Circulating Tumor DNA as a Predictive Biomarker for Clinical Outcomes With Margetuximab and Pembrolizumab in Pretreated HER2-Positive Gastric/ Gastroesophageal Adenocarcinoma.

PURPOSE: To assess the ability of circulating tumor DNA (ctDNA)-based testing to identify patients with HER2 (encoded by ERBB2)-positive gastric/gastroesophageal adenocarcinoma (GEA) who progressed on or after trastuzumab-containing treatments were treated with combination therapy of anti-HER2 and anti-PD-1 agents. METHODS: ctDNA analysis was performed retrospectively using plasma samples collected at study entry from 86 patients participating in the phase 1/2 CP-MGAH22-05 study (NCT02689284). RESULTS: Objective response rate (ORR) was significantly higher in evaluable ERBB2 amplification-positive vs - negative patients based on ctDNA analysis at study entry (37% vs 6%, respectively; P = .00094). ORR was 23% across all patients who were evaluable for response. ERBB2 amplification was detected at study entry in 57% of patients (all HER2 positive at diagnosis), and detection was higher (88%) when HER2 status was determined by immunohistochemistry fewer than 6 months before study entry. ctDNA was detected in 98% (84/86) of patients tested at study entry. Codetected ERBB2-activating mutations were not associated with response. CONCLUSIONS: Current ERBB2 status may be more effective than archival status at predicting clinical benefit from margetuximab plus pembrolizumab therapy. ctDNA testing for ERBB2 status prior to treatment will spare patients from repeat tissue biopsies, which may be reserved for reflex testing when ctDNA is not detected.

Stimulation of glioma cell motility by expression, proteolysis, and release of the L1 neural cell recognition molecule.

BACKGROUND: Malignant glioma cells are particularly motile and can travel diffusely through the brain parenchyma, apparently without following anatomical structures to guide their migration. The neural adhesion/recognition protein L1 (L1CAM; CD171) has been implicated in contributing to stimulation of motility and metastasis of several non-neural cancer types. We explored the expression and function of L1 protein as a stimulator of glioma cell motility using human high-grade glioma surgical specimens and established rat and human glioma cell lines. RESULTS: L1 protein expression was found in 17 out of 18 human high-grade glioma surgical specimens by western blotting. L1 mRNA was found to be present in human U-87/LacZ and rat C6 and 9L glioma cell lines. The glioma cell lines were negative for surface full length L1 by flow cytometry and high resolution immunocytochemistry of live cells. However, fixed and permeablized cells exhibited positive staining as numerous intracellular puncta. Western blots of cell line extracts revealed L1 proteolysis into a large soluble ectodomain (~180 kDa) and a smaller transmembrane proteolytic fragment (~32 kDa). Exosomal vesicles released by the glioma cell lines were purified and contained both full-length L1 and the proteolyzed transmembrane fragment. Glioma cell lines expressed L1-binding alphavbeta5 integrin cell surface receptors. Quantitative time-lapse analyses showed that motility was reduced significantly in glioma cell lines by 1) infection with an antisense-L1 retroviral vector and 2) L1 ectodomain-binding antibodies. CONCLUSION: Our novel results support a model of autocrine/paracrine stimulation of cell motility in glioma cells by a cleaved L1 ectodomain and/or released exosomal vesicles containing L1. This mechanism could explain the diffuse migratory behavior of high-grade glioma cancer cells within the brain.

MUC1 expression is repressed by protein inhibitor of activated signal transducer and activator of transcription-y.

Mucin 1 (MUC1) is a transmembrane glycoprotein that modulates the interaction between the embryo and the uterine epithelial cell surface. MUC1 also is a tumor marker and has been implicated in the protection of cancer cells from immune cell attack as well as in cell signaling in some tumors. We and others have shown that MUC1 expression is activated by progesterone (P), TNF-alpha, and interferon-gamma (IFN-gamma). Here we demonstrate that MUC1 expression is down-regulated by overexpression of members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family, PIAS1, PIAS3, PIASxalpha, PIASxbeta, and PIASy, in human uterine epithelial cell lines HES and HEC-1A and in a breast cancer cell line, T47D. Treatments with P, TNF-alpha, and IFN-gamma were unable to overcome the repression by PIASy. PIASy repression of basal, P-, and TNF-alpha-stimulated MUC1 promoter activity was not dependent on the PIASy sumoylation domain. In contrast, PIASy suppression of IFN-gamma-activated MUC1 promoter activity was dependent on the PIASy sumoylation domain. PIASy and P receptor B were localized to the nucleus upon P treatment, and small interfering RNA knockdown of PIASy resulted in an increase in P-mediated stimulation of MUC1 protein expression. Overexpression of PIASy did not affect P receptor B binding to the MUC1 promoter but surprisingly led to a loss of nuclear receptor corepressor (NCoR), which was recruited to the promoter in response to P. Collectively, these data indicate that PIASy may be a useful target for down-regulation of MUC1 expression in various contexts.

Tumor necrosis factor alpha stimulates MUC1 synthesis and ectodomain release in a human uterine epithelial cell line.

Regulation of MUC1 expression and removal is a salient feature of embryo implantation, bacterial clearance, and tumor progression. In some species, embryo implantation is accompanied by a transcriptional decline in uterine epithelial expression of MUC1. In other species, MUC1 is locally removed at blastocyst attachment sites, suggesting a proteolytic activity. Previously, we demonstrated that MUC1 is proteolytically released from the surface of a human uterine epithelial cell line, HES, and identified TNFalpha converting enzyme/a disintegrin and metalloprotease 17 as a constitutive and phorbol ester-stimulated MUC1 sheddase. The aims of the current study were to test the ability of soluble factors elevated during the periimplantation interval in vivo to stimulate ectodomain shedding of MUC1 from HES uterine epithelial cells and to characterize the nature of this proteolytic activity(ies). We identified TNFalpha as a prospective endogenous stimulus of MUC1 ectodomain release and of MUC1 and TNFalpha converting enzyme/a disintegrin and metalloprotease 17 expression. Moreover, we established that TNFalpha-stimulated MUC1 shedding occurs independently of increased de novo protein synthesis and demonstrated that the TNFalpha-induced increase in MUC1 gene expression is mediated through the kappaB site in the MUC1 promoter. Finally, we determined that the TNFalpha-sensitive MUC1 sheddase is inhibited by the metalloprotease inhibitor, TNFalpha protease inhibitor (TAPI), and the endogenous tissue inhibitor of metalloprotease-3. Collectively, these studies provide the initial in vitro characterization of a putative physiological stimulus of MUC1 ectodomain release and establish the nature of the metalloproteolytic activity(ies) involved.

Synergistic stimulation of MUC1 expression in normal breast epithelia and breast cancer cells by interferon-gamma and tumor necrosis factor-alpha.

The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in human breast cancers. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. We demonstrate that MUC1 expression in T47D breast cancer cells and normal human mammary epithelial cells (HMEC) is enhanced by tumor necrosis factor-alpha (TNF-alpha) in the presence of interferon-gamma (IFN-gamma). MUC1 responsiveness to these cytokines was modest in T47D cells and robustly induced in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that a kappaB site at -589/-580 and the STAT-binding element at -503/-495 and were required for cooperative stimulation by TNFalpha and IFN-gamma. Binding of NFkappaB p65 to the MUC1 kappaB site was induced by TNF-alpha treatment, as demonstrated by electrophoretic mobility shift assay. Specific mutation of the kappaB site prevented binding of NFkappaB p65 and blocked TNF-alpha stimulation of MUC1 promoter activity. Collectively, these studies demonstrate synergistic stimulation of MUC1 expression by TNF-alpha and IFN-gamma that is mediated by independent actions of NFkappaB p65 and STAT1alpha upon kappaB and STAT sites, respectively, in the MUC1 promoter. Strong induction of MUC1 expression by these proinflammatory cytokines is clearly evident in normal mammary epithelium. In contrast, breast tumor cells appear to override normal regulatory responses via as yet undefined cis-elements.

Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening.

High density cDNA microarray screening was used to determine changes in gene expression occurring during the transition between the early luteal (prereceptive) and mid-luteal (receptive) phases in human endometrium. Of approximately 12,000 genes profiled, 693 (5.8%) displayed >2-fold differences in relative levels of expression between these stages. Of these, 370 genes (3.1%) displayed decreases ranging from 2- to >100-fold while 323 genes (2.7%) displayed increases ranging from 2- to >45-fold. Many genes correspond to mRNAs encoding proteins previously shown to change in a similar manner between the proliferative and mid-luteal phases, serving as one validation of the microarray screening results. In addition, novel genes were identified. Genes encoding cell surface receptors, adhesion and extracellular matrix proteins and growth factors accounted for 20% of the changes. Several genes were studied further by Northern blot analyses. These results confirmed that claudin-4/Clostridium perfringens enterotoxin (CPE) receptor and osteopontin (OPN) mRNA increased approximately 4- and 12-fold respectively, while betaig-H3 (BIGH3) decreased >80% during the early to mid-luteal transition. Immunostaining also revealed strong specific staining for claudin-4/CPE, EP(1) and prostaglandin receptor in epithelia, and leukotriene B4 receptor in both epithelia and stroma, at the mid-luteal stage. Collectively, these studies identify multiple new candidate markers that may be used to predict the receptive phase in humans. Some of these gene products, e.g. OPN, may play direct roles in embryo-uterine interactions during the implantation process.