Chromosomal Variations and Clinical Features of Acute Lymphoblastic Leukemia in the North Indian Population: A Cross-Sectional Study.

BACKGROUND: The key prognostic markers in acute lymphoblastic leukemia (ALL) include age, leukocyte count upon diagnosis, immunophenotype, and chromosomal abnormalities. Furthermore, there was a correlation between cytogenetic anomalies and specific immunologic phenotypes of ALL, which in turn had varied outcomes. The objective of this study was to examine the occurrence of cytogenetic abnormalities in individuals diagnosed with acute lymphoblastic leukemia. METHODS: The study employed a cross-sectional design to investigate genetic evaluation and clinical features in 147 ALL patients between March 2021 and August 2022. Demographic data (like age and sex), clinical manifestations, and hematological parameters were collected. Cytogenetic analysis (G-banding) was performed to identify chromosomal abnormalities. The mean±SD and analysis of variance (ANOVA) were used to assess associations and differences among variables using SPSS Version 24 (IBM Corp., Armonk, NY, USA). RESULTS: The study shows male n=85 and female n=62 in ALL patients, with prevalent clinical manifestations: fever n=100 (68.03%), pallor n=123 (83.67%), and lymphadenopathy n=65 (44.22%). The hematological parameters like hemoglobin (Hb) (6.14±2.5 g/dl), total leukocyte count (TLC) (1.7±1.05 cell/mm3), and platelet count (1.2±0.11 lac/mm3) show a significant variation (P<0.05) in patients aged 30-50 years. In addition, chromosomal abnormalities, particularly 46, XX, t(9;22), were prevalent, emphasizing the genetic heterogeneity of ALL. CONCLUSION: The study shows a male predominance with ALL, prevalent clinical manifestations, and significant hematological parameter variations in the 30-50 age group. Chromosomal abnormalities, notably 46, XX, t(9;22), underscore the genetic complexity of the disease, which necessitates tailored therapeutic interventions informed by genetic profiles.

Manufacture of CD22 CAR T cells following positive versus negative selection results in distinct cytokine secretion profiles and γδ T cell output.

Chimeric antigen receptor T cells (CART) have demonstrated curative potential for hematological malignancies, but the optimal manufacturing has not yet been determined and may differ across products. The first step, T cell selection, removes contaminating cell types that can potentially suppress T cell expansion and transduction. While positive selection of CD4/CD8 T cells after leukapheresis is often used in clinical trials, it may modulate signaling cascades downstream of these co-receptors; indeed, the addition of a CD4/CD8-positive selection step altered CD22 CART potency and toxicity in patients. While negative selection may avoid this drawback, it is virtually absent from good manufacturing practices. Here, we performed both CD4/CD8-positive and -negative clinical scale selections of mononuclear cell apheresis products and generated CD22 CARTs per our ongoing clinical trial (NCT02315612NCT02315612). While the selection process did not yield differences in CART expansion or transduction, positively selected CART exhibited a significantly higher in vitro interferon-γ and IL-2 secretion but a lower in vitro tumor killing rate. Notably, though, CD22 CART generated from both selection protocols efficiently eradicated leukemia in NSG mice, with negatively selected cells exhibiting a significant enrichment in γδ CD22 CART. Thus, our study demonstrates the importance of the initial T cell selection process in clinical CART manufacturing.

Turning the tide: From fibrosis to regeneration following anti-fibrogenic cell vaccination.

Fibrosis, the pathological end stage of chronic inflammatory diseases, results from extracellular matrix deposition by fibrogenic fibroblasts. In this issue of Cell Stem Cell, Sobecki et al. (2022) develop a novel vaccination-based immunotherapy against fibrogenic progenitor-restricted antigens, leading to the regression of fibrosis in concert with liver and lung regeneration.

IL-9 Abrogates the Metastatic Potential of Breast Cancer by Controlling Extracellular Matrix Remodeling and Cellular Contractility.

IL-9 is produced by Th9 cells and is classically known as a growth-promoting cytokine. Although protumorigenic functions of IL-9 are described in T cell lymphoma, recently, we and others have reported anti-tumor activities of IL-9 in melanoma mediated by mast cells and CD8+ T cells. However, involvement of IL-9 in invasive breast and cervical cancer remains unexplored. In this study, we demonstrate IL-9-dependent inhibition of metastasis of both human breast (MDA-MB-231 and MCF-7) and cervical (HeLa) tumor cells in physiological three-dimensional invasion assays. To dissect underlying mechanisms of IL-9-mediated suppression of invasion, we analyzed IL-9-dependent pathways of cancer cell metastasis, including proteolysis, contractility, and focal adhesion dynamics. IL-9 markedly blocked tumor cell-collagen degradation, highlighting the effects of IL-9 on extracellular matrix remodeling. Moreover, IL-9 significantly reduced phosphorylation of myosin L chain and resultant actomyosin contractility and also increased focal adhesion formation. Finally, IL-9 suppressed IL-17- and IFN-γ-induced metastasis of both human breast (MDA-MB-231) and cervical (HeLa) cancer cells. In conclusion, IL-9 inhibits the metastatic potential of breast and cervical cancer cells by controlling extracellular matrix remodeling and cellular contractility.

Robust Antitumor Activity and Low Cytokine Production by Novel Humanized Anti-CD19 CAR T Cells.

Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop life-threatening toxicities associated with cytokine release syndrome which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR T cells by humanizing the framework region of single-chain variable fragment (scFv) derived from a murine FMC63 mAb and combining it with CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain (h1CAR19-8BBζ). Docking studies followed by molecular dynamics simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared with murine scFv (mCAR19). Ex vivo studies with CAR T cells generated from healthy donors and patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable antitumor activity and proliferation. More importantly, h1CAR19-8BBζ T cells produced lower levels of cytokines (IFNγ, TNFα) upon antigen encounter and reduced the induction of IL6 cytokine from monocytes than mCAR19-8BBζ T cells. There was a comparable proliferation of h1CAR19-8BBζ T cells and mCAR19-8BBζ T cells upon repeated antigen encounter. Finally, h1CAR19-8BBζ T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.

The Th9 Axis Reduces the Oxidative Stress and Promotes the Survival of Malignant T Cells in Cutaneous T-Cell Lymphoma Patients.

Immune dysfunction is critical in pathogenesis of cutaneous T-cell lymphoma (CTCL). Few studies have reported abnormal cytokine profile and dysregulated T-cell functions during the onset and progression of certain types of lymphoma. However, the presence of IL9-producing Th9 cells and their role in tumor cell metabolism and survival remain unexplored. With this clinical study, we performed multidimensional blood endotyping of CTCL patients before and after standard photo/chemotherapy and revealed distinct immune hallmarks of the disease. Importantly, there was a higher frequency of "skin homing" Th9 cells in CTCL patients with early (T1 and T2) and advanced-stage disease (T3 and T4). However, advanced-stage CTCL patients had severely impaired frequency of skin-homing Th1 and Th17 cells, indicating attenuated immunity. Treatment of CTCL patients with standard photo/chemotherapy decreased the skin-homing Th9 cells and increased the Th1 and Th17 cells. Interestingly, T cells of CTCL patients express IL9 receptor (IL9R), and there was negligible IL9R expression on T cells of healthy donors. Mechanistically, IL9/IL9R interaction on CD3+ T cells of CTCL patients and Jurkat cells reduced oxidative stress, lactic acidosis, and apoptosis and ultimately increased their survival. In conclusion, coexpression of IL9 and IL9R on T cells in CTCL patients indicates the autocrine-positive feedback loop of Th9 axis in promoting the survival of malignant T cells by reducing the oxidative stress. IMPLICATIONS: The critical role of Th9 axis in CTCL pathogenesis indicates that strategies targeting Th9 cells might harbor significant potential in developing robust CTCL therapy.

Hydrogel scaffold with substrate elasticity mimicking physiological-niche promotes proliferation of functional keratinocytes.

High numbers of autologous human primary keratinocytes (HPKs) are required for patients with burns, wounds and for gene therapy of skin disorders. Although freshly isolated HPKs exhibit a robust regenerative capacity, traditional methodology fails to provide a sufficient number of cells. Here we demonstrated a well characterized, non-cytotoxic and inert hydrogel as a substrate that mimics skin elasticity, which can accelerate proliferation and generate higher numbers of HPKs compared to existing tissue culture plastic (TCP) dishes. More importantly, this novel method was independent of feeder layer or any exogenous pharmaceutical drug. The HPKs from the hydrogel-substrate were functional as demonstrated by wound-healing assay, and the expression of IFN-γ-responsive genes (CXCL10, HLADR). Importantly, gene delivery efficiency by a lentiviral based delivery system was significantly higher in HPKs cultured on hydrogels compared with TCP. In conclusion, our study provides the first evidence that cell-material mechanical interaction is enough to provide a rapid expansion of functional keratinocytes that might be used as autologous grafts for skin disorders.

Corrigendum: Lymphocytes in Cellular Therapy: Functional Regulation of CAR T Cells.

[This corrects the article DOI: 10.3389/fimmu.2018.03180.].

Differential Influence of IL-9 and IL-17 on Actin Cytoskeleton Regulates the Migration Potential of Human Keratinocytes.

T cells mediate skin immune surveillance by secreting specific cytokines and regulate numerous functions of keratinocytes, including migration during homeostasis and disease pathogenesis. Keratinocyte migration is mediated mainly by proteolytic cleavage of the extracellular matrix and/or by cytoskeleton reorganization. However, the cross-talk between T cell cytokines and actomyosin machinery of human primary keratinocytes (HPKs), which is required for cytoskeleton reorganization and subsequent migration, remains poorly examined. In this study, we describe that IL-9 profoundly reduced the actin stress fibers, inhibited contractility, and reduced the cortical stiffness of HPKs, which resulted in inhibition of the migration potential of HPKs in an adhesion- and MMP-independent manner. Similarly, IL-9 inhibited the IFN-γ-induced migration of HPKs by inhibiting the actomyosin machinery (actin stress fibers, contractility, and stiffness). IL-17A increased the actin stress fibers, promoted cellular contractility, and increased proteolytic collagen degradation, resulting in increased migration potential of HPKs. However, IL-9 inhibited the IL-17A-mediated HPKs migration. Mechanistically, IL-9 inhibited the IFN-γ- and IL-17A-induced phosphorylation of myosin L chain in HPKs, which is a major regulator of the actomyosin cytoskeleton. Finally, in addition to HPKs, IL-9 inhibited the migration of A-431 cells (epidermoid carcinoma cells) induced either by IFN-γ or IL-17A. In conclusion, our data demonstrate the influence of T cell cytokines in differentially regulating the actomyosin cytoskeleton and migration potential of human keratinocytes, which may have critical roles in skin homeostasis and pathogenesis of inflammatory diseases as well as skin malignancies.

Lymphocytes in Cellular Therapy: Functional Regulation of CAR T Cells.

Lymphocytes especially autologous T cells have been used for the treatment of numerous indications including cancers, autoimmune disorders and infectious diseases. Very recently, FDA approved Chimeric Antigen Receptor T cells (CAR T cells) therapy for relapse and refractory CD19+ B cell acute lymphoblastic leukemia (r/r B-ALL) and r/r diffuse large B cell lymphoma (r/r DLBCL) upon their remarkable success in multiple Phase I-II clinical trials. While CAR T cells are considered as major breakthrough in the field of cancer immunotherapy, the regulation of CAR T cells remains poorly understood. In this review we will discuss the strategies that regulate the CAR T cells efficacy and persistence with focus on roles of different structural component of CAR construct. Different domains of CAR construct, for example, antigen binding domain, hinge, transmembrane, and signaling domain as well as immune-regulatory cytokines have significant impact on CAR T cell efficacy. Finally, this review will highlight the strategies that will promote CAR T cells efficacy and will reduce the toxicity.

B16 Lung Melanoma Model to Study the Role of Th9 Cells in Cancer.

T cell therapy has shown remarkable promise in multiple malignancies including melanoma and Acute Lymphoblastic Leukemia (ALL). Recent data demonstrated the differential efficacy of various subsets of T-helper cells in tumor regression. Th9 cells, the new member of T helper cell family, possess superior anti-tumor activity compared to Th1 and Th2 cells in murine model of melanoma. Therefore, it is important to examine the anti-tumor activity of specific subsets of Th cells in tumor models. Here, we describe the methodology of examining the anti-tumor activity of Th9 cells in murine model of melanoma.

Microdeletions on 6p22.3 are associated with mesomelic dysplasia Savarirayan type.

INTRODUCTION: Mesomelic dysplasias are a group of skeletal disorders characterised by shortness of the middle limb segments (mesomelia). They are divided into 11 different categories. Among those without known molecular basis is mesomelic dysplasia Savarirayan type, characterised by severe shortness of the middle segment of the lower limb. OBJECTIVE: To identify the molecular cause of mesomelic dysplasia Savarirayan type. METHODS AND RESULTS: We performed array comparative genomic hybridisation in three unrelated patients with mesomelic dysplasia Savarirayan type and identified 2 Mb overlapping de novo microdeletions on chromosome 6p22.3. The deletions encompass four known genes: MBOAT1, E2F3, CDKAL1 and SOX4. All patients showed mesomelia of the lower limbs with hypoplastic tibiae and fibulae. We identified a fourth patient with intellectual disability and an overlapping slightly larger do novo deletion also encompassing the flanking gene ID4. Given the fact that the fourth patient had no skeletal abnormalities and none of the genes in the deleted interval are known to be associated with abnormalities in skeletal development, other mutational mechanisms than loss of function of the deleted genes have to be considered. Analysis of the genomic region showed that the deletion removes two regulatory boundaries and brings several potential limb enhancers into close proximity of ID4. Thus, the deletion could result in the aberrant activation and misexpression of ID4 in the limb bud, thereby causing the mesomelic dysplasia. CONCLUSIONS: Our data indicate that the distinct deletion 6p22.3 is associated with mesomelic dysplasia Savarirayan type featuring hypoplastic, triangular-shaped tibiae and abnormally shaped or hypoplastic fibulae.

Deletion of 16q24.1 supports a role for the ATP2C2 gene in specific language impairment.

A 10-year-old boy presented with a history of significant delay in language acquisition as well as receptive and expressive language impairment that persisted into elementary school. In school, he exhibited difficulty with reading comprehension, telling and understanding narratives, and making inferences. Other aspects of his neurodevelopment were normal, with no history of significant medical concerns. He did not have hearing impairment, oromotor dysfunction, or specific neurologic abnormalities. He did not meet testing criteria for autism. Chromosomal microarray analysis and quantitative polymerase chain reaction determined that he had a de novo 159-kilobase deletion of chromosome 16q24.1 that included the ATP2C2 gene. ATP2C2 is a known candidate gene for specific language impairment and is postulated to have neurobiological significance in memory-related circuits. Our patient's language deficits were consistent with a global type of specific language impairment impacting language comprehension, formulation, semantics, syntax, and phonology attributed to his de novo chromosome deletion.

Clinical utility of chromosomal microarray analysis in the diagnosis and management of monosomy 7 mosaicism.

There have been dramatic improvements in our ability to more accurately diagnose the underlying genetic causes of developmental delay/intellectual disability; however, there is less known about the treatment trajectory and whether or not patient management and outcomes have changed due to the information gained from genetic testing. Here we report a case study of a 20-month-old male first referred to the genetics clinic in 2008 for interhemispheric cysts, agenesis of the corpus callosum, left cortical dysplasia, and developmental delay of unknown etiology. The diagnostic work-up for this patient included chromosomal microarray which detected >20% mosaicism for monosomy 7, which raised concern for a possible myelodysplastic syndrome. The clone was not detected in stimulated peripheral blood cultures and his karyotype was reported as a normal male. Because of this microarray finding, he was referred to pediatric hematology/oncology where he was confirmed to have a pre-symptomatic diagnosis of myelodysplastic syndrome and was treated with chemotherapy and a bone-marrow transplant. This case illustrates the clinical utility of microarray testing and the importance of long-term follow-up to assess patient outcomes.

Neurodevelopmental delays and macrocephaly in 17p13.1 microduplication syndrome.

Microduplication of chromosome 17p13.1 is a rarely reported chromosome abnormality associated with neurodevelopmental delays. We describe two unrelated patients with overlapping microduplications of chromosome 17p13.1. The first patient is a 2-year-old male who presented with neurodevelopmental delays and macrocephaly. He was found to have a de novo 788 kb copy gain of 17p13.2p13.1 and a de novo 134 kb copy gain of 17p13.1. These duplications include multiple candidate genes, including EFNB3, NLGN2, DLG4, GABARAP, and DULLARD, which may be responsible for neurodevelopmental delays in affected individuals. The second patient is a 29-year-old female with mild intellectual disability and relative macrocephaly. She was found to have a 62.5 kb copy gain of chromosome 17p13.1 that includes the DLG4, GABARAP, and DULLARD genes. The DLG4, GABARAP, and DULLARD genes included in the microduplications of both our patients appear to be candidate genes for neurodevelopmental delays and macrocephaly in individuals with 17p13.1 microduplication syndrome.

The historical Coffin-Lowry syndrome family revisited: identification of two novel mutations of RPS6KA3 in three male patients.

Coffin-Lowry syndrome (CLS) is a rare X-linked dominant disorder characterized by intellectual disability, craniofacial abnormalities, short stature, tapering fingers, hypotonia, and skeletal malformations. CLS is caused by mutations in the Ribosomal Protein S6 Kinase, 90 kDa, Polypeptide 3 (RPS6KA3) gene located at Xp22.12, which encodes Ribosomal S6 Kinase 2 (RSK2). Here we analyzed RPS6KA3 in three unrelated CLS patients including one from the historical Coffin-Lowry syndrome family and found two novel mutations. To date, over 140 mutations in RPS6KA3 have been reported. However, the etiology of the very first familial case, which was described in 1971 by Lowry with detailed phenotype and coined the term CLS, has remained unknown. More than 40 years after the report, we succeeded in identifying deposited fibroblast cells from one patient of this historic family and found a novel heterozygous 216 bp in-frame deletion, encompassing exons 15 and 16 of RPS6KA3. Drop episodes in CLS patients were reported to be associated with truncating mutations deleting the C-terminal kinase domain (KD), and only one missense mutation and one single basepair duplication involving the C-terminal KD of RSK2 in the patients with drop episode have been reported thus far. Here we report the first in-frame deletion in C-terminal KD of RPS6KA3 in a CLS patient with drop episodes.

Acquired microcephaly in blepharophimosis-ptosis-epicanthus inversus syndrome because of an interstitial 3q22.3q23 deletion.

BACKGROUND: Blepharophimosis-ptosis-epicanthus inversus syndrome is an autosomal dominant condition because of mutations or deletions of the FOXL2 gene. Microcephaly is not associated with FOXL2 mutations but has been reported in individuals with chromosome 3q deletions, which include the FOXL2 gene and other contiguous genes. The ATR gene has been reported as a candidate gene for microcephaly in individuals with contiguous deletion of chromosome 3q involving the FOXL2 gene. PATIENT: We describe a girl with blepharophimosis-ptosis-epicanthus inversus syndrome along with acquired microcephaly and intellectual disability. RESULTS: Our patient had a deletion of chromosome 3q22.2q23, which does not include the ATR gene but does include the PIK3CB gene as a candidate gene for microcephaly. CONCLUSION: We propose that the PIK3CB gene included in our patient's chromosome 3q deletion may be the gene responsible for microcephaly and other patients with blepharophimosis-ptosis-epicanthus inversus syndrome because of a chromosome 3q deletion.

Clinical and genomic evaluation of 201 patients with Phelan-McDermid syndrome.

This study is the first to describe age-related changes in a large cohort of patients with Phelan-McDermid syndrome (PMS), also known as 22q13 deletion syndrome. Over a follow-up period of up to 12 years, physical examinations and structured interviews were conducted for 201 individuals diagnosed with PMS, 120 patients had a focused, high-resolution 22q12q13 array CGH, and 92 patients' deletions were assessed for parent-of-origin. 22q13 genomic anomalies include terminal deletions of 22q13 (89 %), terminal deletions and interstitial duplications (9 %), and interstitial deletions (2 %). Considering different age groups, in older patients, behavioral problems tended to subside, developmental abilities improved, and some features such as large or fleshy hands, full or puffy eyelids, hypotonia, lax ligaments, and hyperextensible joints were less frequent. However, the proportion reporting an autism spectrum disorder, seizures, and cellulitis, or presenting with lymphedema or abnormal reflexes increased with age. Some neurologic and dysmorphic features such as speech and developmental delay and macrocephaly correlated with deletion size. Deletion sizes in more recently diagnosed patients tend to be smaller than those diagnosed a decade earlier. Seventy-three percent of de novo deletions were of paternal origin. Seizures were reported three times more often among patients with a de novo deletion of the maternal rather than paternal chromosome 22. This analysis improves the understanding of the clinical presentation and natural history of PMS and can serve as a reference for the prevalence of clinical features in the syndrome.

22q13.2q13.32 genomic regions associated with severity of speech delay, developmental delay, and physical features in Phelan-McDermid syndrome.

PURPOSE: Phelan-McDermid syndrome is a developmental disability syndrome with varying deletions of 22q13 and varying clinical severity. We tested the hypothesis that, in addition to loss of the telomeric gene SHANK3, specific genomic regions within 22q13 are associated with important clinical features. METHODS: We used a customized oligo array comparative genomic hybridization of 22q12.3-terminus to obtain deletion breakpoints in a cohort of 70 patients with terminal 22q13 deletions. We used association and receiver operating characteristic statistical methods in a novel manner and also incorporated protein interaction networks to identify 22q13 genomic locations and genes associated with clinical features. RESULTS: Specific genomic regions and candidate genes within 22q13.2q13.32 were associated with severity of speech/language delay, neonatal hypotonia, delayed age at walking, hair-pulling behaviors, male genital anomalies, dysplastic toenails, large/fleshy hands, macrocephaly, short and tall stature, facial asymmetry, and atypical reflexes. We also found regions suggestive of a negative association with autism spectrum disorders. CONCLUSION: This work advances the field of research beyond the observation of a correlation between deletion size and phenotype and identifies candidate 22q13 loci, and in some cases specific genes, associated with singular clinical features observed in Phelan-McDermid syndrome. Our statistical approach may be useful in genotype-phenotype analyses for other microdeletion or microduplication syndromes.

Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders.

Autism spectrum disorders (ASDs) include three main conditions: autistic disorder (AD), pervasive developmental disorder, not otherwise specified (PDD-NOS), and Asperger syndrome. It has been shown that many genes associated with ASDs are involved in the neuroligin-neurexin interaction at the glutamate synapse: NLGN3, NLGN4, NRXN1, CNTNAP2, and SHANK3. We screened this last gene in two cohorts of ASD patients (133 patients from US and 88 from Italy). We found 5/221 (2.3%) cases with pathogenic alterations: a 106 kb deletion encompassing the SHANK3 gene, two frameshift mutations leading to premature stop codons, a missense mutation (p.Pro141Ala), and a splicing mutation (c.1820-4 G>A). Additionally, in 17 patients (7.7%) we detected a c.1304+48C>T transition affecting a methylated cytosine in a CpG island. This variant is reported as SNP rs76224556 and was found in both US and Italian controls, but it results significantly more frequent in our cases than in the control cohorts. The variant is also significantly more common among PDD-NOS cases than in AD cases. We also screened this gene in an independent replication cohort of 104 US patients with ASDs, in which we found a missense mutation (p.Ala1468Ser) in 1 patient (0.9%), and in 8 patients (7.7%) we detected the c.1304+48C>T transition. While SHANK3 variants are present in any ASD subtype, the SNP rs76224556 appears to be significantly associated with PDD-NOS cases. This represents the first evidence of a genotype-phenotype correlation in ASDs and highlights the importance of a detailed clinical-neuropsychiatric evaluation for the effective genetic screening of ASD patients.