High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαβ-Large Granular Lymphocytic Leukemia

Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor β chain (TRBC1) expression for assessing Tαβ-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαβ cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1− Tαβ-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1− Tαβ-LGL ranged between 0.36 and 571 cells/μL (3.2–91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/μL (<0.9% or >96% TRBC1+ cells). Among the distinct TCRVβ families, the CD28− effector-memory and terminal-effector polyclonal Tαβ cells ranged between 0 and 25 TRBC1+ or TRBC1− cells/μL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1− cells/μL, representing <1.6% or >98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαβ-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαβ cells. However, in the absence of lymphocytosis or in the case of TαβCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαβ-LGL-expressing individual TCRVβ families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Effect of Daratumumab-Containing Induction on CD34+ Hematopoietic Stem Cells before Autologous Stem Cell Transplantation in Multiple Myeloma

Introduction: Daratumumab (Dara) is an anti-CD38 monoclonal antibody representing a novel treatment agent for multiple myeloma (MM). Nonetheless, several studies have reported a Dara-related impairment of CD34+ hematopoietic stem cell (HSC) mobilization and post-autologous stem cell transplantation (ASCT) complications, including low yields of mobilized HSCs and delayed neutrophil engraftment. Impact of Dara on the mobilization process and HSCs remains poorly understood even though sufficient yields of CD34+ cells are necessary for a successful ASCT and subsequent patient recovery. Aims: To compare the effect of the Dara-containing (Dara-Bortezomib-Dexamethasone [D-VCd]) and conventional (Bortezomib-Thalidomide-Dexamethasone [VTd]) therapy on CD34+ HSCs. Methods: Transplant eligible MM patients were treated with D-VCd or VTd induction regimen followed by a cyclophosphamide + G-CSF mobilization and a high-dose melphalan D -1 before ASCT. Flow cytometry (FCM) screening of CD34+ subsets was performed in the bone marrow (BM) or apheresis product (AP) at three consecutive time points: 1) diagnostic BM (DG), 2) mobilization AP (MOB), 3) a day prior ASCT BM (D-1). Furthermore, RNA sequencing (RNAseq) of sorted CD34+ cells was performed on total RNA with ribo-depletion protocol in AP after the induction. D-VCd samples had lower RNA yields thus the D-VCd or VTd groups were processed as independent batches. Results: Clinical data revealed no significant differences in mobilization (p &gt;0.050) likely due to a small cohort sizes (D-VCd n=5 vs VTd n=9), though a trend towards worse performance in D-VCd was observed. Median CD34+ cell yield was 3.08 vs 10.56 x 10 6/kg. Platelet recovery of &gt;20x10 9/L was D+14 vs D+12 (range: 11-18 vs 10-16). Neutrophil recovery of &gt;0.5x10 9/L was D+12 in both groups (range: 11-17 vs 11-12). In FCM analysis, DG (n=14), MOB D-VCd (n=5) vs VTd (n=9), D-1 D-VCd (n=7) vs VTd (n=15) were compared. CD34+ frequency (Fig. 1A) difference in MOB D-VCd vs VTd was insignificant (median: 1.15% vs 1.89%), whereas CD34+ fraction dropped in D-1 D-VCd (median: 0.52% vs 0.72%, p=0.027), albeit there was no significant reduction in D-1 D-VCd vs initial DG (median: 0.52% vs 0.45%). Differences in the distribution of certain HSC subsets were detected in the CD34+ pool (Fig. 1B-E). Frequency of multipotent progenitors (MPPs) (Fig. 1B) was increased in MOB D-VCd (median: 82.1% vs 66.2%, p=0.004). Frequency of lympho-myeloid-primed progenitor + granulocyte-monocyte progenitor (LMPP+GMP) (Fig. 1C) subset was reduced in D-VCd in both MOB (median: 1.7% vs 16.9%, p=0.042) and D-1 (median: 5.3% vs 14.0%; p=0.026). Erythro-myeloid progenitors (EMPs) (Fig. 1D) were reduced in MOB D-VCd (median: 10.7% vs 19.5%, p=0.042), while the frequency of EMPs increased in D-1 D-VCd (median: 20.8% vs 12.4%, p=0.045). No considerable differences were found in the expression of adhesion molecules CD44/HCAM or CD184/CXCR4. CD38 was strongly diminished in the whole D-VCd CD34+ fraction of MOB and D-1. To understand whether the differences in the mobilization efficacy after D-VCd induction were reflected in the expression profile of mobilized CD34+ cells, differential expression analysis was performed. Overall 133 significantly deregulated genes (p&lt;0.05; log fold change &gt;(-)1) between cohorts (D-VCd n=5 vs VTd n=5) were revealed (Fig. 2). Pathway analysis showed cellular response and localization as the most deregulated categories. The list of deregulated genes contained 25% of non-coding RNAs, some of which were linked to a protein localization in the cell (RN7SL1/2). The expression of adhesion molecules was inspected independently. Out of 59 HSC hallmark genes, only 8 were significantly altered in D-VCd. Interestingly, the main homing molecule CXCR4 seemed to be downregulated in D-VCd, while integrins A3 and B4 were upregulated. Conclusions: Despite the limited cohort sizes, a prospective trend of delayed neutrophil and platelet recovery was observed after D-VCd therapy. FCM analysis revealed a significant reduction of CD34+ subsets responsible, among others, for a reconstitution of neutrophils and megakaryocytes. A strong signal in transcriptome data which would potentially explain differential mobilization in D-VCd cohort was not detected, nevertheless, several genes with adhesive/homing and stem cell differentiation function were indeed altered. The results warrant further investigation. Figure 1 Figure 1. Disclosures Hajek: BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Evaluation of Mushrooms Based on FT-IR Fingerprint and Chemometrics

Edible mushrooms have been recognized as a highly nutritional food for a long time, thanks to their specific flavor and texture, as well as their therapeutic effects. This study proposes a new, simple approach based on FT-IR analysis, followed by statistical methods, in order to differentiate three wild mushroom species from Romanian spontaneous flora, namely, Armillaria mellea, Boletus edulis, and Cantharellus cibarius. The preliminary data treatment consisted of data set reduction with principal component analysis (PCA), which provided scores for the next methods. Linear discriminant analysis (LDA) managed to classify 100% of the three species, and the cross-validation step of the method returned 97.4% of correctly classified samples. Only one A. mellea sample overlapped on the B. edulis group. When kNN was used in the same manner as LDA, the overall percent of correctly classified samples from the training step was 86.21%, while for the holdout set, the percent rose to 94.74%. The lower values obtained for the training set were due to one C. cibarius sample, two B. edulis, and five A. mellea, which were placed to other species. In any case, for the holdout sample set, only one sample from B. edulis was misclassified. The fuzzy c-means clustering (FCM) analysis successfully classified the investigated mushroom samples according to their species, meaning that, in every partition, the predominant species had the biggest DOMs, while samples belonging to other species had lower DOMs.

Mushroom’s Evaluation Based on FT-IR Fingerprint and Chemometrics

Edible mushrooms have been recognized as highly nutritional food for a long time, due to their specific flavor, texture and also for therapeutic effects. This study proposes a new simple approach, based on FT-IR analysis, followed by statistical methods, in order to differentiate three wild mushrooms species from Romanian spontaneous flora, namely Armillaria mellea, Boletus edulis and Cantharellus cibarius. The preliminary data treatment consisted of data set reduction with principal component analysis (PCA), which provided scores for the next methods. Linear discriminant analysis (LDA) manage to 100% classify the three species and the cross validation step of the method returned 97.4% of correctly classified samples. Only one A. mellea sample overlapped on B. edulis group. When kNN was used in the same manner as LDA, the overall percent of correctly classified samples from the training step was 86.21%, while for holdout set the percent raised at 94.74%. The lowered values obtained for the training set was due to one C. cibarius sample, two B. edulis and five A. mellea, which were placed to other species. Anyway, for holdout sample set, only one sample from B. edulis was misclassified. The fuzzy c-means clustering (FCM) analysis successfully classified investigated mushroom samples according to their species, meaning that in every partition the predominant specie had the biggest DOMs, while samples belonging to other specie had lower DOMs.

Intraoperative Digital Analysis of Ablation Margins (DAAM) by Fluorescent Confocal Microscopy to Improve Partial Prostate Gland Cryoablation Outcomes

Partial gland cryoablation (PGC) aims at destroying prostate cancer (PCa) foci while sparing the unaffected prostate tissue and the functionally relevant structures around the prostate. Magnetic Resonance Imaging (MRI) has boosted PGC, but available evidence suggests that ablation margins may be positive due to MRI-invisible lesions. This study aimed at determining the potential role of intraoperative digital analysis of ablation margins (DAAM) by fluoresce confocal microscopy (FCM) of biopsy cores taken during prostate PGC. Ten patients with low to intermediate risk PCa scheduled for PGC were enrolled. After cryo-needles placement, 76 biopsy cores were taken from the ablation margins and stained by the urologist for FCM analysis. Digital images were sent for “real-time” pathology review. DAAM, always completed within the frame of PGC treatment (median time 25 min), pointed out PCa in 1/10 cores taken from 1 patient, thus prompting placement of another cryo-needle to treat this area. Standard HE evaluation confirmed 75 cores to be cancer-free while displayed a GG 4 PCa in 7% of the core positive at FCM. Our data point out that IDAAM is feasible and reliable, thus representing a potentially useful tool to reduce the risk of missing areas of PCa during PGC.

GENOME SIZE DETERMINATION OF CUCUMBER (CUCUMIS SATIVUS), HONEYDEW (CUCUMIS MELO INODORUS) AND ROCK MELON (CUCUMIS MELO CANTALUPENSIS) VIA FLOW CYTOMETRY

The family of Cucurbitaceae consists of species with economical and nutritional value. Morphologically, there are only few differences between Cucumis species. The interspecific and intraspecific variation in the genome size of the Cucumis species are not discovered yet. Due to this, this study aims to determine the genome size of C. sativus, C. melo inodorus and C. melo cantalupensis using flow cytometry (FCM) method. Nuclei suspension of selected Cucumis species were extracted using LBO1 lysis buffer by manual chopping technique and stained by propidium iodide priot to FCM analysis. Genome size of C. sativus, C. melo inodorus (Honeydew) and C. melo cantalupensis (Rockmelon) were determined by using Glycine max (Soybean) as an external reference standard (2C = 2.5 pg). This study found that the genome size of C. sativus, C. melo inodorus and C. melo cantalupensis estimated to be 2.83 pg, 3.00 pg and 3.47 pg respectively. The genome size data obtained from this study can be used in future genome studies as well as species characterization.

Pemetaan untuk Strategi Dakwah di Kota Semarang Menggunakan Pendekatan Data Mining (Mapping for Da'wah Strategy in Semarang City Using Data Mining Approach)

This paper aims to explore the potential of da'wah in the city of Semarang with a data mining approach. The data mining approach is carried out by implementing the fuzzy c-means (FCM) algorithm in order to obtain the optimum number of clusters in the potential clustering of da'wah in the city of Semarang. The data used in this study from the Ministry of Religion of the Republic of Indonesia and the Central Statistics Agency (BPS) of Semarang City. The results of the FCM analysis show that the optimum number of clusters is two clusters, where the sub-district in the second cluster is an area with a high potential for da'wah. This study provides information that in effective da'wah activities, certainty and clarity is needed regarding the targets of da'wah through mapping of da'wah in the form of clustering potential da'wah. This can be a consideration of dakwah strategies for the successful implementation of da'wah studies so that an increase in the target behavior of da'wah can be achieved. The application of FCM to get the optimum cluster of potential da'wah in order to produce da'wah mapping is novelty in the field of Islamic studies, especially the science of da'wah.

Tumor microenviromental reaction would diminish incomparable accuracy and swiftness of flow cytometric diagnosis of primary central nervous system lymphoma

Background Primary central nervous system lymphoma (PCNSL), a relatively rare brain tumor, bears a dire prognosis. On occasion, rapid progression of the tumor makes immediate diagnosis and initiation of therapy imperative. To achieve swift diagnosis, we adopt flow cytometry (FCM) in addition to conventional histopathology. The aim of this study is to reveal utility and drawbacks of FCM diagnosis for PCNSL. Methods Patients with suspected PCNSL on neuroradiological findings and received both FCM and histological diagnosis between August 2015 and April 2020 were retrospectively enrolled into the study. Tumor samples were collected by craniotomy with either of endoscope or microscope. The patients’ electronic medical records were investigated, and histological findings, results of FCM, and other clinical data were evaluated. Results Twenty seven patients met the eligibility criteria. Twenty three patients (11 males and 12 females) were diagnosed with PCNSL by histological confirmation, and 22 cases were B-cell type lymphoma and 1 was T-cell type. Median age at diagnosis was 65. FCM analysis showed lymphoma pattern in 20 cases, but in the other 3 lymphoma cases (FCM discordant: FCM-D) and 4 non-lymphomatous tumor cases, FCM results did not show lymphoma pattern (sensitivity: 86.4%, specificity: 100%). Analysis of FCM-D cases showed infiltration of T lymphocytes or astrocytes into the tumor tissue, indicating tumor microenvironmental reaction, were observed, and it is assumed that those reactions deceived FCM diagnosis. Conclusions Despite some disadvantages, diagnosis of PCNSL by FCM is rapid and reliable.

Circle RNA PLEC Acts as a Sponge of Microrna-198 to Promote Gastric Carcinoma Cell Resistance to Paclitaxel and Tumorigenesis

Gastric carcinoma (GC) is one of the most frequent type of malignancy all over the world. The resistance of Paclitaxel (PTX) has become the obstacle of the prognosis of GC, and the underlying mechanism of is not clear. Previous study showed that GC-related circRNAs have been identified via microarray analysis and bioinformatics analysis, and we discovered that circPLEC (hsa_circ_0085923) remarkably upregulated in GC cells. The molecular mechanism of circPLEC in PTX-resistance GC cells still needs to explore.The expression of circPLEC in GC cells, PTX-resistant GC cells and GC tissues was analyzed by qRT-PCR. CircPLEC was knocked down in GC cells by transfecting shRNA, and then we used the CCK-8 assay, transwell, and FCM analysis to verify the effect in circPLEC in PTX-resistant GC cells. Additionally, we used luciferase reporter assays to confirm the relationship among circPLEC, miR-198 and MUC19.In present study, qRT-PCR exhibited that circPLEC were upregulated in PTX-resistant GC tissues and cells, indicating that circPLEC boost the PTX resistance of GC. circPLEC downregulation could weaken the GC resistance to PTX and the ability of tumorigenesis, migration and invasion, and promote the apoptosis of PTX-resistant GC cells. miR-198 inhibitor could revise the effect of circPLEC downregulation in PTX-resistant GC cells, and MUC19 downregulation could weaken the GC resistance to PTX and the tumorigenesis, and improve the apoptosis of PTX-resistant GC cells. In summary, circPLEC acts as a sponge of miR-198 to promote the PTX resistance and tumorigenesis of GC cells by regulating MUC19 expression.

Nuclear Genome Size Determination Of Christia Vespertilionis Via Flow Cytometry

Christia vespertilionis (butterfly wing plant) is an ornamental plant originated from South East Asia with reported usage in traditional medicine practice and potential as an anticancer and antitumor. This research aims to estimate the genome size of C. vespertilionis via flow cytometry (FCM) method. The research was conducted with the optimisation of nuclear suspension preparation followed by the genome size estimation. Two chopping techniques [manual chopping (MC) and BDTM Medimachine (MM)] and two lysis buffers (Otto and LBO1) were tested. Otto buffer with manual chopping was found to be the most suitable method, generated fine DNA peak with minimum debris background, and coefficient of variation (CV) value less than 3%. Five replicates of the FCM analysis were made for the genome size determination. The estimated genome size of C. vespertilionis was found to be 3.22 pg by using Glycine max cv. Polanka (2C=2.5pg) as an external reference standard. Further comparison with other Christia species was not possible due to the lack of data on genome size. The genome size data of C. vespertilionis can be useful for future morphology and genetics studies of Christia species.