Normal Cell Populations Research Articles

Tumor cell and immune cell profiles in primary human glioblastoma: Impact on patient outcome.

The distribution and role of tumor‐infiltrating leucocytes in glioblastoma (GBM) remain largely unknown. Here, we investigated the cellular composition of 55 primary (adult) GBM samples by flow cytometry and correlated the tumor immune profile with patient features at diagnosis and outcome. GBM single‐cell suspensions were stained at diagnosis (n = 44) and recurrence following radiotherapy and chemotherapy (n = 11) with a panel of 8‐color monoclonal antibody combinations for the identification and enumeration of (GFAP+CD45−) tumor and normal astrocytic cells, infiltrating myeloid cells —i.e. microglial and blood‐derived tumor‐associated macrophages (TAM), M1‐like, and M2‐like TAM, neutrophils. and myeloid‐derived suppressor cells (MDSC)— and tumor‐infiltrating lymphocytes (TIL) —i.e. CD3+T‐cells and their TCD4+, TCD8+, TCD4−CD8−, and (CD25+CD127lo) regulatory (T‐regs) subsets, (CD19+CD20+) B‐cells, and (CD16+) NK‐cells—. Overall, GBM samples consisted of a major population (mean ± 1SD) of tumor and normal astrocytic cells (73% ± 16%) together with a significant but variable fraction of immune cells (24% ± 18%). Within myeloid cells, TAM predominated (13% ± 12%) including both microglial cells (10% ± 11%) and blood‐derived macrophages (3% ± 5%), in addition to a smaller proportion of neutrophils (5% ± 9%) and MDSC (4% ± 8%). Lymphocytes were less represented and mostly included TCD4+ (0.5% ± 0.7%) and TCD8+ cells (0.6% ± 0.7%), together with lower numbers of TCD4−CD8− T‐cells (0.2% ± 0.4%), T‐regs (0.1% ± 0.2%), B‐lymphocytes (0.1% ± 0.2%) and NK‐cells (0.05% ± 0.05%). Overall, three distinct immune profiles were identified: cases with a minor fraction of leucocytes, tumors with a predominance of TAM and neutrophils, and cases with mixed infiltration by TAM, neutrophils, and T‐lymphocytes. Untreated GBM patients with mixed myeloid and lymphoid immune infiltrates showed a significantly shorter patient overall survival versus the other two groups, in the absence of gains of the EGFR gene (p = 0.02). Here we show that immune cell infiltrates are systematically present in GBM, with highly variable levels and immune profiles. Patients with mixed myeloid and T‐lymphoid infiltrates showed a worse outcome.

An optimization technique for solving a class of nonlinear fractional optimal control problems: Application in cancer treatment

This paper proposes an optimization method for solving a general form of nonlinear fractional optimal control problems (NFOCP) governed by nonlinear fractional dynamical systems and mixed conditions. A model for cancer treatment based on the NFOCP is developed, by including the population of immune cells, tumor cells, normal cells, fat cells, chemotherapeutic and immunotherapeutic drug concentrations. The aim behind the NFOCP is to find the optimal doses of chemotherapeutic and immunotherapeutic drugs which minimize the difference between the numbers of tumor and normal cells. The proposed approach includes inserting weight constants for the tumor and normal cells in the cost functional so that the normal cell population is large minimizing, therefore, the tumor burden. The method approximates the solutions of the NFOCP using the generalized shifted Legendre polynomials (GSLP). Operational matrices of integer and fractional derivatives are derived for the GSLP. The Gaussian quadrature rule and the Lagrange multipliers method reduce the NFOCP to a system of algebraic equations. Additionally, the error analysis of the algorithm is considered. Numerical examples demonstrate the effectiveness of the method.

The role of high mobility group protein B3 (HMGB3) in tumor proliferation and drug resistance.

The high mobility group protein B (HMGB) family (including HMGB1, HMGB2, HMGB3, and HMGB4) can regulate the mechanisms of DNA replication, transcription, recombination, and repair, and act as cytokines to mediate responses to infection, injury, and inflammation. HMGB1/2/3 has a high similarity in sequence and structure, while HMGB4 has no acidic C-terminal tail. Among them, HMGB3 can regulate the self-renewal and differentiation of normal hematopoietic stem cell population, but the decrease of its expression is easy to induce leukemia. Up-regulation of its expression promotes tumor development and chemotherapy resistance through a variety of mechanisms, and non-coding RNA can regulate to promote tumor cell proliferation, invasion, and migration and inhibit cancer cell apoptosis.

Alternative approaches to overcome chemoresistance to apoptosis in cancer.

Apoptosis, or programmed cell death, is a form of regulated cell death (RCD) that is essential for organogenesis and homeostatic maintenance of normal cell populations. Apoptotic stimuli activate the intrinsic and/or extrinsic pathways to induce cell death due to perturbations in the intracellular and extracellular microenvironments, respectively. In patients with cancer, the induction of apoptosis by anticancer drugs and radiation can produce cancer cell death. However, tumor cells can adapt and become refractory to apoptosis-inducing therapies, resulting in the development of clinical resistance to apoptosis. Drug resistance facilitates the development of aggressive primary tumors that eventually metastasize, leading to therapy failure and mortality. To overcome the resistance to apoptosis to neoadjuvant chemotherapy or targeted therapy, alternative targets of RCD can be induced in apoptosis-resistant cancer cells. Alternatively, cell death can be independent of apoptosis and this strategy could be utilized to develop novel anti-cancer therapies. This chapter discusses approaches that could be employed to overcome clinical resistance to apoptosis in cancer cells.

The Role of β-Arrestins in Regulating Stem Cell Phenotypes in Normal and Tumorigenic Cells.

β-Arrestins (ARRBs) are ubiquitously expressed scaffold proteins that mediate inactivation of G-protein-coupled receptor signaling, and in certain circumstances, G-protein independent pathways. Intriguingly, the two known ARRBs, β-arrestin1 (ARRB1) and β-Arrestin2 (ARRB2), seem to have opposing functions in regulating signaling cascades in several models in health and disease. Recent evidence suggests that ARRBs are implicated in regulating stem cell maintenance; however, their role, although crucial, is complex, and there is no universal model for ARRB-mediated regulation of stem cell characteristics. For the first time, this review compiles information on the function of ARRBs in stem cell biology and will discuss the role of ARRBs in regulating cell signaling pathways implicated in stem cell maintenance in normal and malignant stem cell populations. Although promising targets for cancer therapy, the ubiquitous nature of ARRBs and the plethora of functions in normal cell biology brings challenges for treatment selectivity. However, recent studies show promising evidence for specifically targeting ARRBs in myeloproliferative neoplasms.

Inhibition of the Wnt Signalling Pathway: An Avenue to Control Breast Cancer Aggressiveness.

Breast cancer (BC) is the most common tumour in women. Although the introduction of novel therapeutic approaches in clinical practice has dramatically improved the clinical outcome of BC patients, this malignant disease remains the second leading cause of cancer-related death worldwide. The wingless/integrated (Wnt) signalling pathway represents a crucial molecular node relevantly implicated in the regulation of normal somatic stem cells as well as cancer stem cell (CSC) traits and the epithelial–mesenchymal transition cell program. Accordingly, Wnt signalling is heavily dysregulated in BC, and the altered expression of different Wnt genes is significantly associated with cancer-related aggressive behaviours. For all these reasons, Wnt signalling represents a promising therapeutic target currently under clinical investigation to achieve cancer eradication by eliminating CSCs, considered by most to be responsible for tumour initiation, relapse, and drug resistance. In this review, we summarized the current knowledge on the Wnt signalling pathway in BC and have presented evidence implicating the suitability of Wnt targeting in an attempt to improve the outcome of patients without affecting the normal somatic stem cell population.

Jasmonates induce Arabidopsis bioactivities selectively inhibiting the growth of breast cancer cells through CDC6 and mTOR.

Summary Phytochemicals are used often in vitro and in vivo in cancer research. The plant hormones jasmonates (JAs) control the synthesis of specialized metabolites through complex regulatory networks. JAs possess selective cytotoxicity in mixed populations of cancer and normal cells.Here, direct incubation of leaf explants from the non‐medicinal plant Arabidopsis thaliana with human breast cancer cells, selectively suppresses cancer cell growth. High‐throughput LC‐MS identified Arabidopsis metabolites. Protein and transcript levels of cell cycle regulators were examined in breast cancer cells.A synergistic effect by methyljasmonate (MeJA) and by compounds upregulated in the metabolome of MeJA‐treated Arabidopsis leaves, on the breast cancer cell cycle, is associated with Cell Division Cycle 6 (CDC6), Cyclin‐dependent kinase 2 (CDK2), Cyclins D1 and D3, indicating that key cell cycle components mediate cell viability reduction. Bioactives such as indoles, quinolines and cis‐(+)‐12‐oxophytodienoic acid, in synergy, could act as anticancer compounds.Our work suggests a universal role for MeJA‐treatment of Arabidopsis in altering the DNA replication regulator CDC6, supporting conservation, across kingdoms, of cell cycle regulation, through the crosstalk between the mechanistic target of rapamycin, mTOR and JAs. This study has important implications for the identification of metabolites with anti‐cancer bioactivities in plants with no known medicinal pedigree and it will have applications in developing disease treatments

Normal or reactive minor cell populations in bone marrow and peripheral blood mimic minimal residual leukemia by flow cytometry.

Measurable residual disease (MRD) is a strong independent poor prognostic factor for acute leukemia. Multiparameter flow cytometry (FCM) is a commonly used MRD detection method. However, FCM MRD detection is not well standardized, and the interpretation is subjective. There are normal/reactive minor cell populations in bone marrow (BM) and peripheral blood (PB), which could be confused with MRD. The FCM data of 231 BM and 44 PB pediatric samples performed in a recent 15-month period were retrospectively reviewed. These samples were from 56 B-lymphoblastic leukemia (B-ALL) patients, 11 T-lymphoblastic leukemia (T-ALL) patients, 28 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) patients, 44 cytopenia/leukocytosis patients, and five patients with mycosis fungoides. There were over 10 normal or reactive minor cell populations identified with certain phenotypes mimicking MRD of acute leukemia. These mimickers included CD19+ NK cells, CD22+ basophils, CD22+ dendritic cells (DCs), and plasma cells for B-ALL MRD; CD4/8 double-negative T cells, CD4/8 double-positive T cells, cytoplasmic CD3+ NK cells, CD2- T cells, CD7- T cells, CD5- gamma delta T cells, CD56+ NKT cells for T-ALL MRD; CD33+ NK cells, CD117+ NK cells, basophils, plasmacytoid DCs, non-classical monocytes, CD56+ and/or CD61+ monocytes for AML MRD. These data confirm the presence of a variety of normal/reactive minor cell populations that could mimic MRD of acute leukemia by FCM. Recognizing these MRD mimickers is important for correct FCM MRD interpretation.

A Phase 1/1b Safety Study of Prgn-3006 Ultracar-T™ in Patients with Relapsed or Refractory CD33-Positive Acute Myeloid Leukemia and Higher Risk Myelodysplastic Syndrome

A Phase 1/1b Safety Study of Prgn-3006 Ultracar-T™ in Patients with Relapsed or Refractory CD33-Positive Acute Myeloid Leukemia and Higher Risk Myelodysplastic Syndrome

Abstract IA21: Stem cells play a role in human leukemia from origin to relapse

Abstract Individual cancer cells exhibit functional heterogeneity of many cancer hallmarks including the capacity for sustaining long-term clonal maintenance, a stemness property involving self-renewal. Our studies established that only rare AML cells possessed such leukemia stem cell (LSC) properties and that AML is a cellular hierarchy. LSC were found to be highly relevant to human disease as gene signatures specific to LSC were much more predictive of patient response to therapy and overall survival compared to the bulk non-LSC AML cells. We have now provided insights into how LSC develop during leukemogenesis. Through sequencing of purified populations of normal blood cells, AML cells, and xenografts from paired diagnosis/relapse AML samples, we tracked the full arc of leukemia development in humans: from the cell of origin (an HSC) that acquires the first genetic mutation; to pre-leukemic clonal expansion of HSC; the generation of genetically diverse LSC; and finally to the cellular origin of relapse (rare LSC subclones) within the diagnosis sample. Similar studies have been undertaken in B-ALL where relapse-fated subclones were found within the diagnosis sample before exposure to chemotherapy. These clones possess distinct epigenetic and metabolic properties that underlie their partially resistance to chemotherapy and they possess stemness signatures that drive their ability to regenerate relapse disease. These studies resulted in identification of novel therapeutic targets of relapse-fated subclones to begin envisioning approaches to eradicate relapse before it develops. Finally, the identification of clonally expanded pre-leukemic HSPC in the diagnosis blood sample of many AML samples predicted that it should be possible to identify individuals who are at risk for progressing to AML long before AML develops. We genotyped individuals from the general population who were enrolled in the large cohort (EPIC) who eventually developed AML and compared them to the enrollment sample of others who never progressed to AML. We have found a clear signature that is able to predict with high accuracy those individuals who have age related clonal hematopoiesis who progress to AML, almost 10 years prior to AML development. This is distinct from individuals who have benign clonal hematopoiesis who never progress. These new findings offer the potential for future intervention to permit AML prevention trials. Citation Format: John E. Dick. Stem cells play a role in human leukemia from origin to relapse [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA21.

Immunomodulatory role of black tea in the mitigation of cancer induced by inorganic arsenic

We present a model analysis of the tumor and normal cell growth under the influence of a carcinogenic agent, an immunomdulator (IM) and variable influx of immune cells including relevant interactions. The tumor growth is facilitated by carcinogens such as inorganic arsenic while the IM considered here is black tea (Camellia sinesnsis). The model with variable influx of immune cells is observed to have considerable advantage over the constant influx model, and while the tumor cell population is greatly mitigated, normal cell population remains above healthy levels. The evolution of normal and tumor cells are computed from the proposed model and their local stabilities are investigated analytically. Numerical simulations are performed to study the long term dynamics and an estimation of the effects of various factors is made. This helps in developing a balanced strategy for tumor mitigation without the use of chemotherapeutic drugs that usually have strong side-effects.

Abstract 3829: Dclk1 expressing crypt epithelial cells become tumor initiating stem cells in an inflammation associated environment

Abstract Background: Chronic intestinal inflammation is a major risk factor for the development of CRC and consequently individuals suffering from inflammatory bowel diseases (IBD) are at increased risk. However, the molecular mechanism by which inflammation initiates colonic tumorigenesis in the cell type is unclear and the regulatory factors that mediate the complex interplay between inflammation and the colonic tumor initiation are unknown. Doublecortin-like kinase 1 (Dclk1) is a microtubule-associated kinase, which marks TSCs in colon cancer, and is predominantly expressed in a unique intestinal epithelial cell type termed the tuft cells. Tuft cells are chemosensory cells that regulate host immunity and inflammation and is highly increased in CRC and, ablation of Dclk1 expressing cells in the ApcMin/+ mice completely regressed the tumors without affecting the normal stem cell population. Our aim is to elucidate the molecular mechanism that is responsible for inflammation-associated colon cancer and to determine the role of Dclk1 expressing tuft cells in this process. Experimental Procedure: YMCA non-malignant colonic epithelial cells were used. Loss and gain of Dclk1 expression in the YMCA cells were carried with siRNA transfection and lentivirus overexpression vector infection respectively. Dclk1 expressing cells were selected for stable cell generation. DSS and LPS were used. Cell self-renewal, secondary and tertiary organoid generation, survival, DNA damage, DNA damage response (DDR) and apoptosis were assessed. Protein and mRNA expressions by western-blot and RT-PCR. Results: Here, we report that increased expression of Dclk1 is the key to the generation of tumor-initiating cells under the influence of an inflammatory environment. We observed increased self-renewal of Dclk1+ YMCA cells treated with DSS and further increase in self-renewal was observed with DSS+LPS treatment. Silencing Dclk1 failed to initiate self-renewal under the influence of DSS and LPS treatment. Dclk1+ YMCA cells displayed enhanced inflammatory mediators, survival with colony formation and increased survival pathways (AKT/mTOR/Beta-catenin/NFkB pathways) and DDR (ATM/ATR) with DSS or LPS treatment. However, the DNA damage and cell apoptosis were not increased in vector control cells and significantly increased in Dclk1 knockdown cells after DSS or LPS treatment. Conclusion: Our data demonstrate that the increased expression of Dclk1 is associated with tumor-initiating features, and survival ability in an inflammatory environment, which is important to understand the cell type and inflammation associated mechanisms in tumor initiation. Based on these findings we proposed that specific targeting of Dclk1-expressing colonic crypt epithelial cells has the potential to result in ablation of tumor initiation. Citation Format: Parthasarathy Chandrakesan, Janani Panneerselvam, Dongfeng Qu, Chinthalapally Rao, Michael Bronze, Courtney Houchen. Dclk1 expressing crypt epithelial cells become tumor initiating stem cells in an inflammation associated environment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3829.

Abstract 5051: RenMab Mouse: A leading platform for fully human antibody generation

Abstract With the development of immune-oncology, therapeutic antibodies have been proven to be extraordinarily effective for cancer treatment. Conventional human antibody discovery process can be divided into steps including target selection, target validation, screening preparation, hits generation, leads selection, lead optimization, and clinical candidate selection. To accelerate antibody development process, Biocytogen has developed RenMab Mouse, a fully human antibody mouse whose entire variable regions were replaced by human Ig heavy chain and κ light chain through Biocytogen's unique Mb-scale chromosome engineering technology. RenMab Mouse provides a remarkably efficient therapeutic antibody discovery platform for fully human antibody generation, characterization, and rapid in vivo efficacy screening. We have characterized RenMab Mouse with the following features. 1. The entire mouse variable regions were replaced with complete human genome DNA in situ for complete human & mouse regulatory elements. The gene regulation of RenMab mouse is highly consistent with that of human. 2. Mouse constant region remains to ensure normal immune cell population, development and maturation. Immune system of RenMab Mouse has been proven to be almost identical to that of wild type mice. RenMab Mouse showed normal antibody immune responses to antigens. 3. RenMab Mouse generates a highly diverse repertoire of fully human antibody variable regions through V(D)J recombination. This capability can lead to improved hit rates in antibody drug discovery. In conclusion, RenMab Mouse, a fully human antibody mouse developed by Biocytogen's chromosome engineering technology, has been proven to exhibit normal immune cell profile and rapid immune response. Combined with existing inventory of single, double or triple target humanized mouse models and a world-leading proprietary gene editing platform, Biocytogen is dedicated to providing a one-stop solution for antibody discovery and development from target validation to IND application. Citation Format: Gary Ng, Huizhen Zhao, Yabo Zhang, Lili Liu, Hui Lu, Shuwen Huang, Yuelei Shen. RenMab Mouse: A leading platform for fully human antibody generation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5051.

Antibody-Based Targeted Interventions for the Diagnosis and Treatment of Skin Cancers.

Cutaneous malignancies most commonly arise from skin epidermal cells. These cancers may rapidly progress from benign to a metastatic phase. Surgical resection represents the gold standard therapeutic treatment of non-metastatic skin cancer while chemo- and/or radiotherapy are often used against metastatic tumors. However, these therapeutic treatments are limited by the development of resistance and toxic side effects, resulting from the passive accumulation of cytotoxic drugs within healthy cells. This review aims to elucidate how the use of monoclonal Antibodies (mAbs) targeting specific Tumor Associated Antigens (TAAs) is paving the way to improved treatment. These mAbs are used as therapeutic or diagnostic carriers that can specifically deliver cytotoxic molecules, fluorophores or radiolabels to cancer cells that overexpress specific target antigens. mAbs raised against TAAs are widely in use for e.g. differential diagnosis, prognosis and therapy of skin cancers. Antibody-Drug Conjugates (ADCs) particularly show remarkable potential. The safest ADCs reported to date use non-toxic photo-activatable Photosensitizers (PSs), allowing targeted Photodynamic Therapy (PDT) resulting in targeted delivery of PS into cancer cells and selective killing after light activation without harming the normal cell population. The use of near-infrared-emitting PSs enables both diagnostic and therapeutic applications upon light activation at the specific wavelengths. Antibody-based approaches are presenting an array of opportunities to complement and improve current methods employed for skin cancer diagnosis and treatment.

Ultrathin glass fiber microprobe for electroporation of arbitrary selected cell groups

A new type of ultrathin fiber microprobe for selective electroporation is reported. The microprobe is 10 cm long and has a diameter of 350 µm. This microprobe is a low cost tool, which allows electroporation of an arbitrary selected single cell or groups of cells among population with use of a standard microscope and cell culture plates. The microprobe in its basic form contains two metal microelectrodes made of a silver-copper alloy, running along the fiber, each with a diameter of 23 µm. The probe was tested in vitro on a population of normal and cancer cells. Successful targeted electroporation was observed by means of accumulation of trypan blue (TB) dye marker in the cell. The electroporation phenomenon was also verified with propidium iodide and AnnexinV in fluorescent microscopy.

Single-cell RNA sequencing demonstrates the molecular and cellular reprogramming of metastatic lung adenocarcinoma

Advanced metastatic cancer poses utmost clinical challenges and may present molecular and cellular features distinct from an early-stage cancer. Herein, we present single-cell transcriptome profiling of metastatic lung adenocarcinoma, the most prevalent histological lung cancer type diagnosed at stage IV in over 40% of all cases. From 208,506 cells populating the normal tissues or early to metastatic stage cancer in 44 patients, we identify a cancer cell subtype deviating from the normal differentiation trajectory and dominating the metastatic stage. In all stages, the stromal and immune cell dynamics reveal ontological and functional changes that create a pro-tumoral and immunosuppressive microenvironment. Normal resident myeloid cell populations are gradually replaced with monocyte-derived macrophages and dendritic cells, along with T-cell exhaustion. This extensive single-cell analysis enhances our understanding of molecular and cellular dynamics in metastatic lung cancer and reveals potential diagnostic and therapeutic targets in cancer-microenvironment interactions.

Pomegranate Seeds Extract Possesses a Protective Effect against Tramadol-Induced Testicular Toxicity in Experimental Rats.

Tramadol is a centrally acting opioid analgesic that is extensively used. The chronic exposure to tramadol induces oxidative stress and toxicity especially for patients consuming it several times a day. Previously, we and others reported that tramadol induces testicular damage in rats. This study was conducted to investigate the possible protective effect of pomegranate seed extract (PgSE) against tramadol-induced testicular damage in adult and adolescent rats. Male rats were orally treated with tramadol or in a combination with PgSE for three weeks. Testes were then dissected and analyzed. Histological and ultrastructural examinations indicated that tramadol induced many structural changes in the testes of adult and adolescent rats including hemorrhage of blood vessels, intercellular spaces, interstitial vacuoles, exfoliation of germ cells in lumen, cell apoptosis, chromatin degeneration of elongated spermatids, and malformation of sperm axonemes. Interestingly, these abnormalities were not observed in tramadol/PgSE cotreated rats. The morphometric analysis revealed that tramadol disrupted collagen metabolism by elevating testicular levels of collagen fibers but that was protected in tramadol/PgSE cotreatment at both ages. In addition, DNA ploidy revealed that S phase of the cell cycle was diminished when adult and adolescent rats were treated with tramadol. However, the S phase had a normal cell population in the cotreated adult rats, but adolescent rats had a lower population than controls. Furthermore, the phytochemistry of PgSE revealed a high content of total polyphenols and total flavonoids within this extract; besides, the DPPH free radical scavenging activity was high. In conclusion, this study indicated that PgSE has a prophylactic effect against tramadol-induced testicular damage in both adult and adolescent ages, although the tramadol toxicity was higher in adolescent age to be completely protected. This prophylactic effect might be due to the high antioxidant compounds within the pomegranate seeds.

Characterization of a Rare Mosaicism in Autosomal Translocation of t(5;21) Using Conventional Cytogenetics and FISH Methods

Background:Mosaicism of a normal cell population and an unbalanced autosomal chromosome rearrangement is rarely seen. If the abnormal cell line contributes to a minor part of soma, the phenotype is expected to be normal. Case Report:We report a 29-year-old woman who had balance chromosomal translocation of 46,XX,t(5;21) with a two-year-old affected girl, characterized by mental retardation, dystrophia, hearing impartment, and dysphagia. Methods and Results:Cytogenetic investigation revealed a low mosaic unbalanced translocation of 46,XX,t(5;21)/ 46,XX, which was confirmed by FISH analysis. Studying 200 metaphases and interphases of peripheral blood sample revealed 70% partial monosomy of 21q22 and partial trisomy of 5q(35.3) and 30% of normal pattern. Conclusion:In rare cases such as this study, parents with balanced translocation with no phenotypes may lead to a mosaic unbalanced translocation with abnormal phenotypes in offspring, which underscores the need for prenatal karyotyping and genetics counseling.

Malignant T-Cells and Normal Intratumoral T-Cells Have Similar Expression of Immune Checkpoint Molecules in Angioimmunoblastic T-Cell Lymphoma

Malignant T-Cells and Normal Intratumoral T-Cells Have Similar Expression of Immune Checkpoint Molecules in Angioimmunoblastic T-Cell Lymphoma

Tumor Circulating Plasma Cells Detected By Flow Cytometric Single Platform Method Correlate with Clinical Response to Therapy and Unfavorable Patients' Characteristics

Background: In multiple myeloma (MM), different clinical parameters and molecular prognostic factors can predict disease course and response to therapy. The classification of myeloma patients includes laboratory parameters associated with higher tumor activity, resistance to therapy and proliferative competence. Tumor circulating plasma cells (TCPC) in MM patients showed a strong correlation with a more aggressive disease. Aim: For the first time, we quantified the amounts of TCPC with single platform flow cytometric method and evaluated their relationship with patients' baseline characteristics and response to therapy before maintenance. Methods: Whole peripheral blood samples from 413 newly diagnosed MM patients ≤65 years enrolled in the UNITO-MM-01/FORTE trial were collected. Patients were randomized [1:1:1; stratification: International Staging System (ISS) and age] to ARM A: carfilzomib-cyclophosphamide-dexamethasone (KCyd) followed by melphalan 200 mg/m2 and autologous stem-cell transplantation (MEL200-ASCT) and consolidation with 4 KCyd; ARM B: carfilzomib-lenalidomide-dexamethasone (KRd) followed by MEL200-ASCT and 4 KRd; ARM C: 12 KRd cycles. Enrollment was completed in March 2017; data cut-off was November 30, 2018. For the single platform tube, the antibody combination CD38PC7/CD138PC5.5/ CD45KO/CD56PE/CD19PB was mixed with 100µL of EDTA peripheral blood, dispensed with reverse pipetting, and incubated for 15 min, added with 500µL of lysing solution and, after 15 min, 100µL of flow count fluorospheres were dispensed with reverse pipetting and cells acquired with Navios flow cytometer. Intracytoplasmic tube was set up to confirm the clonality of CPC. Results: Circulating plasma cells (CPC) were quantified in 413 samples, with median values of 0.03% (range: 0-51%) and 2.37/mm3 (range: 0-6272/mm3). White blood cells were 5710/mm3 (range: 1752-26102/mm3); total events acquired 1285000 (range: 40000-2000000); median CPC events were 58 (range: 0-441000); cellular events acquired were 190000 (range: 4428-1300000). In 390 out of 413 samples (94.4%), CPC were detected; 272 samples (66%) showed TCPC with a median of 1.24/mm3 (range 0.06- 6272/mm3). Patients were sorted according to different baseline characteristics and the medians of absolute TCPC were compared. The most statistically significant differences (p<0.001) were: haemoglobin (Hb) <10 (12.9/mm3) vs. ≥10 (0.81/mm3); ISS I (0.30/mm3) vs. ISS II (2.85/mm3) vs. ISS III (5.14/mm3); R-ISS I (0.25/mm3) vs. II (2.76/mm3) vs. III (7.45/mm3); albumin <3.5g/dL (2.76/mm3) vs. ≥3.5g/dL (1.05/mm3); β2-microglobulin <3.5mg/dL (0.67/mm3) vs. 3.5mg/dL-5.5mg/dL (3.88/mm3) vs. >5.5mg/dL (16.47/mm3); lactate dehydrogenase (LDH) ≤upper limit of normal (ULN, 1.14/mm3) vs. >ULN (7.36/mm3); plasma cells (PC) in biopsy <60% (0.60/mm3) vs. ≥60%(2.76/mm3); with (3.18/mm3) vs. without amp1q (1.18/mm3); Morgan risk standard (1.21/mm3) vs. high (3.00/mm3). Finally, we compared the absolute number of TCPC and the quality of response at the end of consolidation therapy. Higher values of TCPC were related to worst response: <partial response (PR, 4.23/mm3) vs. ≥PR (1.23/mm3); <very good PR (VGPR, 2.91/mm3) vs. ≥VGPR (1.20/mm3); <complete response (CR, 1.95/mm3) vs. ≥CR (1.09/mm3); <stringent CR (sCR, 1.71/mm3) vs. ≥sCR (1.00/mm3), p<0.05. Conclusions: Single-platform flow cytometry is a simple method to quantify TCPC, present in almost all peripheral blood from MM patients; a high number is related to poor clinical response to therapy and helps in identifying high-risk patients. Moreover, it allows the discrimination between normal and pathological plasma cell population in peripheral blood. However, a longer follow up is needed to evaluate how TCPC can affect survival in patients with MM. Disclosures Musto: Amgen: Honoraria; Celgene: Honoraria. Gay:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Boccadoro:Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding. Omedé:Janssen: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: This presentation includes discussion of off-label use of a drug or drugs for the treatment of multiple myeloma.