State of the Art Cell Detection in Bone Marrow Whole Slide Images.

CONTEXT: Diseases of the hematopoietic system such as leukemia is diagnosed using bone marrow samples. The cell type distribution plays a major role but requires manual analysis of different cell types in microscopy images. AIMS: Automated analysis of bone marrow samples requires detection and classification of different cell types. In this work, we propose and compare algorithms for cell localization, which is a key component in automated bone marrow analysis. SETTINGS AND DESIGN: We research fully supervised detection architectures but also propose and evaluate several techniques utilizing weak annotations in a segmentation network. We further incorporate typical cell-like artifacts into our analysis. Whole slide microscopy images are acquired from the human bone marrow samples and annotated by expert hematologists. SUBJECTS AND METHODS: We adapt and evaluate state-of-the-art detection networks. We further propose to utilize the popular U-Net for cell detection by applying suitable preprocessing steps to the annotations. STATISTICAL ANALYSIS USED: Evaluations are performed on a held-out dataset using multiple metrics based on the two different matching algorithms. RESULTS: The results show that the detection of cells in hematopoietic images using state-of-the-art detection networks yields very accurate results. U-Net-based methods are able to slightly improve detection results using adequate preprocessing - despite artifacts and weak annotations. CONCLUSIONS: In this work, we propose, U-Net-based cell detection methods and compare with state-of-the-art detection methods for the localization of hematopoietic cells in high-resolution bone marrow images. We show that even with weak annotations and cell-like artifacts, cells can be localized with high precision.

In oxygen-deprived tumor cells ERp57 provides radioprotection and ensures proliferation via c-Myc, PLK1 and the AKT pathway.

The disulfide isomerase ERp57, originally found in the endoplasmic reticulum, is located in multiple cellular compartments, participates in diverse cell functions and interacts with a huge network of binding partners. It was recently suggested as an attractive new target for cancer therapy due to its critical role in tumor cell proliferation. Since a major bottleneck in cancer treatment is the occurrence of hypoxic areas in solid tumors, the role of ERp57 in cell growth was tested under oxygen depletion in the colorectal cancer cell line HCT116. We observed a severe growth inhibition when ERp57 was knocked down in hypoxia (1% O2) as a consequence of downregulated c-Myc, PLK1, PDPK1 (PDK1) and AKT (PKB). Further, irradiation experiments revealed also a radiosensitizing effect of ERp57 depletion under oxygen deprivation. Compared to ERp57, we do not favour PDPK1 as a suitable pharmaceutical target as its efficient knockdown/chemical inhibition did not show an inhibitory effect on proliferation.

Overcoming hypoxia-induced resistance of pancreatic and lung tumor cells by disrupting the PERK-NRF2-HIF-axis.

Hypoxia-induced resistance of tumor cells to therapeutic treatment is an unresolved limitation due to poor vascular accessibility and protective cell adaptations provided by a network, including PERK, NRF2, and HIF signaling. All three pathways have been shown to influence each other, but a detailed picture remains elusive. To explore this crosstalk in the context of tumor therapy, we generated human cancer cell lines of pancreatic and lung origin carrying an inducible shRNA against NRF2 and PERK. We report that PERK-related phosphorylation of NRF2 is only critical in Keap1 wildtype cells to escape its degradation, but shows no direct effect on nuclear import or transcriptional activity of NRF2. We could further show that NRF2 is paramount for proliferation, ROS elimination, and radioprotection under constant hypoxia (1% O2), but is dispensable under normoxic conditions or after reoxygenation. Depletion of NRF2 does not affect apoptosis, cell cycle progression and proliferation factors AKT and c-Myc, but eliminates cellular HIF-1α signaling. Co-IP experiments revealed a protein interaction between NRF2 and HIF-1α and strongly suggest NRF2 as one of the cellular key factor for the HIF pathway. Together these data provide new insights on the complex role of the PERK-NRF2-HIF-axis for cancer growth.

Tumor cells rely on the thiol oxidoreductase PDI for PERK signaling in order to survive ER stress.

Upon ER stress cells activate the unfolded protein response through PERK, IRE1 and ATF6. Remarkable effort has been made to delineate the downstream signaling of these three ER stress sensors after activation, but upstream regulation at the ER luminal site still remains mostly undefined. Here we report that the thiol oxidoreductase PDI is mandatory for activation of the PERK pathway in HEK293T as well as in human pancreatic, lung and colon cancer cells. Under ER stress, depletion of PDI selectively abrogated eIF2α phosphorylation, induction of ATF4, CHOP and even BiP. Furthermore, we could demonstrate that PDI prevented degradation of activated PERK by the 26S proteasome and therefore contributes to maintained PERK signaling. As a result of decreased PERK activity, PDI depleted cells showed an increased vulnerability to ER stress induced by chemicals or ionizing radiation in 2D as well as in 3D culture models. We conclude that PDI is an obligatory regulator of the PERK pathway with future therapy implications.

Autophagy induced by ionizing radiation promotes cell death over survival in human colorectal cancer cells.

Autophagy is commonly described as a cell survival mechanism and has been implicated in chemo- and radioresistance of cancer cells. Whether ionizing radiation induced autophagy triggers tumor cell survival or cell death still remains unclear. In this study the autophagy related proteins Beclin1 and ATG7 were tested as potential targets to sensitize colorectal carcinoma cells to ionizing radiation under normoxic, hypoxic and starvation conditions. Colony formation, apoptosis and cell cycle analysis revealed that knockdown of Beclin1 or ATG7 does not enhance radiosensitivity in HCT-116 cells. Furthermore, ATG7 knockdown led to an increased survival fraction under oxygen and glutamine starvation, indicating that ionizing radiation indeed induces autophagy which, however, leads to cell death finally. These results highlight that inhibition of autophagic pathways does not generally increase therapy success but may also lead to an unfavorable outcome especially under amino acid and oxygen restriction.

Pembrolizumab as Palliative Immunotherapy in Malignant Pleural Mesothelioma.

INTRODUCTION: There is no approved second-line treatment for malignant pleural mesothelioma (MPM). On the basis of promising early results, pembrolizumab was used off-label in Switzerland and Australia. We investigated outcomes in association with clinicopathological features and expression of programmed death ligand 1 (PD-L1). METHODS: Registry data in Australia and Switzerland were pooled. Patient characteristics, including age, sex, histological subtype, and previous treatments were captured. Outcomes were assessed locally. PD-L1 expression was categorized as negative (<5%), intermediate (5%-49%), and high (≥50%). RESULTS: A total of 93 patients (48 from Switzerland and 45 from Australia) were treated; 68 patients (73%) had epithelioid MPM, and 67 (72%) had an Eastern Cooperative Oncology Group performance status of 0 or 1. Pembrolizumab was the second-line treatment in 48 of 93 patients (52%). PD-L1 expression results were available for 66 patients (71%). Most (68%) were negative, 18% were intermediate, and 14% were high for PD-L1 expression. In the full cohort, the overall response rate (ORR) was 18%, the median progression-free survival (mPFS) was 3.1 months, and the median overall survival was 7.2 months. In patients with an Eastern Cooperative Oncology Group performance status of 0 or 1 and only one previous systemic treatment (n = 35), the ORR was 37%, the mPFS was 3.7 months, and the median overall survival was 10.2 months. The nonepitheloid histological subtype showed an improved ORR (24% versus 16% [p = 0.54) and mPFS (5.6 versus 2.8 months [p = 0.02]). Compared with intermediate and negative PD-L1 expression, high PD-L1 expression was associated with an improved ORR (44% versus 42% versus 11% [p = 0.01]) and mPFS (6.2 versus 3.9 versus 2.7 months [p = 0.04]). Toxicity was as expected. CONCLUSION: These real-world data demonstrate similar response rates but inferior survival compared with those in early-phase trials. High PD-L1 expression and nonepitheloid histological subtype were associated with greater activity. Anti-PD-L1 immunotherapy is a reasonable second-line therapy in patients with MPM.

PDI is an essential redox-sensitive activator of PERK during the unfolded protein response (UPR).

Endoplasmic reticulum (ER) stress leads to activation of the unfolded protein response (UPR) that results in transient suppression of protein translation to allow recovery but leads to cell death when stress cannot be resolved. Central to initiation of the UPR is the activation of the ER transmembrane kinase protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). Here we report that the thiol oxidoreductase ERp57 and protein disulfide isomerase-A1 (PDI), which belong to the same family of luminal ER oxidoreductases, have strikingly opposing roles in the regulation of PERK function. In HCT116 colon carcinoma cells, lentiviral depletion of ERp57 resulted in oxidation of PDI and activation of PERK, whereas depletion or chemical inhibition of PDI reduced PERK signaling and sensitized the cancer cells to hypoxia and ER stress. We conclude that oxidized PDI acts as a PERK activator, whereas ERp57 keeps PDI in a reduced state in the absence of ER stress. Thus, our study defines a new interface between metabolic redox signaling and PERK-dependent activation of the UPR and has the potential to influence future cancer therapies that target PERK signaling.

Depletion of the thiol oxidoreductase ERp57 in tumor cells inhibits proliferation and increases sensitivity to ionizing radiation and chemotherapeutics.

Rapidly growing tumor cells must synthesize proteins at a high rate and therefore depend on an efficient folding and quality control system for nascent secretory proteins in the endoplasmic reticulum (ER). The ER resident thiol oxidoreductase ERp57 plays an important role in disulfide bond formation. Lentiviral, doxycycline-inducible ERp57 knockdown was combined with irradiation and treatment with chemotherapeutic agents. The knockdown of ERp57 significantly enhanced the apoptotic response to anticancer treatment in HCT116 colon cancer cells via a p53-dependent mechanism. Instead of a direct interaction with p53, depletion of ERp57 induced cell death via a selective activation of the PERK branch of the Unfolded Protein Response (UPR). In contrast, apoptosis was reduced in MDA-MB-231 breast cancer cells harboring mutant p53. Nevertheless, we observed a strong reduction of proliferation in response to ERp57 knockdown in both cell lines regardless of the p53 status. Depletion of ERp57 reduced the phosphorylation activity of the mTOR-complex1 (mTORC1) as demonstrated by reduction of p70S6K phosphorylation. Our data demonstrate that ERp57 is a promising target for anticancer therapy due to synergistic p53-dependent induction of apoptosis and p53-independent inhibition of proliferation.

Strabismus might be a risk factor for amblyopia recurrence.

BACKGROUND: Previous reports have suggested that one-fourth of amblyopic subjects present with recurrence of amblyopia even after successful primary treatment. Risk factors for amblyopia recurrence are insufficiently studied, but therapy weaning has recently been suggested as a method for reducing the risk of recurrence. METHODS: In the second phase of a prospective study examining the results of treatment for amblyopia, all 35 children with successful primary amblyopia treatment were put on maintenance therapy. Maintenance therapy consisted of low-intensity patching, atropine, or blurring filter, and all children were regularly examined up to at least 8 years of age. Mean age at start of maintenance therapy was 4.3 years (range, 2-7 years). RESULTS: Of the 35 cases with successful primary treatment, 6 cases deteriorated >or=0.2 logMAR, which was considered recurrence of amblyopia. Two of these cases had a second recurrence. Notably, all six subjects presenting with recurrence had microstrabismus. This finding was just outside statistical significance at the 95% confidence level (p = 0.06), but the sample size was small. All but one of the recurrences appeared within 6 months after successful primary treatment. Age at successful primary treatment or initial interocular difference of visual acuity did not affect the risk of recurrence. CONCLUSIONS: Recurrence of amblyopia occurred in 17% of patients despite maintenance therapy and was associated with microstrabismus. The majority of recurrences occurred within the first 6 months after primary treatment.