BACKGROUND/AIM: Invariant natural killer T-cells (iNKT) stimulated by CD1d-binding glycolipids have been shown to exert antitumor effects by a number of studies in a mouse model. Breast cancer is a devastating disease, with different types of breast cancer recurring locally or distant as metastatic/advanced disease following initial treatment. The aim of this study was to examine the tumoricidal effect of a CD1d-binding glycolipid, called 7DW8-5, against a highly invasive human breast cancer cell line both in vitro and in vivo.
MATERIALS AND METHODS: Parental MDA-MB-231 cells and MDA-MB-231 cells transduced with human CD1d were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), followed by loading with glycolipids. After co-culturing with human iNKT cells, the cells were permeabilized and stained with Alexa Flour 647-conjugated antibody to active caspase-3, and analyzed using a BD LSR II. For the in vivo tumoricidal effect, MDA-MB-231 cells transduced with human CD1d and luciferase genes were injected into the mammary fat pad of female NOD/SCID/IL2rγnull (NSG) mice, followed by the injection of human iNKT cells with or without 7DW8-5, and the levels of luminescence were analyzed with whole-body imaging.
RESULTS: Human iNKT cells could kill CD1d-expressing human breast cancer cells in vitro in the presence of 7DW8-5, but not α-GalCer. As for in vivo, the adoptive transfer of human iNKT cells into tumor-challenged NSG mice significantly inhibited the growth of CD1d+ MDA-MB-231 human breast cancer cells in the presence of 7DW8-5.
CONCLUSION: CD1d-binding, glycolipid-based iNKT-cell therapy is suggested as a potent and effective treatment against breast cancer in humans.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) of the breast is characterized by its unique morphology and frequent nodal metastasis. However, the mechanism for development of this unique subtype has not been clearly elucidated. The aim of this study was to obtain a better understanding of IMPC.
METHODS: Using representative cases of mixed IMPC, mRNA expression in the micropapillary area and usual invasive area was compared. Then, immunohistochemical analyses for 294 cases (76 invasive carcinomas with a micropapillary feature [ICMF] and 218 invasive carcinomas without a micropapillary feature [ICNMF]) were conducted. Clinicopathological analyses were also studied.
RESULTS: DNA microarray analyses for mixed IMPC showed that BC-1514 (C21orf118) was commonly upregulated in the micropapillary area. CAMK2N1, CD1d, PJA2, RPL5, SAMD13, TCF4, and TXNIP were commonly downregulated in the micropapillary area. Immunohistochemically, we confirmed that BC-1514 was more upregulated in ICMF than in ICNMF. CD1d and PJA2 were more downregulated in ICMF than ICNMF. All patients with cases of PJA2 overexpression survived without cancer recurrence during the follow-up period, although the differences for disease-free (p = 0.153) or overall survival (p = 0.272) were not significant.
CONCLUSIONS: The CD1d- and PJA2-related tumour microenvironment might be crucial for IMPC. Further study of the immune microenvironment and micropapillary features is warranted.

T cells expressing CD19-specific chimeric Ag receptors (CARs) produce high remission rates in B cell lymphoma, but frequent disease recurrence and challenges in generating sufficient numbers of autologous CAR T cells necessitate the development of alternative therapeutic effectors. Vα24-invariant NKTs have intrinsic antitumor properties and are not alloreactive, allowing for off-the-shelf use of CAR-NKTs from healthy donors. We recently reported that CD62L

Interdigitating dendritic cell sarcoma (IDCS) is an aggressive neoplasm and is an extremely rare disease, with a challenging diagnosis. Etiology of IDCS is also unknown and most studies with only case reports. In our case, immunohistochemistry showed that the tumor cells were positive for S100, CD45, and CD68, but negative for CD1a and CD21. This study aimed to investigate the causative factors of IDCS by sequencing the protein-coding regions of IDCS. We performed whole-exome sequencing with genomic DNA from blood and sarcoma tissue of the IDCS patient using the Illumina Hiseq 2500 platform. After that, we conducted Sanger sequencing for validation of sarcoma-specific variants and gene ontology analysis using DAVID bioinformatics resources. Through comparing sequencing data of sarcoma with normal blood, we obtained 15 nonsynonymous single nucleotide polymorphisms (SNPs) as sarcoma-specific variants. Although the 15 SNPs were not validated by Sanger sequencing due to tumor heterogeneity and low sensitivity of Sanger sequencing, we examined the function of the genes in which each SNP is located. Based on previous studies and gene ontology database, we found that POLQ encoding DNA polymerase theta enzyme and FNIP1 encoding tumor suppressor folliculin-interacting protein might have contributed to the IDCS. Our study provides potential causative genetic factors of IDCS and plays a role in advancing the understanding of IDCS pathogenesis.

T cells that are genetically modified to express chimeric antigen receptors (CARs) specific for CD19 show great promise for the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL). The first U.S. Food and Drug Administration approval of a cellular cancer therapy in 2017, Novartis's CD19-targeting CAR T-cell product Kymriah™ within the context of relapsed/refractory pediatric ALL, followed rapidly by approval of Kite's Yescarta™ and, more recently, Kymriah™ for diffuse large B-cell indications in adults, highlights the pace of progress made in this field. In this review, we will consider the latest evidence from CAR T-cell therapy for B-lineage ALL. We discuss the barriers to CAR T-cell therapy for ALL patients and give a perspective on the strategy we have taken to date to widen access to CAR T-cell therapy for UK pediatric patients with high-risk ALL.

TNF-related, apoptosis-inducing ligand (TRAIL) apoptotic pathway constitutes a promising therapeutic target due to high selectivity and low toxicity of TRAIL targeting agents when administered in combination therapies. 106 colorectal cancers were examined for: relative mRNA expression of TRAIL pathway genes, decoy receptors TRAIL-R3 and TRAIL-R4 promoter methylation and the presence of KRAS, NRAS, BRAF mutations. Elevated mRNA levels were observed in 26%, 15%, 13%, 12% and 10% of the cases for TRAIL-R4, TRAIL-R3, TRAIL-R2, TRAIL-R1 and TRAIL genes respectively. Reduced mRNA levels were detected in 77%, 65%, 64%, 60% and 37% of the cases for TRAIL, TRAIL-R2, TRAIL-R3, TRAIL-R1 and TRAIL-R4 genes respectively. TRAIL-R3 and TRAIL-R4 promoter methylation was detected in 55% and 16% of the analysed samples respectively. TRAIL-R1, TRAIL-R2 elevated relative mRNA levels inversely correlated with tumor stage (p = .036, p = .048). Strong linear correlations of TRAIL receptors' mRNA levels were found: TRAIL-R1/TRAIL-R2 (R = 0.653, p < .001), TRAIL-R2/TRAIL-R3 (R = 0.573, p < .001). Finally, relative expression of TRAIL was correlated with KRAS, BRAF and NRAS mutation status, defining an inverse correlation between increased TRAIL expression and the absence of mutations in Mitogen-activated protein kinase (MAPK) pathway. In conclusion, simultaneous analysis of TRAIL pathway membrane components, pointed towards a significant deregulation of mRNA expression in colorectal tumours. Death receptor overexpression was an indicator of a less aggressive phenotype. The multiple expression patterns of TRAIL pathway components in colorectal tumours underscore the importance of patient selection in order to achieve maximum efficiency with TRAIL targeted therapy.

BACKGROUND/AIMS: To investigate the cellular effects and clinical significance of microRNA-223 (miR-223) in breast cancer by targeting stromal interaction molecule1 (STIM1).
METHODS: Breast cancer cell lines (T47D, MCF-7, SKB-R3, MDA-MB-231 and MDA-MB-435) and a normal breast epithelial cell line (MCF-10A) were prepared for this study. MiR-223 mimics, anti-miR-223 and pcDNA 3.1-STIM1 were transiently transfected into cancer cells independently or together, and then RT-qPCR was performed to detect the expressions of miR-223 and STIM1 mRNA, dual-luciferase reporter assay was conducted to examine the effects of miR-223 on STIM1, Western blotting was used to measure the expressions of the STIM1 proteins, MTT and Trans-well assays were performed to detect cell proliferation and invasion. Finally, the correlation of miR-223 and STIM1 was investigated by detecting with ISH and IHC in breast cancer specimens or the corresponding adjacent normal tissues.
RESULTS: Compared with normal cells and tissues, breast cancer tissues and cells exhibited significantly lower expression of miR-223, but higher expression of STIM1. MiR-223 could inhibit the proliferation and invasiveness of breast cancer cells by negatively regulating the expressions of STIM1. Reimplantation with STIM1 partially rescued the miRNA-223-induced inhibition of breast cancer cells. Clinical data revealed that high expression of STIM1 and miR-223 was respectively detrimental and beneficial factor impacting patient's disease-free survival (DFS) rather than overall survival (OS). Moreover, Pearson correlation analysis also confirmed that STIM1 was inversely correlated with miR-223.
CONCLUSION: MiR-223 inhibits the proliferation and invasion of breast cancer by targeting STIM1. The miR-223/STIM1 axis could possibly be a potential therapeutic target for treating breast cancer patients.

Redox adaptation plays an important role in cancer cells drug resistance. The antioxidant response is principally mediated by the transcription factor Nrf2, that induces the transcriptional activation of several genes involved in GSH synthesis, chemoresistance, and cytoprotection. YAP is emerging as a key mediator of chemoresistance in a variety of cancers, but its role in controlling the antioxidant status of the cells is yet elusive. Here, we show that impairing YAP protein expression reduced GSH content and Nrf2 protein and mRNA expression in bladder cancer cells. Moreover, in YAP knocked down cells the expression of FOXM1, a transcription factor involved in Nrf2 transcription, was down-regulated and the silencing of FOXM1 reduced Nrf2 expression. On the other hand, the silencing of Nrf2, as well as the depletion of GSH by BSO treatment, inhibited YAP expression, suggesting that cross-talk exists between YAP and Nrf2 proteins. Importantly, we found that silencing either YAP or Nrf2 enhanced sensitivity of bladder cancer cells to cytotoxic agents and reduced their migration. Furthermore, the inhibition of both YAP and Nrf2 expressions significantly increased cytotoxic drug sensitivity and synergistically reduced the migration of chemoresistant bladder cancer cells. These findings provide a rationale for targeting these transcriptional regulators in patients with chemoresistant bladder cancer, expressing high YAP and bearing a proficient antioxidant system.

PURPOSE: Chronic inflammation is a key component in the development and progression of colorectal cancer (CRC). A notable hallmark of the inflammation process is the release of pro-inflammatory cytokines by infiltrating cells of the immune system. Defects in dendritic cells (DCs) recruitment, maturation and cytokine release are a hallmark of the CRC strategy to escape immune surveillance.The purpose of our study was to evaluate the possible role of IL-12B polymorphism in the promoter region of the IL-12B gene (rs17860508) as a genetic factor contributing to the risk for CRC development. Additionally, we aimed to evaluate the influence of this polymorphism on DCs infiltration in tumor microenvironment.
METHODS: IL-12Bpro polymorphism was genotyped by Amplification Refractory Mutation System- Polymerase Chain Reaction (ARMS-PCR). Immunohistochemistry was performed for DCs infiltration.
RESULTS: Statistically significant correlation between the expression of S100 and CD1a DCs and the 11- genotype of the studied polymorphism was found. No statistically significant difference in genotype distribution between cases and controls was observed (p=0.163). Analysis of the overall survival (OS) of genotyped patients revealed a tendency among the carriers of the 22-genotype to have shorter survival of 36 months versus the 11- and 12-cariers- 61 months (log rank, p=0.117).
CONCLUSIONS: The IL-12Bpro polymorphism does not constitute a risk factor for CRC development. However, genotype-11 might have a complex role in the recruitment and maturation of DCs in tumor microenvironment.

An important goal of vaccination against viruses and virus-driven cancers is to elicit cytotoxic CD8

