Combined Immunodeficiency Disease Research Articles

Analysis of efficacy and prognosis of allogeneic hematopoietic stem cell transplantation for the treatment of combined immunodeficiency

Objective: To evaluate the efficacy of allogeneic hematopoietic stem cell transplantation for the treatment of combined immunodeficiency (CID) and explore prognostic risk factors. Methods: In this retrospective cohort study, clinical characteristics, laboratory tests and prognosis of 73 CID children who underwent allogeneic hematopoietic stem cell transplantation from February 2014 to April 2022 in the Children's Hospital of Fudan University were analyzed. Based on the subtypes of diseases, all patients were divided into severe combined immunodeficiency disease (SCID) group and other CID group. Based on the types of donors, all patients were divided into matched sibling donor group, matched unrelated donor group, unrelated cord blood group, and haploidentical donor group. Kaplan-Meier method and Log-Rank test were used to analyze the survival data. Cox regression was used to analyze prognostic factors. Results: Among the 73 patients, there were 61 (84%) males and 12 (16%) females. Fifty-five (75%) patients were SCID, and 18 (25%) patients were other CID. Donor source included 2 (3%) matched sibling donors (MSD), 3 (4%) matched unrelated donors (MUD), 64 (88%) unrelated cord blood (UCB), and 4 (5%) haploidentical donors. The age at transplant was 10.7 (5.9, 27.5) months, and the follow-up time was 36.2 (2.5, 62.9) months. The 3-year overall survival rate of 73 patients with CID was (67±6) %. No significant difference was found in the 3-year overall survival rates between patients with SCID (55 cases) and other CID (18 cases) ((64±7) % vs. (78±10) %, χ2=1.31, P=0.252). And no significant difference was found in the 3-year overall survival rates among patients who received MSD or MUD (5 cases), UCB (64 cases), and haploidentical donor (4 cases) transplant (100% vs. (66±6)% vs. (50±25) %, χ2=2.30, P=0.317). Cox regression analysis showed that the medical history of sepsis (HR=2.55, 95%CI 1.05-6.20, P=0.039) and hypoalbuminemia at transplant (HR=2.96, 95%CI 1.14-7.68, P=0.026) were independent risk factors for the prognosis of allogeneic hematopoietic stem cell transplantation in pediatric patients with CID. Conclusions: Allogeneic hematopoietic stem cell transplantation is an effective treatment for CID. The medical history of sepsis and hypoalbuminemia at transplant were risk factors for prognosis. Enhancing infection prevention and nutritional intervention before transplant can improve patient prognosis.

41 Specifications of ACMG/AMP Variant Curation Guidelines for the Analysis of FOXN1 Sequence Variants: Recommendations by ClinGen’s Severe Combined Immunodeficiency Disease Variant Curation Expert Panel

41 Specifications of ACMG/AMP Variant Curation Guidelines for the Analysis of FOXN1 Sequence Variants: Recommendations by ClinGen’s Severe Combined Immunodeficiency Disease Variant Curation Expert Panel

Abstract 2835: Comparison of xenograft and syngeneic cancer cachexia models for preclinical drug testing

Abstract Cancer cachexia is a wasting syndrome characterized primarily by loss of skeletal muscle and adipose tissue mass and contributes greatly to overall cancer-related mortality (for recent review see Argiles 2023). An estimated 25-75% of cancer patients develop cachexia which leads to impacts on therapeutic tolerability, well-being, and eventually overall survival (Bossi 2021). Some preclinical mouse and rat cancer models manifest cachexia as they progress. In this study we established a syngeneic (immune competent) and a xenograft (immune incompetent) tumor model of cachexia with the intention of comparing the models with respect to typical study endpoints and to provide a platform for testing novel therapeutic agents aimed at inhibiting or reversing the effects of cachexia. The Colon26 syngeneic mouse colon adenocarcinoma model was reported as an experimental cancer cachexia model by Tanaka, et. al. in 1990. The model was developed in male BALB/c × DBA/2 F1 (first generation cross) mice. We typically implant Colon26 in female BALB/c mice for preclinical drug testing of anticancer agents and noticed a strong tendency for mice bearing Colon26 tumors to develop signs of cachexia on study, but not always consistently. A review of the literature showed there are clear gender differences with male cancer patients having higher prevalence of cachexia and greater muscle loss compared with female patients (Zhong 2020). Based on this we implanted Colon26 tumors in male BALB/c mice to establish the cachexia model. The HT1080 human fibrosarcoma model HT1080 was characterized as a cachexic model by Bernardo, et. al. in female severe combined immunodeficiency disease (SCID) mice. We developed the HT1080 model in male SCID mice based on the literature and previous experience. Both models were characterized by multiple endpoints including overall body weight loss, skeletal muscle weight loss, reduction of adipose tissue, and cardiac muscle loss, as well as tracking a broad panel of relevant cytokine levels at study endpoint. Both models generate robust wasting of skeletal muscle and adipose tissue providing an opportunity to assess novel anti-cachexia therapies. Citation Format: Kyle Draheim, Diana Gietl, Karyn Shinn, William Durham, Fei Zhao, Caitlin Thompson, Alexis Baldwin, Glenn Harris, Chassidy Hall, Patrick Fadden, Anya Avrutskaya. Comparison of xenograft and syngeneic cancer cachexia models for preclinical drug testing [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 2835.

Disseminated Bacillus Calmette-Guerin Infection: The Harbinger of Severe Combined Immunodeficiency Disease

Disseminated Bacillus Calmette-Guerin Infection: The Harbinger of Severe Combined Immunodeficiency Disease

Novel homozygous CARD11 variants in two patients with combined immunodeficiency and atopic skin disease

BackgroundCaspase recruitment domain family, member 11 (CARD11) is an important protein which plays a fundamental role in the activation of NF-κβ pathway in lymphocytes. CARD11 deficiency can be inherited in either autosomal dominant or autosomal recessive forms and present with different phenotypes including combined immunodeficiency, atopic dermatitis, and other variable manifestations. The present report describes clinical phenotypes and immunological defects of two unrelated patients with missense homozygous variants in CARD11 presenting with combined immunodeficiency (CID) and atopic skin disease resembling that reported in dominant negative CARD11 deficiency. The patients underwent next generation sequencing, immunophenotyping of T and B subsets by flow cytometry, T cell stimulation, and evaluation of CARD11 expression.ResultsBoth patients had features suggesting CID including repeated pneumoniae with ICU admissions, chronic diarrhea, and itchy atopic skin disease. Patient-1 has homozygous missense variant in the C terminal domain (c.2839G > A, p.Glu947Lys), and patient-2 has homozygous variant in the inhibitory domain (c.1073C > G, p.Pro568Arg). Both have profound defects in Tregs with normal recent thymic emigrants, memory, and naïve CD4+ T cells. However, in response to stimulation, T cells failed to upregulate the expression of CD25. CARD11 expression by flow cytometry was decreased rather than abolished as previously described in patients with autosomal recessive CARD11 deficiency. B cells showed marked deficiency of switched memory and increase in transitional B cells.ConclusionMissense variants causing CARD11 deficiency may affect the protein function rather than the expression and can result in a phenotype combining the atopic skin disease and the features of CID.

Periodontal disease in patients with WHIM syndrome.

WHIM (warts, hypogammaglobulinaemia, infections and myelokathexis) syndrome is a rare combined primary immunodeficiency disease caused by gain-of-function (GOF) mutations in the chemokine receptor CXCR4 and includes severe neutropenia as a common feature. Neutropenia is a known risk factor for periodontitis; however, a detailed periodontal evaluation of a WHIM syndrome cohort is lacking. This study aimed to establish the evidence base for the periodontal status of patients with WHIM syndrome. Twenty-two adult WHIM syndrome patients and 22 age- and gender-matched healthy volunteers (HVs) were evaluated through a comprehensive medical and periodontal examination. A mouse model of WHIM syndrome was assessed for susceptibility to naturally progressing or inducible periodontitis. Fourteen patients with WHIM syndrome (63.6%) and one HV (4.5%) were diagnosed with Stage III/IV periodontitis. No WHIM patient presented with the early onset, dramatic clinical phenotypes typically associated with genetic forms of neutropenia. Age, but not the specific CXCR4 mutation or absolute neutrophil count, was associated with periodontitis severity in the WHIM cohort. Mice with a Cxcr4 GOF mutation did not exhibit increased alveolar bone loss in spontaneous or ligature-induced periodontitis. Overall, WHIM syndrome patients presented with an increased severity of periodontitis despite past and ongoing neutrophil mobilization treatments. GOF mutations in CXCR4 may be a risk factor for periodontitis in humans.

Effect of Cell Density of a Methacrylic Acid-Based Hydrogel Implant on Embedded Islet Function and Viability.

Subcutaneous delivery of islets in a methacrylic acid-based hydrogel may offer a functional cure for type 1 diabetes. Here we show in mice that the hydrogel is able to provide sufficient vasculature to support islet function and viability, when islets are used at a low islet volume fraction (i.e., cell density). The Krogh cylinder model was used to mathematically estimate the effect of implant volume, for a fixed islet dose (600 islet equivalents [IEQ]), on the minimum vessel density required to maintain sufficient pO2 within the graft. Modeling suggested that 200 μL implants would have low enough islet densities and enough vessels to have islets remain viable, but that 50 μL implants would not; this was confirmed experimentally through measurement of glucose level in streptozotocin-induced diabetic severe combined immunodeficiency disease (SCID/bg) mice, comparing 200 and 50 μL implants, both with 600 IEQ. Vessel densities were ∼20-30 vessels/mm2 independent of implant volume and vessels were sufficient to increase subcutaneous oxygen tension, as measured with microcapsules containing oxygen sensitive material (a platinum [Pt] porphyrin); both these results were determined without cells. These results are useful in thinking about the scale-up of this system to humans: to maintain a low islet density (∼0.5%), many more islets will require attention to the subcutaneous implant configuration to satisfy the oxygen needs of the cells.

Development of a HPLC-MS/MS Method to Assess the Pharmacokinetics and Tumour Distribution of the Dimethylarginine Dimethylaminohydrolase 1 Inhibitors ZST316 and L-257 in a Xenograft Model of Triple-Negative Breast Cancer in Mice.

We describe the development and validation of an HPLC-MS/MS method to assess the pharmacokinetics and tumour distribution of ZST316, an arginine analogue with inhibitory activity towards dimethylarginine dimethylaminohydrolase 1 (DDAH1) and vasculogenic mimicry, and its active metabolite L-257 in a xenograft model of triple-negative breast cancer (TNBC). The method proved to be reproducible, precise, and highly accurate for the measurement of both compounds in plasma and tumour tissue following acute and chronic (five days) intraperitoneal administration of ZST316 (30 mg/Kg daily) in six-week-old severe combined immunodeficiency disease (SCID) mice inoculated with MDA-MB-231 TNBC cells. ZST316 was detected in tumour tissue and plasma after 1 h (6.47 and 9.01 μM, respectively) and 24 h (0.13 and 0.16 μM, respectively) following acute administration, without accumulation during chronic treatment. Similarly, the metabolite L-257 was found in tumour tissue and plasma after 1 h (15.06 and 8.72 μM, respectively) and 24 h (0.17 and 0.17 μM, respectively) following acute administration of ZST316, without accumulation during chronic treatment. The half-life after acute and chronic treatment ranged between 4.4-7.1 h (plasma) and 4.5-5.0 h (tumour) for ZST316, and 4.2-5.3 h (plasma) and 3.6-4.9 h (tumour) for L-257. The results of our study demonstrate the (a) capacity to accurately measure ZST316 and L-257 concentrations in plasma and tumour tissue in mice using the newly developed HPLC-MS/MS method, (b) rapid conversion of ZST316 into L-257, (c) good intra-tumour penetration of both compounds, and (d) lack of accumulation of both ZST316 and L-257 in plasma and tumour tissue during chronic administration. Compared to a previous method developed by our group to investigate ZST316 in plasma, the main advantages of the new method include a wider range of linearity which reduces the need for dilutions and the combined assessment of ZST316 and L-257 in plasma and tumour tissue which limits the required amount of matrix. The new HPLC-MS/MS method is useful to investigate the in vivo effects of ZST316 and L-257 on vasculogenic mimicry, tumour mass, and metastatic burden in xenograft models of TNBC.

MiR-455-3p mediates PPARα through UBN2 to promote apoptosis and autophagy in acute myeloid leukemia cells

Acute myeloid leukemia (AML) is one of the deadliest hematologic malignancies, and its targeted therapy has developed slowly. The molecular mechanism of the pathophysiology of the disease remains to be clarified. The aim of our study was to probe the specific regulatory mechanism of miR-455-3p in AML. This study measured the levels of miR-455-3p and ubinuclein-2 (UBN2) in AML cell lines, evaluated cell viability with CCK-8, used flow cytometry to estimate the cell cycle and apoptosis, detected cell apoptosis and autophagy-related protein levels by Western blotting, and added 50 μM chloroquine (CQ) to evaluate the relationship between autophagy and AML. In animal experiments, HL-60 cells were injected into male non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice through the tail vein to determine survival time and observe the degree of liver and spleen damage in the mice. miR-455-3p was prominently reduced in the peripheral blood and AML cell lines, and UBN2 showed high expression. The transfected miR-455-3p mimic effectively restrained the activity of AML cells, whereas overexpression of UBN2 or the addition of the autophagy inhibitor CQ reversed the effect of miR-455-3p. The interaction between UBN2 and peroxisome proliferator-activated receptor alpha (PPARα) was confirmed by coimmunoprecipitation, and overexpression of PPARα reversed the promoting effect of UBN2 knockdown on apoptosis and autophagy in AML cells. In conclusion, miR-455-3p mediates PPARα protein expression through UBN2, exacerbating AML cell apoptosis and autophagy. This study found that miR-455-3p plays an important role in AML cell apoptosis and autophagy, which may provide novel insights for the treatment of AML diseases.

JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation.

Janus tyrosine kinase 3 (JAK3) is primarily expressed in immune cells and is needed for signaling by the common gamma chain (γc) family of cytokines. Abnormal JAK3 signal transduction can manifest as hematological disorders, e.g., leukemia, severe combined immunodeficiency (SCID) and autoimmune disease states. While regulatory JAK3 phosphosites have been well studied, here a functional proteomics approach coupling a JAK3 autokinase assay to mass spectrometry revealed ten previously unreported autophosphorylation sites (Y105, Y190, Y238, Y399, Y633, Y637, Y738, Y762, Y824, and Y841). Of interest, Y841 was determined to be evolutionarily conserved across multiple species and JAK family members, suggesting a broader role for this residue. Phospho-substitution mutants confirmed that Y841 is also required for STAT5 tyrosine phosphorylation. The homologous JAK1 residue Y894 elicited a similar response to mutagenesis, indicating the shared importance for this site in JAK family members. Phospho-specific Y841-JAK3 antibodies recognized activated kinase from various T-cell lines and transforming JAK3 mutants. Computational biophysics analysis linked Y841 phosphorylation to enhanced JAK3 JH1 domain stability across pH environments, as well as to facilitated complementary electrostatic JH1 dimer formation. Interestingly, Y841 is not limited to tyrosine kinases, suggesting it represents a conserved ubiquitous enzymatic function that may hold therapeutic potential across multiple kinase families.

Disseminated tuberculosis complicating Bacillus Calmette-Guérin (BCG) vaccine as only presentation of severe combined immunodeficiency (SCID): A report of 3 cases.

Severe combined immunodeficiency disease (SCID) is a rare primary immunodeficiency disease, usually manifest in the first six months of life with failure to thrive, oral thrush, recurrent respiratory infection, and chronic diarrhea. In three male patients, we describe an unusual presentation of SCID. They are an outcome of consanguineous marriage; all received the BCG vaccine at birth. All three cases presented with regional lymphadenopathy at three months, progressing to generalized lymphadenopathy treated with anti-tuberculous. The first and second cases were twins. The first had an uneventful history until 33 months when he developed multiple Suppurative Tuberculous lymphadenitis confirmed by biopsy. The second and the third cases were diagnosed with Disseminated Tuberculosis at 24 months as they developed fever, anemia, weight loss, tuberculous peritonitis, and lymphadenopathy confirmed by biopsy. After investigations, the first case was diagnosed as CD4, CD16 lymphopenic SCID, the second one as CD4, CD8, CD19, CD16 lymphopenic SCID with hypogammaglobulinemia and the third case as CD3, CD4, CD8 lymphopenic SCID with hypogammaglobulinemia. They received anti-Tuberculous medications, prophylactic Trimethoprim/Sulfamethoxazole, and Immunoglobulin infusion. When writing this abstract, the patients were alive and had no other bacterial, viral, or fungal infections. The twins are three years old, and the third case is 30 months old. SCID may not exhibit the classical manifestation of recurrent infections. It may present only as a complication of the BCG vaccine, alarming to maintain high susceptibility in such patients, especially in a developing country, specifically in Sudan, where the BCG vaccine is usually given at birth.

Severe CD8+ T Lymphopenia in WHIM Syndrome Caused by Selective Sequestration in Primary Immune Organs.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an ultra-rare combined primary immunodeficiency disease caused by heterozygous gain-of-function mutations in the chemokine receptor CXCR4. WHIM patients typically present with recurrent acute infections associated with myelokathexis (severe neutropenia due to bone marrow retention of mature neutrophils). Severe lymphopenia is also common, but the only associated chronic opportunistic pathogen is human papillomavirus and mechanisms are not clearly defined. In this study, we show that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses invitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow.

Resolution of IgA vasculitis nephritis in patient with activated PI3Kdelta syndrome after treatment with leniolisib

IntroductionActivated PI3K-delta syndrome is a combined immunodeficiency disease with a heterogenous immune phenotype. Common clinical manifestations include recurrent respiratory infections, persistent herpesvirus infections, lymphoproliferative disease, and autoimmunity. We describe the case of a patient with APDS who developed IgA vasculitis nephritis that responded to steroids, plasmapheresis, rituximab, and initiation of a new selective PI3Kdelta inhibitor, leniolisib. Case description19-year-old male with APDS (pathogenic variant in PI3KCD, p.E81K) complicated by recurrent ear and sinus infections, marginal zone B-cell hyperplasia, and EBV lymphadenitis who presented to the hospital with hematuria and fatigue. Initial work-up revealed acute kidney injury (Cr 1.72 mg/dL) with gross hematuria and nephrotic range proteinuria. A renal biopsy was performed which demonstrated IgA dominant immune complex glomerulonephritis (10% focal cellular crescents). The patient was started on empiric high-dose steroids. However, due to worsening renal injury (Cr peak to 4.1 mg/dL) and proteinuria, plasmapheresis and rituximab were administered. Additionally, leniolisib, a new oral PI3Kdelta inhibitor, was initiated at 70 mg twice daily. The patient’s renal function began to stabilize after administration of these therapies, and he was discharged on daily prednisone and leniolisib. On outpatient follow-up over the course of six months, he demonstrated normalization of his creatinine and improvement of his proteinuria. ConclusionsGlomerulonephritis has been reported as an autoimmune renal manifestation in APDS. This case highlights the first reported instance of treatment response to leniolisib and immunomodulatory agents in an APDS patient with IgA vasculitis nephritis. While leniolisib is an unapproved selective PI3K-delta inhibitor, its use should be considered in the management of APDS patients with significant autoimmune complications.

Recommendations from the ClinGen SCID VCEP: Implementation of ACMG/AMP Variant Curation Guidelines for Severe Combined Immunodeficiency Disease

BackgroundThe Clinical Genome Resource (ClinGen) is a National Institutes of Health (NIH)-funded program that aims to improve our understanding of the relationship between genetic variations and human health. ClinGen has formed Variant Curation Expert Panels (VCEPs) that provide evidence-based interpretations of genomic variants associated with specific diseases. The ClinGen Severe Combined Immunodeficiency Disease (SCID) VCEP was created to develop specifications-rules for SCIDassociated genes because the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines from 2015 are not gene-specific. The panel has focused on IL2RG, JAK3, ADA, DCLRE1C, IL7R, RAG1, and RAG2 to aid in precise diagnosis, clinical management, and genetic counseling.Methods and Results: Of the 28 ACMG/AMP criteria, the SCID VCEP adapted 18 rules to the 7 selected genes and determined that 10 rules were not applicable (with slight differences between each gene). The key specifications of the rules included setting thresholds for minor allele frequency, developing a method for counting probands and segregations, reviewing functional assays, and determining phenotypic characteristics specific to each gene. Pilot testing of the rules modifications demonstrated appropriate performance as determined by the VCEP. ConclusionsThe VCEP will curate 5,460 variants using these gene-specific rules and categorize them into five groups: pathogenic, likely pathogenic, uncertain significance, likely benign, and benign. The pathogenicity classifications will be published in ClinVar with 3-star review status. This standardized and evidence-based interpretation of genetic variants associated with SCID is crucial to ensure accurate diagnosis, effective clinical management and informed genetic counseling for affected individuals and their families.

POLD3 deficiency is associated with severe combined immunodeficiency, neurodevelopmental delay, and hearing impairment

Combined immunodeficiency diseases (CID) represent the most severe forms of inborn errors of immunity. Defective T cell development and/or function, leading to an impairment in adaptive immunity are responsible for these diseases. The DNA polymerase δ complex is important for genome duplication and maintenance and consists of the catalytic subunit POLD1, and the accessory subunits POLD2 and POLD3 which stabilizes the complex. Mutations in POLD1 and POLD2 have been recently shown to be associated with a syndromic CID characterized by T cell lymphopenia with or without intellectual deficiency and sensorineural hearing loss. Here we report a homozygous POLD3 variant (NM_006591.3; p.Ile10Thr) in a Lebanese patient, the product of a consanguineous family, presenting with a syndromic severe combined immunodeficiency (SCID) with neurodevelopmental delay and hearing loss. The homozygous POLD3Ile10Thr variant abolishes POLD3 as well as POLD1 and POLD2 expression. Our findings implicate POLD3 deficiency as a novel cause of syndromic SCID.

Abstract 132: The molecular basis of an evolutionary arms race between cancer cells and their host

Abstract Genetic and nongenetic changes can drive abnormal characteristics of cancer cells, including resistance to tumor growth. Through selective breeding of animals with the slowest growing tumor at 11 days following implantation over ten generations, we evolved resistance to tumor growth in two contrasting strains of mice: severe combined immunodeficiency disease (SCID) and immuno-competent C57BL/6. We found that the two strains evolved different strategies of resistance to the cancer. In the immunocompromised mice, skin fibroblasts infiltrated into and around the tumor to impede growth, while an enhanced immune response to tumor antigens, slowed tumor growth in the immunocompetent mice. However, when the experiment was extended beyond 11 days, tumor growth rapidly increased so that, at 28 days, it was comparable in size to the control animals. We investigated the molecular-level mechanisms underlying these resistance traits in SCID mice. We identified transcriptomic changes using RNA-Seq in both skin and tumor samples to compare the evolved mice (EV) with the original strain (WT) at 11 days and 28 days. We used DESeq2 and two-way ANOVAs on the normalized data to identify significant differences in gene expression. We adjusted p-values for multiple testing with the Benjamini-Hochberg false discovery correction and used a q value of less than 0.1 (and/or a greater than two-fold change) to determine the significance for differential expression levels. Comparing transcriptomic data from skin fibroblasts from evolved (EV) and non-evolved (WT) SCID mice at day 11 following implantation (i.e., the point of maximal tumor suppression) revealed 98 differentially expressed genes (DEGs). EV SCID fibroblasts showed increased expression of MicroRNAs, including MiR-7 and Mir5125, and acid metabolic pathways, such as Carbonic anhydrase III. A comparison of the tumor cells from day 11 and day 28 demonstrated substantial epigenetic evolution with 1353 DEGs. The tumor cells at day 28 adapted to the host response by increasing expression of genes for extracellular matrix (ECM) remodeling, mainly the metalloproteases gene family. Here we used selective breeding to allow evolution to reveal potential host strategies to suppress tumor growth. However, we find that, over time, tumor cells can evolve successful counter strategies to overcome host resistance. Our results provide novel insights into key dynamics of somatic evolution during carcinogenesis and highlight the critical roles of epigenetic modification and inheritance in these somatic eco-evolutionary dynamics. Citation Format: Arig A. Ibrahim-Hashim, Robert A. Gatenby, Christina L. Richards, Joel S. Brown. The molecular basis of an evolutionary arms race between cancer cells and their host [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 132.

Abstract 5932: Clonal mapping phylogenetic and transcriptomic analysis of an M36 PDX amputation lung metastatic osteosarcoma model

Abstract Introduction: Osteosarcoma is a primary malignant bone tumor characterized by the production of spindle cells resulting in immature bone formation. It has been revealed that metastatic cancers including osteosarcoma have a subset of cells with different phenotypic and genetic signatures conferring advantage to drive progression and drug resistance within the intra-tumorally heterogeneous population. However, the lack of a defined in vivo model that can identify the rare genetic subpopulation and recapitulate clonal evolution has been a challenge. In this study, we used an established model to track and identify the clonal subpopulations and assess the genetic signatures that confer specific advantages selecting for uncontrolled growth, adaptation and colonization in the lungs. Methods: We injected barcoded M36 PDX osteosarcoma cells intratibially in ten severe combined immunodeficiency disease (SCID) mice to track the clonal population/subpopulation of cells that metastasize the established primary tumors to the lungs. Limb amputation was performed on the tumor bearing leg upon detection of palpable tumor. Lung tissue was harvested post-amputation following death from metastasis or euthanasia. DNA and RNA from frozen tissues was extracted and analyzed by PCR, NGS, WES and RNA sequencing to map clonality, mutational and evolutionary profiles. Results: Limb amputation was performed in 7 mice with all mice surviving the surgery. The mean mouse survival following amputation was 46.9 days and the longest survival was 167 days. Four (40%) of the primary tumor bearing mice had lung metastasis. A Shannon-Wiener and Jaccard similarity index show a diversity in clonal architecture between the primary tumors of all mice injected on the same day and a diversity in clonal population in both the primary tumors and their matched lung metastatic nodules. Clonal mapping further revealed 21 unique clones shared in all primary tumors examined. However, the clonal frequencies and distribution varied within each primary tumor and across the tumor samples. Interestingly, clonal frequencies increased in the lung metastatic nodules when compared to their matched primary tumors. Four distinct clones that were present in all the primary tumors were also found in all lung metastatic samples. These unique clones, here in called “seeding clones” may play an important role in the lung metastatic process. Conclusions: We previously showed that the barcoded osteosarcoma PDX models are inter and intratumorally heterogenous both in vitro and in vivo. In the current model, we identify the multiple tumorigenic seeding clones that potentially act as drivers in the lung metastatic process. Furthermore, we show the dynamic evolutionary relationships between the clones by reconstructing a phylogenetic map of the M36 PDX tumor model. Funding: Swim Across America, Foster Foundation and the Barbara Epstein Foundation. Citation Format: Sylvester Jusu, Zhongting Zhang, Zhang Wendong, Zhaohui Xu, Michael Roth, Jonathan Gill, Richard Gorlick. Clonal mapping phylogenetic and transcriptomic analysis of an M36 PDX amputation lung metastatic osteosarcoma model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5932.

Viral Vectors in Gene Therapy: Where Do We Stand in 2023?

Viral vectors have been used for a broad spectrum of gene therapy for both acute and chronic diseases. In the context of cancer gene therapy, viral vectors expressing anti-tumor, toxic, suicide and immunostimulatory genes, such as cytokines and chemokines, have been applied. Oncolytic viruses, which specifically replicate in and kill tumor cells, have provided tumor eradication, and even cure of cancers in animal models. In a broader meaning, vaccine development against infectious diseases and various cancers has been considered as a type of gene therapy. Especially in the case of COVID-19 vaccines, adenovirus-based vaccines such as ChAdOx1 nCoV-19 and Ad26.COV2.S have demonstrated excellent safety and vaccine efficacy in clinical trials, leading to Emergency Use Authorization in many countries. Viral vectors have shown great promise in the treatment of chronic diseases such as severe combined immunodeficiency (SCID), muscular dystrophy, hemophilia, β-thalassemia, and sickle cell disease (SCD). Proof-of-concept has been established in preclinical studies in various animal models. Clinical gene therapy trials have confirmed good safety, tolerability, and therapeutic efficacy. Viral-based drugs have been approved for cancer, hematological, metabolic, neurological, and ophthalmological diseases as well as for vaccines. For example, the adenovirus-based drug Gendicine® for non-small-cell lung cancer, the reovirus-based drug Reolysin® for ovarian cancer, the oncolytic HSV T-VEC for melanoma, lentivirus-based treatment of ADA-SCID disease, and the rhabdovirus-based vaccine Ervebo against Ebola virus disease have been approved for human use.

Clinical and Genetic Characteristics of BCG Disease in Chinese Children: a Retrospective Study.

Summarize the characteristics of a large cohort of BCG disease and compare differences in clinical characteristics and outcomes among different genotypes and between primary immunodeficiency disease (PID) and patients without identified genetic etiology. We collected information on patients with BCG disease in our center from January 2015 to December 2020 and divided them into four groups: chronic granulomatous disease (CGD), Mendelian susceptibility to mycobacterial disease (MSMD), severe combined immunodeficiency disease (SCID), and gene negative group. A total of 134 patients were reviewed, and most of them had PID. A total of 111 (82.8%) patients had 18 different types of pathogenic gene mutations, most of whom (91.0%) were classified with CGD, MSMD, and SCID. CYBB was the most common gene mutation (52/111). BCG disease behaves differently in individuals with different PIDs. Significant differences in sex (P < 0.001), age at diagnosis (P = 0.013), frequency of recurrent fever (P = 0.007), and vaccination-homolateral axillary lymph node enlargement (P = 0.039) and infection severity (P = 0.006) were noted among the four groups. The CGD group had the highest rate of males and the oldest age at diagnosis. The MSMD group had the highest probability of disseminated infection (48.3%). The course of anti-tuberculosis treatment and the survival time between patients with PID and without identified genetic etiology were similar. Greater than 80% of BCG patients have PID; accordingly, gene sequencing should be performed in patients with BCG disease for early diagnosis. BCG disease behaves differently in patients with different types of PID. Patients without identified genetic etiology had similar outcomes to PID patients, which hints that they may have pathogenic gene mutations that need to be discovered.

Co-Expression Pattern of Artemis Serine 516 Phosphorylation with Indicators Involving in Differentiation, Proliferation and Apoptosis of Human Anagen Hair Follicles: An Immunofluorescence Double-Staining Study.

Artemis belongs to the SNM1 gene family, and plays a role in repairing ionizing-radiation-induced DNA double-strand breaks and variable (diversity) joining recombination. S534, S538, S516, S645 represent four most rapid phosphorylation sites in Artemis, and serine phosphorylation at amino acid 516 is closely associated with activation. Artemis mutation is perceived as contributing to Omenn syndrome, which manifest features of severe combined immunodeficiency disease, associated with lymphadenopathy, hepatosplenomegaly, erythroderma and baldness. In addition, Artemis phosphorylated at serine 516 (Artemis S516-P) was expressed in scalp hair follicles (HF) as well as other skin appendages, and its expression level is important to mouse hair cycling. However, whether Artemis participated in the regulation of HF growth still unclear. Using immunofluorescence double-staining, we assessed the association between Artemis S516-P with proliferation, apoptosis, and differentiation markers in normal adult anagen scalp HF. The results of double-staining immunofluorescence revealed overlapping expression pattern for Artemis S516-P and keratin16, similar pattern for c-myc and p21, while presenting opposite trends for keratin 10, phospho-p53, Bax, Bcl-2 and keratin 14. Our study provides the clues that Artemis may play roles in regulation of differentiation, proliferation, apoptosis and cell cycling during HF growth and development.