Clonal Cell Populations Research Articles

Immunoglobulin light chain amyloidosis

SummaryImmunoglobulin light chain (AL) amyloidosis is a rare and underdiagnosed life-threatening systemic disease, primarily caused by insoluble depositions of misfolded monoclonal light chains. The monoclonal light chain paraprotein originates from a small clonal B‑cell or a clonal plasma cell population. If left undetected the paraprotein can induce a number of complications based on organ damage. The most dangerous and life-threatening organ dysfunction emerges from cardiac involvement. Thus, patients overall survival depends on early detection. Establishing the correct diagnosis and clear characterization of the amyloid-forming protein, staging, risk assessment and treatment are crucial and depend on a highly experienced interdisciplinary, multiprofessional team.

Chromosomal coordination and differential structure of asynchronous replicating regions

Stochastic asynchronous replication timing (AS-RT) is a phenomenon in which the time of replication of each allele is different, and the identity of the early allele varies between cells. By taking advantage of stable clonal pre-B cell populations derived from C57BL6/Castaneous mice, we have mapped the genome-wide AS-RT loci, independently of genetic differences. These regions are characterized by differential chromatin accessibility, mono-allelic expression and include new gene families involved in specifying cell identity. By combining population level mapping with single cell FISH, our data reveal the existence of a novel regulatory program that coordinates a fixed relationship between AS-RT regions on any given chromosome, with some loci set to replicate in a parallel and others set in the anti-parallel orientation. Our results show that AS-RT is a highly regulated epigenetic mark established during early embryogenesis that may be used for facilitating the programming of mono-allelic choice throughout development.

Abstract IA007: The mutational landscape of normal human endometrial epithelium

Abstract Human endometrium is a highly dynamic tissue that undergoes numerous cycles of breakdown, rapid repair and remodelling in response to the oscillating levels of oestrogen and progesterone. The marked regenerative capacity of the tissue’s epithelial compartment is maintained by the intra-glandular adult stem cells (ASCs) that are retained during menstruation. Although the endometrial epithelial ASCs were first described over a decade ago, they remain poorly characterised in comparison to their counterparts in other tissues, such as the small and arge intestine. In particular, the size of the stem cell pool within individual glands, clonal dynamics and genomic landscape are largely unknown. In this study, we laser-capture microdissected 257 morphologically normal endometrial glands from pre- and post-menopausal women (aged 19 to 81). In selected donors, we also obtained samples of normal endocervical and Fallopian tubular epithelium. Isolated cellular material was subjected to our modified library preparation protocol and subsequent whole genome sequencing (WGS). Analysis of the WGS data identified that the majority of the glands were clonal cell populations sharing a recent common ancestor. Somatic mutations accumulated at a rate of 29 substitutions per year. Total mutation burden in the normal endometrial epithelium is higher than in other normal cells and manyfold lower than in endometrial cancers. Overall, despite the heterogeneity in age, reproductive history and BMI, we found relatively homogenous mutational processes to be operative in this tissue with only occasional outliers. Remarkably, we not only identify recurrent acquisitions of certain cancer-associated mutations, particularly those that are advantageous to cell growth, proliferation and migration, but also show that such events occur early in life, potentially even before adolescence. Using a generalised linear mixed effect model, we found that age has a positive association with the accumulation of driver mutations, while parity has a negative association. Over time, the mutant ASCs serve as a reservoir for the acquisition of further driver mutations to the extent that in some cases, the entire sampled endometrium becomes ‘neoplastic’ on the genomic level while still retaining the apparently normal phenotype. Our data identify a distinct mutational landscape in normal endometrium that is in keeping with the presence of early positive selection in this highly regenerative tissue. Citation Format: Luiza Moore. The mutational landscape of normal human endometrial epithelium [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA007.

Methylation status of galectin-12 gene promoter in acute myeloid leukemia

Intro duction Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder, characterized by a block in differentiation of hematopoiesis, which results in the growth of a clonal population of neoplastic cells or blasts. In a previous study, the expression profiling of cancer-related galectins in AML showed upregulated expression of galectin-12 leading to cell cycle arrest. This observation pointed to the possibility that galectin-12 in AML could be a tumor-suppressor gene and a potential prognostic marker. Objective The aim was to study the methylation status of galectin-12 gene promoter in AML and its correlation with the expression of the gene and its potential effect on overall survival of patients. Patients and methods This study was carried out on 28 newly diagnosed AML cases. Expression of gal-12 gene was detected by reverse transcriptase-PCR, and the methylation status was examined by bisulfite genomic sequencing. Results Our results showed that 23/28 (82.1%) cases were negative for mRNA expression. The same group (82.1%) showed hypermethylated CpG island in the promoter region of LGALS12. Conclusion There is an association between LGALS12 expression and promoter hypermethylation [refers to the addition of a methyl group (CH3) covalently to the base cytosine (C) in the dinucleotide 5[Combining Acute Accent]-CpG-3[Combining Acute Accent]]. The mean survival of patients with unmethylated LGALS12 gene is significantly higher than methylated gene.

Significance of Tumor Growth Modeling in the Behavior of Homogeneous Cancer Cell Populations: Are Tumor Growth Models Applicable to Both Heterogeneous and Homogeneous Populations?

The ability to predict and slow the spread of cancer in the human body is a task that medical professionals have been trying to accomplish for many years. Being able to give factual basis to the use of certain growth models for application in not just heterogeneous, but also homogeneous cancer cell populations is imperative to treatment research as using mathematical analysis to predict the dynamics of tumor growth allows professionals to simulate how tumors might behave in the human body. This study follows the process of single-cloning and the growth of a homogeneous cell population in a superficial environment over the course of six weeks with the end goal of showing which of five tumor growth models commonly used to predict heterogeneous cancer cell population growth (Exponential, Logistic, Gompertz, Linear, and Bertalanffy) would also best exemplify that of homogeneous cell populations. We hypothesized that the Gompertz, Linear, and Bertalanffy models would provide the best fit to the homogeneous cancer (clonal) cell population growth data while models such as the exponential and logistic model, which are most commonly associated with the growth of heterogeneous cancer cell populations in natural environments (i.e. malignant tumors), would veer off the growth data. It was shown that Gompertz and Linear functions provided the best fit for this population, while exponential and Logistic functions fell slightly behind. The data collection and analysis for this research was performed through the University of Michigan Research Labs and Solver by Frontline Systems.

Transcriptional behavior of the HIV-1 promoter in context of the BACH2 prominent proviral integration gene

Chronic infection with human immunodeficiency virus (HIV)-1 is characterized by accumulation of proviral sequences in the genome of target cells. Integration of viral DNA in patients on long-term antiretroviral therapy selectively persists at preferential loci, suggesting site-specific crosstalk of viral sequences and human genes. This crosstalk likely contributes to chronic HIV disease through modulation of host immune pathways and emergence of clonal infected cell populations. To systematically interrogate such effects, we undertook genome engineering to generate Jurkat cell models that replicate integration of HIV-1 long terminal repeat (LTR) sequences at the BTB and CNC Homolog 2 (BACH2) integration locus. This locus is a prominent HIV-1 integration gene in chronic infection, found in 30 % of long-term treated patients with mapped proviral integrations. Using five clonal models carrying an LTR-driven reporter at different BACH2 intergenic regions, we here show that LTR transcriptional activity is repressed in BACH2 regions associated with proviral-DNA integrations in vivo but not in a control region. Our data indicates that this repression is in part epigenetically regulated, particularly through DNA methylation. Importantly, we demonstrate that transcriptional activity of the LTR is independent of BACH2 gene transcription and vice versa in our models. This suggests no transcriptional interference of endogenous and HIV-1 promoters. Taken together, our study provides first insights into how activity of HIV-1 LTR sequences is regulated at the BACH2 locus as prominent example for a recurrently-detected integration gene in chronic infection. Given the importance of integration-site dependent virus/host crosstalk for chronic HIV disease, our findings for the BACH2 locus have potential implications for future therapeutic strategies.

