Identification of a novel immune prognostic model in gastric cancer.

PURPOSE: The tumor immune microenvironment (TIME) is now considered as an important factor during gastric cancer (GC) development. This study identified a novel immune-related risk model for predicting prognosis and assessing the immune status of GC patients. METHODS: Transcriptomic data were obtained from the TCGA database. The differential expressed immune-related genes (IRGs) were identified through the ImmPort portal. Enrichment analysis was performed to explore the potential molecular mechanism of these IRGs. By the Cox regression analysis, we constructed the immune prognostic model. Then, the association between the model and the immune microenvironment was estimated. The model was validated in the GSE84433 dataset. RESULTS: Totally, we identified 222 differentially expressed IRGs. These IRGs were closely correlated with immune response and immune signaling pathways. Through the Cox regression analysis, we developed the immune prognostic model based on the expression of seven IRGs (CXCL3, NOX4, PROC, FAM19A4, RNASE2, IGHD2-15, CGB5). Patients were stratified into two groups according to immune-related risk scores. Survival analysis indicated that the prognosis of high-risk patients was poorer than low-risk patients. Moreover, the immune-related risk score was an independent prognostic biomarker. More importantly, we found that the infiltration level of immunosuppressive cells and the expression of inhibitory immune checkpoints were higher in high-risk patients. The immune microenvironment tended to be a suppressive status in patients with high-risk scores. CONCLUSION: This study demonstrated that our model had predictive value for prognosis and the TIME in GC. It might be a robust tool to improve personalized patient management.

Leukemia stem cell immunophenotyping tool for diagnostic, prognosis, and therapeutics.

The existence of cancer stem cells is debatable in numerous solid tumors, yet in leukemia, there is compelling evidence of this cell population. Leukemic stem cells (LSCs) are altered cells in which accumulating genetic and/or epigenetic alterations occur, resulting in the transition between the normal, preleukemic, and leukemic status. These cells do not follow the normal differentiation program; they are arrested in a primitive state but with high proliferation potential, generating undifferentiated blast accumulation and a lack of a mature cell population. The identification of LSCs might guide stem cell biology research and provide key points of distinction between these cells and their normal counterparts. The identification and characterization of the main features of LSCs can be useful as tools for diagnosis and treatment. In this context, the aim of the present review was to connect immunophenotype data in the main types of leukemia to further guide technical improvements.

Dual-targeted therapeutic strategy combining CSC-DC-based vaccine and cisplatin overcomes chemo-resistance in experimental mice model.

BACKGROUND AND PURPOSE: Emerging evidence suggests that one of the main reasons of chemotherapy treatment failure is the development of multi-drug resistance (MDR) associated with cancer stem cells (CSCs). Our aim is to identify a therapeutic strategy based on MDR-reversing agents. MATERIALS AND METHODS: CSC-enriched Ehrlich carcinoma (EC) cell cultures were prepared by drug-resistant selection method using different concentrations of cisplatin (CIS). Cell cultures following drug exposure were analyzed by flow cytometry for CSC surface markers CD44+/CD24-. We isolated murine bone marrow-derived dendritic cells (DCs) and then used them to prepare CSC-DC vaccine by pulsation with CSC-enriched lysate. DCs were examined by flow cytometry for phenotypic markers. Solid Ehrlich carcinoma bearing mice were injected with the CSC-DC vaccine in conjunction with repeated low doses of CIS. Tumor growth inhibition was evaluated and tumor tissues were excised and analyzed by real-time PCR to determine the relative gene expression levels of MDR and Bcl-2. Histopathological features of tumor tissues excised were examined. RESULTS AND CONCLUSION: Co-treatment with CSC-DC and CIS resulted in a significant tumor growth inhibition. Furthermore, the greatest response of downregulation of MDR and Bcl-2 relative gene expression were achieved in the same group. In parallel, the histopathological observations demonstrated enhanced apoptosis and absence of mitotic figures in tumor tissues of the co-treatment group. Dual targeting of resistant cancer cells using CSC-DC vaccine along with cisplatin represents a promising therapeutic strategy that could suppress tumor growth, circumvent MDR, and increase the efficacy of conventional chemotherapies.

To Cure or Not to Cure: Consequences of Immunological Interactions in CML Treatment.

Recent clinical findings in chronic myeloid leukemia (CML) patients suggest that the number and function of immune effector cells are modulated by tyrosine kinase inhibitors (TKI) treatment. There is further evidence that the success or failure of treatment cessation at least partly depends on the patients immunological constitution. Here, we propose a general ODE model to functionally describe the interactions between immune effector cells with leukemic cells during the TKI treatment of CML. In total, we consider 20 different sub-models, which assume different functional interactions between immune effector and leukemic cells. We show that quantitative criteria, which are purely based on the quality of model fitting, are not able to identify optimal models. On the other hand, the application of qualitative criteria based on a dynamical system framework allowed us to identify nine of those models as more suitable than the others to describe clinically observed patterns and, thereby, to derive conclusion about the underlying mechanisms. Additionally, including aspects of early CML onset, we can demonstrate that certain critical parameters, such as the strength of immune response or leukemia proliferation rate, need to change during CML growth prior to diagnosis, leading to bifurcations that alter the attractor landscape. Finally, we show that the crucial parameters determining the outcome of treatment cessation are not identifiable with tumor load data only, thereby highlighting the need to measure immune cell number and function to properly derive mathematical models with predictive power.

Breast Tumor Cells Highly Resistant to Drugs Are Controlled Only by the Immune Response Induced in an Immunocompetent Mouse Model.

BACKGROUND: The tumor cells responsible for metastasis are highly resistant to chemotherapy and have characteristics of stem cells, with a high capacity for self-regeneration and the use of detoxifying mechanisms that participate in drug resistance. In vivo models of highly resistant cells allow us to evaluate the real impact of the immune response in the control of cancer. MATERIALS AND METHODS: A tumor population derived from the 4T1 breast cancer cell line that was stable in vitro and highly aggressive in vivo was obtained, characterized, and determined to exhibit cancer stem cell (CSC) phenotypes (CD44+, CD24+, ALDH+, Oct4+, Nanog+, Sox2+, and high self-renewal capacity). Orthotopic transplantation of these cells allowed us to evaluate their in vivo susceptibility to chemo and immune responses induced after vaccination. RESULTS: The immune response induced after vaccination with tumor cells treated with doxorubicin decreased the formation of tumors and macrometastasis in this model, which allowed us to confirm the immune response relevance in the control of highly chemotherapy-resistant ALDH+ CSCs in an aggressive tumor model in immunocompetent animals. CONCLUSIONS: The antitumor immune response was the main element capable of controlling tumor progression as well as metastasis in a highly chemotherapy-resistant aggressive breast cancer model.

Transcription Factor Networks derived from Breast Cancer Stem Cells control the immune response in the Basal subtype.

Breast cancer is the most common cancer in women worldwide and metastatic dissemination is the principal factor related to death by this disease. Breast cancer stem cells (bCSC) are thought to be responsible for metastasis and chemoresistance. In this study, based on whole transcriptome analysis from putative bCSC and reverse engineering of transcription control networks, we identified two networks associated with this phenotype. One controlled by SNAI2, TWIST1, BNC2, PRRX1 and TBX5 drives a mesenchymal or CSC-like phenotype. The second network is controlled by the SCML4, ZNF831, SP140 and IKZF3 transcription factors which correspond to immune response modulators. Immune response network expression is correlated with pathological response to chemotherapy, and in the Basal subtype is related to better recurrence-free survival. In patient-derived xenografts, the expression of these networks in patient tumours is predictive of engraftment success. Our findings point out a potential molecular mechanism underlying the balance between immune surveillance and EMT activation in breast cancer. This molecular mechanism may be useful to the development of new target therapies.

Isolation, cultivation and characterization of CD133+ stem cells from human glioblastoma.

OBJECTIVE: To establish the method of isolation and culture of human glioblastoma neurospheres, and the purification of their stem cells, followed by the process of obtaining tumor subspheres, immunophenotypically characterizing this clonogenic set. METHODS: Through the processing of glioblastoma samples (n=3), the following strategy of action was adopted: (i) establish primary culture of glioblastoma; (ii) isolation and culture of tumor neurospheres; (iii) purify cells that initiate tumors (CD133+) by magnetic separation system (MACS); (iv) obtain tumor subspheres; (v) study the expression of the markers nestin, CD133, and GFAP. RESULTS: The study successfully described the process of isolation and culture of glioblastoma subspheres, which consist of a number of clonogenic cells immunophenotypically characterized as neural, which are able to initiate tumor formation. CONCLUSION: These findings may contribute to a better understanding of the process of gliomagenesis.

Targeting cancer stem cells with monoclonal antibodies: a new perspective in cancer therapy and diagnosis.

This review discusses some of the impacts that biotechnology, genomics and nanotechnology convergence should have on future cancer management, in particular, the development of innovative diagnostic and therapeutic approaches based on monoclonal antibodies (mAbs) and cancer stem cells. Emergent therapeutic strategies in cancer have been focusing on the use of mAbs to stimulate an immune response against tumors, to block signaling pathways, or to refine delivery of cytotoxic agents. Now that cancer stem cells are being identified and characterized in different tumor types, their relevance to cancer physiopathology is becoming evident, making them natural targets for mAb development. Cancer stem cells are postulated to be responsible for tumor development, metastasis and relapse after conventional therapies. Therefore, mAbs targeting specific antigens and related pathways altered in cancer stem cells should facilitate earlier diagnosis through molecular imaging techniques and more efficient destruction of tumor initiating cells, thus improving clinical outcome.

Preliminary report on tumor stem cell/B cell hybridoma vaccine for recurrent glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM), the most aggressive glioma, presents with a rapid evolution and relapse within the first year, which is attributed to the persistence of tumor stem cells (TSC) and the escape of immune surveillance. Mixed leukocyte culture (MLC) cytoimplant has been shown to function as a powerful intratumor pro-inflammatory cytokine pump. Tumor B-cell hybridoma (TBH) vaccines have been shown to function as antigen-presenting cells. We evaluated the toxicity and efficiency of each treatment alone and in combination. PATIENTS AND METHODS: In an open study, 12 consecutive patients were evenly divided into 3 groups, each group receiving 3 different treatments. Patients in Group 1 were treated, after diagnosis, with debulking surgery (DS)+radiotherapy (Rx), and after the first relapse underwent DS+MLC treatment. Patients in Group 2 were similarly treated but after the first relapse underwent DS+MLC+TBH. Finally, patients in Group 3 were similarly treated but after the first relapse underwent DS+TBH. Nestin PAP stain assessed TSC participation in TBH. RESULTS: Treatment with MLC had strong and rapid therapeutic effects, but was limited in duration and induced various degrees of brain inflammation. Treatment with MLC+TBH acted synergistically, provoking a rapid, strong and lasting therapeutic response but also generating different degrees of brain inflammation. A lasting therapeutic effect without generating high degrees of brain inflammation occurred in patients treated with TBH vaccine alone. CONCLUSION: TSC vaccine consisting of TBH alone seems to have potent adjuvant reactions overcoming both persistence of tumor stem cells and immune escape of GBM without provoking an encephalitic reaction.

Antigenic differences between metastatic cells in bone marrow and primary tumours and the anti-MUC1 humoral immune response induced in breast cancer patients.

The dissemination of a malignant neoplasia is a complex process, which requires a set of molecules that remains unknown. It has been suggested that mucins and their carbohydrate-associated antigens may be implicated in tumour spreading which may be also influenced by an anti-MUC1 immune response. In this pilot study, we report the pattern of carbohydrate and peptidic MUC1-associated epitopes on carcinoma cells isolated from bone marrow (BM), taking into account primary tumour histopathologic features. We also bring information about the anti-MUC1 humoral response in these patients. Seventeen patients with invasive breast carcinoma were included. A sample of the primary tumour, a serum sample and a BM aspirate were obtained from each patient. Clinical features studied were tumour size, number of metastatic nodes, histological type and disease stage. Standard immunohistochemistry was performed with antigenic retrieval using different monoclonal antibodies (MAbs): anti carbohydrate antigens: Lewis x (KM380), sLewis x (KM93), Lewis y (C14) and Tn, anti-MUC1 peptide core MAbs: C595, HMFG2 and SM3, anti-cytokeratins, anti-protoncogenes ErbB2 and ErbB3 (IgG) MAbs and also anti-CD34 and anti-CD45 MAbs. ELISA techniques were employed to study circulating MUC1 as well as free and complexed anti-MUC1 antibodies. Immunohistochemical results showed that carbohydrate antigenic expression increases in BM neoplastic cells compared to the original tumours. However, we were not able to demonstrate that a humoral immune response to MUC1 has been induced in these patients. Finally, the employed procedures allow the selective immortalisation of micrometastatic carcinoma cells since short-term cell lines were established.

[Immunophenotyping of acute lymphoblastic leukemia in Mexican children]. / Inmunofenotipo de la leucemia aguda linfoblástica en niños mexicanos.

PURPOSE: To analyse the immunophenotype of leukaemic cells in a group of children diagnosed of lymphoblastic leukaemia in order to assess the frequency of the different immunologic subtypes. PATIENTS AND METHODS: In the period comprised between APR 1987 and MAR 1995, 402 Mexican children were studied in a prospective way. Conventional immunological markers were used, either associated to or specific for B, T, myelo-monocytic or megakaryocytic-platelet cell populations. RESULTS: Five major immunologic subtypes were disclosed, showing a series of specific surface markers: null-ALL, 5%; early pre-B, 7.5%; common, 74.6%; B-cell, 3.5%, and T-cell, 9.4%. A net predominance of B-cell precursor CD10- ALL was found in children under one year of age, and of CD10+ B-cells beyond that age. Although there was only slight predominance of male sex, the prevalence of B and TALL in males was not confirmed. CONCLUSIONS: These results show that the incidence of the different immunologic subtypes of lymphoblastic leukaemias and their distribution according to age and sex are closely similar to those reported among Caucasians in other parts of the world.

[Monocytoid B-lymphocytic leukemia]. / Leucemia de linfocitos monocitoides B.

The recently described monocytoid B-cell lymphoma is a low-grade lymphoma presenting most frequently in elderly women and commonly associated with autoimmune diseases. Leukaemic expression of this disease has been reported in advanced stages. A case of monocytoid lymphocytosis without lymph node enlargement is presented herein. A 60-year old woman complaining of easy bruises was found to have a 2-cm splenomegaly. Her laboratory data included the following: haemoglobin, 125 g/L; haematocrit, 0.35 L/L; white cell count, 29 x 10(9)/L with 32% PMN, 3% stabs, 2% myelocytes, 1% metamyelocytes, 30% lymphocytes and 32% atypical mononucleated cells showing wide, pale cytoplasm neatly contoured and oval nucleus with monocytoid features. The basal coagulation study showed prothrombin 50%, APTT 40 seconds, fibrinogen 68 mg/dL and FDP between 80 and 160 ng/dL. Splenomegaly without lymph-node enlargement was found on CT scan. The bone-marrow biopsy showed a 68% monocytoid lymphocytic infiltration, acid-phosphatase positive and tartrate-sensitive, without fibrosis. Bone-marrow and peripheral immunophenotype showed those cells to be CD22, CD 19 and CD11 positive, while T and CD25 markers were absent. The patient was treated with alpha-2b interferon at a dose of 3MU three times a week for 6 months, with general improvement and regression of the leukaemic expression. Eleven months after diagnosis she died of a central nervous system haemorrhage. The morphological, immunological and cytochemical features of the monocytoid lymphocytes in this case are commented, along with their variable behaviour. A review of the literature is also carried out, attention being laid on the onset and the response to therapy of B-cell monocytoid lymphomas as the singularity of this case lies on its exclusively leukaemic onset. It is concluded that an interrelationship between monocytoid B-lymphocytic leukaemia and B-cell monocytoid lymphoma might possibly exist, such as that between chronic lymphocytic leukaemia and diffuse lymphocytic lymphoma.

Differentiation antigens of human germ cell tumours: distribution of carbohydrate epitopes on glycolipids and glycoproteins analyzed using PDMP, an inhibitor of glycolipid synthesis.

Human and mouse embryonal carcinoma (EC) cells are characterized by their expression of cell surface carbohydrate antigens, present in both glycolipids and glycoproteins. These antigens disappear upon differentiation and are replaced with other antigens. We have used the inhibitor of glucosyl ceramide synthetase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), to study the distribution of carbohydrate epitopes between glycolipids and glycoproteins. PDMP inhibited the expression of glycolipid antigens, but not glycoprotein antigens assayed by immunofluorescence and thin layer chromatography. In the case of SSEA1, we observed expression on both glycolipids and glycoproteins. Resistance to PDMP inhibition suggests that glycoproteins carry the immunodominant form of SSEA1 on the cell surface of differentiated human EC cells and undifferentiated murine EC cells.

Effect of human interleukin 3 on the susceptibility of fresh leukemia cells to interleukin-2-induced lymphokine activated killing activity.

Pretreatment of acute myeloblastic leukemia cells with the hemopoietic growth factor interleukin 3 (IL3) increased their susceptibility to lymphokine activated killing (LAK) but did not affect their constitutive resistance to native natural killer activity. In addition, IL3 treatment did not alter the LAK cell-mediated killing of CD34+ hemopoietic progenitors present in normal bone marrow. Increased 3H-thymidine uptake was generally observed after IL3 treatment. However, failure to proliferate in response to IL3, observed in some cases, did not prevent changes in LAK susceptibility. Enhanced lysis of IL3-treated leukemic cells was accompanied by a moderate increase of the effector-target binding. Increased LAK susceptibility was already observed at 18 h, while optimal cytolysis and expression of the cell adhesion molecule (CAM) LFA-3 (CD58) by IL3-treated AML cells were concomitantly observed at later culture times. In contrast, the CAM ICAM-1 (CD54) was not modulated by IL3, nor were significant changes in the expression of either CAMs observed in normal hemopoietic cells. Blocking experiments with the anti-CD58 monoclonal antibody demonstrated a variable neutralizing effect on the IL3-induced increase of LAK activity, depending on the leukemia cell studied. The effect described here, together with the known role of IL3 in normal hemopoiesis makes it a factor of potential therapeutic value for the treatment of leukemic patients.