Pattern Of Antigen Expression Research Articles

Comparative Evaluation of the Immunophenotypic Profile of Acute Lymphoblastic Leukaemia in Adult and Paediatric Age Groups in Iraq

Background: Immunophenotyping is a primary tool for investigating acute lymphoblastic leukemia (ALL). Acute lymphoblastic leukemia accounts for 75 - 80% of pediatric leukemia cases and 14% of adult leukemia cases. However, the prognostic factors, treatment strategies, and pathogenic pathways differ between these groups. Objectives: This study aims to compare the pattern of antigen expression between adult and pediatric ALL patients in the Iraqi population and to evaluate the frequency of aberrant myeloid antigen expression in both groups. Methods: A total of 131 patients with de-novo ALL were studied from May 2014 to May 2015. The diagnosis of ALL was confirmed by morphology and immunophenotyping of peripheral blood and bone marrow aspirate specimens using a panel of fluorochrome-labeled antibodies in 6 - colored flow cytometry. Results: The B-ALL phenotype was prevalent in 44 (68.7%) of adult patients compared to 51 (76.1%) in the pediatric group. CD10 expression was present in 49 (76.6%) of adults, mostly with B-ALL. The pediatric group had lower CD10 expression in T-ALL (P = 0.039). CD20 expression was significantly lower in adults compared to children. In T-ALL, significant differences between adult and pediatric groups were observed in CD1a and CD2 expression. Aberrant myeloid antigen expression was present in 29 (45.3%) adults and 30 (44.8%) pediatric patients, with CD33 and CD13 being the most prevalent in both groups. Conclusions: There was an increased T-cell lineage and aberrant antigen expression in both adult and pediatric ALL cases in this Iraqi sample. Adults with ALL tend to display higher levels of CD20 and lower levels of CD1a and CD2 compared to children, markers associated with poorer prognoses.

Prevalence and distribution of serological characteristics of weak ABO subgroups in the Chinese population

ObjectivesThis study aimed to determine the true frequency of weak ABO subgroups and to investigate the serological characteristics of various subgroup alleles in the Chinese population. MethodsA total of 2,945,643 blood samples were collected from January 2009 to December 2017. After initial screening and re-examination using automatic blood group analyzers, all ABO-discrepant samples were confirmed by standard serological analysis and molecular detection by DNA sequencing. The true frequency of weak ABO subgroups was determined by the number of ABO subgroup donors and the missed detection rate. The ABO antigen expressions corresponding to subgroup alleles were analyzed by the agglutination intensity of red blood cells. ResultsThe detection rate of ABO subgroups was 0.031 % (927/2,945,643). Considering the missed detection rate (27.81 %), the true frequency of ABO subgroups in the Chinese population was 0.044 %. The three most common genetic variations among blood donors in Shanghai were BA.04, BW.12 and BA.02. BW.03 showed the weakest B antigen expression (6.00 ± 1.97) and Bvar-1 the strongest (9.20 ± 1.10). ConclusionMany ABO subgroups were missed. BA.04 was found to be the most common subgroup allele in the Chinese population. Different ABO subgroup alleles exhibit different ABO antigen expression patterns.

Assessment of hepatitis c core antigen in epithelial salivary gland neoplasms (ex-vivo study)

BackgroundSalivary gland neoplasms (SGNs) pose a challenge to both pathologists and clinicians. Despite research, the etiology of these neoplasms remains unclear. This study aimed to identify any potential association between the presence of hepatitis C virus (HCV) at the protein or gene level and epithelial salivary gland neoplasms.MethodsFormalin-fixed paraffin-embedded (FFPE) blocks of epithelial salivary gland neoplasms were retrieved from the archives of the Oral and Maxillofacial Pathology Department, Faculty of Dentistry, Cairo University within the 5-year period from 2016 to 2020. Immunohistochemistry was used to assess HCV core antigen, while reverse transcription polymerase chain reaction was employed for the evaluation of HCV RNA.ResultsA total of 44 specimens were collected, 28 of which were benign neoplasms and 16 were malignant neoplasms. There was a statistically significant difference in HCV positivity between the two groups (P-value = 0.036). Benign tumors showed a statistically significant lower percentage of positive cases than malignant tumors. The localization of staining was also evaluated, revealing various patterns of HCV core antigen expression, including diffuse cytoplasmic, patchy cytoplasmic, nuclear, and a combination of nuclear and cytoplasmic expression. There was no statistically significant difference between the expression patterns in benign and malignant tumors (P-value = 0.616). Given that Pleomorphic Adenoma and Mucoepidermoid Carcinoma were the predominant tumor types in this study, four cases were selected for RNA detection. HCV RNA was detected in all cases using RT-PCR.ConclusionsHCV core antigen is frequently detected in SGNs and is suggested to be a potential risk factor for the development of these neoplasms. Further studies are required to discover other biomarkers, their roles, and the pathways associated with HCV in SGNs.

Cancer/testis antigen expression and co-expression patterns in Gastroesophageal Adenocarcinoma.

