Six induced pluripotent stem cell lines from fibroblasts of individuals with CLN3-related conditions.

Primary fibroblasts from six individuals with CLN3-related conditions were used to generate induced pluripotent stem cell (iPSC) lines CHDTRi001-B, CHDTRi002-B, CHDTRi003-A, CHDTRi004-B, CHDTRi005-A, and CHDTRi006-E through the expression of four reprogramming factors: human OCT3/4, KLF4, SOX2, and c-MYC. The iPSC lines were characterized to confirm their pluripotency via immunocytochemistry, flow cytometry, and teratoma formation. Genomic stability, cell line identity, and CLN3 genotype were confirmed. These iPSC lines may be used as participant-derived experimental models for further investigation of CLN3, a rare, fatal, pediatric, blindness and neurodegenerative lysosomal disorder with no cure.

Immune Checkpoint Inhibition-related Neuroinflammation Disrupts Cognitive Function.

Combinatorial blockade of Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and Programmed Cell Death Protein 1 (PD-1) significantly improve the progression-free survival of individuals with metastatic cancers, including melanoma. In addition to unleashing anti-tumor immunity, combination immune checkpoint inhibition (ICI) disrupts immune-regulatory networks critical for maintaining homeostasis in various tissues, including the central nervous system (CNS). Although ICI- and cancer-related cognitive impairments (CRCI) in survivors are increasingly becoming evident, our understanding of ICI-induced immune-related adverse effects (IREA) in the CNS remains incomplete. Here, our murine melanoma model reveals that combination ICI impairs hippocampal-dependent learning and memory, as well as memory consolidation processes. Mechanistically, combination ICI disrupted synaptic integrity, and neuronal plasticity, reduced myelin, and further predisposed CNS for exaggerated experimental autoimmune encephalomyelitis. Combination ICI substantially altered both lymphoid and myeloid cells in the CNS. Neurogenesis was unaffected, however, microglial activation persisted for two-months post- ICI, concurrently with cognitive deficits, which parallels clinical observations in survivors. Overall, our results demonstrate that blockade of CTLA-4 and PD-1 alters neuro-immune homeostasis and activates microglia, promoting long-term neurodegeneration and driving cognitive impairments. Therefore, limiting microglial activation is a potential avenue to mitigate CNS IRAE while maintaining the therapeutic benefits of rapidly evolving ICIs and their combinations. SIGNIFICANCE: Despite the superior therapeutic efficacy of immune checkpoint inhibition (ICI) for cancers, its undesired effects on brain function are not fully understood. Here, we demonstrate that combination ICI elevates neuroinflammation, activates microglia, leading to detrimental neurodegenerative and neurocognitive sequelae.

Complement C5a receptor 1 blockade reverses cognitive deficits following cranial radiation therapy for brain cancer.

Cranial radiation therapy (RT) for brain cancers leads to an irreversible decline in cognitive function without an available remedy. Radiation-induced cognitive deficits (RICD) are particularly a pressing problem for the survivors of pediatric and low grade glioma (LGG) patients who often live long post-RT. Radiation-induced elevated neuroinflammation and gliosis, triggered by the detrimental CNS complement cascade, lead to excessive synaptic and cognitive loss. Using intact and brain cancer-bearing mouse models, we now show that targeting anaphylatoxin complement C5a receptor (C5aR1) is neuroprotective against RICD. We used a genetic knockout, C5aR1 KO mouse, and a pharmacologic approach, employing the orally active, brain penetrant C5aR1 antagonist PMX205, to reverse RICD. Irradiated C5aR1 KO and WT mice receiving PMX205 showed significant neurocognitive improvements in object recognition memory and memory consolidation tasks. C5aR1 inhibition reduced microglial activation, astrogliosis, and synaptic loss in the irradiated brain. Importantly, C5aR1 inhibition in the syngeneic, orthotopic astrocytoma, and glioblastoma-bearing mice protected against RICD without interfering with the therapeutic efficacy of RT to reduce tumor volume in vivo . PMX205 is currently in clinical trials for amyotrophic lateral sclerosis (ALS). Thus, C5aR1 inhibition is a translationally feasible approach to address RICD, an unmet medical need.

CD81 and Its Relationship to Treatment Response in Patients With Acute Myeloid Leukemia at a Hospital in Hanoi, Vietnam.

BACKGROUND: Acute myeloid leukemia (AML) has the proliferation of poorly differentiated immature myeloid cells. New studies on immune markers also consider them as one of the factors that affect the prognosis or the patient's ability to respond to drugs. Our study was designed to determine the rate of remission and mortality, and the ability to respond to drugs in newly diagnosed AML patients with positive CD81. METHODS: A total of 50 patients diagnosed with AML (excluding acute promyelocytic leukemia) underwent immunophenotyping analysis using flow cytometry. Following the initial diagnosis, the patients received induction therapy, followed by three cycles of consolidation therapy. The patients were then followed up for a period of six months. The treatment efficacy was assessed at two timepoints: on day 28 after the first chemotherapy course and on day 28 after the fourth chemotherapy course. RESULTS: Out of the 50 newly diagnosed AML patients, 40 (80%) were found to be CD81 positive. This CD81-positive group had a high mortality rate after the first course of chemotherapy (17.5%) and after the fourth course of chemotherapy (52.5%), while no patients died in the CD81-negative group. The CD81-positive group had a worse drug response rate with 22.5% and 18.2% in CD81 positive group versus 30% and 40% in the CD81-negative group achieving complete remission after the first course and fourth course, respectively. CONCLUSIONS:  The CD81 immunological marker was found to be highly prevalent among AML patients in Vietnam. Overexpression of CD81 in patients with AML is associated with an unfavorable prognosis, characterized by higher mortality rates and poorer treatment response.

A Case of Atypical Hairy Cell Leukemia With CD10+ and CD38+: Diagnosis and Treatment.

Hairy cell leukemia (HCL) is a rare disease of mature B-cell neoplasms. Its name comes from the hair-like strands surrounding the cytoplasm of the cells, which are observed on peripheral blood or bone marrow smears. Leukemic cells mainly involve the spleen, peripheral blood, and bone marrow. The classical immunophenotyping of HCL includes overexpression of the B-cell surface antigens such as CD19, CD20, and CD22 and co-expression of CD25, CD103, CD11c, and CD123. Other markers including CD5, CD10, and CD38 are usually negative, in which CD38 is considered a poor prognostic factor. Herein, we report a case of HCL with atypical morphology and abnormal expression of both CD38 and CD10.

Flow-Cytometry in the Diagnosis of Diffuse Large B-Cell Lymphoma Based on Stomach Tissue Samples: A Case Report.

Morphology and immunohistochemistry on node, tissue, and bone marrow biopsies are frequently used in lymphoma diagnosis to characterize the stage and subtype of diseases. Multicolor flow cytometry technology is a novel technique for the analysis of immunological markers to identify lymphoma on fresh tissue when immunohistochemical staining is ambiguous. We report a case of a patient diagnosed with diffuse large B-cell lymphoma by flow cytometry on a stomach tissue biopsy.

CD38 - Negative Anaplastic Plasma Cell Myeloma: A Rare Case Report.

CD38 is a glycoprotein that is highly and uniformly expressed in plasma cells in multiple myeloma. A panel of CD38 and CD138/CD19/CD45/CD56/CD117 markers is considered the immunophenotypic diagnosis of plasma cell myeloma. Expression of the CD38 marker may fade or weaken compared with the CD138 marker in plasma cells after chemotherapy treatment. Herein we present a rare case of CD38-negative multiple myeloma that was initially misdiagnosed as acute leukemia.

Bimodal expression of potential drug target CLL-1 (CLEC12A) on CD34+ blasts of AML patients.

OBJECTIVES: This study aims to retrospectively assess C-lectin-like molecule 1 (CLL-1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL-1 bimodal associations with leukemia and patient-specific characteristics. METHODS: Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL-1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL-1-negative subpopulation of CLL-1 bimodal AML samples. RESULTS: The frequency of a bimodal pattern of CLL-1 expression of CD34+ blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL-1 expression was most prevalent in patients with MDS-related AML (P = .011), ELN adverse risk (P = .002), NPM1 wild type (WT, P = .049), FLT3 WT (P = .035), and relatively low percentages of leukemia-associated immunophenotypes (P = .006). Additional immunophenotyping analysis revealed the CLL-1- subpopulation may consist of pre-B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)-leukemic mutations were detected in both CLL-1+ and CLL-1- subfractions of bimodal samples (N = 3). CONCLUSIONS: C-lectin-like molecule 1 bimodality occurs in about 25% of AML patients and the CLL-1- cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL-1-targeted therapies and warrant further investigation.

Venetoclax Increases Intratumoral Effector T Cells and Antitumor Efficacy in Combination with Immune Checkpoint Blockade.

The antiapoptotic protein BCL2 plays critical roles in regulating lymphocyte development and immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL2 is critical for antitumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL2, would influence the antitumor activity of immune checkpoint inhibitors (ICI). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the antitumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T-cell function in response to antigen stimuli in vitro and did not antagonize T-cell activation induced by anti-PD-1. Furthermore, we demonstrate that the antiapoptotic family member BCL-XL provides a survival advantage in effector T cells following inhibition of BCL2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy. SIGNIFICANCE: The antiapoptotic oncoprotein BCL2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development, and immune system regulation. Here we demonstrate that venetoclax, the first FDA/European Medicines Agency-approved BCL2 inhibitor, unexpectedly can be combined preclinically with immune checkpoint inhibitors to enhance anticancer immunotherapy, warranting clinical evaluation of these combinations.This article is highlighted in the In This Issue feature, p. 1.

BCL2 Expression in First-Line Diffuse Large B-Cell Lymphoma Identifies a Patient Population With Poor Prognosis.

INTRODUCTION: The prognostic value of B-cell lymphoma 2 (BCL2) expression in de novo diffuse large B-cell lymphoma (DLBCL) treated with immunochemotherapy is of interest to define a target patient population for clinical development of BCL2 inhibitors. We aimed to develop a reproducible immunohistochemistry algorithm and assay to determine BCL2 protein expression and assess the prognostic value of BCL2 in newly diagnosed DLBCL cohorts. PATIENTS AND METHODS: The prospectively defined algorithm incorporated BCL2 staining intensity and percentage of BCL2-positive cells. Functionally relevant cutoffs were based on the sensitivity of lymphoma cell lines to venetoclax. This assay was highly reproducible across laboratories. The prognostic impact of BCL2 expression was assessed in DLBCL patients from the phase 3 MAIN (n = 230) and GOYA (n = 366) trials, and a population-based registry (n = 310). RESULTS: Approximately 50% of tumors were BCL2 positive, with a higher frequency in high International Prognostic Index (IPI) and activated B-cell-like DLBCL subgroups. BCL2 expression was associated with poorer progression-free survival in the MAIN study (hazard ratio [HR], 1.66; 95% confidence interval [CI], 0.81-3.40; multivariate Cox regression adjusted for IPI and cell of origin). This trend was confirmed in the GOYA and registry cohorts in adjusted multivariate analyses (GOYA: HR, 1.72; 95% CI, 1.05-2.82; registry: HR, 1.89; 95% CI, 1.29-2.78). Patients with BCL2 immunohistochemistry-positive and IPI-high disease had the poorest prognosis: 3-year progression-free survival rates were 51% (GOYA) and 37% (registry). CONCLUSION: Findings support use of our BCL2 immunohistochemistry scoring system and assay to select patients with BCL2-positive tumors for future studies.

Genomic Analysis of Circulating Tumor Cells at the Single-Cell Level.

Circulating tumor cells (CTCs) have a great potential for noninvasive diagnosis and real-time monitoring of cancer. A comprehensive evaluation of four whole genome amplification (WGA)/next-generation sequencing workflows for genomic analysis of single CTCs, including PCR-based (GenomePlex and Ampli1), multiple displacement amplification (Repli-g), and hybrid PCR- and multiple displacement amplification-based [multiple annealing and loop-based amplification cycling (MALBAC)] is reported herein. To demonstrate clinical utilities, copy number variations (CNVs) in single CTCs isolated from four patients with squamous non-small-cell lung cancer were profiled. Results indicate that MALBAC and Repli-g WGA have significantly broader genomic coverage compared with GenomePlex and Ampli1. Furthermore, MALBAC coupled with low-pass whole genome sequencing has better coverage breadth, uniformity, and reproducibility and is superior to Repli-g for genome-wide CNV profiling and detecting focal oncogenic amplifications. For mutation analysis, none of the WGA methods were found to achieve sufficient sensitivity and specificity by whole exome sequencing. Finally, profiling of single CTCs from patients with non-small-cell lung cancer revealed potentially clinically relevant CNVs. In conclusion, MALBAC WGA coupled with low-pass whole genome sequencing is a robust workflow for genome-wide CNV profiling at single-cell level and has great potential to be applied in clinical investigations. Nevertheless, data suggest that none of the evaluated single-cell sequencing workflows can reach sufficient sensitivity or specificity for mutation detection required for clinical applications.

A benchtop system to assess the feasibility of a fully independent and implantable brain-machine interface.

OBJECTIVE: State-of-the-art invasive brain-machine interfaces (BMIs) have shown significant promise, but rely on external electronics and wired connections between the brain and these external components. This configuration presents health risks and limits practical use. These limitations can be addressed by designing a fully implantable BMI similar to existing FDA-approved implantable devices. Here, a prototype BMI system whose size and power consumption are comparable to those of fully implantable medical devices was designed and implemented, and its performance was tested at the benchtop and bedside. APPROACH: A prototype of a fully implantable BMI system was designed and implemented as a miniaturized embedded system. This benchtop analogue was tested in its ability to acquire signals, train a decoder, perform online decoding, wirelessly control external devices, and operate independently on battery. Furthermore, performance metrics such as power consumption were benchmarked. MAIN RESULTS: An analogue of a fully implantable BMI was fabricated with a miniaturized form factor. A patient undergoing epilepsy surgery evaluation with an electrocorticogram (ECoG) grid implanted over the primary motor cortex was recruited to operate the system. Seven online runs were performed with an average binary state decoding accuracy of 87.0% (lag optimized, or 85.0% at fixed latency). The system was powered by a wirelessly rechargeable battery, consumed ∼150 mW, and operated for >60 h on a single battery cycle. SIGNIFICANCE: The BMI analogue achieved immediate and accurate decoding of ECoG signals underlying hand movements. A wirelessly rechargeable battery and other supporting functions allowed the system to function independently. In addition to the small footprint and acceptable power and heat dissipation, these results suggest that fully implantable BMI systems are feasible.

Electrocorticographic Activity of the Brain During Micturition.

Current therapies for neurogenic bladder do not allow spinal cord injury patients to regain conscious control of urine storage or voiding. Novel neural technologies may provide means to improve or restore the connection between the brain and the bladder; however, the specific brain areas and their underlying neural activities responsible for micturition must be better understood in order to design such technologies. In this retrospective study, we analyzed electrocorticographic (ECoG) data obtained from epilepsy patients who underwent ECoG grid implantation for epilepsy surgery evaluation, in the hopes of determining specific electrophysiological activity associated with micturition. Our results indicate modulation of the delta (δ, 0.1-4 Hz) and low-gamma (\gamma, 25-50 Hz) activity in the peri-Sylvian area and the inferior temporal lobe. These findings suggest involvement of the insular cortex and the uncinate fasciculus in micturition, important structures related to sensation and decision making. To date, this is the first known study utilizing ECoG data to elucidate the electrophysiological activity of the brain associated with bladder control and sensation.

Spontaneously regressing brain lesions in Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a metabolic disorder caused by an inborn error of cholesterol synthesis that affects the development of many organ systems. Malformations in the central nervous system typically involve midline structures and reflect abnormal growth and differentiation of neurons and supporting cells. Despite these defects in central nervous system development, brain tumor formation has only rarely been reported in association with SLOS. We present three individuals with SLOS and lesions in the basal ganglia or brainstem detected by MRI that were concerning for tumor formation. However, the individuals' clinical and neurological course remained stable, and the lesions regressed after several years. These lesions have similarities to spongiotic changes observed in individuals with neurofibromatosis type 1 (NF1). Notably, impaired activity of small GTPases is present in both SLOS and NF1, perhaps giving mechanistic insight into the formation of these lesions.

Characterization of electrocorticogram high-gamma signal in response to varying upper extremity movement velocity.

The mechanism by which the human primary motor cortex (M1) encodes upper extremity movement kinematics is not fully understood. For example, human electrocorticogram (ECoG) signals have been shown to modulate with upper extremity movements; however, this relationship has not been explicitly characterized. To address this issue, we recorded high-density ECoG signals from patients undergoing epilepsy surgery evaluation as they performed elementary upper extremity movements while systematically varying movement speed and duration. Specifically, subjects performed intermittent pincer grasp/release, elbow flexion/extension, and shoulder flexion/extension at slow, moderate, and fast speeds. In all movements, bursts of power in the high-[Formula: see text] band (80-160 Hz) were observed in M1. In addition, the amplitude of these power bursts and the area of M1 with elevated high-[Formula: see text] activity were directly proportional to the movement speed. Likewise, the duration of elevated high-[Formula: see text] activity increased with movement duration. Based on linear regression, M1 high-[Formula: see text] power amplitude and duration covaried with movement speed and duration, respectively, with an average [Formula: see text] of [Formula: see text] and [Formula: see text]. These findings indicate that the encoding of upper extremity movement speed by M1 high-[Formula: see text] activity is primarily linear. Also, the fact that this activity remained elevated throughout a movement suggests that M1 does not merely generate transient instructions for a specific movement duration, but instead is responsible for the entirety of the movement. Finally, the spatial distribution of high-[Formula: see text] activity suggests the presence of a recruitment phenomenon in which higher speeds or increased muscle activity involve activation of larger M1 areas.

Biomarker analyses from a placebo-controlled phase II study evaluating erlotinib±onartuzumab in advanced non-small cell lung cancer: MET expression levels are predictive of patient benefit.

PURPOSE: In a recent phase II study of onartuzumab (MetMAb), patients whose non-small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit. EXPERIMENTAL DESIGN: Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA. RESULTS: A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean≥5 copies/cell by FISH); however, benefit was maintained in "MET IHC-positive"/MET FISH-negative patients (HR, 0.37; P=0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P=0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P=0.09) in favor of onartuzumab treatment. CONCLUSIONS: MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers.

Sensitivity and specificity of upper extremity movements decoded from electrocorticogram.

Electrocorticogram (ECoG)-based brain computer interfaces (BCI) can potentially be used for control of arm prostheses. Restoring independent function to BCI users with such a system will likely require control of many degrees-of-freedom (DOF). However, our ability to decode many-DOF arm movements from ECoG signals has not been thoroughly tested. To this end, we conducted a comprehensive study of the ECoG signals underlying 6 elementary upper extremity movements. Two subjects undergoing ECoG electrode grid implantation for epilepsy surgery evaluation participated in the study. For each task, their data were analyzed to design a decoding model to classify ECoG as idling or movement. The decoding models were found to be highly sensitive in detecting movement, but not specific in distinguishing between different movement types. Since sensitivity and specificity must be traded-off, these results imply that conventional ECoG grids may not provide sufficient resolution for decoding many-DOF upper extremity movements.

High heregulin expression is associated with activated HER3 and may define an actionable biomarker in patients with squamous cell carcinomas of the head and neck.

PURPOSE: Tumors with oncogenic dependencies on the HER family of receptor tyrosine kinases (RTKs) often respond well to targeted inhibition. Our previous work suggested that many cell lines derived from squamous cell carcinomas of the head and neck (SCCHNs) depend on autocrine signaling driven by HER2/3 dimerization and high-level co-expression of HRG. Additionally, results from a Phase I trial of MEHD7495A, a dual-action antibody that blocks ligand binding to EGFR and HER3, suggest that high-level HRG expression was associated with clinical response in SCCHN patients. Here we explore the hypothesis that high-level HRG expression defines a subpopulation of SCCHNs with activated HER3. EXPERIMENTAL DESIGN: qRT-PCR expression profiling was performed on >750 tumors of diverse origin, including >150 therapy-naïve, primary, and recurrent SCCHNs. Activated HER3, defined by immunoprecipitation of phospho-HER3, was compared to HRG expression in SCCHN samples. Paracrine versus autocrine expression was evaluated using RNA-in situ hybridization. RESULTS: SCCHN tumors express the highest levels of HRG compared to a diverse collection of other tumor types. We show that high HRG expression is associated with activated HER3, whereas low HRG expression is associated with low HER3 activation in SCCHN tumors. Furthermore, HRG expression is higher in recurrent SCCHN compared to patient-matched therapy naïve specimens. CONCLUSIONS: HRG expression levels define a biologically distinct subset of SCCHN patients. We propose that high-level expression of HRG is associated with constitutive activation of HER3 in SCCHN and thus defines an actionable biomarker for interventions targeting HER3.