Characterization of a novel Hodgkin cell-line, HD-MyZ, HD-Myr, with myelomonocytic features mimicking Hodgkin's disease in severe combined immunodeficient mice

A novel Hodgkin cell line, designated HD-MyZ, was established from the pleural effusion of a 29-yr-old patient with Hodgkin's disease (HD) of nodular sclerosing type. The majority of cells grow adherently and display typical morphological characteristics of Reed-Sternberg (RS) and Hodgkin (H) cells, i.e., large multi- and mononucleated cells with prominent nucleoli. Immunofluorescence analysis revealed a myelomonocytoid immunophenotype (expression of CD13 and CD68, and lack of lymphoid markers). HD-MyZ cells strongly expressed restin, a recently described intermediate filament-associated protein, the expression of which is restricted to H cells, RS cells, and in vitro cultivated peripheral blood monocytes. In addition mRNA expression of c-fms (colony-stimulating factor 1 receptor) could be induced in HD-MyZ cells by phorbol myristate acetate (PMA) stimulation. Southern blot analysis did not detect rearrangement of T cell receptor beta and immunoglobulin H loci, thus demonstrating the lack of lymphoid commitment. HD-MyZ cells were also devoid of Epstein-Barr virus genomes. HD-MyZ cells constitutively express mRNAs for interleukin 1 alpha (IL-1 alpha), IL-1 beta, IL-5, IL-6, IL-7, IL-8, IL-10, IL-1 receptor (type I), and IL-6 receptor. Stimulation of cells with PMA increased mRNA expression as well as the secretion of IL-1 beta, IL-6, and IL-8, and induced the de novo expression of IL-8 receptors. Xenotransplantation into severe combined immunodeficient (SCID) mice by intravenous or subcutaneous inoculation led to development of disseminated tumors with infiltrative and destructive growth. In addition lymphadenopathy, pleural effusion, and infiltration of spleen were observed. Morphological and immunological analysis of tumor cells revealed the same features as HD-MyZ cells. This cell line might be an important tool for understanding the pathogenesis and biology of HD. In addition the SCID mice model might prove helpful in developing new therapeutic strategies

Blocking of transcription factor E2F/DP by dominant-negative mutants in a normal breast epithelial cell line efficiently inhibits apoptosis and induces tumor growth in scid mice

The transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis

Exploiting liver immunity for the prevention of hepatic metastases

Over the last decade immunotherapy has progressively gained a significant clinical interest for cancer treatment. The actual benefit of active immunotherapy, namely cancer vaccines, has been repeatedly claimed as effective in several settings and prompted prospective investigations (phase III clinical trials) currently ongoing in patients with prostate carcinoma, melanoma, and lung cancer, while the first vaccine for the treatment of metastatic prostate carcinoma has been recently approved by the FDA [1,2]. Adoptive immunotherapy, based on the administration of antibodies (Ab) targeting tumor receptors or molecules expressed in the microenvironment, has also entered clinical practice in several cancers such as breast carcinoma, colorectal cancer, and haematological tumors [3], whereas adoptive transfer of ex vivo activated tumor-specific T cells has shown dramatic efficacy in metastatic melanoma [4], a known benchmark-tumor for immune manipulations both in animal and human models. Ab-therapy of cancer was initially conceived to directly eliminate tumor cells by interfering with the activity of either receptors involved in cancer cell proliferation (e.g. members of the HER family) or molecules regulating tumor blood supply (such in the case of anti-VEGF Ab) [5]. However, the clinical efficacy of broadly used Ab was subsequently found to rely, at least in part, on the contribution of the immune system through the activation of antibody-dependent cell cytotoxicity (ADCC) mediated by cellular components of innate immunity [5,6]. NK cells, monocytes, macrophages, and granulocytes can indeed bind to Ab thanks to the expression of specific receptors for the invariant portion of this molecule (the so called Fc portion) and be recruited to attack target cells, hence contributing to the therapeutic potential of Abbased therapies. Evidence proving such mechanism has been collected in preclinical studies, showing that anti-tumor antibodies, like trastuzumab, have reduced activity when administered in FcR knock-out mice or used in a FcR-binding defective form. Similarly in clinical setting, breast cancer trials have reported immune infiltrate in cancer lesions of Ab-treated patients, and better efficacy in subjects expressing defined FcR genotypes [6]. More recently, the anti-tumor activity of such strategy has been also potentiated through bi-specific engineered single-chain Ab designed to tether cytotoxic T lymphocytes to cancer cells, a

