoietic stem cell transplantation (HCT) recipients are at increased risk of CH, but the mechanisms driving the emergence and expansion of CH clones are largely unknown. Furthermore, the prevalence of CH in pediatric HCT recipients, in whom the long-term consequences of CH are particularly relevant, remains to be defined. Aim To determine the prevalence and risk factors of CH in long-term survivors of HCT at pediatric age. Methods We included survivors of HCT at pediatric age, with a minimum survival of 5 years after HCT. CH was assessed by targeted, error-corrected sequencing of mutations in 27 cancer driver genes in whole blood, at a variant allele frequency detection threshold of 1%. HSC age was calculated by the sum of the age of the donor and the interval after HCT. In case the age of a donor was unknown (n=53), the median age for donors of the same donor relation were used (i.e., 12.6 years for sibling donors and 28.7 for matched unrelated donors). Clinical characteristics were compared using the Wilcoxon signed ranked test for continuous variables and Chi-squared or Fisher's exact test for categorical variables. Results Here, we report the interim results of the first 120 HCT survivors in this study. The median age of the survivors was 20.4 years (range 6.7-51.8), and the median interval after HCT was 11.8 years (range 5.2-38.2). Indications for HCT were hemato-oncology (n=95), bone marrow failure (n=22), or immune deficiency (n=3). The median calculated age of the HSCs was 31 years (range 5.2-61.8). CH was present in 13.3% of survivors (n=16). Mutated genes were DNMT3A (n=15), TET2 (n=4) and MPL (n=1), with three individuals having more than one driver mutation. The median variant allele frequency of the CH clones was 2.2%, but allele frequencies up to 30% were found. No differences were observed in recipient age or interval after HCT between individuals with or without CH (Figure 1A). However, the age of the HSCs was significantly higher in individuals with CH compared to those without (38.6 versus 26.3 years, p = 0.01, Figure 1B). Surprisingly, we detected CH in two recipients of umbilical cord blood grafts, at an interval of 11 and 15 years after HCT. Conclusion Clonal hematopoiesis is common in long-term survivors of pediatric HCT. After HCT, the prevalence of CH is related to the age of the transplanted HSCs, rather than the age of the survivor. The presence of CH in cord blood HSCs within two decades after HCT suggests that HCT-related factors may drive the de novo emergence and/or outgrowth of donor-derived CH. Ongoing research is aimed at investigating how factors such as stem cell source and post-transplant complications affect clonal expansion after HCT. Furthermore, we are investigating the prevalence of CH in an age-matched control cohort using the same detection pipeline, enabling direct comparison of the prevalence of CH in HCT survivors compared to the general population.

Post-vaccination SARS-Cov-2 T-cell receptor repertoires in patients with multiple sclerosis and related disorders

BackgroundAttenuation in post-vaccination SARS-CoV-2 humoral responses has been demonstrated in people treated with either anti-CD20 therapies or sphingosine-1-phosphate (S1P) receptor modulators. In the setting of disease modifying therapy (DMT) use, humoral response may not correlate with effective immunity, and analysis of vaccine-mediated SARS-CoV-2-specific memory T-cell responses is crucial. While vaccination in patients treated with anti-CD20 agents leads to deficient antibody production, emerging data from live cell assays suggests intact T-cell responses to vaccination. We evaluated post-vaccination SARS-CoV-2 T-cell receptor (TCR) repertoires in DMT-treated patients using the ImmunoSeqR assay, an assay that does not require live cells. MethodsAdults 18–80 years old without prior COVID-19, with neuroimmune conditions, who had been vaccinated with two doses of Pfizer-BioNTech or Moderna mRNA vaccines at least 3 weeks and up to 6 months prior, were recruited. Whole blood was obtained for immunosequencing, and matched serum was obtained for humoral analysis. Immunosequencing of the CDR3 regions of human TCRβ chains was completed using the immunoSEQR Assay (Adaptive Biotechnologies). TCR sequences were mapped across a set of TCR sequences reactive to SARS-CoV-2. Clonal diversity (breadth) and frequency (depth) of TCRs specific to SARS-CoV-2 spike protein were calculated and relationships with clinical variables were assessed. ResultsForty patients were recruited into the study, aged 25–77, and 27 female. 37 had MS, 2 had neuromyelitis optica spectrum disorder (NMOSD), and 1 had hypophysitis. Subjects treated with anti-CD20 agents and S1P receptor modulators had severely attenuated humoral responses, but SARS-CoV-2-spike-specific TCR clonal depth and breadth were robust across all treatment classes except S1P modulators. No spike-specific or non-spike-specific SARS-CoV-2-associated TCRs were found in those treated with S1P modulators (p = 0.002 for both breadth and depth). Subjects treated with fumarates exhibited somewhat lower spike TCR breadth than subjects treated with other or no DMTs (median 2.27 × 10^-5 for fumarates and 4.96 × 10^-5 for all others, p = 0.008), but no statistically significant difference was demonstrated with spike TCR depth. No other significant associations with DMT type were found. We found no significant correlations between depth or breadth and age, duration of treatment, type of vaccination, or time interval since vaccination. ConclusionThis is the first study to characterize post-vaccination SARS-CoV-2 TCR repertoires in DMT-treated individuals. We demonstrated a dichotomous response to SARS-CoV-2 vaccination in anti-CD20-treated patients, with severely attenuated humoral response but intact TCR depth and breadth. It is unclear to what degree each arm of the adaptive immune system impacts post-vaccine immunity, both from the standpoint of incidence of post-vaccine infections and that of infection severity, and further clinical studies are necessary. S1P modulator-treated subjects exhibited both severely attenuated humoral responses and absent spike-specific TCR depth and breadth, information which is crucial for counseling of patients on these agents. Our methodology can be used in larger studies to determine the benefit of repeated vaccination doses, including those that are modified to better target modern or seasonal variants, without the use of live cell assays.

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes

BackgroundStreptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase.MethodsBacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry.ResultsHere, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation.ConclusionsOur findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

Breaking down the tumor immune infiltration within pediatric sarcomas.

Immunotherapies are a promising therapeutic option, yet for a variety of reasons, these treatments have achieved limited success against sarcomas. The immunosuppressive tumor microenvironment (TME) of sarcomas as well as lack of predictive biomarkers, decreased T-cell clonal frequency, and high expression of immunosuppressive infiltrating cells has thus far prevented major success using immunotherapies. By breaking down the TME into its individual components and understanding how the various cell types interact with each other as well as in the context of the complex immune microenvironment, can lead to effective therapeutic immunotherapy treatments, potentially improving outcomes for those with metastatic disease.