Gastroesophageal adenocarcinoma (GEAC) poses a significant challenge due to its poor prognosis and limited treatment options. Recently, Cancer/testis antigens (CTAs) have emerged as potential therapy targets due to their high expression in tumor cells and their immunogenic nature. We aimed to explore the expression and co-expression of CTAs in GEAC. We analyzed 63 GEAC patients initially and validated our findings in 329 patients from The Cancer Genome Atlas (TCGA) database. CTA expression was measured after RNA sequencing, while clinical information, including survival outcomes and treatment details, was collected from an institutional database. Co-expression patterns among CTAs were determined using Pearson correlation analysis. The majority of the study cohort were male (87%), Caucasian (94%), and had stage IV disease (64%). CTAs were highly prevalent, ranging from 58-19%. The MAGE gene family showed the highest expression, consistent across both cohorts. The correlation matrix revealed a distinct cluster of significantly co-expressed genes, including MAGEA3, NY-ESO-1, and others (0.27 ≤ r ≤ 0.73). Survival analysis revealed that individual CTAs were associated with poorer survival outcomes in patients not receiving immunotherapy while showing potential for improved survival in those undergoing immunotherapy, although these findings lacked robust reliability. Our study provides a comprehensive characterization of CTA expression and co-expression in GEAC. The strong correlation among CTAs like MAGE, NY-ESO-1, and GAGE suggests a potential for therapies targeting multiple CTAs simultaneously. Further research, including prospective trials, is warranted to assess the prognostic value of CTAs and their suitability as therapeutic targets.

Unlocking the Potential: Biomarkers of Response to Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) have reshaped the cancer treatment landscape across a variety of different tumor types. ADCs' peculiar pharmacologic design combines the cytotoxic properties of chemotherapeutic agents with the selectivity of targeted therapies. At present, the approval of many ADCs used in clinical practice has not always been biomarker-driven. Indeed, predicting ADCs' activity and toxicity through the demonstration of specific biomarkers is still a great unmet need, and the identification of patients who can derive significant benefit from treatment with ADCs may often be uncertain. With the lack of robust predictive biomarkers to anticipate primary, intrinsic resistance to ADCs and no consolidated biomarkers to aid in the early identification of treatment resistance (ie, acquired resistance), the determination of precise biologic mechanisms of ADC activity and safety becomes priority in the quest for better patient-centric outcomes. Of great relevance, whether the target antigen expression is a determinant of ADCs' primary activity is still to be clarified, and available data remain quite controversial. Antigen expression assessment is typically performed on tissue biopsy, hence only providing information on a specific tumor site, therefore unable to capture heterogeneous patterns of tumor antigen expression. Quantifying the expression of the target antigen across all tumor sites would help better understand tumor heterogeneity, whereas molecularly characterizing tumor-intrinsic features over time might provide information on resistance mechanisms. In addition, toxicity can represent a critical concern, since most ADCs have a safety profile that resembles that of chemotherapies, with often unique adverse events requiring special management, possibly because of the differential in pharmacokinetics between the small-molecule agent versus payload of a similar class (eg, deruxtecan conjugate-related interstitial lung disease). As such, the identification of robust predictive biomarkers of safety and activity of ADCs has the potential to improve patient selection and enrich the population of patients most likely to derive a substantial clinical benefit, especially in those disease settings where different ADCs happen to be approved in competing clinical indications, with undefined biomarkers to make precise decision making and unclear data on how to sequence ADCs. At this point, the identification of clinically actionable biomarkers in the space of ADCs remains a top research priority.

EXPRESSION PATTERNS OF CD90, CD117, CD123, AND CD147 IN CONTINUOUS CELL LINES OF HUMAN MATURE B-CELL TUMORS

Summary. Aim: to analyze expression patterns of CD90, CD117, CD123, and CD147 in vitro in different continuous lines of human mature B-cell neoplasms. Objects and methods: expression patterns of the surface antigens on cells of the continuous lines of Burkitt lymphoma (Namalwa and BJAB), Hodgkin lymphoma (КМ-Н2), and follicular lymphoma (Car-1) were analyzed by flow cytometry with monoclonal antibodies against CD90, CD117, CD123, and CD147. Results: BJAB and Namalwa cells did not express CD90 and CD123, while a small fraction of cells (less than 2%) was CD117-positive. КМ-Н2 and Сar-1 cells did not express any of three above mentioned antigens. The percentage of CD147-positive cells in continuous lines of human mature B-cell neoplasms amounted to 80–100%. Conclusions: CD117 expression in cell populations of Burkitt lymphoma continuous cell lines is rather rare. CD90 and CD123 are not expressed in all cell lines of human mature B-cell neoplasms under study. At the same time, ubiquitous pattern of CD147 has been evident.

Thrombocytopenia and Non-Expression of Tn And TF Antigens Define Non-Metastasizing Breast Cancer in Serial Transplants of Dimethyl Benz(A)Anthracene Induced Mammary Tumours in Sprague Dawley Rats

Functional mammary tumours were generated as serial transplants of dimethyl benz(a)anthracene (DMBA) in immunosuppressed Sprague Dawley rats (SD). These serial transplants were evaluated for their cellular behaviour and biomarkers. Breast cancer was induced by 10 mg/kg of 7, 12 dimethylbenzanthracene (DMBA) in 10 female SD rats. Following tumour development, the primary tumour was passaged into apparently healthy rats for first and second serial transplants. Cellular behaviour and biomarkers of transplants included growth of tumour, haematological indices, expression pattern of Tn and TF antigens and TNF-alpha. An elevation of TNF-α, non-induction of Tn antigen with thrombocytopenia were observed in the serial transplants derived from a malignant tumour induced by 7, 12, DMBA in female Sprague Dawley rats. Furthermore, neutrophil to lymphocyte and platelet to lymphocyte ratios were found to be significantly low in animals from the first serial transplant while being significantly high in animals of the second serial transplant group. Results indicate that DMBA-induced mammary tumours in Sprague Dawley rats undergo divergent cellular behaviour and antigenic expressions during serial transplantation.

Precision Immunotherapy Utilizing Adapter CAR-T Cells (AdCAR-T) in Metastatic Breast Cancer Leads to Target Specific Lysis.

A frequent symptom of metastasized breast cancer (BC) includes the development of malignant pleural effusion (MPE), which contains malignant cells derived from the primary tumor site. The poor prognosis of MPE in metastasized BC indicates the necessity for dependable precision oncology and the importance of models representing the heterogenous nature of metastatic BC. In this study, we cultured MPE-derived metastatic tumor cells from four advanced BC patients using organoid technology. We assessed the expression of tumor-associated antigens on MPE-derived organoid lines by flow cytometry (FC). Based on an individual antigen expression pattern, patient-derived organoids were treated with adapter CAR-T cells (AdCAR-T) and biotinylated monoclonal antibodies targeting CD276, HER2, EGFR, TROP2, or EpCAM. Co-culture assays revealed specific organoid lysis by AdCAR-T depending on individual antigen expression patterns. Our results demonstrate that MPE-derived organoids can serve as a reliable tool for assessing the efficacy of AdCAR-T on metastatic BC in a patient-individualized manner. This approach could potentially be applied in a preclinical setting to instruct therapy decisions. Further, our study demonstrates the feasibility of precision immunotherapy utilizing AdCAR-T to target patient-individualized antigen patterns.

The Laip-Based-Dfn Approach Is Superior in Terms of Useful MRD Results As Compared to the Laip Approach after Cycle II in Acute Myeloid Leukemia

Clinical decision-making based on measurable residual disease (MRD) in acute myeloid leukemia (AML) is steadily increasing; therefore, accurate MRD results are imperative. Multi-parameter flow cytometry (MFC) is often used due to wide availability, particularly when no suitable mutation is present. Two approaches are most common in MFC-MRD: 1. Leukemia-associated immunophenotype (LAIP) approach, in which the aberrant phenotype is identified at diagnosis and tracked during follow-up. 2. Different-from-Normal (DfN) approach, in which aberrant surface antigen expression patterns not present in normal bone marrow (empty spaces in 2D dot plots) are identified at follow-up. The LAIP approach is sensitive, but does not consider immunophenotypic shifts and cannot be used when no LAIP is identified at diagnosis or when a diagnosis sample is unavailable. The DfN approach can detect these shifts, but ample expertise is needed to determine a malignant population. In the ELN-DAVID AML-MRD guidelines 2022, it is recommended to use the combination of the LAIP approach and the DfN approach to use the advantages of both approaches. In the HOVON-SAKK132 (HO132), the MRD was based on the combination of MFC-MRD and NPM1 RQ-PCR. Initially, the MFC-MRD in this study was based on the LAIP approach. However, the complete MFC panel (four 8-color tubes) published by Cloos et al. (J.Vis.Exp, 2018) was used for measuring MRD during follow-up. Therefore in this study, we had the unique opportunity to retrospectively analyze the samples after induction cycle 2 (C2) of patients in complete remission (CR/CRi) and determine additional MRD results with the LAIP-based-DfN approach. The original LAIP approach encompasses determining the percentages of the LAIPs in the follow-up sample, which were also identified at diagnosis. For the LAIP-based-DfN approach, we followed the same approach, but determined the percentages of all possible LAIPs in the MFC-MRD panel at diagnosis and after C2. An emerging LAIP is defined as a LAIP that is present in the follow-up but was not found at diagnosis. MRD positivity was defined as ≥ 0.1% LAIP cells of white blood cells. Patients with an emerging LAIP < 0.1% or no LAIPs in follow-up would result in MRD negativity for LAIP-based-DfN, but was only possible when at least 500,000 cells were acquired in all four tubes. At diagnosis, we received 884 samples of 905 patients (98%), in which we found a LAIP ≥ 10% of CD45dim blasts in 83% of the samples (Figure 1A). After C2, we received 615 samples, for which we had MFC-MRD results for 79% (487/615) by the LAIP approach. When we applied the LAIP-based-DfN approach, we scored 69 of 615 extra MRD results: an emerging LAIP ≥ 0.1% (MRDpos, N = 14), no LAIPs (MRDneg, N = 25) or an emerging LAIP < 0.1% (MRDneg, N = 30). When including these results in the data of the LAIP approach, the percentage of patients with an MRD result increased from 79% (487/615) to 90% (556/615), and the MRD positive percentage increased from 13% (81/615) to 19% (118/615)(Figure 1B). The LAIPs of emerging clones most often included CD56, CD15, and CD22. To assess the prognostic value in the HO132, we combined the reanalyzed LAIP-based-DfN MFC-MRD with the NPM1-MRD (N = 567). In these analyses, overall survival (OS) hazard ratio (HR) 95% confidence interval (CI) 1.92 (1.44-2.56) and relapse-free survival (RFS) HR 95% CI 1.7 (1.31-2.19) were similar to the original LAIP based/ NPM1 approach: OS HR: 95%CI 2.02 (1.48-2.76) and RFS HR: 95%CI 1.78 (1.34-2.35), respectively. In conclusion, the LAIP-based-DfN approach adds additional MRD results while retaining the prognostic value. Since HOVON-SAKK centers are highly compliant with the protocol and 98% of diagnosis samples were available, the LAIP-based-DfN approach can offer additional value for studies with limited access to diagnosis samples.

Treatment with Blinatumomab in Combination with Venetoclax for Mixed Phenotype Acute Leukemia

Background: Mixed phenotype acute leukemia (MPAL) is a rare and heterogeneous type of acute leukemia that displays an ambiguous pattern of antigen expression to a degree that assigning a single lineage of origin is not possible due to which, understanding of the pathophysiology of the disease, diagnosis and treatment are limited. The lack of well-established optimal treatment for MPAL has paved path for requirement of a more effective and less toxic approach. Blinatumomab, a bispecific CD19-directed CD3 T-cell engager (BiTE) antibody and venetoclax, a potent, highly selective B-cell leukemia/lymphoma-2 (BCL2) inhibitor was approved for the treatment relapsed/refractory (r/r) B-acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), respectively. Here, we present 6 patients with CD19-positive MPAL that are successfully treated with blinatumomab in combination with venetoclax and/or azacytidine in front line setting. Methods: All 6 patients met the criteria for MPAL as defined by European Group for Immunological Characterization of Acute Leukemias (EGIL) or World Health Organization (WHO 2022). Flow cytometry and polymerase chain reaction (PCR) was used to assess MRD and BCR::ABL1 or KMT2A gene, respectively. As pre-phase treatment, patients received glucocorticoid for 7 days. Blinatumomab at a dose of 9 mg/day for 1 week followed by 28 mg/day for 3 weeks combined with venetoclax at a dose of 100 mg/day for 14 to 21 days and/or azacytidine at a dose of 75 mg/m 2/day was given as induction therapy subcutaneously for 7 days. The patient with BCR::ABL1 received blinatumomab in combination with olverembatinib. Intrathecal (IT) chemotherapy with cytarabine and methotrexate was administered to central nervous system (CNS) as prophylaxis treatment. Results: Of the six patients included five were male and only one was female; five were diagnosed with B-cell/myeloid MPAL, while one with B-cell/T-cell MPAL. Abnormalities in KMT2A gene was observed in three patients while one patient had abnormality in BCR::ABL1 gene, two patients were negative for both gene abnormalities. After one cycle of blinatumomab combination treatment, all the six patients achieved complete response (CR) with full hematologic recovery. In 5 patients, MRD was <0.1% of these 3 had MRD-negative remission (<0.01%) and two patients had molecular remission. Patients were switched to chemotherapy post induction therapy, after one cycle of post-reinduction, the patients who still had the CD19-positive flow MRD or persistent molecular MRD received sequencing chemotherapy and blinatumomab. The treatment and MRD was presented in table 1. The median follow-up time was 10 months. Except 2 patients, all other patients achieved continuous MRD and molecular remission, of which two patients underwent an allogeneic hematopoietic stem cell transplant (allo-HSCT). The most frequent grade 3 or 4 adverse events (AEs) observed were anemia (6 patients), neutropenia (6 patients) and thrombocytopenia (4 patients). No AEs led to treatment interruptions and no serious AEs were reported. Conclusion: The results provide preliminary evidence of effective and safe treatment with blinatumomab and venetoclax combination as first-line therapy in MPAL resulting in the achievement of normal bone marrow function with less toxicity than using conventional chemotherapy.

Expression Pattern of Tumor-associated Antigens in Neuroblastoma: Association With Cytogenetic Features and Survival.

The prognosis of high-risk and relapsed neuroblastoma (NB) patients remains poor. The identification of tumor-associated markers is important for differential diagnosis, prognosis, and the development of targeted therapies. The aim of the study was to determine the expression profile of nine most common NB antigens and assess their association with clinicopathological characteristics and patient survival. Tumor samples from 86 patients with NB were evaluated for the expression of tumor-associated antigen (TAA) using quantitative PCR. Twenty-one patients with benign tumors and 17 healthy donors were assigned as controls. Overexpression of tyrosine hydroxylase (TH), PHOX2B, PRAME, GPC2, B7-H3, and Survivin is the most typical for NB. Positive expression of MAGEA3, MAGEA1, and NY-ESO-1 at low levels was detected in 54%, 48%, and 52%, respectively, and was not NB specific. Higher TH expression was observed in samples without MYCN-amplification, while higher expression of Survivin, PHOX2B, and GPC2 was significantly associated with the presence of 1p.36 deletion. Overexpression of TH, PHOX2B, and MAGEA1 was associated with better event-free (EFS) and overall survival (OS). Survivin overexpression was associated with poor EFS but had no impact on OS. Multivariate analysis confirmed Survivin as independent marker for poor survival, and PHOX2B and MAGEA1 for better survival. High expression of TH, PHOX2B, and MAGEA1 genes are favorable prognostic factors for OS and EFS, whereas high expression of Survivin is associated with an increased risk of relapse or progression.