Long-term survival of patients with relapsed/refractory acute lymphoblastic leukemia treated with blinatumomab

Background: Blinatumomab is a CD19 BiTE (bispecific T-cell engager) immuno-oncology therapy that mediates the lysis of cells expressing CD19. Methods: A pooled analysis of long-term follow-up data from 2 phase 2 studies that evaluated blinatumomab in heavily pretreated adults with Philadelphia chromosome-negative, relapsed/refractory B-cell precursor acute lymphoblastic leukemia was conducted. Results: A total of 259 patients were included in the analysis. The median overall survival (OS) among all patients, regardless of response, was 7.5 months (95% confidence interval [CI], 5.5-8.5 months); the median follow-up time for OS was 36.0 months (range, 0.3-60.8 months). The median relapse-free survival (RFS) among patients who achieved a complete remission (CR) or complete remission with partial hematologic recovery (CRh) in the first 2 cycles (n = 123) was 7.7 months (95% CI, 6.2-10.0 months); the median follow-up time for RFS was 35.0 months (range, 9.5-59.5 months). OS and RFS plateaued with 3-year rates of 17.7% and 23.4%, respectively. The cumulative incidence function of the time to relapse, with death not due to relapse considered a competing risk, for patients who achieved a CR/CRh within 2 cycles of treatment also plateaued with a 3-year relapse rate of 59.3%. For patients who achieved a CR/CRh with blinatumomab followed by allogeneic hematopoietic stem cell transplantation while in continuous CR, the median OS was 18.1 months (95% CI, 10.3-30.0 months) with a 3-year survival rate of 37.2%. Conclusions: These data suggest that long-term survival is possible after blinatumomab therapy. Lay Summary: Immuno-oncology therapies such as blinatumomab activate the patient's own immune system to kill cancer cells. This study combined follow-up data from 2 blinatumomab-related clinical trials to evaluate long-term survival in patients with relapsed and/or refractory B-cell precursor acute lymphoblastic leukemia at high risk for unfavorable outcomes. Among patients who achieved a deep response with blinatumomab, one-third lived 3 years or longer. These findings suggest that long-term survival is possible after treatment with blinatumomab

Predicting microenvironment in CXCR4- and FAP-positive solid tumors - a pan-cancer machine learning workflow for theranostic target structures

Simple Summary Imaging based on positron emission tomography (PET) is a crucial part of up-to-date cancer care. For this purpose, PET employs and marks target structures at the cellular surface. Recently, C-X-C Motif Chemokine Receptor 4 (CXCR4) and Fibroblast Activation Protein Alpha (FAP) emerged as clinically relevant PET targets. However, it is unclear whether high levels of CXCR4 and FAP represent distinct cancer states—especially in solid tumors. Therefore, we established a machine learning model based on 9242 samples from 29 different cancer entities. Our analysis revealed that—in most solid tumors—high levels of CXCR4 were associated with immune cells infiltrating these tumors. Instead, FAP-positive tumors were characterized by high amounts of tumor vessels. Our machine learning approach potentially can identify the Achilles’ heel of tumors in a non-invasive manner—by performing PET without having to obtain tumor tissue beforehand. Abstract (1) Background: C-X-C Motif Chemokine Receptor 4 (CXCR4) and Fibroblast Activation Protein Alpha (FAP) are promising theranostic targets. However, it is unclear whether CXCR4 and FAP positivity mark distinct microenvironments, especially in solid tumors. (2) Methods: Using Random Forest (RF) analysis, we searched for entity-independent mRNA and microRNA signatures related to CXCR4 and FAP overexpression in our pan-cancer cohort from The Cancer Genome Atlas (TCGA) database—representing n = 9242 specimens from 29 tumor entities. CXCR4- and FAP-positive samples were assessed via StringDB cluster analysis, EnrichR, Metascape, and Gene Set Enrichment Analysis (GSEA). Findings were validated via correlation analyses in n = 1541 tumor samples. TIMER2.0 analyzed the association of CXCR4 / FAP expression and infiltration levels of immune-related cells. (3) Results: We identified entity-independent CXCR4 and FAP gene signatures representative for the majority of solid cancers. While CXCR4 positivity marked an immune-related microenvironment, FAP overexpression highlighted an angiogenesis-associated niche. TIMER2.0 analysis confirmed characteristic infiltration levels of CD8+ cells for CXCR4-positive tumors and endothelial cells for FAP-positive tumors. (4) Conclusions: CXCR4- and FAP-directed PET imaging could provide a non-invasive decision aid for entity-agnostic treatment of microenvironment in solid malignancies. Moreover, this machine learning workflow can easily be transferred towards other theranostic targets