Anion exchanger 2 (AE2, encoded by SLC4A2) is a sodium-independent chloride/bicarbonate transporter and implicated in the regulation of intracellular pH and membrane potential. Previous studies have linked AE2 to the tumorigenesis of various cancers. Here, AE2 was identified as an up-regulated protein in ovarian cancer tissues compared to adjacent non-tumor lesions based on quantitative proteomics analysis. AE2 mRNA was also overexpressed in human ovarian cancer samples, and that AE2 overexpression correlated with the shortened survival time of ovarian cancer patients. Short-hairpin RNA-mediated knockdown of AE2 in A2780 and SK-OV-R3 cells inhibited cell growth and induced cell cycle G1 phase arrest. In nude mice, its stable knockdown inhibited the tumorigenicity of A2780 cells. Gene set enrichment analysis on The Cancer Genome Atlas dataset identified that the cell cycle process and mTOR pathway were correlatively with the AE2 expression. Expression of key regulators of G1/S transition (Cyclin D1 and CDK4), and phosphorylation levels of p70S6K were notably reduced in AE2 knockdown cells. Moreover, experiments with mTOR inhibitor suggested that AE2 may promote cell cycle progression through mTOR/p70S6K1 pathway. Together, our results suggest up-regulated AE2 promotes ovarian cancer tumorigenesis by activating mTOR/p70S6K1 pathway and implicate the potential application of AE2 in cancer therapy.

Folliculotropic mycosis fungoides (FMF), a variant of mycosis fungoides (MF) with distinct clinical features, is characterized by infiltration of malignant T cells in hair follicles. This raises the hypothesis that antigens in the hair follicle may contribute to the pathogenesis of FMF. T-cell receptor β gene (TRB) sequences as well as dendritic cell subsets in patients with FMF (n = 21) and control patients with MF (n = 20) were studied to explore this hypothesis. A recurrent usage of the TRB junctional genes TRBJ2-1 and TRBJ2-7 was found in patients with FMF compared with those with MF. These genes contribute to an amino acid motif in the complementarity-determining region 3 (CDR3) of the T-cell receptor. This motif was previously found in T cells stimulated by lipids bound to CD1 on antigen-presenting cells. Additional immunohistochemical analysis revealed abundant CD1c- and CD1a- expressing dendritic cells in FMF. The combined findings support a role for lipid-antigen stimulation in FMF.

Peptidylarginine deiminase 4 (PADI4), a new histone modification enzyme, which converts both arginine and monomethyl-arginine to citrulline, has gained massive attention in recent years as a potential regulator of gene transcription. Recent studies have shown that arginine residues R2, R8, R17, and R26 in the H3 tail and R3 in the H4 tail can be deiminated by PADI4. This kind of histone post-translational modification has the potential to antagonize histone methylation and coordinate with histone deacetylation to regulate gene transcription. PADI4 also deiminates non-histone proteins, such as p300, NPM1, ING4, RPS2, and DNMT3A. PADI4 has been shown to involve in cell apoptosis and differentiation. Moreover, PADI4 can interact with tumor suppressor p53 and regulate the transcriptional activity of p53. Dysregulation of PADI4 is implicated in a variety of diseases, including rheumatoid arthritis, tumor development, and multiple sclerosis. A wide variety of PADI4 inhibitors have been identified. Further understanding of PADI4 functions may lead to novel diagnostic and therapeutic approaches in these diseases. This review summarizes the recent progress in the study of the regulation mechanism of PADI4 on gene transcription and the major physiological functions of PADI4 in human diseases.

BACKGROUND: TSPO translocator protein, encoded in humans by the Tspo gene plays a crucial role in mitochondria mediated apoptosis and necrotic cell death through its association with Mitochondrial Permeability Transition pore (MPTP). It has been shown that this function can be exploited as a potential treatment for human Glioblastoma Multiforme. In this study, a novel robust fragment based QSAR model has been developed for a series of 4-phenylquinazoline-2-carboxamides experimentally known to be ligands for TSPO, thus triggering apoptotic mechanism cascade.
RESULTS: Model developed showed satisfactory statistical parameters for the experimentally reported dataset (r
CONCLUSION: The high binding affinity and the presence of essential structural features in these compounds make them an ideal choice for the consideration as potent anti-GBM drugs. Activity predicted by GQSAR model reinforces their potential as worthy candidates for drugs against GBM. The detailed analysis carried out in this study provides a substantial basis for the prospective design and development of novel 4-phenylquinazoline-2-carboxamide compounds as TSPO ligands capable of inducing apoptosis in cancer cells.