Trimeric SARS-CoV-2 Spike Proteins Produced from CHO Cells in Bioreactors Are High-Quality Antigens

The spike protein of the pandemic human corona virus is essential for its entry into human cells. In fact, most neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) are directed against the Virus-surface exposed spike protein, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus spikes are large heavily glycosylated homo-trimeric complexes, with inherent instability. The poor manufacturability now threatens the availability of these proteins for vaccines and diagnostic tests. Here, we outline scalable, Good Manufacturing Practice (GMP) compliant, and chemically defined processes for the production of two cell-secreted stabilized forms of the trimeric spike proteins (Wuhan and D614G variant). The processes are chemically defined and based on clonal suspension-CHO cell populations and on protein purification via a two-step scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2-specific-immunoglobulin molecules was found to exceed that of spike fragments (Spike subunit-1, S1 and Receptor Binding Domain, RBD). The process described here will enable production of sufficient high-quality trimeric spike protein to meet the global demand for SARS-CoV-2 diagnostic tests and potentially vaccines.

Subcellular Singlet Oxygen and Cell Death: Location Matters.

We developed a tool for targeted generation of singlet oxygen using light activation of a genetically encoded fluorogen-activating protein complexed with a unique dye molecule that becomes a potent photosensitizer upon interaction with the protein. By targeting the protein receptor to activate this dye in distinct subcellular locations at consistent per-cell concentrations, we investigated the impact of localized production of singlet oxygen on induction of cell death. We analyzed light dose-dependent cytotoxic response and characterized the apoptotic vs. necrotic cell death as a function of subcellular location, including the nucleus, the cytosol, the endoplasmic reticulum, the mitochondria, and the membrane. We find that different subcellular origins of singlet oxygen have different potencies in cytotoxic response and the pathways of cell death, and we observed that CT26 and HEK293 cell lines are differentially sensitive to mitochondrially localized singlet oxygen stresses. This work provides new insight into the function of type II reactive oxygen generating photosensitizing processes in inducing targeted cell death and raises interesting mechanistic questions about tolerance and survival mechanisms in studies of oxidative stress in clonal cell populations.

Myelodysplasia and Bone Marrow Manifestations of Somatic UBA1 Mutated Autoinflammatory Disease

Somatic mutations in UBA1 in hematopoietic stem cells and myeloid cells have recently been described and are associated with adult-onset severe autoinflammatory diseases including relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, and giant cell arteritis. This newly defined syndrome is named VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic). We performed clinicopathologic, cytogenetic, flow cytometric and molecular bone marrow assessment of 15 patients diagnosed with VEXAS and peripheral cytopenias. All patients were male, with a median age of 64y (IQR 45-80) and all had somatic missense mutations affecting the p.Met41 residue in UBA1 with variant allele frequencies (VAF) over 20% from peripheral blood or bone marrow samples and with lineage restriction to mature myeloid cells. Germline mutations at this residue are not reported in any public databases. Peripheral blood findings included macrocytic anemia in all patients (100%), thrombocytopenia in 9/15, and neutropenia in 1/15. All bone marrow aspirates (15/15; 100%) showed prominent vacuolization of both myeloid and erythroid precursors with few vacuoles noted in mature cells. Of the 9 patients tested for serum copper levels all were normal or borderline low. Marrow was hypercellular with granulocytic and megakaryocytic hyperplasia in 12/15 (80%). Definitive WHO criteria for MDS was met in 5/15 (40%) with 3 cases of MDS-MLD, and 2 of MDS-SLD. The remaining cases were suspicious for MDS with low or borderline levels of dyspoiesis that did not exceed greater than 10% of cells in respective lineages meeting criteria for clonal cytopenia of undetermined significance (CCUS). Of the MDS cases, dysplasia was seen in megakaryocytes (5/5), myeloid (2/5) and erythroid (1/5) precursors. Cytogenetic abnormalities were detected in 2/5 MDS patients involving t(3;12)(q21;q13) in one case, and del (5q)/del (13q) in another case. Next generation sequencing analyses were performed in 9/15 patients and showed mutations in DNMT3A in 2/5 MDS cases (VAF 43% and 44%), CSF1R in 1/5 MDS (VAF 3.1%), GNA11 in 1/5 MDS (VAF3.3%) and EZH2 mutation (VAF 22%) in one of the patients without overt MDS. Clonal plasma cell or B-cell populations were diagnosed in 6/15 patients with 3 plasma cell dyscrasias, and 2 cases of monoclonal B-cell lymphocytosis. The most common abnormalities detected by bone marrow flow cytometry analysis were severely reduced or absent B-cell precursors, inverted CD4:CD8 ratios and abnormal expression of CD56 on monocytes. In summary, hematologic disorders were identified in 10/15 VEXAS patients, including both myeloid and lymphoid clonal disease comprising MDS, plasma cell dyscrasias, and monoclonal B cell lymphocytosis. Based on the presence of UBA1 somatic mutations in hematopoietic cells of all VEXAS patients, the patients without evidence of overt neoplasia in this study met criteria for CCUS. Importantly, all patients had characteristic vacuoles in myeloid and erythroid precursors in the marrow without evidence of copper deficiency. These findings suggest that vacuolization of hematopoietic precursors in marrow of cytopenic patients, particularly in male patients and in the setting of systemic inflammation and normal copper levels, should prompt evaluation for somatic UBA1 mutations. In addition to severe autoinflammatory disease manifestations, VEXAS patients appear to confer increased risk for development of both myeloid and lymphoid/plasma cell neoplasia and require surveillance for disease progression. Figure Disclosures Young: Novartis: Research Funding.

Hematological Complications of Donor Origin Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplant (HSCT) offers curative therapy for some malignant hematological disease and marrow failure states. Post-transplant donor cell pathology, which might become more prevalent in long term survivors of allogeneic HSCT, warrants further study. We report five recipients of allogeneic HSCT from our institution who developed donor cell abnormalities, discuss potential mechanisms underlying these disorders and propose potential avenues for future study. Case 1 involves a 43-year-old female who developed leukopenia and adenopathy and was subsequently found to have diffuse large B-cell lymphoma. She received a matched unrelated donor (MUD) SCT from a 23-year-old male and subsequently developed a reactive lymphocytosis. In case 2, a 44-year-old male with myelodysplastic syndrome (MDS) received an allogenic peripheral blood (PB) SCT from his 50-year-old sister and later developed donor-cell derived acute myeloid leukemia (AML) with chimerism studies demonstrating 100% donor cells in CD3, CD33/66 and CD56. Case 3 involved a 72-year-old female with AML who received a PBSCT from her 65-year-old brother and subsequently developed MDS with peripheral blood chimerism studies demonstrating 100% donor cells in all three compartments. Case 4 involved a 40-year-old female with AML who achieved molecular remission following chemotherapy and underwent a double umbilical cord transplant, subsequently developing donor-cell derived AML with chimerism studies negative for host cells. Finally, case 5 highlighted a 54-year-old male with AML who underwent a MUDSCT from a 44-year-old male and passed away from progressive leukemia. Peripheral blood demonstrated only donor cells with near identity of phenotypic and molecular studies. It remains possible that no malignant cells exited the marrow. Nonetheless, this case in particular highlights the difficulty in precisely diagnosing donor cell leukemia (DCL). DCL correlates with increasing donor age, suggesting that age-related mutation in hematopoietic stem cells might be the cause. Notably, the average donor age in our case studies was 53 years old. Hematopoiesis is maintained by both hematopoietic stem and progenitor cells and during the normal aging process becomes clonally restricted. Clonal hematopoiesis of indeterminate potential (CHIP) describes the presence of a clonal blood cell population associated with a driver mutation at a VAF ≥ 2%, in the absence of severe cytopenia or a WHO defined disorder. These mutations are found in 5% of individuals greater than 50 years of age, with the most common gene mutations being found inDNMT3A, ASXL1,andTET2. The rate of development of overt neoplasia in patients with CHIP is 0.5% to 1% per year and these patients are 4 to 15 times more likely to develop a hematologic malignancy. CHIP mutations might confer a survival advantage allowing these clones to expand under the influence of a selection pressure such as chemotherapy or immunosuppression. These surviving clones might be the origin of DCL. DCL is estimated to account for approximately 1-5% of all post-transplant leukemia. When transplanted, expansion of these clones as required for repopulation of the hematopoietic stem cell niche might accelerate the acquisition of secondary mutations providing surviving mutated clones a survival advantage leading to malignancy. An open question remains particularly as it concerns older donors >55 years, as to whether they should be screened by next generation sequencing (NGS) for the presence of mutations known to be associated with CHIP or frank hematologic malignancy. If the incidence of DCL increases, consideration should be given to subjecting older donors to these screening methods. When multiple donors of different age are available preference can be assigned to younger donors to avoid possibly transmitting CHIP. Disclosures Fazal: BMS:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Jansen:Speakers Bureau;Novartis:Consultancy, Speakers Bureau;Celgene:Speakers Bureau;Gilead/Kite:Consultancy, Speakers Bureau;Glaxosmith Kline:Consultancy, Speakers Bureau;Agios:Consultancy, Speakers Bureau;Takeda:Consultancy, Speakers Bureau;Incyte Corporation:Consultancy, Honoraria, Speakers Bureau;Karyopham:Speakers Bureau;Amgen:Consultancy, Speakers Bureau;Stemline:Consultancy, Speakers Bureau;Jazz Pharma:Consultancy, Speakers Bureau.

Clonal Hematopoiesis in Late-Stage Non-Small-Cell Lung Cancer and Its Impact on Targeted Panel Next-Generation Sequencing.

Somatic mutations derived from the expansion of clonal populations of blood cells (clonal hematopoiesis of indeterminate potential, or CHIP) may be detected in sequencing of cell-free DNA (cfDNA) samples. We evaluated the potential implications of CHIP in targeted sequencing of plasma samples using matched peripheral blood mononuclear cells (PBMCs) from patients with lung cancer to identify potential CHIP-associated mutations. A total of 332 plasma and corresponding PBMC samples were collected predose, cycle 1 day 1 (C1D1), from the randomized, phase III study (OAK) comparing atezolizumab versus docetaxel in previously treated patients with non-small-cell lung cancer (NSCLC). The samples were analyzed with the AVENIO ctDNA Surveillance Kit (for research use only; not for use in diagnostic procedures), a 198-kb next-generation sequencing panel targeting cancer-related genes. CHIP variants were assessed by analyzing both plasma and PBMC sequencing data. A range of zero to eight CHIP variants (median = one) was detected per cfDNA sample. Most of these variants were not in the Database of Single Nucleotide Polymorphisms (dbSNP). The number of CHIP variants was positively associated with age, and TP53 was the most frequently mutated gene. Furthermore, the allele frequency was less variable over time for CHIP variants than for tumor-derived variants. CHIP-derived mutations are present in late-stage NSCLC. However, not all plasma samples had CHIP mutations detected with targeted panel sequencing. Paired PBMC sequencing analysis may be needed to remove CHIP variants for comprehensive genomic profiling using plasma samples to identify true somatic mutations.

Profiling the Non-genetic Origins of Cancer Drug Resistance with a Single-Cell Functional Genomics Approach Using Predictive Cell Dynamics.

Non-genetic heterogeneity observed in clonal cell populations is an immediate cause of drug resistance that remains challenging to profile because of its transient nature. Here, we coupled three single-cell technologies to link the predicted drug response of a cell to its own genome-wide transcriptomic profile. As a proof of principle, we analyzed the response to tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) in HeLa cells to demonstrate that cell dynamics can discriminate the transient transcriptional states at the origin of cell decisions such as sensitivity and resistance. Our same-cell approach, named fate-seq, can reveal the molecular factors regulating the efficacy of a drug in clonal cells, providing therapeutic targets of non-genetic drug resistance otherwise confounded in gene expression noise. A record of this paper's transparent peer review process is included in the Supplemental Information.

A non-viral genome editing platform for site-specific insertion of large transgenes

BackgroundThe precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA—despite being successful in E. coli and animal models—still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies.MethodsPreviously, we have demonstrated the utility of our phage λ-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a ‘gene of interest’. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII.ResultsThe two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of ~ 10 kb and of a DUX4 reporter cassette of ~ 7 kb in hESCs.ConclusionWe present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications.

Extent and context dependence of pleiotropy revealed by high-throughput single-cell phenotyping.

Pleiotropy-when a single mutation affects multiple traits-is a controversial topic with far-reaching implications. Pleiotropy plays a central role in debates about how complex traits evolve and whether biological systems are modular or are organized such that every gene has the potential to affect many traits. Pleiotropy is also critical to initiatives in evolutionary medicine that seek to trap infectious microbes or tumors by selecting for mutations that encourage growth in some conditions at the expense of others. Research in these fields, and others, would benefit from understanding the extent to which pleiotropy reflects inherent relationships among phenotypes that correlate no matter the perturbation (vertical pleiotropy). Alternatively, pleiotropy may result from genetic changes that impose correlations between otherwise independent traits (horizontal pleiotropy). We distinguish these possibilities by using clonal populations of yeast cells to quantify the inherent relationships between single-cell morphological features. Then, we demonstrate how often these relationships underlie vertical pleiotropy and how often these relationships are modified by genetic variants (quantitative trait loci [QTL]) acting via horizontal pleiotropy. Our comprehensive screen measures thousands of pairwise trait correlations across hundreds of thousands of yeast cells and reveals ample evidence of both vertical and horizontal pleiotropy. Additionally, we observe that the correlations between traits can change with the environment, genetic background, and cell-cycle position. These changing dependencies suggest a nuanced view of pleiotropy: biological systems demonstrate limited pleiotropy in any given context, but across contexts (e.g., across diverse environments and genetic backgrounds) each genetic change has the potential to influence a larger number of traits. Our method suggests that exploiting pleiotropy for applications in evolutionary medicine would benefit from focusing on traits with correlations that are less dependent on context.

An immortalized human adipose-derived stem cell line with highly enhanced chondrogenic properties

Human adipose-derived stem cells (ASCs) are a commonly used cell type for cartilage tissue engineering. However, donor-to-donor variability, cell heterogeneity, inconsistent chondrogenic potential, and limited expansion potential can hinder the use of these cells for modeling chondrogenesis, in vitro screening of drugs and treatments for joint diseases, or translational applications for tissue engineered cartilage repair. The goal of this study was to create an immortalized ASC line that showed enhanced and consistent chondrogenic potential for applications in cartilage tissue engineering as well as to provide a platform for investigation of biological and mechanobiological pathways involved in cartilage homeostasis and disease. Starting with the ASC52telo cell line, a hTERT-immortalized ASC line, we used lentivirus to overexpress SOX9, a master regulator of chondrogenesis, and screened several clonal populations of SOX9 overexpressing cells to form a new stable cell line with high chondrogenic potential. One clonal line, named ASC52telo-SOX9, displayed increased GAG and type II collagen synthesis and was found to be responsive to both mechanical and inflammatory stimuli in a manner similar to native chondrocytes. The development of a clonal line such as ASC52telo-SOX9 has the potential to be a powerful tool for studying cartilage homeostasis and disease mechanisms in vitro, and potentially as a platform for in vitro drug screening for diseases that affect articular cartilage. Our findings provide an approach for the development of other immortalized cell lines with improved chondrogenic capabilities in ASCs or other adult stem cells.

Heritable gene expression variability and stochasticity govern clonal heterogeneity in circadian period.

A ubiquitous feature of the circadian clock across life forms is its organization as a network of cellular oscillators, with individual cellular oscillators within the network often exhibiting considerable heterogeneity in their intrinsic periods. The interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock's entrainability, but our knowledge of mechanisms governing period heterogeneity within circadian clock networks remains largely elusive. In this study, we aimed to explore the principles that underlie intercellular period variation in circadian clock networks (clonal period heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 to 28 h. We report that a single parent clone can produce progeny clones with a wide distribution of circadian periods, and this heterogeneity, in addition to being stochastically driven, has a heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our clone panel, we found that inheritance of expression patterns in at least three clock genes might govern clonal period heterogeneity in circadian clock networks. Furthermore, we provide evidence suggesting that heritable epigenetic variation in gene expression regulation might underlie period heterogeneity.

Diverse patterns of antibody variable gene repertoire disruption in patients with amyloid light chain (AL) amyloidosis.

Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. AL amyloidosis is caused by a misfolded light chain produced by a clonal population of plasma cells. Disease status currently is defined by measuring the absolute quantity of serum free light chain protein, but this measurement often fails to identify the subclinical presence of clonal cells that may merit additional therapy. Next generation sequencing has the sensitivity to measure the relative amount of dominating light chains within the repertoire of a patient, and this technique is in clinical use to identify clonal populations of plasma cells for multiple myeloma, a related disorder. In this proof-of-concept study, we used bone marrow aspirates of AL amyloidosis positive patients and used reverse transcription of the antibody transcriptome followed by next generation sequencing to identify antibody variable-diversity-joining gene sequences for patients with immunoglobulin light chain amyloidosis, and demonstrate that this technology can be used to identify the dominant clone. The data also reveal differing patterns of overall antibody repertoire disruption in different patients. This method merits further study in larger prospective studies to establish its utility in detecting residual disease for patients with immunoglobulin light chain amyloidosis.

Single-cell analysis of clonal maintenance of transcriptional and epigenetic states in cancer cells.

Propagation of clonal regulatory programs contributes to cancer development. It is poorly understood how epigenetic mechanisms interact with genetic drivers to shape this process. Here we combine single-cell analysis of transcription and DNA methylation with a Luria-Delbrück experimental design to demonstrate the existence of clonally stable epigenetic memory in multiple types of cancer cells. Longitudinal transcriptional and genetic analysis of clonal colon cancer cell populations reveals a slowly drifting spectrum of epithelial-to-mesenchymal transcriptional identities that is seemingly independent of genetic variation. DNA methylation landscapes correlate with these identities but also reflect an independent clock-like methylation loss process. Methylation variation can be explained as an effect of global trans-acting factors in most cases. However, for a specific class of promoters, in particular cancer testis antigens (CTA), de-repression is correlated with and likely driven by loss of methylation in cis. This study indicates how genetic sub-clonal structure in cancer cells can be diversified by epigenetic memory.

Molecular profiling and quantitative image analysis reveal spatial intratumor heterogeneity in TNBC.

e12536 Background: Triple negative breast cancer (TNBC) is a molecularly complex and heterogeneous subtype with distinct biological features and clinical behavior. Extensive intra-tumor heterogeneity is suspected to be a major cause of therapeutic failure. Spatially-distinct parts of the tumor harbor diverse and divergent clonal populations of cancer cells. Thus it is likely increasing the likelihood that some of those clones resist treatment, expand in numbers and eventually, repopulate the tumor leading to recurrence and spread. Therefore, a deeper understanding of this complexity is fundamental to gaining insights into this clinically important issue. In this study, we used RNA sequencing and machine learning approaches to examine the molecular and phenotypic/cellular profiles of various tumor samples from the same patient tumor to reveal spatial intra-tumor heterogeneity in TNBCs. Methods: We used 34 samples (2-4 samples from each patient tumor) from a total of 11 unique TNBC patients. RNA-sequencing was performed to quantify differential gene expression and machine learning (ML) approaches (deep learning regression modeling) were used to quantify the percentage of tumor and tumor-infiltrating lymphocytes (TILs) in the H&E stained tissue sections. The extent of concordance/discordance between the multiple tumor samples that originated from the same patient was analyzed by analyzing the intra- and inter-patient variance of normalized tumor cell, TIL % and gene expression. We also performed pathway analysis to identify signaling pathways dysregulated within (intra-tumoral heterogeneity) and between tumors (inter-tumoral heterogeneity) and performed molecular subtype analysis. Results: We observed that gene expression variance as higher within-patient (intra-tumor) compared to between-patient (inter-tumor). Tumor samples from 70% of patients showed different molecular subtypes representing extensive intra-tumor heterogeneity. Our ML-based image analysis showed that intra-patient tumor cell and TILs density/percentage variance was greater than inter-patient variance. In addition, patients with high within-patient gene expression variability had a high tumor and TIL variance. Among the within-patient expression variability, the genes associated with the PLK1 and Notch signaling were enriched. Conclusions: Our results suggest that TNBCs exhibit higher intra-tumor gene expression and cellular variance compared to inter-tumor gene expression and cellular variance suggesting higher intra-tumor heterogeneity in TNBCs.

MON-441 Primary Thyroid Maltoma: A Rare Non-hodgkin Malignancy

Introduction: Thyroid lymphoma is a rare malignancy; only 2% of extranodal lymphomas arise within the thyroid gland. Furthermore, lymphomas represent no more than 2% of all malignant thyroid tumors and almost always non-Hodgkin type. Case: A 58-year-old female with a medical history of rheumatoid arthritis treated with Golimumab for years. Exam revealed a right thyroid lobe enlargement with bilateral palpable nodules. Labs include TSH of 1.4 mU/L, Free T4 of 1.76 ng/dL, normal anti-thyroid peroxidase antibodies, but elevated anti-thyroglobulin. US of the thyroid showed right-sided, hyperechoic nodule measuring 1.3cm, and left-sided solid hypoechoic nodule measuring 2.2cm. US-guided FNA of the right nodule yielded atypical cells of undetermined significance (2017 Bethesda category III). FNA of the left nodule showed severe, chronic thyroiditis, with small clonal population of cells and suspicion for lymphoma. She underwent total thyroidectomy with left central lymph node dissection. Microscopic evaluation showed a predominant lymphocytic population with plasmacytoid/plasma cells. The histological finding was consistent with extranodal marginal zone lymphoma (EMZL) of mucosa-associated lymphoid tissue (MALT lymphoma or MALToma). However, thyroid tissues were negative for clonal immunoglobulin gene rearrangements (both immunoglobulin heavy and light chains). Immunohistochemistry demonstrated that the tumor cells were CD19, CD 20, CD 22, PAX5, CD78A positive, highlighting predominantly small to medium-sized B- cell lymphocytes, along with plasmacytoid/plasma cells (MUM1 and CD 75A positive). Furthermore, occasional reactive germinal centers (CD10, BCL6 positive, BCL2 negative) were noted. In conclusion, these features were consistent with a low grade, non-Hodgkin’s lymphoma of MALT type. Discussion: EMZL is a clinically indolent non-Hodgkin lymphoma, and growing evidence suggests that numerous cases originate in the background of chronic immune stimulation. MALToma is associated with Hashimoto’s thyroiditis or other immune diseases. Whereas half of EMZL can still arise without a Hashimoto thyroiditis background. Characteristics of the MALToma include reactive follicles, with the neoplastic cells invading the marginal zone (small lymphocytes, marginal zone B-cells, and plasma cells). The key findings are the presence of clonality (light chain restriction), and confirmation of B-cell origin (presence of B-cell markers). The immunophenotype is confirmed by immunohistochemistry and flow cytometry. Localized EMZL of the thyroid can be effectively treated with radiation alone. Surgery is usually used for diagnostic biopsy only. Chemotherapy is usually reserved for those with advanced stages