A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells

Wepreviously reported the association of elevated levels of themultifunctional transcription factor, CCCTC binding factor (CTCF), in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecularmechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs) within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell deathwere observed only in breast cancer cells depleted of CTCF.We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1,WT1, EGR1, and c-Myc) was generally increased in non- breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis. © 2013 Neoplasia Press, Inc

Molecular response with blinatumomab in relapsed/refractory B-cell precursor acute lymphoblastic leukemia

Minimal residual disease (MRD), where leukemic cell levels are lower than the morphologic detection threshold, is the most important prognostic factor for acute lymphoblastic leukemia (ALL) relapse during first-line chemotherapy treatment and is standard of care in treatment monitoring and decision making. Limited data are available on the prognostic value of MRD response after relapse. We evaluated the relationship between MRD response and outcomes in blinatumomab-treated adults with relapsed/refractory (R/R) B-cell precursor ALL. Of 90 patients with complete remission (CR) or CR with partial hematologic recovery (CRh), 64 (71.1%) achieved a complete MRD response (no detectable individual rearrangements of immunoglobulin/T-cell receptor genes by polymerase chain reaction [PCR] at a minimum sensitivity level of 10-4). Eleven patients had MRD <10-4. Therefore, overall, 75 (83.3%) experienced an MRD response (no detectable MRD or detectable MRD) measured by PCR within the first 2 treatment cycles. Overall survival (OS) and relapse-free survival (RFS) were significantly longer in patients who achieved CR/CRh and MRD response (median, 20.6 and 9.0 months, respectively) compared with CR/CRh patients without MRD response (median, 12.5 and 2.3 months, respectively). In conclusion, longer durations of OS and RFS associated with MRD response support the value of achieving MRD response and its use as a prognostic factor for blinatumomab treatment in R/R ALL. This trial was registered at www.clinicaltrials.gov as #NCT01466179

Immunoreactivity of CD99 in Non-Hodgkin's Lymphoma: Unexpected Frequent Expression in ALK-positive Anaplastic Large Cell Lymphoma

To verify the spectrum of CD99-expressing lymphoid malignancy, an immunohistochemical study for CD99 was carried out in 182 cases of non-Hodgkin's lymphoma, including 21 lymphoblastic lymphomas, 11 small lymphocytic lymphomas, 9 mantle cell lymphomas, 12 follicular lymphomas, 37 diffuse large B cell lymphomas, 18 Burkitt's lymphomas, 28 NK/T-cell lymphomas, 8 angioimmunoblastic T-cell lymphomas, 23 peripheral T-cell lymphomas, unspecified, and 15 systemic anaplastic large cell lymphomas. CD99 was positive in all T-lymphoblastic lymphomas and in 60% of B-lymphoblastic lymphomas. Majority of T and NK cell lymphomas were negative for CD99, except anaplastic large cell lymphomas (ALCLs). Eight of 15 cases (54%) of ALCLs reacted with anti CD99 antibody. Seven of 10 (70%) ALK positive ALCLs expressed CD99, whereas only 1 of 5 (20%) ALK negative ALCLs were positive. Of the mature B-cell lymphomas, 5.4% (2/37) of diffuse large B cell lymphomas and 11.1% (2/18) of Burkitt's lymphomas expressed CD99. In conclusion, CD99 is infrequently expressed in mature B and T cell lymphomas, except ALK-positive ALCL. High expression of CD99 in ALK-positive ALCL is unexpected finding and its biologic and clinical significances have yet to be clarified

On-target restoration of a split T cell-engaging antibody for precision immunotherapy

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (V-L) or variable heavy (V-H) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies