Clonal Cell Populations Research Articles

Light-chain amyloidosis with dysphagia as the main symptom: a case report

BackgroundImmunoglobulin light-chain amyloidosis is a relatively rare condition with a worldwide incidence of 5.1–12.8 cases per million person-years (Baker, 2022). It is characterized by a clonal population of immunoglobulin-secreting cells that produce a monoclonal light chain of κ or λ type as either an intact molecule or a fragment.Case presentationA 69-year-old East Asian (Chinese) male patient who presented with progressive dysphagia visited multiple hospitals repeatedly for more than 2 years and was finally diagnosed with immunoglobulin light-chain amyloidosis.ConclusionsOtolaryngologists should consider immunoglobulin light-chain amyloidosis when encountering suspicious clinical manifestations and intervene early to avoid misdiagnosis.

Genetic variation drives cancer cell adaptation to ECM stiffness

The progression of many solid tumors is accompanied by temporal and spatial changes in the stiffness of the extracellular matrix (ECM). Cancer cells adapt to soft and stiff ECM through mechanisms that are not fully understood. It is well known that there is significant genetic heterogeneity from cell to cell in tumors, but how ECM stiffness as a parameter might interact with that genetic variation is not known. Here, we employed experimental evolution to study the response of genetically variable and clonal populations of tumor cells to variable ECM stiffness. Proliferation rates of genetically variable populations cultured on soft ECM increased over a period of several weeks, whereas clonal populations did not evolve. Tracking of DNA barcoded cell lineages revealed that soft ECM consistently selected for the same few variants. These data provide evidence that ECM stiffness exerts natural selection on genetically variable tumor populations. Soft-selected cells were highly migratory, with enriched oncogenic signatures and unusual behaviors such as spreading and traction force generation on ECMs with stiffness as low as 1 kPa. Rho-regulated cell spreading was found to be the directly selected trait, with yes-associated protein 1 translocation to the nucleus mediating fitness on soft ECM. Overall, these data show that genetic variation can drive cancer cell adaptation to ECM stiffness.

Histone H3-K27 and H3-K9 methylation does not predict lifespan in divergent populations of Drosophila melanogaster

A universal theory of aging remains elusive despite decades of research. Traditional gerontological research has focused on genetic alterations as the driving unit of the aging processes. Recent breakthroughs in cellular reprogramming, transgenerational epigenetic inheritance, and molecular aging clocks have motivated theories proposing that the disruption of epigenetic regulation is the proximal cause of aging. The claim that aging is fundamentally an epigenetic process, however, is largely based on studies using clonal cell populations and genetically identical organisms. Consequently, there has been limited experimental evidence demonstrating the correlation between epigenetic differences and longevity differences among genetically diverse populations. To address this gap in literature we leveraged laboratory populations of the model organism Drosophila melanogaster. Using sister populations with evolved differences in longevity after more than 1 500 generations of divergent life history selection, we measured the methylation at the H3K27 and H3K9 sites which have previously been shown to become dysregulated with age. Here we report that there were no differences in the amount of H3K27me3 or H3K9me2 between 3 replicate population pairs of the divergent lines. These results do not support that epigenetic dysregulation is the underlying cause of aging. Future studies investigating additional indicators of epigenetic dysregulation are required to clarify the role of epigenetics in aging.

The energy nanosensor AMPfret, expressed stably in HEK cells, provides insights into toxic stress responses and heterogeneity within clonal cell populations

The energy nanosensor AMPfret, expressed stably in HEK cells, provides insights into toxic stress responses and heterogeneity within clonal cell populations

The landscape of somatic mutations in human atherosclerotic plaques indicates the presence of large clonal cell populations

The landscape of somatic mutations in human atherosclerotic plaques indicates the presence of large clonal cell populations

Characterization of latently infected EBV+ antibody-secreting B cells isolated from ovarian tumors and malignant ascites.

Intra-tumoral B cells mediate a plethora of immune effector mechanisms with key roles in anti-tumor immunity and serve as positive prognostic indicators in a variety of solid tumor types, including epithelial ovarian cancer (EOC). Several aspects of intra-tumoral B cells remain unclear, such as their state of activation, antigenic repertoires, and capacity to mature into plasma cells. B lymphocytes were isolated from primary EOC tissue and malignant ascites and were maintained in cell culture medium. The stably maintained cell lines were profiled with flow cytometry and B cell receptor sequencing. Secreted antibodies were tested with a human proteome array comprising more than 21,000 proteins, followed by ELISA for validation. Originating tumor samples were used for spatial profiling with chip cytometry. Antibody-secreting B lymphocytes were isolated from the ovarian tumor microenvironment (TME) of four different EOC patients. The highly clonal cell populations underwent spontaneous immortalization in vitro, were stably maintained in an antibody-secreting state, and showed presence of Epstein-Barr viral (EBV) proteins. All originating tumors had high frequency of tumor-infiltrating B cells, present as lymphoid aggregates, or tertiary lymphoid structures. The antigens recognized by three of the four cell lines are coil-coil domain containing protein 155 (CCDC155), growth factor receptor-bound protein 2 (GRB2), and pyruvate dehydrogenase phosphatase2 (PDP2), respectively. Anti-CCDC155 circulating IgG antibodies were detected in 9 of 20 (45%) of EOC patients' sera. Tissue analyses with multiparameter chip cytometry shows that the antibodies secreted by these novel human B cell lines engage their cognate antigens on tumor cells. These studies demonstrate that within the tumor-infiltrating lymphocyte population in EOC resides a low frequency population of antibody-secreting B cells that have been naturally exposed to EBV. Once stably maintained, these novel cell lines offer unique opportunities for future studies on intratumor B cell biology and new target antigen recognition, and for studies on EBV latency and/or viral reactivation in the TME of non-EBV related solid tumors such as the EOC.

Mitoclone2: an R package for elucidating clonal structure in single-cell RNA-sequencing data using mitochondrial variants.

Clonal cell population dynamics play a critical role in both disease and development. Due to high mitochondrial mutation rates under both healthy and diseased conditions, mitochondrial genomic variability is a particularly useful resource in facilitating the identification of clonal population structure. Here we present mitoClone2, an all-inclusive R package allowing for the identification of clonal populations through integration of mitochondrial heteroplasmic variants discovered from single-cell sequencing experiments. Our package streamlines the investigation of this phenomenon by providing: built-in compatibility with commonly used tools for the delineation of clonal structure, the ability to directly use multiplexed BAM files as input, annotations for both human and mouse mitochondrial genomes, and helper functions for calling, filtering, clustering, and visualizing variants.

An in Vitro triple screen model for human mineralocorticoid receptor activity

The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron and other tissues. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. We developed an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussia luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45 nM and 0.30 nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6 nM and 56.0 nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20 nM (CV1-MRLuc) to ∼2000 nM, while the EC50 for aldosterone remained unchanged. The addition of 18β-glycyrrhetinic acid (18β-GA), a HSD11B2 inhibitor, restored the potency of cortisol back to ∼70 nM in CV1-hMRLuc-HSD11B2 cells. Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts.

Loss of heterozygosity impacts MHC expression on the immune microenvironment in CDK12-mutated prostate cancer

BackgroundIn prostate cancer (PCa), well-established biomarkers such as MSI status, TMB high, and PDL1 expression serve as reliable indicators for favorable responses to immunotherapy. Recent studies have suggested a potential association between CDK12 mutations and immunotherapy response; however, the precise mechanisms through which CDK12 mutation may influence immune response remain unclear. A plausible explanation for immune evasion in this subset of CDK12-mutated PCa may be reduced MHC expression.ResultsUsing genomic data of CDK12-mutated PCa from 48 primary and 10 metastatic public domain samples and a retrospective cohort of 53 low-intermediate risk primary PCa, we investigated how variation in the expression of the MHC genes affected associated downstream pathways. We classified the patients based on gene expression quartiles of MHC-related genes and categorized the tumors into “High” and “Low” expression levels. CDK12-mutated tumors with higher MHC-expressed pathways were associated with the immune system and elevated PD-L1, IDO1, and TIM3 expression. Consistent with an inflamed tumor microenvironment (TME) phenotype, digital cytometric analyses identified increased CD8 + T cells, B cells, γδ T cells, and M1 Macrophages in this group. In contrast, CDK12-mutated tumors with lower MHC expression exhibited features consistent with an immune cold TME phenotype and immunoediting. Significantly, low MHC expression was also associated with chromosome 6 loss of heterozygosity (LOH) affecting the entire HLA gene cluster. These LOH events were observed in both major clonal and minor subclonal populations of tumor cells. In our retrospective study of 53 primary PCa cases from this Institute, we found a 4% (2/53) prevalence of CDK12 mutations, with the confirmation of this defect in one tumor through Sanger sequencing. In keeping with our analysis of public domain data this tumor exhibited low MHC expression at the RNA level. More extensive studies will be required to determine whether reduced HLA expression is generally associated with primary tumors or is a specific feature of CDK12 mutated PCa.ConclusionsThese data show that analysis of CDK12 alteration, in the context of MHC expression levels, and LOH status may offer improved predictive value for outcomes in this potentially actionable genomic subgroup of PCa. In addition, these findings highlight the need to explore novel therapeutic strategies to enhance MHC expression in CDK12-defective PCa to improve immunotherapy responses.

Abstract 2012: Extrachromosomal DNA promotes drug resistance in pancreatic ductal adenocarcinoma cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in Western Countries and has been predicted to become the second leading cause of cancer-related deaths by 2030. Difficulties in early detection as well as strong chemoresistance of the disease prevent the successful treatment of PDAC patients. Although we have previously identified CYP3A5 as a mediator of paclitaxel resistance, further targets remain elusive. Thus, we set out to identify these CYP3A5-independent mechanisms which could lead to the identification of novel biomarkers and drug targets to improve PDAC treatment. For this project, pancreatic cancer cell lines were derived from treatment-naive PDAC samples and exposed to increasing paclitaxel concentrations to induce resistance. The cells were characterized on a genomic and transcriptomic level using short-read sequencing as well as optical mapping to characterize potential candidate genes in vitro. In our results, multi-drug resistance protein 1 (ABCB1) was highly upregulated in our differential gene expression analysis and confirmed as a CYP3A5-independent mediator of acquired paclitaxel resistance. Its overexpression was mediated by extrachromosomal DNA (ecDNA) amplifications upon drug treatment. These circular DNAs were characterized by short-read sequencing as well as long-read optical mapping, which revealed the structures of the most abundant molecules. Furthermore, we investigated the biogenesis of the ecDNA. Current models propose different events such as chromothripsis or breakage-fusion-bridge cycles to initiate ecDNA formation, however, knowledge about the underlying mechanisms remains limited. To study the ecDNA dynamics, we generated ecDNA-negative cell clones from a paclitaxel resistant PDAC cell line. Interestingly, our clonal cell population appears to show the capacity for ecDNA de novo generation and quickly adapts to changing selective pressure such as therapy. This finding might unveil new insights into the triggers of ecDNA formation and its dynamic regulation. In conclusion, our results suggest the clinical relevance of ABCB1 as a mediator of paclitaxel resistance in PDAC and we currently validate our findings in patient samples. However, ABCB1 inhibition is associated with severe side effects. Instead, the regulatory pathways associated with enhanced ABCB1 expression and the formation of ecDNAs might provide therapeutic potential. Additionally, the characterization of ecDNA de novo generation might unveil new insights into how cancer cells adapt to changing selective pressures. Citation Format: Tim Vorberg, Manuel Reitberger, Bernardo Rodriguez-Martin, Maja Starostecka, Ornella Kossi, Vanessa Vogel, Jan Korbel, Andreas Trumpp, Martin Sprick. Extrachromosomal DNA promotes drug resistance in pancreatic ductal adenocarcinoma cells [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 2012.

Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing.

CRISPR/Cas9 genome editing is a widely used tool for creating genetic knock-ins, which allow for endogenous tagging of genes. This is in contrast with random insertion using viral vectors, where expression of the inserted transgene changes the total copy number of a gene in a cell and does not reflect the endogenous chromatin environment or any trans-acting regulation experienced at a locus. There are very few protocols for endogenous fluorescent tagging in macrophages. Here, we describe a protocol to design and test CRISPR guide RNAs and donor plasmids, to transfect them into RAW 264.7 mouse macrophage-like cells using the Neon transfection system and to grow up clonal populations of cells containing the endogenous knock-in at various loci. We have used this protocol to create endogenous fluorescent knock-ins in at least six loci, including both endogenously tagging genes and inserting transgenes in the Rosa26 and Tigre safe harbor loci. This protocol uses circular plasmid DNA as the donor template and delivers the sgRNA and Cas9 as an all-in-one expression plasmid. We designed this protocol for fluorescent protein knock-ins; it is best used when positive clones can be identified by fluorescence. However, it may be possible to adapt the protocol for non-fluorescent knock-ins. This protocol allows for the fairly straightforward creation of clonal populations of macrophages with tags at the endogenous loci of genes. We also describe how to set up imaging experiments in 24-well plates to track fluorescence in the edited cells over time. Key features • CRISPR knock-in of fluorescent proteins in RAW 264.7 mouse macrophages at diverse genomic loci. • This protocol is optimized for the use of the Neon transfection system. • Includes instructions for growing up edited clonal populations from single cells with one single-cell sorting step and efficient growth in conditioned media after cell sorting. • Designed for knocking in fluorescent proteins and screening transfected cells by FACS, but modification for non-fluorescent knock-ins may be possible.

Abstract PR009: Tick tock trees: Reconstructing the evolutionary dynamics of human tissues using fluctuating methylation clocks

Abstract The development of cancer from healthy somatic cells is fundamentally an evolutionary process. Understanding this process is vital to improving its clinical management and developing new treatment strategies. Tracing individual human cells’ fates would simultaneously advance somatic evolutionary theory and our understanding of normal tissue function and its change through development, aging, and cancer progression. We cannot use human cell-labeling techniques in vivo. Still, population genetic and phylogenetic models can leverage the information recorded in heritable alterations to reconstruct the unobserved somatic cells’ history. Most such methods are limited by the few parameters they can estimate and restricted to neoplastic samples due to the lack of adequate evolutionary signal in healthy somatic cells. Gabbutt et al. recently ameliorated this limitation by introducing fluctuating methylation clocks (FMCs)—tissue-specific CpG sites that randomly change their methylation state—and a model that uses them to reconstruct the evolution of a clonal stem-cell population. In this application, all FMCs in a bulk sample are interrogated using a single standard methylation array to yield estimates of rates of methylation, demethylation and cell replacement, and the size of the stem-cell niche. This technique is especially suited to studying stem-cell niche dynamics in healthy or pre-malignant tissues organized in clonal cell populations where the whole population can be sampled together, like crypts (e.g., intestine, Barrett’s esophagus) and glands (e.g., endometrium). We have developed a multi-sample extension of this model that simultaneously reconstructs the evolutionary dynamics of stem cells within a niche and the niches within the tissue. This phylodynamic model produces additional estimates like ancestral relationships between sampled clonal populations, their calendar divergence times, the number of effective evolutionary niches, and how some of these parameters change over time. Here, we will present the method, demonstrate its validity, characterize its accuracy under a broad simulation parameter space, and showcase its application in analyzing human data from the following tissues: colon, small intestine, endometrium, and Barrett’s esophagus. We will also discuss the current model's limitations and future improvements that will discern between mechanisms of stem-cell-niche reproduction and incorporate geographical information. The stochastic nature of neoplastic progression makes it unlikely for any particular somatic alteration to be highly predictive of its outcome. This can explain why developing reliable traditional biomarkers has been so difficult. Alternatively, evolutionary biomarkers measure the characteristics of the process itself and thus should apply to all neoplasms. Parametric phylogenetic reconstruction models like the one introduced here will enable us to develop such universal biomarkers. Our ultimate goal is for evolutionary biomarkers to join evolutionary therapies in the revolution of the clinical management of cancer. Citation Format: Diego Mallo, Pablo Bousquets-Muñoz, Heather E. Grant, Darryl Shibata, Trevor A. Graham, Carlo C. Maley, Calum Gabbutt. Tick tock trees: Reconstructing the evolutionary dynamics of human tissues using fluctuating methylation clocks [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 PR009.

Abstract A019: The role of tumor transcriptional variability in evading acute CD8+ T cell exposure in melanoma

Abstract Throughout melanoma progression and immunotherapy treatment, melanoma cells often develop an ability to evade the immune system. Thus, there is a pressing need to identify and target the melanoma cell-intrinsic mechanisms underlying immune evasion. Our laboratory has pioneered the identification of transcriptional pathways that underlie melanoma progression. We have found that within even clonal melanoma cell populations, subclones of cells demonstrate tremendous heterogeneity in their transcriptional state. We previously demonstrated that these varying transcriptional states correspond to varying sensitivities of melanoma cells to stressors such as targeted therapies, and that these states can be stable across multiple cell divisions. We hypothesize that varying transcriptional states also correspond to cells that have differing abilities to evade CD8+ T cells, which are major effector cells against melanoma. To study the interactions between melanoma cells and CD8+ T cells, we engineered an ovalbumin antigen and OT-I T cell receptor system using mouse melanoma cell lines. We combined this system with DNA barcoding, which enables us to track subclones of cells undergoing immune selection. When cocultured together in vitro, ovalbumin-expressing melanoma cells are effectively killed by OT-I cells. However, we find that subclones of melanoma cells consistently possess different sensitivity to OT-I cells, and these differences are not due to their genetic background nor their ability to express and present ovalbumin. We then leveraged our DNA barcoding system with single cell RNA sequencing (scRNA-seq) to link the lineage and transcriptional state of single melanoma cells that persist through OT-I coculture. By expanding on these preliminary results, we aim to identify novel melanoma cell targets to decrease immune evasion. Citation Format: Raymond W.S. Ng, Sydney M Shaffer. The role of tumor transcriptional variability in evading acute CD8+ T cell exposure in melanoma [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 A019.

HIV Expression in Infected T Cell Clones.

The principal barrier to an HIV-1 cure is the persistence of infected cells harboring replication-competent proviruses despite antiretroviral therapy (ART). HIV-1 transcriptional suppression, referred to as viral latency, is foremost among persistence determinants, as it allows infected cells to evade the cytopathic effects of virion production and killing by cytotoxic T lymphocytes (CTL) and other immune factors. HIV-1 persistence is also governed by cellular proliferation, an innate and essential capacity of CD4+ T cells that both sustains cell populations over time and enables a robust directed response to immunological threats. However, when HIV-1 infects CD4+ T cells, this capacity for proliferation can enable surreptitious HIV-1 propagation without the deleterious effects of viral gene expression in latently infected cells. Over time on ART, the HIV-1 reservoir is shaped by both persistence determinants, with selective forces most often favoring clonally expanded infected cell populations harboring transcriptionally quiescent proviruses. Moreover, if HIV latency is incomplete or sporadically reversed in clonal infected cell populations that are replenished faster than they are depleted, such populations could both persist indefinitely and contribute to low-level persistent viremia during ART and viremic rebound if treatment is withdrawn. In this review, select genetic, epigenetic, cellular, and immunological determinants of viral transcriptional suppression and clonal expansion of HIV-1 reservoir T cells, interdependencies among these determinants, and implications for HIV-1 persistence will be presented and discussed.

Maxizyme-mediated suppression of chikungunya virus replication and transmission in transgenic Aedes aegypti mosquitoes.

Chikungunya virus (CHIKV) is an emerging mosquito-borne pathogen of significant public health importance. There are currently no prophylactic vaccines or therapeutics available to control CHIKV. One approach to arbovirus control that has been proposed is the replacement of transmission-competent mosquitoes with those that are refractory to virus infection. Several transgene effectors are being examined as potentially useful for this population replacement approach. We previously demonstrated the successful use of hammerhead ribozymes (hRzs) as an antiviral effector transgene to control CHIKV infection of, and transmission by, Aedes mosquitoes. In this report we examine a maxizyme approach to enhance the catalytic activity and prevent virus mutants from escaping these ribozymes. We designed a maxizyme containing minimized (monomer) versions of two hRzs we previously demonstrated to be the most effective in CHIKV suppression. Three versions of CHIKV maxizyme were designed: Active (Mz), inactive (ΔMz), and a connected CHIKV maxizyme (cMz). The maxizymes with their expression units (Ae-tRNA val promoter and its termination signal) were incorporated into lentivirus vectors with selection and visualization markers. Following transformation, selection, and single-cell sorting of Vero cells, clonal cell populations were infected with CHIKV at 0.05 and 0.5 MOI, and virus suppression was assessed using TCID50-IFA, RT-qPCR, and caspase-3 assays. Five transgenic mosquito lines expressing cMz were generated and transgene insertion sites were confirmed by splinkerette PCR. Our results demonstrate that Vero cell clones expressing Mz exhibited complete inhibition of CHIKV replication compared to their respective inactive control version or the two parent hRzs. Upon oral challenge of transgenic mosquitoes with CHIKV, three out of the five lines were completely refractory to CHIKV infection, and all five lines tested negative for salivary transmission. Altogether, this study demonstrates that maxizymes can provide a higher catalytic activity and viral suppression than hRzs.

Random Allelic Expression in Inherited Retinal Disease Genes.

Inherited retinal diseases (IRDs) are a significant contributor to visual loss in children and young adults, falling second only to diabetic retinopathy. Understanding the pathogenic mechanisms of IRDs remains paramount. Some autosomal genes exhibit random allelic expression (RAE), similar to X-chromosome inactivation. This study identifies RAE genes in IRDs. Genes in the Retinal Information Network were cross-referenced with the recent literature to identify expression profiles, RAE, or biallelic expression (BAE). Loss-of-function intolerance (LOFI) was determined by cross-referencing the existing literature. Molecular and biological pathways that are significantly enriched were evaluated using gene ontology. A total of 184 IRD-causing genes were evaluated. Of these, 31 (16.8%) genes exhibited RAE. LOFI was exhibited in 6/31 (19.4%) of the RAE genes and 18/153 (11.8%) of the BAE genes. Brain tissue exhibited BAE in 107/128 (83.6%) genes for both sexes. The molecular pathways significantly enriched among BAE genes were photoreceptor activity, tubulin binding, and nucleotide/ribonucleotide binding. The biologic pathways significantly enriched for RAE genes were equilibrioception, parallel actin filament bundle assembly, photoreceptor cell outer segment organization, and protein depalmitoylation. Allele-specific expression may be a mechanism underlying IRD phenotypic variability, with clonal populations of embryologic precursor cells exhibiting RAE. Brain tissue preferentially exhibited BAE, possibly due to selective pressures against RAE. Pathways critical for cellular and visual function were enriched in BAE, which may offer a survival benefit.

Concurrent BCR::-ABL1 and CBFB Rearrangement in De Novo Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is known for its genetic heterogeneity. Several recurrent genetic variants have been characterized including AML-defining chromosomal rearrangements involving the core binding factor (CBF), such as inv(16)/t(16;16)/ CBFB:: MYH11. 1 Less common is a subset of AML characterized by the Philadelphia chromosome (Ph+), generating a BCR::ABL1 fusion protein. 2 The Ph+ AML subtype is associated with poor prognosis and treatment resistance. 3,4 Co-occurrence of CBFB rearrangement and BCR::ABL1 fusion is rare, representing a subgroup with unique clinical and molecular features. To date, there have been less than 30 cases reported in the literature. 5-8 Case Description A 62-year-old male presented to hospital with headaches, visual changes, weakness, and a nodular rash on his back, with leukocytosis, thrombocytopenia, and 42.5% circulating blasts. A bone marrow (BM) aspirate and biopsy was diagnostic of AML with 56% myeloblasts. RT-PCR was positive for an e19a2 BCR::ABL1 fusion transcript coding for p230 kDa fusion protein (p230) and CBFB: :MYH11 fusion gene transcript. Cytogenetics identified a complex karyotype with an inv(16) and a Ph chromosome (46,XY,t(1;14)(p32;q24),t(9;22)(q34;q11.2),inv(16)(p13.1q22)[10]), consistent with concurrent BCR::ABL1 and CBFB::MYH11 rearrangements within the same clonal population of cells. Treatment consisted of hydroxyurea for cytoreduction, 7+3 induction, one cycle of low dose cytarabine consolidation, and oral azacytidine maintenance, in combination with dasatinib. He did not receive Gemtuzumab ozogamicin (GO) given patient preference and comorbidities. Post-induction course was complicated by febrile neutropenia due to Enterococcus Faceium bacteremia, pre-renal acute kidney injury, and dasatinib-related complications including rash and transaminitis. These issues resolved by holding dasatinib for 15 days. Complete morphologic remission (CR) was obtained by Day 33 post-induction and no fusion gene transcripts were detectable by nested RT-PCR from BM aspirate on day 49 post-induction, nor day 76 post-consolidation The patient was ineligible for an allogeneic stem cell transplant given his poor performance status. At present, the patient remains alive and well, continuing on maintenance with oral azacytidine and dasatinib. Discussion We present the only case, to our knowledge, of a concurrent BCR::ABL1 p230 isoform and CBFB::MYH11 with de novo AML. Based on our literature search, 26 cases have been reported. BCR::ABL1 and CBFB::MYHII rearrangements in de novo AML potentially confers an intermediate to favorable prognosis with outcomes comparable to those of CBF-AML. Studies suggest that this entity may be more akin to AML with inv(16) rather than CML-BP with inv(16), thereby retaining favorable prognosis. 5,9-11 There are no formal guidelines for management of de novo AML with concurrent BCR::ABL1 and CBFB rearrangements. Traditional chemotherapy treatment strategies for Ph+ AML are often ineffective in these cases. The presence of BCR::ABL1 fusion transcript raises the possibility of utilizing TKIs targeting the BCR::ABL1 kinase domain. In our case, the patient achieved rapid CR with induction chemotherapy and dasatinib, but experienced multiple complications, precluding further intensive chemotherapy and allogeneic stem cell transplantation. This case raises a number of questions, including the optimal timing, combination, and duration of TKI therapy in the context of dual rearrangement de novo AML. This patient subgroup may benefit from intensive chemotherapy regimens and TKIs. Prospective monitoring of these cases is warranted to assess outcomes in comparison to Ph+ AML without coinciding CBFB rearrangement. This case report underscores the importance of comprehensive genetic profiling in Ph+ AML to determine the genetic landscape and individualize therapeutic strategies.

Identification of Autoantibodies Associated with Connective Tissue Disorders in Idiopathic Multicentric Castleman Disease

Background: Idiopathic multicentric Castleman disease (iMCD) is a rare, atypical lymphoproliferative disorder with significant morbidity and mortality and unknown etiology. While anti-interleukin-6 (IL-6) therapy with siltuximab is effective in 34-50% of iMCD patients, disease pathophysiology remains largely unknown. A fundamental question is whether iMCD is most appropriately considered an infectious disease caused by an as-yet-unknown pathogen, an autoimmune disease caused by autoantibodies, an autoinflammatory disease caused by germline mutations, or a neoplastic disease caused by somatic mutations in a clonal cell population. While limited evidence exists for each of these hypotheses, the pathogen hypothesis has been the most systematically evaluated to date. In this study, we quantified the levels of autoantibodies that may be involved in iMCD pathology. Methods: We constructed two custom bead-based protein arrays to screen serum samples for IgG autoantibodies from iMCD patients (n = 101) and healthy controls (HC, n = 30). The Connective Tissue Disease (CTD) array consisted of 52 antigens associated with traditional CTDs including in Scleroderma, Myositis/Overlap Syndromes, Systemic Lupus Erythematosus (SLE)/Sjögren's, GI/Endocrine, DNA-Associated, and Inflammation/Stress. The Anti-Cytokine Autoantibody (ACA) array included 38 cytokines, chemokines, and cell surface proteins. Serum samples came from the University of Pennsylvania (n = 38), University of Arkansas for Medical Sciences (n = 45), and Osaka University (Japan, n = 18). A “positive” autoantibody result in any sample was defined as greater than five standard deviations (SD) above the average mean fluorescent intensity (MFI) of the HC and at least 3,000 MFI. Fisher's exact tests were used to determine associations between autoantibody positivity and disease status. Results: To evaluate assay precision, we investigated anti-IL-6 antibodies given that a large portion (38%) of iMCD patients were on anti-IL-6 therapy. As expected, 40 iMCD samples were positive for anti-IL-6 antibodies, the most common autoantibody detected in iMCD subjects. IL-6 was then removed from further analyses. Next, we evaluated the proportion of CTD autoantibody positive iMCD patients. Overall, 46 (46%) iMCD subjects were positive for a CTD autoantibody compared to five (17%) in HCs (OR 4.1, P = 0.005) (Figure 1A). When investigating autoantibody prevalence in CTD subcategories, Myositis/Overlap Syndromes (OR 7.5, P = 0.026) and SLE/Sjögren's (OR 6.2, P = 0.073) autoantibodies trended higher in iMCD patients, but these associations did not survive multiple testing correction. The most common Myositis/Overlap Syndrome autoantibodies were anti-Mi2 (iMCD: 7/101 vs HC: 0/30), and anti-SRP54 (7/101 vs 0/30). The most common SLE/Sjögren's autoantibodies were anti-La/SSB (9/101 vs 0/30) and anti-Ro60 (5/101 vs 0/30). For the custom ACA array, there were significantly more ACA positive iMCD subjects (n = 40, 40%) than HC (n = 3, 10%; OR 5.8, P = 0.002) (Figure 1B). The most common ACAs in iMCD were anti-OSM (9/101 vs 0/30), anti-TNF (6/101 vs 0/30), anti-ITM2B (6/101 vs 0/30), and IFNe (5/101 vs 1/30). Conclusions: IgG autoantibodies associated with CTDs, such as anti-Mi2, anti-SRP54, anti-La, anti-Ro, and anti-Histone 3; and ACAs, such as anti-OSM, anti-TNF, and anti-ITM2B were common in iMCD patients. While autoantibodies suggest autoimmune involvement, the presence of autoantibodies does not directly implicate autoimmunity as a pathological mechanism. For example, Hodgkin's disease and other post-infectious etiologies can present with elevated autoantibodies. Although not surviving multiple hypothesis correction, myositis and SLE autoantibodies trended higher in iMCD patients. While most of these autoantibodies have not been reported, anti-Ro and and anti-La antibodies have been reported previously in iMCD case reports. The presence of anti-OSM is interesting, as OSM is a member of the IL-6 family. Two hypothesized mechanisms include that autoantibodies may be non-neutralizing leading to increased half-life and cytokine activity or autoantibodies are neutralizing and lack of negative feedback from OSM may lead to increased signaling via other IL-6-family cytokines. More research is needed to understand the role of these autoantibodies and, if pathogenic, whether iMCD should be considered an autoimmune disease.

Outcomes Associated with Clone-Directed Therapies for Monoclonal Gammopathy of Renal Significance

Background: Monoclonal Gammopathy of Renal Significance (MGRS) is a category of diseases not meeting criteria for cancer, in which a clonal population of B-lymphoid or plasma cells produces a nephrotoxic protein leading to kidney injury. Informal consensus has arisen that therapy targeting the causal clone is most likely to be efficacious, and the primary goal of therapy is to prolong renal survival. Light chain (AL) amyloidosis has been extensively studied, yet there is a paucity of data describing outcomes associated with clone-directed therapy for the various subtypes of non-AL MGRS. Methods: This was a retrospective analysis of patients treated at two National Cancer Institute (NCI)-designated cancer centers in the United States. Medical records of adult patients seen between January 2010 and March 2023 were screened for MGRS. Patients were eligible if they had renal biopsy-confirmed histopathologic findings compatible with MGRS. AL amyloidosis was excluded. The primary outcome was renal survival, defined as time from diagnosis of MGRS to requiring renal replacement therapy (RRT) or renal transplant, with patients censored at last follow up or death. Baseline characteristics were described, including 24-hour proteinuria, estimated glomerular filtration rate (eGFR), whether a clonal population of B cells or plasma cells could be identified on histopathologic examination of marrow or lymph node biopsy, concentration of monoclonal protein, and involved serum free light chain concentration. Initial therapy was reported, although differences in outcomes across therapeutic strategies were not compared. Baseline clinical characteristics between patients who required RRT and those who did not were compared. Baseline clinical variables and time to RRT were compared across the two most common MGRS subtypes in the cohort: proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) and light chain deposition disease (LCDD). Continuous variables were compared across subgroups using Wilcoxon rank-sum test. Time-to-event outcomes were analyzed according to the Kaplan-Meier method. All analyses were conducted using R version 3.6.1. Results: In total, 65 patients were included, 23 (35%) of whom ultimately required RRT or transplant. Four patients died, all after RRT. Median baseline proteinuria was 2802 mg/24h (IQR 972.5 - 4492.0) and median eGFR was 33.0 mL/min (IQR 23 - 53) for the entire cohort. In all cases proteinuria was mostly albumin. Median baseline involved light chain concentration was 8.3 mg/dL (IQR 3.1 - 26.4). A majority of patients (n=33, 51%) had no identifiable serum monoclonal protein. The two most common MGRS subtypes were PGNMID (n = 26) followed by LCDD (n = 21). Clonal populations thought to be causing MGRS were identified in 41/64 patients (64%), including 37 (58%) with plasma cell clones, 1 (2%) with a lymphoplasmacytic lymphoma clone, and 3 (4.7%) with other B-cell clones. Clones were not identified in 23 (36%). The most common chemotherapy agents included bortezomib (n=42, 64%) and rituximab (n=14, 22%). Compared to patients not requiring RRT, patients requiring RRT had higher baseline proteinuria (median 3531 vs. 1980 mg/24h, p<0.001) and lower eGFR (median 16 v. 44 mL/min, p<0.001); differences in serum free light chain concentrations were not significant ( Figure 1). A clonal population thought to be causing MGRS was identified on bone marrow biopsy in a majority of LCDD patients (n = 20, 95%), while clones were infrequently found in PGNMID (n = 6, 23%). The vast majority (n=20, 95%) of patients with LCDD received bortezomib-based therapy. Initial therapy in patients with PGNMID was more evenly divided: 9 (35%) received bortezomib-based therapy, 10 (38%) received rituximab-based therapy, and 6 (23%) received other regimens. Despite differences in therapeutic approach, patients with LCDD and in patients with PGNMID had similar risk of RRT (cumulative risk 35% v. 43%, p=0.54; HR 0.83 (95% CI 0.52-1.33); p =0.44) ( Figure 2). Conclusion: Patients with MGRS remain at high risk of requiring RRT even with clone-directed therapy. Despite differences in pathogenic clones and therapeutic strategies, outcomes of patients with PGNMID and LCDD are similar. Because the severity of baseline renal function across MGRS histologies predicts renal survival, early diagnosis and treatment is likely critical to improving renal outcomes.

Clonal Hematopoiesis and Inflammation in the Vasculature (CHIVE), a Prospective, Longitudinal Clonal Hematopoiesis Cohort and Biorepository

Introduction Clonal hematopoiesis (CH), the expansion of a clonal population of hematopoietic cells, is an age-associated phenomenon leading to increased risk of both hematologic malignancy and non-malignant organ dysfunction. There have been significant advances in our understanding of the underlying pathophysiology of CH in recent years. However, additional studies are needed to better understand clinical trajectories and optimal care of patients with CH. To address this gap, the prospective, longitudinal CHIVE (Clonal Hematopoiesis and Inflammation in the VasculaturE) registry and biorepository was created to support multidisciplinary CH clinics. Methods Adults without active hematological malignancy who were clinically identified to have CH or who were at risk for CH were sequentially enrolled utilizing a multidisciplinary referral approach between October 2020 to April 2023 at Vanderbilt University Medical Center (VUMC). Research samples were procured at the time of routine clinical blood draws or bone marrow biopsies and stored in a newly established biorepository, equipped to store DNA, plasma, and viably cryopreserved cells to enable a spectrum of downstream applications. Specimens were sequenced on a highly cost efficient (~$6/sample) targeted next generation sequencing capture panel that identified mutations residing in the 24 most frequently mutated CHIP genes.The VUMC CH clinic, which serves as a referral pool, were included in Arm A of CHIVE (n=57). We also sought out a population at risk for CH which included patients over 18 years of age with a history of solid tumor, cardiovascular disease, renal disease, rheumatologic disease, or diabetes, who were included in Arm B (n=122). Patients from Arm A or B were included in the CH-positive (CH+) group if they met criteria for CH on the research assay at any time during their enrollment in the study. All other genotyped patients who were not found to have CH by the research assay, regardless of their clinical CH status at study enrollment, were included in the CH-negative (CH-) group.Anthropometric data and clinical assessments, including laboratories and cardiovascular studies obtained in the course of routine clinical care were extracted from the electronic health record, and recorded and maintained in the CHIVE database. Results 261 patients were approached for consent to enroll in CHIVE and 179 were ultimately enrolled. More than one half (54%) of all participants had a CH mutation (CH+) and of those, 49.5% were female. Median age of CH+ patients was 71.9 (IQR 64.0-77.5). DNMT3A (n=44) and TET2 (n=39) were the most commonly mutated genes, while variant allele fractions (VAF) ranged from 2.1 - 79.8% (Figure 1). 246 samples were serial samples collected from 89 patients at regular intervals. Significantly more CH+ patients had a diagnosis of coronary artery disease compared to CH- patients (55.7% vs 32.9%, p=0.004). CH+ patients also had increased rates of hypertension and heart failure compared to the CH- group (p<0.001, 0.035, respectively). Laboratory evaluation showed CH+ patients to have a lower median white blood cell count, hemoglobin, and platelets, and elevated blood urea nitrogen values compared to CH- patients, differences that did not meet statistical significance. 8.2% of the CH+ patients progressed to frank hematologic malignancy over the course of the 2.5-year study (Table 1). Eight of the 97 CH+ and 2 of the 82 CH- patients died over the course of the study period resulting in a trend toward decreased survival in CH+ patients (p=0.112). Conclusions We demonstrate the feasibility of a prospective, observational study of CH patients utilizing a robust referral network to support both clinical care and translational research. A well-genotyped and phenotyped cohort of CH patients will be critical to future translational research efforts and clinical trials - a key function of our study design. Early clinical findings from our cohort recapitulate large scale retrospective datasets where CH patients are at an increased risk of development of hematologic malignancy, end organ damage, and all-cause mortality. Scaling this resource in collaboration with other centers is underway and will ultimately enable the development of clinical guidelines and treatment strategies for this increasingly recognized patient population.