ABO and Rh Blood Group Antigens and Natural Anti-A and ANTI-B Antibodies in the Neonates

ABO blood group is determined by the presence or absence of A and B antigens on the surface of RBC and of anti-A and anti-B antibodies in the serum. The relatively weak expression of A and B antigens in newborns due to their developing immune systems poses challenges in accurately detecting naturally occurring IgM antibodies against these antigens. This difficulty in immunoserological methods contributes to the potential for errors in determining the blood groups of newborns. Despite this, the Rh antigen expression in newborns remains comparable to that in adults. Nonetheless, various factors contribute to diverse blood typing results in newborns, including the utilization of alternative testing methods. The complexity of blood typing is magnified when using samples from the umbilical vein. Furthermore, compared to adults, the exploration of ABO antigen expression in newborns is limited, and the identification of specific subgroups such as A1 and A2 is even rarer. This underscores the need for standardized testing procedures and further research to enhance our understanding of antigen expression patterns in newborns. Based on the aforementioned details, the primary objective of our study was to delve into specific aspects related to blood group characterization in newborns. This encompassed exploring the expression of A, B, AB, and D antigens on the surface of red blood cells (RBCs) and detecting anti-A and anti-B antibodies in the plasma of newborns. These analyses were conducted using samples obtained from the heels of 208 newborns and were typed by forward and reverse blood typing methods with monoclonal antibodies and srandart erythrocytes. The distribution of phenotypic groups within the ABO system among the newborns was not uniform. The r allele was identified with the highest frequency in the analyzed samples (0.6), while the prevalence of the p allele significantly lags at 0.3. The q allele has the lowest frequency (0.1). In our study, we propose that for the majority of cases (43.94±3.5%) among the studied newborns, there was an absence of naturally occurring anti-A and anti-B antibodies (n=87). In a specific scenario, within the O(I) blood group nwborns, partial synthesis of these antibodies was detected in 14.14±2.4% (n=28). Meanwhile, 41.92±3.5% of the newborns in our study exhibited natural antibodies similar to those found in adults. We didn’t find any difficulties in typing the Rh blood group antigens in the newborns. In conclusion, our study's findings indicate that newborns, in certain instances, exhibit strongly pronounced natural anti-A and anti-B antibodies within the ABO system. However, in the majority of cases, these antibodies are not evident. Majority of cases erythrocyte A and B antigens were weakly expressed and for detecting these images optic microscopes were used.

Association between the Expression Patterns of Hepatitis B Surface Antigen and Hepatitis B Core Antigen with Clinicopathological Parameters and Antiviral Therapy in Liver Biopsies Obtained from Chronically Infected Hepatitis B Positive Omani Patients

Background: Hepatitis B virus (HBV) infection is considered a major global health problem. The main objectives of the current study is to determine the patterns of expression of the hepatitis B surface antigen (HBsAg) and HBV core antigen (HBcAg) in liver tissue samples obtained from hepatitis B virus-infected Omani patients and to associate between the pattern of the expression of HBsAg and HBcAg with the other clinical parameters and anti-viral therapy. Methods : The expression patterns of HBsAg and HBV core antigen HBcAg were determined by immunohistochemistry (IHC), in 58 formalin-fixed paraffin-embedded liver tissue biopsies obtained from chronic hepatitis B Omani patients. The association between positivity for HBV antigens with gender, age group, histological appearances, and antiviral therapy was determined. Results: IHC-positive staining of HBsAg was demonstrated in 28 patients (48.3%) patients, of whom 4 (6.9%) also showed HBcAg expression. The expression pattern of HBsAg was predominantly cytoplasmic and was seen in 25 (89.3%) of patients, whereas expression of HBcAg was nuclear in 3 (75%) of patients. HBsAg and HBcAg IHC positivity were more common among males than females and among those aged 39–58 years (P = 0.130 and 0.569, respectively). The presence of lymphocytic infiltration in the majority of liver biopsies examined in the present study indicates that the liver damage could be attributed to immunologically mediated events, especially because HBV is a non-cytopathogenic virus. No significant statistical association was found between positivity for HBsAg/HBcAg by IHC and antiviral treatment. Conclusions: Determination of the expression patterns of HBV antigens in liver biopsies obtained from chronic hepatitis B Omani patients and the association between these expression patterns with other clinical histopathological parameters and anti-viral therapy, will contribute greatly to a better understanding of the pathogenesis of HBV in this unique cohort group of infected individuals.

Development of Spectral Nano-Flow Cytometry for High-Throughput Multiparameter Analysis of Individual Biological Nanoparticles

Correlated analysis of multiple biochemical parameters at the single-particle level and in a high-throughput manner is essential for insights into the diversity and functions of biological nanoparticles (BNPs), such as bacteria and subcellular organelles. To meet this challenge, we developed a highly sensitive spectral nano-flow cytometer (S-nFCM) by integrating a spectral recording module to a laboratory-built nFCM that is 4-6 orders of magnitude more sensitive in side scattering detection and 1-2 orders of magnitude more sensitive in fluorescence detection than conventional flow cytometers. An electron-multiplying charge-coupled device (EMCCD) was used to acquire the full fluorescence spectra of single BNPs upon holographic grating dispersion. Up to 10,000 spectra can be collected in 1 min with 2.1 nm resolution. The precision, linearity, and sensitivity were examined. Complete discernment of single influenza viruses against the background signal, discrimination of different strains of marine cyanobacteria in a mixed sample based on their spectral properties of natural fluorescence, classification of bacterial categories exhibiting different patterns of antigen expression, and multiparameter analysis of single mitochondria for drug discovery were successfully demonstrated.

A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor.

Mesenchymal stromal cells (MSCs) are promising therapeutic candidates in a variety of diseases due to having immunomodulatory and pro-regenerative properties. In recent years, MSC-derived small extracellular vesicles (sEVs) have attracted increasing interest as a possible alternative to conventional cell therapy. However, translational processes of sEVs for clinical applications are still impeded by inconsistencies regarding isolation procedures and culture conditions. We systematically compared different methods for sEV isolation from conditioned media of ex vivo expanded bone marrow-derived MSCs and demonstrated considerable variability of quantity, purity, and characteristics of sEV preparations obtained by these methods. The combination of cross flow filtration with ultracentrifugation for sEV isolation resulted in sEVs with similar properties as compared to isolation by differential centrifugation combined with ultracentrifugation, the latter is still considered as gold standard for sEV isolation. In contrast, sEV isolation by a combination of precipitation with polyethylene glycol and ultracentrifugation as well as cross flow filtration and size exclusion chromatography resulted in sEVs with different characteristics, as shown by surface antigen expression patterns. The MSC culture requires a growth-promoting supplement, such as platelet lysate, which contains sEVs itself. We demonstrated that MSC culture with EV-depleted platelet lysate does not alter MSC characteristics, and conditioned media of such MSC cultures provide sEV preparations enriched for MSC-derived sEVs. The results from the systematic stepwise evaluation of various aspects were combined with culture of MSCs in a hollow fiber bioreactor. This resulted in a strategy using cross flow filtration with subsequent ultracentrifugation for sEV isolation. In conclusion, this workflow provides a semi-automated, efficient, large-scale-applicable, and good manufacturing practice (GMP)-grade approach for the generation of sEVs for clinical use. The use of EV-depleted platelet lysate is an option to further increase the purity of MSC-derived sEVs.

EXPRESSION PATTERNS OF CD90, CD117, CD123, AND CD147 IN MATURE B CELL NEOPLASMS

Summary. Aim: to analyze the expression patterns of CD90, CD117, CD123, and CD147 on cells of the mature B-cell neoplasms, in particular marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL) and the expression of CD147 on chronic lymphocytic leukemia (CLL) cells. Object and methods: the expression patterns of the surface antigens on the transformed B cells were analyzed in the peripheral blood samples of the MZL, MCL, and CLL patients (34 patients in total) by the flow cytometry using the monoclonal antibodies. Results: at least one of the antigens – CD90, CD117, or CD123 was expressed in 20.0–33.3% of MCL cases and 27.2–54.5% of MZL cases at the level ≥ 1% of the cells in the subpopulation of CD45-positive lymphoid cells. One MCL case demonstrated the high content of CD117-positive cells (31.0%) accompanied by CD90 expression on 3.8% of cells. The high content of the CD147-positive cells was found not only in MZL and MCL but also in CLL. Conclusion: a small fraction of cells of lymphoid neoplasms expresses several antigens of the leukemic stem cells and cells of the myeloid lineage. The ubiquitous pattern of CD147 expression on cells of these neoplasms has been demonstrated. The diagnostic and prognostic significance of the expression of the studied antigens remains unknown necessitating further clinical studies of the clinical features of these diseases taking into account the expression of the atypical markers.

Intrahepatic quantification of HBV antigens in chronic hepatitis B reveals heterogeneity and treatment-mediated reductions in HBV core-positive cells