The role of the unique T-cell population, natural killer T (NKT) cells, which have similar functions to NK cells in pancreatic cancer (PC), is not yet evaluated. To address the regulatory roles of NKT cells on tumour progression through tumour-associated macrophages (TAM) and their production of microsomal prostaglandin E synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) in (Kras)-driven pancreatic tumour (KPT) progression, we crossed CD1d

Breast cancer is one of the most lethal malignancies for women. Retinoic acid (RA) and double-stranded RNA (dsRNA) are considered signaling molecules with potential anticancer activity. RA, co-administered with the dsRNA mimic polyinosinic-polycytidylic acid (poly(I:C)), synergizes to induce a TRAIL (Tumor-Necrosis-Factor Related Apoptosis-Inducing Ligand)- dependent apoptotic program in breast cancer cells. Here, we report that RA/poly(I:C) co-treatment, synergically, induce the activation of Interferon Regulatory Factor-3 (IRF3) in breast cancer cells. IRF3 activation is mediated by a member of the pathogen recognition receptors, Toll-like receptor-3 (TLR3), since its depletion abrogates IRF3 activation by RA/poly(I:C) co-treatment. Besides induction of TRAIL, apoptosis induced by RA/poly(I:C) correlates with the increased expression of pro-apoptotic TRAIL receptors, TRAIL-R1/2, and the inhibition of the antagonistic receptors TRAIL-R3/4. IRF3 plays an important role in RA/poly(I:C)-induced apoptosis since IRF3 depletion suppresses caspase-8 and caspase-3 activation, TRAIL expression upregulation and apoptosis. Interestingly, RA/poly(I:C) combination synergizes to induce a bioactive autocrine/paracrine loop of type-I Interferons (IFNs) which is ultimately responsible for TRAIL and TRAIL-R1/2 expression upregulation, while inhibition of TRAIL-R3/4 expression is type-I IFN-independent. Our results highlight the importance of IRF3 and type-I IFNs signaling for the pro-apoptotic effects induced by RA and synthetic dsRNA in breast cancer cells.

CD1d-restricted invariant natural killer T (iNKT) cells are known as potent early regulatory cells of immune responses. Besides the established roles in the regulation of inflammation and autoimmune disease, studies have shown that iNKT cells have important roles in tumor surveillance and the control of tumor metastasis. Here we found that the absence of iNKT cells markedly decreased the total number of intestinal polyps in APC

Non-coding mutations may drive cancer development. Statistical detection of non-coding driver regions is challenged by a varying mutation rate and uncertainty of functional impact. Here, we develop a statistically founded non-coding driver-detection method, ncdDetect, which includes sample-specific mutational signatures, long-range mutation rate variation, and position-specific impact measures. Using ncdDetect, we screened non-coding regulatory regions of protein-coding genes across a pan-cancer set of whole-genomes (n = 505), which top-ranked known drivers and identified new candidates. For individual candidates, presence of non-coding mutations associates with altered expression or decreased patient survival across an independent pan-cancer sample set (n = 5454). This includes an antigen-presenting gene (

Inhibitor of DNA binding (Id) proteins, including Id1-4, are transcriptional regulators involved in promoting cell proliferation and survival in various cell types. Although upregulation of Id proteins is associated with a broad spectrum of tumors, recent studies have identified that Id3 plays a tumor-suppressor role in the development of Burkitt's lymphoma in humans and hepatosplenic T cell lymphomas in mice. In this article, we report rapid lymphoma development in

Trials with second generation CD19 chimeric antigen receptors (CAR) T-cells report unprecedented responses but are associated with risk of cytokine release syndrome (CRS). Instead, we studied the use of donor Epstein-Barr virus-specific T-cells (EBV CTL) transduced with a first generation CD19CAR, relying on the endogenous T-cell receptor for proliferation. We conducted a multi-center phase I/II study of donor CD19CAR transduced EBV CTL in pediatric acute lymphoblastic leukaemia (ALL). Patients were eligible pre-emptively if they developed molecular relapse (>5 × 10

Autologous T cells engineered to express chimeric antigen receptor against the B cell antigen CD19 (CAR19) are achieving marked leukemic remissions in early-phase trials but can be difficult to manufacture, especially in infants or heavily treated patients. We generated universal CAR19 (UCART19) T cells by lentiviral transduction of non-human leukocyte antigen-matched donor cells and simultaneous transcription activator-like effector nuclease (TALEN)-mediated gene editing of T cell receptor α chain and CD52 gene loci. Two infants with relapsed refractory CD19

Pexa-Vec (pexastimogene devacirpvec; JX-594) has emerged as an attractive tool in oncolytic virotherapy. Pexa-Vec demonstrates oncolytic and immunotherapeutic mechanisms of action. But the determinants of resistance to Pexa-Vec are mostly unknown. We treated hemoatologic malignant cells with Pexa-Vec and examined the gene-expression pattern of sensitive and resistant cells. Human myeloid malignant cell lines (RPMI-8226, IM-9, K562, THP-1) and lymphoid cancer cell lines (MOLT4, CCRF-CEM, Ramos, U937) were treated with Pexa-Vec. Pexa-Vec was cytotoxic on myeloid cell lines in a dose-dependent manner, and fluorescent imaging and qPCR revealed that Pexa-Vec expression was low in RAMOS than IM-9 after 24 hrs and 48 hrs of infection. Gene expression profiles between two groups were analyzed by microarray. Genes with at least 2-fold increase or decrease in their expression were identified. A total of 660 genes were up-regulated and 776 genes were down-regulated in lymphoid cancer cell lines. The up- and down-regulated genes were categorized into 319 functional gene clusters. We identified the top 10 up-regulated genes in lymphoid cells. Among them three human genes (LEF1, STAMBPL1, and SLFN11) strongly correlated with viral replication. Up-regulation of PVRIG, LPP, CECR1, Arhgef6, IRX3, IGFBP2, CD1d were related to resistant to Pexa-Vec. In conclusion, lymphoid malignant cells are resistant to Pexa-Vec and displayed up-regulated genes associated with resistance to oncolytic viral therapy. These data provide potential targets to overcome resistance, and suggest that molecular assays may be useful in selecting patients for further clinical trials with Pexa-Vec.

BACKGROUND: Birt-Hogg-Dubé (BHD) syndrome is a rare inherited autosomal genodermatosis and caused by germline mutation of the folliculin (FLCN) gene, a tumor suppressor gene of which protein product is involved in mechanistic target of rapamycin (mTOR) signaling pathway regulating cell growth and metabolism. Clinical manifestations in BHD syndrome is characterized by fibrofolliculomas of the skin, pulmonary cysts with or without spontaneous pneumothorax, and renal neoplasms. There has been no pulmonary neoplasm reported in BHD syndrome, although the condition is due to deleterious sequence variants in a tumor suppressor gene. Here we report, for the first time to our knowledge, a patient with BHD syndrome who was complicated with a clear cell "sugar" tumor (CCST) of the lung, a benign tumor belonging to perivascular epithelioid cell tumors (PEComas) with frequent causative relation to tuberous sclerosis complex 1 (TSC1) or 2 (TSC2) gene.
CASE PRESENTATION: In a 38-year-old Asian woman, two well-circumscribed nodules in the left lung and multiple thin-walled, irregularly shaped cysts on the basal and medial area of the lungs were disclosed by chest roentgenogram and computer-assisted tomography (CT) during a preoperative survey for a bilateral faucial tonsillectomy. Analysis of the resected tumor showed large polygonal cells with clear cytoplasm proliferating in a solid pattern. Immunohistochemistry revealed that these tumor cells were positive for microphthalmia-transcription factor, S100, and CD1a but negative for HMB45, indicating that the tumor was a CCST. Genetic testing indicated that the patient had a germline mutation on exon 12 of the FLCN gene, i.e., insertion of 7 nucleotides (CCACCCT) (c.1347_1353dupCCACCCT). Direct sequencing of the FLCN exon 12 using genomic DNA obtained from her microdissected CCST cells clearly revealed loss of the wild-type FLCN sequence, which confirmed complete functional loss of the FLCN gene. On the other hand, no loss of heterozygosity around TCS1- or TSC2-associated genetic region was demonstrated.
CONCLUSION: To our knowledge, this is the first report of CCST of the lung in a patient with BHDS, indicating that CCST should be added to the spectrum of pulmonary manifestations of BHDS.

BACKGROUND: Germline mutations in BRCA1 and BRCA2 (BRCA1/2) account for the majority of hereditary breast and/or ovarian cancers. Pakistan has one of the highest rates of breast cancer incidence in Asia, where BRCA1/2 small-range mutations account for 17% of early-onset and familial breast/ovarian cancer patients. We report the first study from Pakistan evaluating the prevalence of BRCA1/2 large genomic rearrangements (LGRs) in breast and/or ovarian cancer patients who do not harbor small-range BRCA1/2 mutations.
MATERIALS AND METHODS: Both BRCA1/2 genes were comprehensively screened for LGRs using multiplex ligation-dependent probe amplification in 120 BRCA1/2 small-range mutations negative early-onset or familial breast/ovarian cancer patients from Pakistan (Group 1). The breakpoints were characterized by long-range PCR- and DNA-sequencing analyses. An additional cohort of 445 BRCA1/2 negative high-risk patients (Group 2) was analyzed for the presence of LGRs identified in Group 1.
RESULTS: Three different BRCA1 LGRs were identified in Group 1 (4/120; 3.3%), two of these were novel. Exon 1-2 deletion was observed in two unrelated patients: an early-onset breast cancer patient and another bilateral breast cancer patient from a hereditary breast cancer (HBC) family. Novel exon 20-21 deletion was detected in a 29-year-old breast cancer patient from a HBC family. Another novel exon 21-24 deletion was identified in a breast-ovarian cancer patient from a hereditary breast and ovarian cancer family. The breakpoints of all deletions were characterized. Screening of the 445 patients in Group 2 for the three LGRs revealed ten additional patients harboring exon 1-2 deletion or exon 21-24 deletion (10/445; 2.2%). No BRCA2 LGRs were identified.
CONCLUSIONS: LGRs in BRCA1 are found with a considerable frequency in Pakistani breast/ovarian cancer cases. Our findings suggest that BRCA1 exons 1-2 deletion and exons 21-24 deletion should be included in the recurrent BRCA1/2 mutations panel for genetic testing of high-risk Pakistani breast/ovarian cancer patients.

An immunotherapeutic strategy is discussed supporting anti-tumor activity toward malignancies overexpressing ganglioside D3. GD3 can be targeted by NKT cells when derived moieties are presented in the context of CD1d. NKT cells can support anti-tumor responses by secreting inflammatory cytokines and through cytotoxicity toward CD1d

Histone deacetylases (HDACs) are a family of enzymes that influence expression of genes implicated in tumor initiation, progression, and anti-tumor responses. In addition to their canonical role in deacetylation of histones, HDACs regulate many non-canonical targets, such as Signal Transducer and Activator of Transcription 3 (STAT3). We hypothesize that tumors use epigenetic mechanisms to dysregulate CD1d-mediated antigen presentation, thereby impairing the ability of natural killer T (NKT) cells to recognize and destroy malignant cells. In this study, we pre-treated CD1d-expressing tumor cells with HDAC inhibitors (HDACi) and assessed CD1d-dependent NKT cell responses to mantle cell lymphoma (MCL). Pre-treatment with Trichostatin-A, a pan-HDACi, rapidly enhanced both CD1d- and MHC class II-mediated antigen presentation. Similarly, treatment of MCL cells with other HDACi resulted in enhanced CD1d-dependent NKT cell responses. The observed changes are due, at least in part, to an increase in both CD1D mRNA and CD1d cell surface expression. Mechanistically, we found that HDAC2 binds to the CD1D promoter. Knockdown of HDAC2 in tumor cells resulted in a significant increase in CD1d-mediated antigen presentation. In addition, treatment with HDACi inhibited STAT3 and STAT3-regulated inflammatory cytokine secretion by MCL cells. We demonstrated that MCL-secreted IL-10 inhibits CD1d-mediated antigen presentation and pre-treatment with TSA abrogates secretion of IL-10 by MCL. Taken together, our studies demonstrate the efficacy of HDACi in restoring anti-tumor responses to MCL through both cell-intrinsic and cell-extrinsic mechanisms and strongly implicate a role for HDACi in enhancing immune responses to cancer.

Multidrug resistance (MDR) among breast cancer cells is the paramount obstacle for the successful chemotherapy. In this study, anti-EGFR antibody h-R3 was designed to self-assembled h-R3-siRNA-PAMAM-complexes (HSPCs) via electrostatic interactions for siRNA delivery. The physicochemical characterization, cell uptake, MDR1 silencing efficiency, cell migration, cell growth and cell apoptosis were investigated. The HSPCs presented lower cytotoxicity, higher cellular uptake and enhanced endosomal escape ability. Also, HSPCs encapsulating siMDR1 knockdowned 99.4% MDR1 gene with up to ∼6 times of enhancement compared to naked siMDR1, increased the doxorubicin accumulation, down-regulated P-glycoprotein (P-gp) expression and suppressed cellular migration in breast cancer MCF-7/ADR cells. Moreover, the combination of anticancer drug paclitaxel (PTX) and siMDR1 loaded HSPCs showed synergistic effect on overcoming MDR, which inhibited cell growth and induced cell apoptosis. This h-R3-mediated siMDR1 delivery system could be a promising vector for effective siRNA therapy of drug resistant breast cancer.

Advanced medullary thyroid cancers (MTCs) are now being treated with drugs that inhibit receptor tyrosine kinases, many of which involved in angiogenesis. Response rates vary widely, and toxic effects are common, so treatment should be reserved for MTCs likely to be responsive to these drugs. RET mutations are common in MTCs, but it is unclear how they influence the microvascularization of these tumors. We examined 45 MTCs with germ-line or somatic RET mutations (RETmut group) and 34 with wild-type RET (RETwt). Taqman Low-Density Arrays were used to assess proangiogenic gene expression. Immunohistochemistry was used to assess intratumoral, peritumoral and nontumoral expression levels of VEGFR1, R2, R3, PDGFRa, PDGFB and NOTCH3. We also assessed microvessel density (MVD) and lymphatic vessel density (LVD) based on CD31-positive and podoplanin-positive vessel counts, respectively, and vascular pericyte density based on staining for a-smooth muscle actin (a-SMA), a pericyte marker. Compared with RETwt tumors, RETmut tumors exhibited upregulated expression of proangiogenic genes (mRNA and protein), especially VEGFR1, PDGFB and NOTCH3. MVDs and LVDs were similar in the two groups. However, microvessels in RETmut tumors were more likely to be a-SMA positive, indicating enhanced coverage by pericytes, which play key roles in vessel sprouting, maturation and stabilization. These data suggest that angiogenesis in RETmut MTCs may be more intense and complete than that found in RETwt tumors, a feature that might increase their susceptibility to antiangiogenic therapy. Given their increased vascular pericyte density, RETmut MTCs might also benefit from combined or preliminary treatment with PDGF inhibitors.

Interleukin-15 (IL-15) is a cell growth-factor that regulates lymphocyte function and homeostasis. Its strong immunostimulatory activity coupled with an apparent lack of toxicity makes IL-15 an exciting candidate for cancer therapy, somehow limited by its short half-life in circulation. To increase IL-15 bioavailability we constructed a recombinant adeno-associated vector expressing murine IL-15 (AAV-mIL15) in the liver. Mice injected with AAV-mIL15 showed sustained and vector dose-dependent levels of IL-15/IL-15Rα complexes in serum, production of IFN-γ and activation of CD8+ T-cells and macrophages. The antitumoral efficacy of AAV-mIL15 was tested in a mouse model of metastatic colorectal cancer established by injection of MC38 cells. AAV-mIL15 treatment slightly inhibits MC38 tumor-growth and significantly increases the survival of mice. However, mIL-15 sustained expression was associated with development of side effects like hepatosplenomegaly, liver damage and the development of haematological stress, which results in the expansion of hematopoietic precursors in the bone marrow. To elucidate the mechanism, we treated IFN-γ receptor-, RAG1-, CD1d- and µMT-deficient mice and performed adoptive transfer of bone marrow cells from WT mice to RAG1-defcient mice. We demonstrated that the side effects of murine IL-15 administration were mainly mediated by IFN-γ-producing T-cells.
CONCLUSIONS: IL-15 induces the activation and survival of effector immune cells that are necessary for its antitumoral activity; but, long-term exposure to IL-15 is associated with the development of important side effects mainly mediated by IFN-γ-producing T-cells. Strategies to modulate T-cell activation should be combined with IL-15 administration to reduce secondary adverse events while maintaining its antitumoral effect.

Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia, known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans.