Grade of Primary Cutaneous Leiomyosarcoma Dictates Risk for Metastatic Spread and Disease-Specific Mortality.

BACKGROUND AND OBJECTIVES: Primary cutaneous leiomyosarcoma (cLMS), a rare, typically intradermal tumor, has previously been reported to exhibit an indolent course of disease with zero-to-low risk of local recurrence or distant metastasis. This study seeks to evaluate recurrence and survival of cLMS patients through study of its clinicopathologic and treatment characteristics. METHODS: All patients included underwent resection of primary cLMS at this institution between 2006 and 2019. A retrospective cohort study analysis of clinicopathologic characteristics, treatment, recurrence, and overall survival was performed. Data was assessed through descriptive statistics and outcome measures assessed by Cox proportional models and log-rank tests. RESULTS: Eighty-eight patients with cLMS were evaluated. The majority were men (n = 68, 77%) and Caucasian (n = 85, 97%), with median age at diagnosis of 66 years (range 20-96). 65% of tumors were located on the extremities, with a median size of 1.3 cm (range .3-15). Assessment revealed low (n = 41, 47%), intermediate (n = 29, 33%), and high (n = 18, 20%) grade tumors, demonstrating extension into subcutaneous tissue in 38/60 (60%), with 3 patients exhibiting extension into muscle (3%). All underwent resection as primary treatment with median 1 cm margins (range .5-2). With median follow-up of 27.5 months (IQR 8-51; range 1-131), no low-grade cases had recurrence or death while there was a recurrence rate of 19.1% (9/47) and death rate of 8.5% (4/47) in intermediate- to high-grade cases. CONCLUSIONS: Primary tumor resection of cLMS provides excellent local control for low-grade tumors as no low-grade cases experienced recurrence. For patients with intermediate- to high-grade tumors, there is potential for local recurrence, distant metastasis, and death, and therefore surveillance following treatment is encouraged.

CX3CR1 deficiency-induced TIL tumor restriction as a novel addition for CAR-T design in solid malignancies.

Advances in the understanding of the tumor microenvironment have led to development of immunotherapeutic strategies, such as chimeric antigen receptor T cells (CAR-Ts). However, despite success in blood malignancies, CAR-T therapies in solid tumors have been hampered by their restricted infiltration. Here, we used our understanding of early cytotoxic lymphocyte infiltration of human lymphocytes in solid tumors in vivo to investigate the receptors in normal, adjacent, and tumor tissues of primary non-small-cell lung cancer specimens. We found that CX3CL1-CX3CR1 reduction restricts cytotoxic cells from the solid-tumor bed, contributing to tumor escape. Based on this, we designed a CAR-T construct using the well-established natural killer group 2, member D (NKG2D) CAR-T expression together with overexpression of CX3CR1 to promote their infiltration. These CAR-Ts infiltrate tumors at higher rates than control-activated T cells or IL-15-overexpressing NKG2D CAR-Ts. This construct also had similar functionality in a liver-cancer model, demonstrating potential efficacy in other solid malignancies.

MicroRNA-155 governs SHIP-1 expression and localization in NK cells and regulates subsequent infiltration into murine AT3 mammary carcinoma.

NK cell migration and activation are crucial elements of tumor immune surveillance. In mammary carcinomas, the number and function of NK cells is diminished, despite being positively associated with clinical outcome. MicroRNA-155 (miR-155) has been shown to be an important regulator of NK cell activation through its interaction with SHIP-1 downstream of inhibitory NK receptor signaling, but has not been explored in regard to NK cell migration. Here, we explored the migratory potential and function of NK cells in subcutaneous AT3 in mice lacking miR-155. Without tumor, these bic/miR-155-/- mice possess similar numbers of NK cells that exhibit comparable surface levels of cytotoxic receptors as NK cells from wild-type (WT) mice. Isolated miR-155-/- NK cells also exhibit equivalent cytotoxicity towards tumor targets in vitro compared to isolated WT control NK cells, despite overexpression of known miR-155 gene targets. NK cells isolated from miR-155-/- mice exhibit impaired F-actin polymerization and migratory capacity in Boyden-chamber assays in response chemokine (C-C motif) ligand 2 (CCL2). This migratory capacity could be normalized in the presence of SHIP-1 inhibitors. Of note, miR-155-/- mice challenged with mammary carcinomas exhibited heightened tumor burden which correlated with a lower number of tumor-infiltrating NK1.1+ cells. Our results support a novel, physiological role for SHIP-1 in the control of NK cell tumor trafficking, and implicate miR-155 in the regulation of NK cell chemotaxis, in the context of mammary carcinoma. This may implicate dysfunctional NK cells in the lack of tumor clearance in mice.

Immune evasion by TGFß-induced miR-183 repression of MICA/B expression in human lung tumor cells.

Immune escape is a hallmark of cancer. In human lung cancer, we have identified a unique microRNA (miR)-based pathway employed by tumor cells to repress detection by immune cells via the NKG2D-MICA/B receptor-ligand system. MICA/B is readily induced by cell transformation and serves as a danger signal and ligand to alert NK and activated CD8+ T cells. However, immunohistochemical analysis indicated that human lung adenocarcinoma and squamous cell carcinoma specimens express little MICA/B while high levels of miR-183 were detected in both tumor types in a TCGA database. Human lung tumor cell lines confirmed the reverse relationship in expression of MICA/B and miR-183. Importantly, a miR-183 binding site was identified on the 3'untranslated region (UTR) of both MICA and MICB, suggesting its role in MICA/B regulation. Luciferase reporter constructs bearing the 3'UTR of MICA or MICB in 293 cells supported the function of miR-183 in repressing MICA/B expression. Additionally, anti-sense miR-183 transfection into H1355 or H1299 tumor cells caused the upregulation of MICA/B. Abundant miR-183 expression in tumor cells was traced to transforming growth factor-beta (TGFß), as evidenced by antisense TGFß transfection into H1355 or H1299 tumor cells which subsequently lost miR-183 expression accompanied by MICA/B upregulation. Most significantly, anti-sense miR-183 transfected tumor cells became more sensitive to lysis by activated CD8+ T cells that express high levels of NKG2D. Thus, high miR-183 triggered by TGFß expressed in lung tumor cells can target MICA/B expression to circumvent detection by NKG2D on immune cells.

S100A9-induced overexpression of PD-1/PD-L1 contributes to ineffective hematopoiesis in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by dysplastic and ineffective hematopoiesis that can result from aberrant expansion and activation of myeloid-derived suppressor cells (MDSCs) within the bone marrow (BM) niche. MDSCs produce S100A9, which mediates premature death of hematopoietic stem and progenitor cells (HSPCs). The PD-1/PD-L1 immune checkpoint impairs immune responses by inducing T-cell exhaustion and apoptosis, but its role in MDS is uncharacterized. Here we report an increased expression of PD-1 on HSPCs and PD-L1 on MDSCs in MDS versus healthy donors, and that this checkpoint is also activated in S100A9 transgenic (S100A9Tg) mice, and by treatment of BM mononuclear cells (BM-MNC) with S100A9. Further, MDS BM-MNC treated with recombinant PD-L1 underwent cell death, suggesting that the PD-1/PD-L1 interaction contributes to HSPC death in MDS. In accordance with this notion, PD-1/PD-L1 blockade restores effective hematopoiesis and improves colony-forming capacity in BM-MNC from MDS patients. Similar findings were observed in aged S100A9Tg mice. Finally, we demonstrate that c-Myc is required for S100A9-induced upregulation of PD-1/PD-L1, and that treatment of MDS HSPCs with anti-PD-1 antibody suppresses the expression of Myc target genes and increases the expression of hematopoietic pathway genes. We conclude anti-PD-1/anti-PD-L1 blocking strategies offer therapeutic promise in MDS in restoring effective hematopoiesis.

Effect of chlorination by-products on the quantitation of microcystins in finished drinking water.

Microcystins are toxic peptides that can be produced by cyanobacteria in harmful algal blooms (HABs). Various analytical techniques have been developed to quantify microcystins in drinking water, including liquid chromatography tandem mass spectrometry (LC/MS/MS), enzyme linked immunosorbent assay (ELISA), and oxidative cleavage to produce 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) with detection by LC/MS/MS, the "MMPB method". Both the ELISA and MMPB methods quantify microcystins by detecting a portion of the molecule common to most microcystins. However, there is little research evaluating the effect of microcystin chlorination by-products potentially produced during drinking water treatment on analytical results. To evaluate this potential, chlorinated drinking water samples were fortified with various microcystin congeners in bench-scale studies. The samples were allowed to react, followed by a comparison of microcystin concentrations measured using the three methods. The congener-specific LC/MS/MS method selectively quantified microcystins and was not affected by the presence of chlorination by-products. The ELISA results were similar to those obtained by LC/MS/MS for most microcystin congeners, but results deviated for a particular microcystin containing a variable amino acid susceptible to oxidation. The concentrations measured by the MMPB method were at least five-fold higher than the concentrations of microcystin measured by the other methods and demonstrate that detection of MMPB does not necessarily correlate to intact microcystin toxins in finished drinking water.

Predicting the migration rate of dialkyl organotins from PVC pipe into water.

Organotins (OTs) are additives widely used as thermal and light stabilizers in polyvinyl chloride (PVC) plastics. OTs can leach into water flowing through PVC pipes. This work examines the leaching rates of two potentially neurotoxic OTs, dimethyl tin (DMT) and dibutyl tin (DBT), from PVC pipe. Water was circulated in a closed loop laboratory PVC pipe system. Using a gas chromatograph-pulsed flame photometric detector (GC-PFPD), the change in concentrations of DMT and DBT in the water in the system was monitored over time and allowed to reach equilibrium. OT concentration as a function of time was analyzed using a mechanistic leaching rate model. The diffusion coefficient for OT in the PVC pipe material, the only unknown model parameter, was found to be 9 × 10(-18) m(2)/s. This value falls within with the range of values estimated from the literature (2 × 10(-18) to 2 × 10(-17) m(2)/s) thus increasing confidence in the leaching rate model.

Synthesis and characterization of mesoporous silica films encapsulating titanium dioxide particles; Photodegradation of 2,4-dichlorophenol.

Degussa P-25 TiO2 encapsulated in a SBA-15 mesoporous thin film silica matrix prepared using a block copolymer templating method. The film structure was characterized using small angle X-ray diffraction (XRD), variable angle spectroscopic ellipsometry (VASE), and polarized light microscopy. The photoactivity of the films was assessed using HPLC and UV-vis spectroscopy to study the photodegradation of 2,4-dichlorophenol, an industrial pollutant, in water under 350 nm irradiation. The unoptimized film was found to be approximately half as photoactive as an equivalent dispersion of TiO2.