Current state of clinical development of TROP2-directed antibody–drug conjugates for triple-negative breast cancer

SummaryAntibody–drug conjugates (ADCs) against numerous molecular targets are currently being developed for the treatment of breast cancer (BCa). While the first ADC directed against Her2, namely trastuzumab–emtansine, was approved several years ago, targeting of TROP‑2, an epithelial cell marker overexpressed in approximately 80% of triple-negative breast cancers (TNBC) has gained interest through positive clinical data reported for the compound sacituzumab–govitecan (SG) resulting from the phase 3 ASCENT trial. This short review summarizes the data that led to approval of SG and to take a closer look at the state of clinical development of other ADCs targeting TROP‑2 in TNBC.

Analysis of The Cells Isolated From Epithelial Cell Rests of Malassez Through Single-cell Limiting Dilution

The epithelial cell rests of Malassez (ERM) play a pivotal role in preventing ankylosis between the alveolar bone and the tooth. Although several functions of ERM has been reported, the mechanism behind preventing dentoalveolar ankylosis remains unclear. In this study, 18 clones were isolated from ERM (CRUDE) using the single-cell limiting dilution method. Among them, ERM-2 and -3, which exhibited the highest and lowest proliferation rates, respectively, were selected. ERM-2, ERM-3, and CRUDE ERM were stained with epithelial markers, including cytokeratin-wide and cytokeratin-19, via immunofluorescence. The qRT-PCR analysis revealed increased expression levels of p75 (ameloblast marker), amelogenin, and sfrp5 (inner enamel epithelial cell marker) in the ERM-2 cells. Alternatively, ameloblastin and ck-14 (outer enamel epithelial cell marker) were highly expressed in ERM-3 cells. The mineralization of human periodontal ligament fibroblast (HPDLF) was inhibited when co-cultured with ERM-2, ERM-3, and CRUDE ERM cells. The addition of an anti-amelogenin antibody restored the mineralization of HPDLF cells. Transplanted rat molar cultured in ERM-2 (high amelogenin secretive clone) cell-derived supernatant resulted in significantly smaller bone formation than those cultured in the CRUDE ERM and ERM-3 cell-derived supernatants. These findings indicate that amelogenin produced by ERM cells might be involved in preventing dentoalveolar ankylosis.

IL-32 induces epithelial-mesenchymal transition by triggering endoplasmic reticulum stress in A549 cells

Background Epithelial-mesenchymal transition (EMT) is a key process in the onset and development of idiopathic pulmonary fibrosis (IPF) with unclear mechanisms. Our previous studies found that bleomycin and tunicamycin could induce ER stress and consequently trigger EMT accompanying with IL-32 overexpression. This study was aimed to investigate the effects of IL-32 on EMT and ER stress to elucidate the pathogenesis of IPF. Methods Human lung adenocarcinoma A549 cells were treated with recombinant human (rh)IL-32, IL-32 siRNA and EMT inducer tunicamycin, or 4-phenylbutyric acid (4-PBA), respectively. Then the cell morphology was observed and the expression of ER-related markers and EMT-related markers were detected by RT-qPCR or western blotting. Results Stimulation of A549 cells with rhIL-32 led to a morphological change from a pebble-like shape to an elongated shape in a portion of the cells, accompanied by down regulated expression of the epithelial cell marker E-cadherin and up regulated expression of the mesenchymal cell markers N-cadherin, Vimentin, and Zeb-1. However, these rhIL-32 induced changes were inhibited by the ER stress inhibitor 4-PBA. Suppression of IL-32 expression with siRNA inhibited TM-induced EMT. Further stimulation of the A549 cells with rhIL-32 demonstrated an increase in the expression of GRP78, although this increase was also inhibited by 4-PBA. Conclusions These results suggest that IL-32 induces EMT in A549 cells by triggering ER stress, and IL-32 may be a novel marker for IPF.

Exploration of the role of serum ghrelin in the diagnosis and treatment of digestive tract malignancies

Objective The incidence of digestive tract malignancies (DTMs) is increasing, early diagnosis is limited, and treatment effects are unsatisfactory. DTMs express ghrelin, which might be involved in tumor formation and development; whether serum ghrelin can provide useful guidance remains unknown. Methods Sera of healthy individuals were obtained from October 2017 through March 2018; serum samples from patients with gastric (GC), colon (CC), and rectal (RC) cancers were collected during the same period. Serum ghrelin was tested by ELISA and correlated with clinicopathology of patients with DTMs. Results Serum ghrelin was higher in patients (GC, 38 patients; CC, 24; RC, 26) than in 69 healthy individuals and decreased significantly after tumor resection. Nutrition Risk Screening 2002 score and neutrophil:lymphocyte ratio affected perioperative serum ghrelin levels. The epithelial cell marker AE1/AE3 (pan keratin) in patients with GC, tumor location in the colon in patients with CC, and age in patients with RC also affected perioperative serum ghrelin. Conclusions Serum ghrelin might provide early warning of occurrence and guide prognosis of DTMs. Ghrelin can be used when screening for nutritional risk and inflammation. The clinicopathological influence on serum ghrelin in patients with DTMs is related to tumor location in the digestive tract.

Increased Levels of Nuclear Factor κB and Fos-Related Antigen 1 in Lung Tissues From Patients With Acute Respiratory Distress Syndrome

Context.—Both nuclear factor κB and Fos-related antigen 1 have been implicated in the pathogenesis of inflammatory lung diseases, including acute lung injury/acute respiratory distress syndrome. Objective.—To evaluate lung tissues from patients with acute respiratory distress syndrome for presence of nuclear factor κB and Fos-related antigen 1. Design.—Lung tissue sections from 5 patients with acute respiratory distress syndrome and sections of normal lung tissues of 4 patients were stained with antibodies against epithelial cell marker (surfactant protein B) and nuclear factor κB or Fos-related antigen 1. Samples were analyzed using confocal laser microscopy. Results.—We have detected significantly increased levels of activated nuclear factor κB and Fos-related antigen 1 in lung tissues from patients with acute respiratory distress syndrome compared with control tissues, suggesting that these transcription factors undergo activation in lungs of patients suffering from acute respiratory distress syndrome. Conclusions.—Our data demonstrate that activated nuclear factor κB and Fos-related antigen 1 are elevated in epithelial cells in lung tissues of patients with acute respiratory distress syndrome.

163. POTENTIAL MARKERS FOR THE PROSPECTIVE ISOLATION OF HUMAN ENDOMETRIAL EPITHELIAL STEM/PROGENITOR CELLS

The human endometrium regenerates each month following menstruation, parturition and in post-menopausal women taking hormone replacement therapy. Adult stem/progenitor cells discovered residing in human and mouse endometrium may be responsible for this regenerative capacity. However, assays used to identify these stem/progenitor cells are retrospective. The aim of this study is to identify surface markers for the prospective isolation of human endometrial epithelial progenitor cells using a panel of 22 antibodies. Flow cytometry and was used in the initial screen and immunohistochemistry was used to reveal the location of marker expression. Multi-colour FACS protocols were developed with promising markers in conjunction with EpCAM (epithelial cell marker) and to exclude endothelial (CD31+), leukocytes (CD45+) and stromal (CD90+) cells. Sorted subpopulations were assessed for clonogenicity and self-renewal activity using in vitro cloning assays. Six antibodies were short-listed. 2D1D12 enriched for progenitor cells that formed epithelial clones in culture (n = 2). The 2D1D12+EpCAM+ fraction produced very few colony-forming units (CFU). 2D1D12–EpCAM+ fraction gave rise to small CFU. However the 2D1D12+EpCAM– population showed the greatest progenitor activity, producing many large CFU that could be serially cloned twice, indicating self renewal activity. This preliminary data suggests that 2D1D12+EpCAM–CD90–CD31–CD45– population may enrich for human endometrial epithelial stem/progenitor cells. Importantly, large CFU have previously been reported to exhibit stem cell properties of self-renewal, differentiation and high proliferative potential (1). Future studies will focus on xenografting this population to assess tissue reconstitution ability in vivo. The identification of endometrial epithelial stem/progenitor cell marker(s) will enable their prospective isolation for further characterisation and will assist in the investigation of their potential role in endometrial proliferative disorders such as endometriosis and endometrial cancer. (1) Gargett CE et al (2009) Biol Reprod 80: 1136–45.

Clinical Value of Immunohistochemically Detected Lymphatic and Vascular Invasions in Clinically Staged Endometrioid Endometrial Cancer

Background:A novel technique to differentiate lymphatic from vascular invasion and to assess the clinicopathological significance in patients with early endometrial cancer.Methods:Dual immunohistochemical techniques against pancytokeratin epithelial cell marker (PCK), D6 lymphatic endothelial marker, and CD31 nonspecific endothelial marker were deployed for differentiation. Seventy-seven patients were included with a median follow-up of 161 months. Tumors with positive evidence of lymphovascular space invasion on PCK-CD31 immunohistochemistry and absence of lymphatic space invasion on PCK-D6 were regarded as cases with vascular space invasion only.Results:Significant association between depth of myometrial invasion, recurrence rate, and hematoxylin and eosin that detected lymphovascular space invasion were noted (P < 0.0001 and P = 0.009, respectively). The 5-year recurrence-free survival was 45% for the group with hematoxylin and eosin evidence of lymphovascular space invasion compared with 89% for the group without (P = 0.0014). Pancytokeratin epithelial cell marker-D6 dual immunostaining detected lymphatic space invasion in 22 (29%) patients. There was significant association between lymphatic space invasion and depth of myometrial invasion (P = 0.046). Lymphatic space invasion detected on immunohistochemistry was present in 8 (72%) of 11 patients with recurrent disease. Of the remaining 49 patients with no evidence of recurrent disease, only 11 (22%) had presented with lymphatic space invasion. Positive association between tumor recurrence rate and lymphatic space invasion was noted (P = 0.003). The 5-year recurrence-free survival was 53% for the group with lymphatic invasion compared with 93% for the group without. This difference was similarly shown to be of significance (P = 0.0009). There were no apparent association between immunohistochemically detected lymphovascular or vascular space invasion and any clinicopathological factor.Conclusions:Lymphatic space invasion detected by using dual immunostaining is of significant value in identifying high-risk patients.

Tracheal occlusion stimulates cell cycle progression and type I cell differentiation in lungs of fetal rats

Fetal tracheal occlusion (TO) has been reported to stimulate lung growth but decreases number and maturation of type II cells, effects that vary with gestational age and duration of TO. We examined effects of a novel method of TO (unipolar microcautery to seal the trachea) produced at 19.5–20 days (d) of gestation in fetal rats; fetuses were delivered at term, 22 d. Controls were sham operated and unoperated littermates. TO increased wet lung weight but not dry lung weight or lung DNA and protein. To evaluate further the effects of TO, we examined the cell cycle regulators, cyclins D1 and A, in fetal lungs. Cyclin D1 increased with TO ( P < 0.005). TO also increased expression of the type I epithelial cell marker RTI40 (mRNA and protein). TO decreased mRNA for surfactant proteins (SP)-A and -C but did not affect protein levels of SP-A and -B and of RTII70, a type II epithelial cell marker. We conclude that TO by microcautery, even of short duration, has diverse pulmonary effects including stimulating increased levels of cyclin D1 with probable cell cycle progression, type I cell differentiation, and possibly inhibiting type II cell function.

Expression of estrogen, progesterone and androgen receptors in the oviduct of developing, cycling and pre-implantation rats

To determine expression and localization of receptors for estrogen (ER), progesterone (PR) and androgen (AR), detailed immunohistochemical evaluations were performed in the Sprague-Dawley rat oviduct during pre- and neonatal development, estrous cycle and pre-implantation period. In addition, real-time RT-PCR studies were conducted to evaluate changes in ERalpha, ERbeta, total PR (PR-A+B), PR-B and AR mRNA expressions. All receptors except for ERbeta were detected in epithelial, and stromal or mesenchymal cells of the fetal and neonatal oviduct, and increased with development. During the estrous cycle and early pregnancy, ERalpha and PR-A+B were expressed in epithelial, stromal and muscle cells throughout the oviduct region, and showed changes in expression predominantly in the isthmus. Only a few epithelial cells in the infundibulum (inf) and ampulla (AMP) showed ERbeta staining. AR was detected in stromal and muscle cells throughout the oviduct region, and in epithelial cells of the inf/AMP. Taken together, ERalpha, PR-A+B and AR were detected in the epithelium of the inf/AMP region, but all of these receptors were expressed in a distinct subset of epithelial cells which were negative for beta-tubulin IV, a ciliated epithelial cell marker. These results contribute to a better understanding of the respective roles of ERs, PRs and AR in the rat oviduct.