Patterns of liver HBV antigen expression have been described but not quantified at single-cell resolution. We applied quantitative techniques to liver biopsies from individuals with chronic hepatitis B and evaluated sampling heterogeneity, effects of disease stage, and nucleos(t)ide (NUC) treatment, and correlations between liver and peripheral viral biomarkers. Hepatocytes positive for HBV core and HBsAg were quantified using a novel four-plex immunofluorescence assay and image analysis. Biopsies were analysed from HBeAg-positive (n= 39) and HBeAg-negative (n= 75) participants before and after NUC treatment. To evaluate sampling effects, duplicate biopsies collected at the same time point were compared. Serum or plasma samples were evaluated for levels of HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), and HBV RNA. Diffusely distributed individual HBV core+ cells and foci of HBsAg+ cells were the most common staining patterns. Hepatocytes positive for both HBV core and HBsAg were rare. Paired biopsies revealed large local variation in HBV staining within participants, which was confirmed in a large liver resection. NUC treatment was associated with a >100-fold lower median frequency of HBV core+ cells in HBeAg-positive and HBeAg-negative participants, whereas reductions in HBsAg+ cells were not statistically significant. The frequency of HBV core+ hepatocytes was lower in HBeAg-negative participants than in HBeAg-positive participants at all time points evaluated. Total HBV+ hepatocyte burden correlated with HBcrAg, HBV DNA, and HBV RNA only in baseline HBeAg-positive samples. Reductions in HBV core+ hepatocytes were associated with HBeAg-negative status and NUC treatment. Variation in HBV positivity within individual livers was extensive. Correlations between the liver and the periphery were found only between biomarkers likely indicative of cccDNA (HBV core+ and HBcrAg, HBV DNA, and RNA). HBV infects liver hepatocyte cells, and its genome can exist in two forms that express different sets of viral proteins: a circular genome called cccDNA that can express all viral proteins, including the HBV core and HBsAg proteins, or a linear fragment that inserts into the host genome typically to express HBsAg, but not HBV core. We used new techniques to determine the percentage of hepatocytes expressing the HBV core and HBsAg proteins in a large set of liver biopsies. We find that abundance and patterns of expression differ across patient groups and even within a single liver and that NUC treatment greatly reduces the number of core-expressing hepatocytes.

Circulating CD22+/CD19-/CD24- progenitors and CD22+/CD19+/CD24- mature B cells: Diagnostic pitfalls for minimal residual disease detection in B-lymphoblastic leukemia.

Multiparametric flow cytometry (MFC) has become a powerful tool in minimal residual disease (MRD) detection in B-lymphoblastic leukemia/lymphoma (B-ALL). In the setting of targeted immunotherapy, B-ALL MRD detection often relies on alterative gating strategies, such as the utilization of CD22 and CD24. It is important to depict the full diversity of normal cell populations included in the alternative B-cell gating methods to avoid false-positive results. We describe two CD22-positive non-neoplastic cell populations in the peripheral blood (PB), including one progenitor population of uncertain lineage and one mature B-cell population, which are immunophenotypic mimics of B-ALL. Using MFC, we investigated the prevalence and phenotypic profiles of both CD22-positive populations in 278 blood samples from 52 patients with B-ALL; these were obtained pre- and post-treatment with CD19 and/or CD22 CAR-T therapies. We further assessed whether these two populations in the blood were exclusively associated with B-ALL or recent anticancer therapies, by performing the same analysis on patients diagnosed with other hematological malignancies but in long-term MRD remission. The progenitor population and mature B-cell population were detected at low levels in PB of 61.5% and 44.2% of B-ALL patients, respectively. Both cell types showed distinctive and highly consistent antigen expression patterns that are reliably distinguishable from B-ALL. Furthermore, their presence is not restricted solely to B-ALL or recent therapy. Our findings aid in building a complete immunophenotypic profile of normal cell populations in PB, thereby preventing misdiagnosis of B-ALL MRD and inappropriate management.

Identification of Phenotypic Subgroups of Acute Myeloid Leukemia, Defined By Differentiation According to Who 2022 Classification

Background: The recently published WHO classification 2022 has extended genetic findings as the diagnostic basis for acute myeloid leukemia with defining genetic abnormalities (AML-DGA) resulting in 13 such entities leaving only a minority of cases not genetically defined, i.e. AML, defined by differentiation (AML-DBD). Clustering of flow cytometrically (FC) analyzed immunophenotypes may help to identify subgroups within AML-DBD allowing to search for additional genetic abnormalities which in future WHO classifications may qualify for AML-DGA. Aim: To identify subgroups within AML-DBD based on immunophenotypes analyzed by FC and to elucidate their genetic background applying panel sequencing. Methods: In a multicenter study comprising four institutions in North America, Europe and Asia cases with AML-DBD according to WHO 2022 classification were selected which had been diagnosed applying broad antigen panels by FC and broad gene panels by NGS. Expression data by FC on 30 surface and cytoplasmic antigens were collected and rated as negative, dim, partial and bright. Cluster analysis was performed to identify subgrops of cases with similar phenotpyes. Patterns of gene mutations within these subgroups were assessed. Results: A total of 234 AML-DBD were identified and included in the study. Nine antigens were found to be differentially expressed and to contribute to defining subgroups: MPO, CD34, CD11b, CD14, CD36, CD45, CD15, CD64 and CD65. Clustering of cases appyling expression categories of these nine antigens resulted in the identification of seven subgroups with clear differences in the antigen expression patterns (figure, panel A). Groups 1 and 3, different from all other groups, both showed bright expression of MPO and CD34 but only group 3 in addition showed bright CD36 expression (figure, panel B). Groups 2 and 4 also brightly expressed CD34 but showed only dim and no expression, respectively, of MPO. Conversely, groups 5 and 6 showed bright MPO expression with heterogeneous and largely no expression, respectively, of CD34. Group 5 exclusively showed bright expression of CD45 and CD64 in line with a monocytic phenotype. Group 7 was characterized by merely no or dim only expression of the nine antigens found differentially expressed between these seven groups. Interestingly, there has been no clear correlation between these seven groups and cytomorphologic findings besides AML with maturation occuring frequently in groups 1 (51% of cases) and 4 (67%) and AML without maturation in group 7 (57%). NGS revealed mutations in seven genes, which are not used by the WHO 2022 classification to define AML-DGA, to occur at different frequencies among the seven subgroups: DDX41(non germline), DNMT3A, FLT3-ITD, IDH1, IDH2, KMT2A-PTD and NRAS. In groups 1 to 4 20% to 39% of cases showed DDX41 mutations but no cases did so in groups 5 to 7. DNMT3A mutation frequencies varied among all groups between 17% and 44%. FLT3-ITD was found in 50% of group 5 but in only 7% to 28% of other groups. Mutations in IDH1 and IDH2 were found in all groups at frequencies ranging from 17% to 44%. While in the majority of groups frequencies of IDH2 mutations exceeded those of IDH1 mutations this was not the case in groups 3 and 5. KMT2A-PTD were found at a particularly high frequency of 44% in group 7 with other groups showing frequencies between 5% and 33%. NRAS mutations were found overall most infrequently with frequencies in groups ranging from 4% to 17%. Conclusions: Clustering of flow cytometric data revelad seven subgroups of AML-DBD with different antigen expression patterns in nine differentially expressed genes. Results of panel sequencing revealed differences in the genetic background of the seven groups but at the same time showed overlaps in mutation patterns between groups. Following the insights in the genetic basis of hematopoietic neoplasms gained in recent years and its application as principles in the WHO classification 2022 it is anticipated that the seven subgroups of AML-DBD identified in the present study feature additional genetic abnormalities besides the mutations described here. Whole genome and transcriptome sequencing will help identifying these additional abnormalities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Longitudinal Multimodal Atlas of AML Surface Antigens Enables Adaptive and Combinatorial Targeting

Acute myeloid leukemia (AML) is a clinically heterogeneous disease characterized by chromosomal abnormalities, gene mutations, and inter- and intra-patient genetic and phenotypic variability. Therapies specifically targeting cell surface antigens are promising treatments for AML, but surface antigen expression variability and potential antigen escape mechanisms may impact efficacy of single-targeting therapies. Delineating surface antigen expression uniformity and density between patients and during disease progression from diagnosis to relapse will enable rational prioritization of AML antigens for adaptive and combinatorial targeting. Here, we elucidate surface antigen expression patterns for candidate therapeutic targets by performing multimodal integrative analysis of AML surface proteins and transcriptomes of matched diagnosis and relapse clinically annotated samples. To identify potential therapeutic AML targets, we compiled a comprehensive list of protein surface targets reported in clinicaltrials.gov, literature, and large proteomics mass spectrometry datasets of AML patient bone marrow samples (deBoer et. al. Cancer Cell 2018; Jayavelu et al, Cancer Cell 2022). To focus on surface antigens and reduce the risk of targeting antigens that may have off-tumor solid tissue toxicity, we then excluded antigens that lack plasma cell membrane localization or are expressed on healthy non-hematologic tissues. We adapted Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) to generate longitudinal single-cell multimodal readouts of both the transcriptome and targeted surface proteins of matched diagnostic and relapsed samples from 42 adults with AML (Figure 1). Antibody derived tags (ADTs) against selected antigens were used at saturating conditions. Single cell libraries were prepared with 10X Genomics 5' Feature Barcoding Library Kit v2 and sequenced on Illumina NextSeq 2000. Cellranger v6.0.0 and Seurat v3 were used for mapping, read quantification, and analysis, respectively. We leveraged AML cell lines with known expression of benchmark antigens to assess the accuracy of the ADT readout by correlating it with antibody bound per cell (ABC) values derived from the traditional flow cytometry-based QuantiBRITE assay. Patient bone marrow data were projected onto low dimensional space using UMAP to visualize the population structure of the data. We observed separation of AML blasts from healthy cell lineages and delineation of blast gating based on CD45 expression. k-nearest neighbors clustering identified phenotypically diverse clusters within AML blasts within each patient sample, enabling quantification of specific antigen-positive clusters and antigen co-expression patterns. We found strong correlation between ADT readout and corresponding ABC values for cell surface protein expression on AML blasts of our benchmark antigens (CD33, CD123, CLL1, and ADGRE2). We then proceeded to systematically rank 87 AML surface antigens in blast populations based on ADT readout. Moreover, transcriptome profiling allowed investigation of antigens with limited antibody availabilities, exploration into novel cell states, and identification of signaling pathways that may associate with the presence of particular surface antigens. Identification and analysis of leukemic stem cells are ongoing. Healthy bone marrow and peripheral blood will be analyzed alongside AML samples to elucidate antigen target expression during normal hematopoiesis. In summary, our multimodal analysis of AML surface antigens informs cell surface clonal heterogeneity of patient AML and builds a reference atlas of expression patterns and intensities of potential surface AML antigen targets. In ongoing work, we are analyzing correlations between clonal surface antigen expression in longitudinal analysis of diagnosis and relapse samples as well as correlation with clinical parameters. This comprehensive surface antigen expression profiling will inform selection of therapeutically targetable and consistently expressed antigens to address AML heterogeneity in future immunotherapy targeting studies. Note: J.E. and M.U. share co-first authorship. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal