Peripheral Blood Samples Of Patients Research Articles

Novel associations between MTDH gene polymorphisms and invasive ductal breast cancer: a case–control study

ObjectiveTo reveal the contributing effects of MTDH gene SNPs in the risk of invasive ductal breast cancer (IDC).Patients and methodsA case–control study was conducted, recruiting a total of 300 cases of IDC and 565 cancer-free controls from East China. Genotyping of three single-nucleotide polymorphisms (SNPs) in the MTDH gene was performed. Genomic DNA was extracted from peripheral blood samples of patients. The three SNPs (rs1311 T > C, rs16896059 G > A, rs2449512 A > G) in the MTDH gene were selected for detection using a TaqMan real-time polymerase chain reaction assay. The association between MTDH and the risk of IDC was analyzed employing an epidemiology case–control study and a multinomial logistic regression model.ResultsAmong the three evaluated SNPs, rs1311 T > C, rs16896059 G > A, and rs2449512 A > G demonstrated a significant association with an increased risk of IDC. Furthermore, stratified analysis revealed that individuals carrying the rs1311 CC genotype, rs16896059 GA/AA genotypes, and rs2449512 GG genotype were more susceptible to developing IDC in subgroups of patients younger than 53 years, without family history of IDC, pre-menopause status, clinical stage 2, high grade, with no distant metastasis or invasion, Her2-positive type, ER positive, PR positive, and Ki67 cells less than 10%. However, carriers of the rs16896059 GA/AA genotypes and rs2449512 GG genotype had an elevate the risk of IDC in patients with tumor size larger than 2 cm, post-menopause status, clinical stage 3, with invasion, lymph node infiltration, ER negative, PR negative, Her2 negative, and Ki67 cells exceeding 10%. Compared to the reference haplotype TGA, haplotypes TAA, TAG, and TGG were significantly associated with an increased IDC risk.ConclusionIn this study, we demonstrated a significant association between MTDH gene polymorphisms and an increased risk of IDC. Moreover, our findings suggested that MTDH gene polymorphisms could serve as a potential biomarker for IDC subtyping and therapeutic selection.

Abstract 3352: A CRISPR-enabled function genomics profiling platform in acute leukemia: Updates from a pilot study of Function (Fx) Heme utilizing fresh patient samples

Abstract Introduction: In this study we utilized a novel primary cell CRISPR/Cas9 profiling approach to identify gene dependencies in fresh acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) patient samples. The primary aim of this prospective study was to demonstrate the feasibility of the CRISPR-based personalized functional genomic testing with fresh patient samples in the clinical setting. Methods: AML and ALL patient peripheral blood samples (10-16 mL) were obtained at time of initial diagnosis and/or at relapse, and if feasible post-treatment. Primary cells were transduced with lentivirus harboring Cas9 enzyme and a ‘drug targets’ sgRNA panel. Transduced cells were harvested at early (input) and Day 8 timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated to calculate gene-level phenotype scores, enabling identification of gene dependencies. Results: 37 patients were included in the study, 23 with AML, 9 with Philadelphia (Ph) (+) ALL, 5 with Ph (-) ALL. The majority of patients had adverse or poor risk disease. The average age of patients was 52.6, range 18-84. 23 were male, 14 female; 20 identified as white, 17 as other. 20 patients were sampled at initial diagnosis, 12 were sampled at time of relapse and 5 at time of refractory disease. Serial samples were collected from 9 patients. 34 patients received treatment following sample collection. The majority of patients received intensive chemotherapy or a hypomethylating agent with venetoclax, however 15 patients received some form of targeted therapy. At the time of the abstract submission, Fx Heme results were available for 23 patients and among these, we identified potential treatment targets in 10 samples (43%). Importantly, 60% of the positive samples have a dependency on a gene clinically targetable by an approved leukemia drug. In addition, our profiling platform enabled the discovery of novel druggable dependencies in 100% of the positive samples. Conclusions: With this pilot study we demonstrated that utilizing small volume fresh patient samples to assess CRISPR-defined gene dependencies on targeted therapies in AML and ALL is feasible, timely, and results in clear Fx Heme dependency gene output. Our data indicates that CRISPR-based comprehensive functional profiling has the potential to add valuable information beyond traditional NGS methods to determine the optimal therapy for patients with AML and ALL. Future directions include translating further into the clinical setting and correlating Fx Heme dependence with response to targeted treatment in acute leukemia patients. Citation Format: Kaitlyn C. Dykes, Tuyet R. Tan, Karl Hodel, Kathleen Tran, Maci Meyers, Leonardo Hagmann, Michele Baltay, Peter Kilfeather, Yali Li, Christian Schmedt, Srinath Sampath, Srihari Sampath, Tiffany N. Tanaka, Stephane Corbel. A CRISPR-enabled function genomics profiling platform in acute leukemia: Updates from a pilot study of Function (Fx) Heme utilizing fresh patient samples [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 3352.

Abstract 6076: Mathematical oncology in the context of early breast cancer detection

Abstract Breast cancer (BC) is the most common cancer in women globally, with approximately 94% of patients initially diagnosed with early-stage disease. However, despite the initial lack of detectable metastases and administration of subsequent treatments, 40% of these patients will develop recurrence over their lifetime. The standard screening method for BC is mammography, with a tissue biopsy to confirm diagnosis. However, only ~60% of all cases are currently diagnosed via this pathway, primarily due to the limited (86.9%) sensitivity. To demonstrate the feasibility of a liquid biopsy (LBx) test for early BC detection and assessment, we employed the third-generation, High Definition Single Cell Assay (HDSCA3.0) to detect and characterize rare cells (e.g., circulating tumor cells [CTCs]) and acellular structures (e.g., oncosomes) in patient peripheral blood samples using immunofluorescent (IF) imaging. We have previously investigated this signal in a cohort of early-stage BC patients (n = 74), late-stage BC patients (n = 26), and age- and gender-matched controls (n = 30) and observed statistically significant differences in levels of total rare cells, CTCs, and oncosomes via a manual approach. For this study, we employed a hybrid methodology, that utilizes both automatic and manual techniques, to investigate the reproducibility of the signal in an expanded validation set. Within this modified approach, we employ an outlier detection algorithm to accelerate the rare event identification and curation process (minimizing the manual portion), a separate machine learning model to classify the events based on their IF expressions, and a suite of applications to enumerate and quantify the result. After implementation, we quantified the results of the fully manual and hybrid approaches to determine the signal loss associated with scale up. Utilizing the hybrid approach, we observed similar results as with the fully manual and were able to stratify the three cohorts with high accuracy. Namely, there was an increase in the total rare cell and CTC populations in late-stage BC patients, and an increase in the oncosome population for the early-stage BC patients. To note, these patients included a subset with invasive lobular carcinoma (ILC; n = 19), which manifests in ~5-15% of BC cases and is considered “mammogram silent” due to the biological development of the disease. Additionally, employing the hybrid approach allowed for analyzing patient samples in less time while minimizing overall signal loss. In summary, we have been able to detect reproducible patterns in the enumeration of rare cells and oncosomes with high accuracy. When utilized at the patient-level, these analytes can power prediction models that are capable of stratifying disease from non-disease. The results presented here demonstrate the utility of a LBx test as a companion to screening mammography in the detection of early-stage BC across various histologies. Citation Format: Jeremy Mason, Stephanie N. Shishido, George Courcoubetis, Dean Tessone, Emmett Liljegren, Amin Naghdloo, James Hicks, Jorge J. Nieva, Peter Kuhn. Mathematical oncology in the context of early breast cancer detection [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 6076.

Decreased human leukocyte antigen-D–related expression on CD14+ monocytes in patients with out-of-hospital cardiac arrest provided target temperature management therapy: a prospective observational study

Decreased human leukocyte antigen-D–related expression on CD14+ monocytes in patients with out-of-hospital cardiac arrest provided target temperature management therapy: a prospective observational study

Peripheral Blood B-Cell Subsets Frequency and Distribution and the BSF-2(IL-6) to CSIF:TGIF(IL-10) Ratio as Severity-Associated Signatures in Primary Open-Angle Glaucoma: A Case-Controlled Study.

Flow cytometry was used to determine B-cell subset frequencies from 30 POAG patients grouped by hierarchical cluster analysis or the mean deviation (MD) of the visual field (VF) and correlated with the patients' clinical and pathological data, as well as with BSF-2(IL-6) and CSIF:TGIF(IL-10), which were quantified in peripheral blood samples of patients and controls by ELISA. The total B-cell frequency was increased in the POAG group in comparison to the control group (n = 30). Frequencies of specific B-cell subsets, such as double-negative (DN) and naïve B-cell subsets, were increased in relation to the severity of the POAG disease. However, the unswitched memory B compartment subset decreased in the POAG group. Other non-typical B-cell subsets such as DN B cells also showed significant changes according to the POAG disease severity course. These differences allow us to identify POAG severity-associated inflammatory clusters in patients with specifically altered B-cell subsets. Finally, ocular parameters, biomarkers of inflammation, and other glaucoma-related or non-clinical scores exhibited correlations with some of these B-cell subpopulations. The severity of the POAG disease course is accompanied by changes in the B-cell subpopulation, namely, DN B cells. Furthermore, the existing relationship of the B-cell subset frequencies with the clinical and the inflammatory parameters BSF-2(IL-6), CSIF:TGIF(IL-10), and the BSF-2(IL-6) to CSIF:TGIF(IL-10) ratio suggests that these B lymphocyte cells could serve as potential molecular bio-markers for assessing POAG disease severity and/or progression.

Analysis of circulating tumor DNA identifies distinct therapeutic response to intraperitoneal and intravenous paclitaxel plus S-1 in gastric cancer patients with peritoneal metastasis.

Circulating tumor DNA (ctDNA) is a promising technique for predicting curative effects and monitoring tumor recurrence. The utility of ctDNA for gastric cancer with peritoneal dissemination remains elusive. To assess the feasibility of ctDNA in predicting tumor response to chemotherapy in gastric cancer with peritoneal dissemination. This was a prospective study. We enrolled 30 patients with gastric cancer peritoneal metastasis, treated with intraperitoneal and intravenous paclitaxel plus S-1. Peripheral blood samples of patients were prospectively collected at baseline, after treatment initiation accompanied by computed tomography scan and disease progression. Mutational profiles from ctDNA were analyzed to evaluate its association with chemotherapeutic response. Tumor protein 53 (TP53) was the most frequently altered gene at baseline blood samples. Although baseline TP53 mutation was not related to therapeutic response, patients with TP53 mutation had worse progression-free survival (PFS) and overall survival (OS). Additionally, baseline ctDNA content fraction (CCF) was found to be significantly lower in responders than non-responders. Meanwhile, patients with high CCF had a trend of worse PFS and OS. Combining TP53 alteration and CCF, the prognosis of TP53-wt patients could be further stratified. Patients with CCF-low_TP53-wt had markedly longer survival than those with CCF-high_TP53-wt. Our study highlighted the significance of ctDNA in predicting potential clinical outcomes in gastric cancer patients during chemotherapy. ChiCTR-IIR-16009802 (Chinese Clinical Trial Registry).

DNase aggravates intestinal microvascular injury in IBD patients by releasing NET-related proteins.

Neutrophils accumulate in the inflammatory mucosa of patients with inflammatory bowel disease (IBD), and excessive release of NETs (neutrophil extracellular traps may be one of the important factors that cause IBD progression. However, the specific mechanism underlying vascular injury caused by NETs remains unclear. Immunofluorescence, ELISA, and flow cytometry were used in this study to detect the expression of NETs and DNase in the tissue and peripheral blood samples of patients with IBD. DSS mouse model was used to detect colon injury and vascular permeability. We found that NETs and DNase levels increased in the colon of patients with IBD. We found an increase in the activity of NET-related MPO released by DNase. DNase released NET-related proteins and damaged vascular endothelial cells invitro. In DSS mouse model, the synchronous increase of DNase and NETs in the colon leads to an increase in vascular injury markers (CD44, sTM). DNase aggravated colon injury and increased vascular permeability invivo, which was inhibited by gentamicin sulfate (GS). GS does not reduce the expression of DNase, but rather reduces the release of NET-related proteins to protect vascular endothelium by inhibiting DNase activity. MPO and histones synergistically damaged the vascular endothelium, and vascular injury can be improved by their active inhibitors. We further found that H2 O2 is an important substrate for MPO induced vascular damage. In conclusion, in IBD, DNase, and NET levels increased synchronously in the lesion area and released NET-related proteins to damage the vascular endothelium. Therefore, targeting DNase may be beneficial for the treatment of IBD.

A Phase 1 Study of Adding Pitavastatin to Venetoclax-Based Therapy in AML and CLL/SLL

Statins (HMG-CoA-reductase inhibitors) are drugs used widely for the treatment of high cholesterol and are generally well-tolerated. There has been increasing interest in repurposing statins for oncology based on the recognition that many cancer cells are “addicted” to the mevalonate biosynthetic pathway (Iannelli et al, 2017; Juarez and Fruman 2021). We have shown that statins increase expression of the pro-apoptotic BCL2 family member PUMA (Lee at al, 2018). We have strong pre-clinical evidence that statins improve responses to the BCL2 inhibitor venetoclax (VEN) in cell line models and ex vivo primary patient specimens from patients with both chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) (Lee et al, 2018). Additionally, retrospective review of patients enrolled into the early clinical trials of VEN in CLL revealed improved responses to VEN (complete remission [CR] and progression-free survival) in the patients who were concurrently on a statin medication (Lee et al, 2018). These retrospective analyses may underestimate the potential of statins for leukemia therapy, as the statin drugs most commonly prescribed for hypercholesterolemia are not pharmacologically optimized for oncology (Abdullah et al., 2018). We developed a phase 1 study of adding pitavastatin (PIT) to VEN-based therapy in patients with AML and CLL/small lymphocytic lymphoma (SLL) (NCT04512105). AML patients were eligible if receiving induction therapy with azacitidine (AZA) and VEN per standard of care. CLL/SLL patients could receive either VEN with obinutuzumab (O) or rituximab. A 3+3 design was employed. Dose level 1 was PIT 2 mg daily, and dose level 2 (DL2) was 4 mg daily. These doses are consistent with the doses used for the treatment of hypercholesterolemia. Those enrolling at DL2 had a creatinine clearance of 60 mL/min or higher based on the prescribing information for pitavastatin. Patients already on statins for the treatment for hypercholesterolemia were allowed; these patients stopped their prescribed statin for 72 hours prior to initiation of PIT. PIT started after patients completed VEN ramp-up and had been on a stable VEN dose for at least 5 days. Primary objectives were assessing safety and tolerability and determining the recommended phase 2 dose of PIT in combination with VEN-based therapies. Secondary objectives included CR rates. Exploratory objectives included performing BH3 profiling on pretreatment and post-treatment blood samples to assess whether add-on treatment with PIT increases apoptotic priming, as seen in preclinical models (Lee et al, 2018). 14 patients signed informed consent. 6 were deemed ineligible. 2 withdrew consent prior to starting PIT therapy. 6 patients received PIT on study. 2 patients had AML and received AZA+VEN. 2 patients had CLL, and 2 patients had SLL. The CLL/SLL patients all received VEN+O. Grade 1 myalgia was reported in one subject on the 4mg PIT dose. 1 patient experienced a grade 1 increase in AST on the 4mg dose. Another patient had grade 1 elevation in bilirubin and alkaline phosphatase at the 4 mg dose. Grade 3-4 toxicities included leukopenia, neutropenia (1 case of febrile neutropenia, grade 3), anemia, and thrombocytopenia. Cycle 1 was defined as the dose-limiting toxicity (DLT) period, and no hematologic toxicities occurred during the DLT period. 1 patient had grade 3 pancreatitis, but this was felt to be unrelated to therapy or disease (also outside the DLT period). No grade 5 toxicities occurred while on study therapy. Toxicities were overall felt to be similar to patients receiving VEN-based therapy in standard clinical practice. All evaluable patients (n=5) achieved clinical CR. 1 patient with SLL is awaiting re-staging at the time of this abstract. Correlative studies are ongoing. In 2 patient peripheral blood samples analyzed, the % blasts (for an AML sample) and % CD5+ B-cells (for a CLL sample) were reduced 24 hours after PIT dosing compared to prior to any treatment and before PIT/after VEN ramp-up. Based on the tolerability of PIT and encouraging clinical outcomes, a phase 2 study is being planned to better assess the efficacy of adding PIT to VEN-based therapies in AML and CLL/SLL.

Enrichment of effector memory T cells in the CD4 and CD8 T cell compartment during chronic graft versus host disease in children

BackgroundDuring allogeneic Hematopoietic stem cell transplantation (HSCT), frequent pathological scenarios include graft versus host disease (GVHD) and viral infections. We hypothesized if exogenous stimulus as alloantigen and viral antigens might impact on central and effector memory T cells in pediatric recipients. Patients and methodsSubjects included 21 pediatric recipients and 20 healthy children (control group). Peripheral blood samples of patients were collected along the first 712 days post-HSCT. T cell phenotyping of naïve, central, and effector memory T cells (TCMs and TEMs, respectively) was conducted using flow cytometry. Viral nucleic acids were detected using real-time PCR. ResultsT cell reconstitution was not reached after 1 year post-HSCT. Chronic GVHD was associated with increased numbers of naïve CD4 T cells (p < 0.05) as well as an increase in TEM and TCM cells of the CD4 (p < 0.0001 and p < 0.05, respectively) and CD8 T cell TEM (p < 0.0001). and TCM (p < 0.001) populations too. Moreover, BK and Epstein–Barr viruses were the main viral pathogens detected (<104 copies), which were associated with a decrease in all T cell compartments. ConclusionDuring chronic GVHD, alloantigen persistence generates TEM cell enrichment among CD4 and CD8 T cells, and viral infections are associated with deficient recovery of T cells after HSCT.

Ultrasensitive Detection of SALL4 Oncoproteins in Peripheral Blood and Bone Marrow Aspirate: Companion Diagnostics and Therapeutic Monitoring

Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 is a tumor driver, as shown by gain-of-function and loss-of-function studies; high SALL4 expression invariably correlates with worse prognosis. These studies established SALL4 as a novel, druggable target, such as in liver, lung, and blood cancers. In myelodysplastic syndrome (MDS), SALL4 can be upregulated by hypomethylating agents (HMAs), correlating with worse patient outcomes. Despite its well-established function on oncogenesis, SALL4 targeting therapies (inhibitors and degraders) are under pre-clinical development. There are currently no FDA/CLIA approved SALL4 diagnostic tests beyond tissue immunohistochemistry. Therefore, there is outstanding clinical need for the development and validation of a less invasive and sensitive SALL4 expression assay via peripheral blood, which can be used for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders. We developed a protein based SALL4 assay, compatible with peripheral blood and bone marrow aspirates samples, using the SIMOA (Single Molecule Array) technology, a bead-based digital enzyme-linked immunosorbent assay (aka digital ELISA). Previously, circulating SALL4 oncoproteins have been detected in the peripheral blood of patients with liver cancer using a conventional ELISA kit, but it is only semi-quantitative with limited sensitivity. We cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations. After antibody screening, we used the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. For quantitative assay development, we successfully expressed and purified full-length SALL4 for the first time, which was used to calibrate the standard curve and determine the assay's limit of quantification. The top bead and detector antibody pair could detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform in two different cell lysis buffers. Assay sensitivity was then tested. The Simoa assay captured endogenous SALL4 expression with greater sensitivity compared to the Western blot. The Western blot's limit of detection for SALL4A protein was 0.08 µg/µL whereas the Simoa assay continued until 3 ng/µL. Pre-analytic steps were then optimized, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. We assessed the effect of sample age for downstream processing and signal recovery as well as the possible matrix effect and interference from the buffy coat and plasma components of the peripheral blood and bone marrow samples. Our data showed no evidence of interference from the peripheral blood components, and interference from bone marrow aspirate can be further optimized. Antibody validation was then conducted. The preliminary data showed that the SALL4 Simoa assay can be used as a proof of principle to monitor SALL4 upregulation on HMA treatment for blood cancers and for SALL4 targeting therapy with two cell lines (SNU398 and H661) for solid cancers. To validate assay accuracy and precision, a retrospective cohort analysis is being conducted from patient peripheral blood samples. We obtained MGB Biobank samples for MDS, acute myeloid leukemia (AML), and liver cancer. We will use these samples to test for digital SALL4 protein detection. Further plans include expanding these analyses to breast and lung cancer. Additionally, we will conduct prospective studies to explore the role of the SALL4 protein biomarker in risk stratification and treatment monitoring, especially in MDS/AML patients undergoing HMA treatments. In conclusion, we have developed an ultrasensitive SALL4 protein expression diagnostic assay for use in monitoring patient treatment response and for guiding targeted therapy by analyzing peripheral blood for both solid and liquid tumors. Future steps include validating the diagnostic assay for specificity and sensitivity in a retrospective cohort analysis and conducting prospective studies to explore the role of the SALL4 protein biomarker for risk stratification, monitoring on HMA treatment, and guiding targeted therapy, ideally filing for approval as a laboratory developed test (LDT) in the near future

Development of Novel Protein-Drug Conjugates for the Treatment of Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid stem cell neoplasm with few effective therapies and poor prognosis. We have developed CCL2-drug conjugates that specifically bind, internalize and eliminate cells of the disease clone via inhibition of the key metabolic enzyme NAMPT. CCL2 is a chemokine which binds CCR2, a receptor expressed predominantly on the classical monocytes canonically expanded in CMML, while CMML stem and progenitors display ~100-fold higher CCR2 expression compared with healthy equivalents ( Ferrall-Fairbanks et al, Blood Cancer Discov 2022 3(6):536-553). CCL2-NAMPTi conjugates thus represent an attractive and novel strategy, co-opting both lineage specificity and therapeutic window to deliver payload drugs for efficient and targeted cell killing. Methods: Four NAMPTi warheads were made in adaptable seven-step synthetic route and covalently conjugated to human CCL2 though TGase. We screened the range of CCL2-NAMPTi conjugates on CCR2-expressing cell lines (THP-1; MV411) for their ability to reduce NAD/NADH and induce cytotoxicity. The lead CCL2-NAMPTi conjugate was identified based on its ability to reduce NAD/NADH levels, induce cell death, and show impermeability when using the unconjugated warhead alone. Plasma stability studies confirmed conjugate stability in mouse and human plasma with no release of unconjugated active warhead. The lead compound was then tested on CD14+ cells isolated from CMML patient peripheral blood (PB); examining NAD/NADH levels, cell viability and induction of caspase 3/8. To better understand its specificity and target cell population, we conducted in vivo studies using fluorescently labelled CCL2 in wild type and CCR2 KO mice. Therapeutic effectiveness of the lead conjugate was evaluated by means of NAD/NADH reduction in THP1 subcutaneous models. Results: All CCL2-NAMPTi tested induced cell death in THP-1 cells (IC50 1.1-7.2 nM) following 72 hours incubation. The lead compound, MM015, effectively reduced NAD/NADH levels in CCR2+ cell lines (THP-1; MV411) 24-48 hours following treatment (IC 50 0.1-0.8 nM), with a corresponding decrease in cell viability at 72 and 96 hours (IC 50 1.1- 2.1 nM). No effect was observed on CCR2- cell lines (Jurkat; PC9). Specificity of the lead conjugate was confirmed by measuring NAD/NADH levels and cytotoxicity in presence of 100-fold excess of unconjugated CCL2. Efficacy studies on primary CMML PB samples confirmed that MM015 exposure specifically reduced NAD/NADH levels (n=6, IC 50 0.5-15 nM) and cell viability (n=6, IC 50 1.9-250 nM), and induced caspase 3/8 activation. Flow cytometry analysis of CMML PB confirmed CCR2 expression was largely restricted to monocytes and precursors, as compared with other immune cell subsets (n=6). Accordingly, efficacy measures in treated whole PB were exclusively seen in the CD14+ population, sparing lymphoid and other lineage cells, demonstrating ability of CCL2-NAMPTi to selectively target the CMML disease clone. In vivo experiments using fluorescently labelled CCL2 confirmed selective uptake of intravenously administered human CCL2 by CCR2+ cells within mouse blood, bone marrow and in THP-1 subcutaneous tumour cells. No uptake of fluorescently labelled CCL2 was observed in CCR2 KO mice, further confirming CCR2 specific uptake. Additionally, measurement of human CCL2 in CCR2 KO mouse peripheral blood by ELISA confirmed CCR2 dependent CCL2 uptake, 1 hour following intravenous administration. In vivo efficacy studies showed consistent reduction of NAD/NADH levels in xenografted tumor cells, and monocytic cells in the murine BM compartment, 24 hours following IV injection of MM015. Conclusion: We have developed a novel drug delivery strategy utilizing CCL2-NAMPTi conjugates to selectively target and eliminate key CMML cell populations. CCL2-NAMPTi can effectively reduce NAD levels and induce subsequent cell death in CCR2-expressing cell lines, primary CMML cells and in vivo models. This innovative approach holds therapeutic promise to address this major unmet clinical need.

SHP2 is involved in the occurrence, development and prognosis of cancer.

Src homology-2 domain-containing protein tyrosine phosphatase (SHP2), encoded by protein tyrosine phosphatase non-receptor type 11 (PTPN11), is widely expressed in several human tissue types, and plays an important role in a variety of diseases. The present study assessed the impact of SHP2 on the occurrence, development and prognosis of solid tumors. The transcriptome sequencing data of 33 cancer types were downloaded from The Cancer Genome Atlas database. Clinical information of the corresponding patients, tumor mutational burden and information pertinent to microsatellite instability were also downloaded. The log-rank test and univariate Cox's regression test were used to evaluate patient survival. The 'ESTIMATE' method was used to assess the tumor microenvironment, and the 'CIBERSORT' algorithm was used to evaluate tumor immune cell infiltration. Spearman's correlation analysis was used to evaluate the correlation between SHP2 expression and the targets identified. ELISA was used to assess the SHP2 expression levels in peripheral blood samples of patients with breast, ovarian, endometrial and cervical cancer. The data indicated that the expression levels of SHP2 were increased in a variety of tumor tissues, and were associated with tumor progression and prognosis. In peripheral blood, the positive rates of SHP2 expression in breast cancer (71.43%) and ovarian cancer (58.82%) were significantly higher than those in the corresponding control groups. However, the positive rates of SHP2 expression in patients with endometrial cancer (31.03%) and cervical cancer (41.30%) were significantly lower than those in the corresponding control groups. Increased SHP2 expression improved overall survival (OS) and disease free survival (DFS) time in patients with kidney renal clear cell carcinoma. However, increased SHP2 expression reduced OS and DFS in patients with urothelial carcinoma, and cervical and endocervical cancer types. Moreover, the elevated expression of SHP2 could also reduce the OS of patients with breast invasive carcinoma, mesothelioma and liver hepatocellular carcinoma. PTPN11 expression was associated with the tumor microenvironment of various tumor types. The tumor mutational burden of various tumor types was associated with microsatellite instability. PTPN11 inhibited T-cell activation and promoted M2 macrophage activation in several tumors. Therefore, SHP2 may be used in the evaluation of tumor progression and prognosis, and it may be an optimal potential biological target for cancer therapy.

Recruitment of myeloid‑derived suppressor cells and regulatory T‑cells is associated with the occurrence of acute myocardial infarction.

The roles of myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs) in acute myocardial infarction (AMI) remain elusive. The present study aimed to analyze the proportions of the granulocytic and monocytic populations of MDSCs (G-MDSCs and M-MDSCs, respectively), and Tregs in the peripheral blood mononuclear cells (PBMCs) of patients with AMI. The present study recruited 34 patients with AMI and 37 healthy controls without clinical signs of myocardial ischemia. PBMCs were isolated from the peripheral blood samples of patients with AMI within 24 h following admission to the hospital and from those of the healthy controls during a physical examination. Two subsets of MDSCs, G-MDSCs (CD15+CD33+CD11b+CD14-HLA-DRlow) and M-MDSCs (CD14+CD15-CD11b+HLA-DRlow), and Tregs (CD3+CD4+CD25highCD127low T-cells) in the PBMCs derived from the patients with AMI and healthy controls were analyzed using flow cytometry. The effects of MDSCs derived from patients with AMI on naïve CD4+ T-cells were examined in the co-culture system. The results revealed that the proportions of G-MDSCs and M-MDSCs were higher in the peripheral blood of patients with AMI than in that of the healthy controls. The patients with AMI had significantly higher numbers of programmed death-ligand (PD-L)1- and PD-L2-positive G-MDSCs and M-MDSCs compared with the healthy controls (P<0.05). The MDSCs could acquire a granulocytic phenotype following AMI, and the G-MDSCs and M-MDSCs would be more likely to express PD-L2 and PD-L1, respectively. The ratios of Tregs to CD4+ T-cells and PD-1+ Tregs in the peripheral blood of patients with AMI were significantly higher than those in the healthy controls (P<0.05). The results of flow cytometry demonstrated an increase in the numbers of inducible Tregs in the co-culture system with the G-MDSCs derived from patients with AMI compared with the G-MDSCs derived from the healthy controls (P<0.01). On the whole, the findings presented herein demonstrate the accumulation of MDSCs, and the upregulation of PD-L1 and PD-L2 expression on the surface of MDSCs in patients with AMI. MDSCs can induce the expansion of Tregs by binding PD-1 on the surface of Tregs, thus playing a crucial role in AMI.

Real world implementation of flow cytometric monocyte subset partitioning for distinguishing chronic myelomonocytic leukaemia from other causes of monocytosis

Monocyte subset partitioning by flow cytometry may be a useful tool in distinguishing chronic myelomonocytic leukaemia (CMML) from other causes of monocytosis, however there has been varying success in real world implementation. Additionally, current assays require an individual tube for analysis despite significant overlap in antibodies used in routine T and NK cell analysis. The objective of this study was to validate a flow cytometry assay for the enumeration of monocyte subsets in our community-based laboratory and compare this to a hybrid panel allowing analysis of monocytes, T cells and NK cells in a single tube. Monocyte subset analysis was performed on peripheral blood samples of patients with monocytosis at the time of bone marrow biopsy or transient monocytosis in the setting of bacteraemia. Cut-offs of >94% classical and <1.13% non-classical monocytes for distinguishing CMML were assessed. Classical monocytes were significantly higher, and non-classical monocytes significantly lower in CMML compared to other causes of monocytosis. The sensitivity and specificity of >94% classical monocytes were 73% [95% confidence interval (CI) 43-90%] and 89% (95% CI 75-96%) regardless of which panel was used. Non-classical monocytes of <1.13% had a sensitivity and specificity of 82% (95% CI 52-97%) and 83% (95% CI 68-92%) with the monocyte panel and 55% (95% CI 28-78%) and 89% (95% CI 75-96%) using the hybrid panel. We have found the estimation of the classical monocyte subset to be the most robust and repeatable variation of this assay with sensitivity and specificity that is clinically useful. A hybrid panel may provide an effective approach to implementing monocyte subsets into practice.

The Role of Circulating Tumor DNA and Cell-Free DNA in the Management of Germ Cell Tumors: A Narrative Review

Liquid biopsy has demonstrated success as a diagnostic, prognostic, and therapy response monitoring tool in various cancers and could represent a rapid and minimally invasive alternative or complementary test for testicular germ cell tumors (GCTs). This article aims to review the current state of the research into circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) in testicular GCTs.Studies have confirmed the presence of ctDNA and cfDNA can be identified in peripheral blood samples of patients with testicular GCTs. Further research has attempted to optimize the methods for ctDNA detection in plasma to improve the sensitivity of these tests; however, a single method with high sensitivity and reliability has yet to be established. Previous studies have employes different methods for detecting cfDNA, including spectrophotometry, capillary electrophoresis, quantitative polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), and whole genome sequencing. These studies have various elements of cfDNA examined such as total cfDNA quantity, methylation patterns, and specific mutations. Additional studies have investigated the efficacy of cfDNA detection in combination with other tests including miRNA analysis.The application of cfDNA as a biomarker has been rapidly expanding in several malignancies. However, there is a relative paucity of research on the clinical utility of cfDNA in testicular cancer, and many questions remain about the significance and feasibility of this biomarker in GCTs. Cell-free DNA shows promise as a biomarker to enhance detection and disease monitoring in testicular cancer, but robust studies are needed to develop an optimal and reproducible method for cfDNA detection in order to determine its clinical application in testicular cancer.

Mitochondrial DNA copy number in autism spectrum disorder and attention deficit hyperactivity disorder: a systematic review and meta-analysis.

Several reports suggest that altered mitochondrial DNA copy number (mtDNA-cn), a common biomarker for aberrant mitochondrial function, is implicated in autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD), but the results are still elusive. A meta-analysis was performed to summarize the current indication and to provide a more precise assessment of the mtDNA-cn in ASD and ADHD. A search in the MEDLINE-PubMed, Scopus, and EMBASE databases was done to identify related studies up to the end of February 2023. The meta-analysis was conducted according to recommendations of the Cochrane Handbook of Systematic Reviews. Fourteen studies involving 666 cases with ASD and ADHD and 585 controls were collected and judged relevant for the systematic review and meta-analysis. The pooled results by a random effects meta-analysis was reported as a geometric mean of the estimated average response ratio and 95% confidence interval. Overall analysis of studies reported differences in mtDNA-cn in blood samples (k = 10) and non-blood samples (brain tissues and oral samples; k = 4) suggested significantly higher mtDNA-cn in patients compared to controls (p = 0.0275). Sub-analysis by stratifying studies based on tissue type, showed no significant increase in mtDNA-cn in blood samples among patients and controls (p = 0.284). Conversely, higher mtDNA-cn was observed in non-blood samples in patients than in controls (p = 0.0122). Further stratified analysis based on blood-cell compositions as potential confounds showed no significant difference in mtDNA-cn in peripheral blood samples of patients comparted to controls (p = 0.074). In addition, stratified analysis of aged-matched ASD and ADHD patients and controls revealed no significant difference in mtDNA-cn in blood samples between patients and controls (p = 0.214), whereas a significant increase in mtDNA-cn was observed in non-blood samples between patients and controls (p < 0.001). Finally, when the mtDNA-cn was analyzed in blood samples of aged-matched patients with ASD (peripheral blood, leukocytes, and PBMCs) or ADHD (peripheral blood), no significant difference in mtDNA-cn was observed between ASD patients and controls (p = 0.385), while a significant increase in mtDNA-cn was found between ADHD patients and controls (p = 0.033). In this first meta-analysis of the evaluation of mtDNA-cn in ASD/ADHD, our results show elevated mtDNA-cn in ASD and ADHD, further emphasizing the implication of mitochondrial dysfunction in neurodevelopmental disorders. However, our results indicate that the mtDNA-cn in blood is not reflected in other tissues in ASD/ADHD, and the true relationship between blood-derived mtDNA-cn and ASD/ADHD remains to be defined in future studies. The importance of blood-cell compositions as confounders of blood-based mtDNA-cn measurement and the advantages of salivary mtDNA-cn should be considered in future studies. Moreover, the potential of mtDNA-cn as a biomarker for mitochondrial malfunction in neurodevelopmental disorders deserves further investigations.

Characterization of monoclonal gammopathy of undetermined significance progression to multiple myeloma through meta-analysis of GEO data

The etiology of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) is still obscure as are the processes that enable the progression of MGUS to MM. Understanding the unique vs. shared transcriptomes can potentially elucidate why individuals develop one or the other. Furthermore, highlighting key pathways and genes involved in the pathogenesis of MM or the development of MGUS to MM may allow the discovery of novel drug targets and therapies.We employed STARGEO platform to perform three separate meta-analysis to compare MGUS and MM transcriptomes. For these analyses we tagged (1) 101 MGUS patient plasma cells from bone marrow samples and 64 plasma cells from healthy controls (2) 383 MM patient CD138+ cells from bone marrow and the 101 MGUS samples in the first analysis as controls (3) 517 MM patient peripheral blood samples and 97 peripheral blood samples from healthy controls. We then utilized Ingenuity Pathway Analysis (IPA) to analyze the unique genomic signatures within and across these samples.Our study identified genes that may have unique roles in MGUS (GADD45RA and COMMD3), but also newly identified signaling pathways (EIF2, JAK/STAT, and MYC) and gene activity (NRG3, RBFOX2, and PARP15) in MGUS that have previously been shown to be involved in MM suggesting a spectrum of molecular overlap. On the other hand, genes such as DUSP4, RN14, LAMP5, differentially upregulated in MM, may be seen as tipping the scales from benignity to malignancy and could serve as drug targets or novel biomarkers for risk of progression. Furthermore, our analysis of MM identified newly associated gene/pathway activity such as inhibition of Wnt-signaling and defective B cell development. Finally, IPA analysis, suggests the multifactorial, oncogenic qualities of IFNγ signaling in MM may be a unifying pathway for these diverse mechanisms and prompts the need for further studies.

Immune Phenotype as a Biomarker for Systemic Lupus Erythematosus.

The treatment of rheumatoid arthritis was revolutionized with the use of molecular-targeted drugs that target immunoregulatory molecules. The success of treatment with these drugs prompted the development of molecular-targeted drugs for systemic lupus erythematosus. However, systemic lupus erythematosus is a disease with high heterogeneous immune abnormalities, and diverse cells or molecules can be treatment targets. Thus, the identification of subpopulations based on immune abnormalities is essential for the development of effective treatment. One analytical method used to identify subpopulations is the immunophenotyping of peripheral blood samples of patients. This analysis evaluates the validity of target molecules for peripheral blood immune cell subsets, which are expected to be developed as biomarkers for precision medicine in which appropriate treatment targets are set for each subpopulation.

Immune cell proportions correlate with clinicogenomic features and ex vivo drug responses in acute myeloid leukemia.

The implementation of small-molecule and immunotherapies in acute myeloid leukemia (AML) has been challenging due to genetic and epigenetic variability amongst patients. There are many potential mechanisms by which immune cells could influence small-molecule or immunotherapy responses, yet, this area remains understudied. Here we performed cell type enrichment analysis from over 560 AML patient bone marrow and peripheral blood samples from the Beat AML dataset to describe the functional immune landscape of AML. We identify multiple cell types that significantly correlate with AML clinical and genetic features, and we also observe significant correlations of immune cell proportions with ex vivo small-molecule and immunotherapy responses. Additionally, we generated a signature of terminally exhausted T cells (Tex) and identified AML with high monocytic proportions as strongly correlating with increased proportions of these immunosuppressive T cells. Our work, which is accessible through a new "Cell Type" module in our visualization platform (Vizome; http://vizome.org/), can be leveraged to investigate potential contributions of different immune cells on many facets of the biology of AML.

HERCULES: A prostate cancer (PC) sequencing panel for parallel analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs).

5036 Background: In metastatic PC, tracking genomic alterations over time and with treatment can predict disease outcomes, guide treatment, and identify novel therapeutic targets. Analysis of peripheral blood ctDNA offers a non-invasive liquid biopsy for genomic profiling, but ctDNA often comprises a small portion of total cell-free DNA (cfDNA) in the blood, requiring high depth of sequencing which may still miss PC-relevant variants. We and others have shown that single CTCs captured from the same blood sample yield additional PC-relevant genomic data when combined with ctDNA profiling, but currently there is no assay that can be applied uniformly to both analytes. Here, we present a new amplicon-based sequencing panel capable of detecting PC-relevant variants in both cfDNA and single CTCs. Methods: PC-relevant genes were curated by overlapping reviews of cBioPortal for cancer genomics, the catalogue of somatic mutations in cancer (COSMIC), and PC-relevant genomic literature. HERCULES targets 36 genes with 258 amplicons covering PC-associated mutations and copy number variations. AmpliSeq HD targeted sequencing technology was applied for analysis of both cfDNA and CTCs. Analytical validation was performed sequentially, first using AcroMetrix and Seracare DNA control samples, then single and pooled LNCap and PC3 PC cells and cfDNA, then 8 patient peripheral blood samples of matched plasma cfDNA and single CTCs captured using the RareCyte platform from participants in SWOG S1802, an ongoing CTEP/CIRB-approved phase 3 trial for men with metastatic PC. Results: In DNA control samples with allele frequencies of 1%, 0.25%, and 0.1%, HERCULES displayed sensitivity of 100%, 75%, and 50%, respectively. In matched single cells and cfDNA from cell lines, HERCULES detected 100% of database-annotated somatic variants in cfDNA and in pools of 2 or more cells, and it identified &gt;90% of variants in single cells. In 16 matched patient samples of cfDNA and single CTCs, with read coverage depths of &gt;50,000x for 20ng cfDNA and &gt;1,500x for single CTCs, HERCULES detected shared variants (mean N per sample = 6.1) as well as unique somatic variants (mean of 1.1 CTC variants not called in the cfDNA, and 4.1 cfDNA variants not called in the CTCs). Conclusions: HERCULES is, to our knowledge, the first PC-specific targeted sequencing panel capable of parallel genomic profiling of both ctDNA and CTCs. In our preliminary analytical validation, it detected somatic variants with high sensitivity in matched cfDNA and CTC samples. With additional validation, HERCULES has the potential to augment conventional ctDNA profiling by illuminating both cell-free and cellular genomic evolution during PC treatment and progression.