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The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5-3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and ZVAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation. [ABSTRACT FROM AUTHOR]

Background: Spine radiosurgery has been performed at Oslo University hospital, The Norwegian Radium Hospital since July 2011. Our approach has been to develop a simple planning technique, where the organs at risk are spared while substantially reducing the planning time. Materials and methods: We have developed a library plan with nine static beams, where each beam is automatically formed to the PTV, but also manually ensuring that a spinal cord planning risk volume (PRV) is excluded in all directions. This is a fast forward planning (FFP) technique compared to regular IMRT or VMAT techniques for spine radiosurgery. In this work ten patient plans using FFP were compared to IMRT and VMAT plans for the identical patients. The dose distributions were compared for the different plans, emphasizing the dose to the target volumes and reduced dose to the surrounding tissue. The planning time consume and treatment time were also registered. Results: We found that the risk organs dose criteria normally were fulfilled within a sufficient margin for FFP, IMRT and VMAT techniques. The dose distribution following FFP yields a steeper gradient close to the spinal cord, but also gives a more heterogeneous dose distribution within the PTV. The GTV dose applying a FFP technique is normally higher than from IMRT and VMAT. For single fraction spine radiosurgery, the average GTV dose was 25.8 Gy for FFP, while the objective for an IMRT or VMAT optimization should exceed 18 Gy to D98%. Planning time for FFP is normally less than 30 minutes, while IMRT or VMAT optimization may last for several hours. Conclusions: The main alternative for this patient group at our hospital has traditionally been 30 Gy in 10 fractions, conventionally planned with one or two opposing fields. Our preliminary findings using this FFP technique suggest a sufficient and comparable dose distribution to those resulting from IMRT or VMAT, while the total time consume is comparable or less than the conventional method. Disclosure: No significant relationships. [ABSTRACT FROM AUTHOR]

Background and Purpose: Lung cancer is the most common cause of brain metastases. Stereotactic radiosurgery (SRS) is increasingly utilized in the treatment of brain metastases, and is in our institution delivered via linear accelerator. We compared median overall survival (mOS) in lung cancer patients who received either SRS alone, SRS combined with whole brain radiation therapy (WBRT) or WBRT alone for 1- 3 cerebral metastases. Material an. Methods: We conducted a retrospective review of lung cancer patients with 1-3 MRI-verified brain metastases of a diameter of 50 mm or less treated with SRS (+/-WBRT) in the period 2007-2011 and an identical group treated with WBRT only in the period 2004-2006. SRS was not offered in our institution until 2007, so the earlier time period for collection of WBRT patients was chosen to avoid a retrospective selection bias for either WBRT or SRS based on prognostic factors. Results: 52 patients were found to fulfill the inclusion criteria in the WBRT only group, whereas 111 consecutive patients were included in the SRS group. Median age and female percentage in the WBRT only patients vs SRS patients were 62 vs 65 years and 46 vs 61 respectively. In the WBRT group, 17 patients (33%) had 1 brain metastasis, whereas in the SRS group, 72 patients (65%) had 1 brain metastasis. 78 patients (70%) received SRS as their only radiation treatment. mOS in the WBRT and SRS groups were 3.1 and 6.0 months respectively (p=0.0055), and for patients with only 1 metastasis, the mOS was 3.4 vs 6.4 months (p=0.009). For patients with 2-3 metastases, the mOS was 2.8 vs 4.4 in the favor of SRS, but this did not reach statistical significance (p=0.29). The combination of WBRT and SRS yielded a non-significant prolongation of mOS compared to SRS alone (7.6 vs 5.5 months, p=0.22), and for patients with 1 metastasis, patients treated with SRS alone achieved similar mOS as those treated with both SRS and WBRT (6.6 vs 6.8 months respectively). Conclusion In our cohort of lung cancer patients, we show that patients with single brain metastasis treated with SRS had a significant survival benefit compared to those treated with WBRT only, and there was also a trend towards longer survival in patients with 2-3 metastases. This confirms and extends findings of previous studies, where patients with solitary cerebral metastases show better survival with combination therapy. Disclosure: No significant relationships. [ABSTRACT FROM AUTHOR]

Background: Six thoracic pathologists reviewed 259 lung neuroendocrine tumours (LNETs) from the lungNENomics project, with 171 of them having associated survival data. This cohort presents a unique opportunity to assess the strengths and limitations of current World Health Organization (WHO) classification criteria and to evaluate the utility of emerging markers., Patients and Methods: Patients were diagnosed based on the 2021 WHO criteria, with atypical carcinoids (ACs) defined by the presence of focal necrosis and/or 2-10 mitoses per 2 mm 2 . We investigated two markers of tumour proliferation: the Ki-67 index and phospho-histone H3 (PHH3) protein expression, quantified by pathologists and automatically via deep learning. Additionally, an unsupervised deep learning algorithm was trained to uncover previously unnoticed morphological features with diagnostic value., Results: The accuracy in distinguishing typical from ACs is hampered by interobserver variability in mitotic counting and the limitations of morphological criteria in identifying aggressive cases. Our study reveals that different Ki-67 cut-offs can categorise LNETs similarly to current WHO criteria. Counting mitoses in PHH3+ areas does not improve diagnosis, while providing a similar prognostic value to the current criteria. With the advantage of being time efficient, automated assessment of these markers leads to similar conclusions. Lastly, state-of-the-art deep learning modelling does not uncover undisclosed morphological features with diagnostic value., Conclusions: This study suggests that the mitotic criteria can be complemented by manual or automated assessment of Ki-67 or PHH3 protein expression, but these markers do not significantly improve the prognostic value of the current classification, as the AC group remains highly unspecific for aggressive cases. Therefore, we may have exhausted the potential of morphological features in classifying and prognosticating LNETs. Our study suggests that it might be time to shift the research focus towards investigating molecular markers that could contribute to a more clinically relevant morpho-molecular classification., Competing Interests: Disclosure Where authors are identified as personnel of the International Agency for Research on Cancer/WHO, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/WHO. The rest of the authors declare no conflict of interest., (Copyright © 2024 World Health Organization. Published by Elsevier Ltd.. All rights reserved.)

Background: Checkpoint inhibitors have proven effectiveness in clinical trials for non-small cell lung cancer (NSCLC) patients, but if this is congruent with routine patient care is discussed. We present real-world experience with the PD1-inhibitor nivolumab in NSCLC., Patients and Methods: Patients with NSCLC were considered eligible for nivolumab treatment after one or more lines of chemotherapy, and when in reasonable performance status (PS) [Eastern Cooperative Oncology Group (ECOG) < 3]. Treatment was given according to guidelines in the two phase III studies, CA209017 and CA209057. Response evaluation was done according to Recist 1.1, and treatment given until unequivocal progression or intolerable toxicity., Results: Fifty-eight patients (30 females) commenced therapy in the period June-August 2015. Median age was 64.6 years (range 32.3-88.2). Twenty-four patients had squamous cell carcinoma and 32 adenocarcinoma, 38 had received two or more prior lines of therapy. Fourteen cases (24%) were in ECOG PS 2. After a medium observation time of 14.3 months, 13 (22%) are still in treatment. Median time to treatment failure (TTF) was 4.0 months, 34% were off treatment during the first two months. Median overall survival (OS) is 11.7 months. There was no difference in TTF or OS among patients with squamous versus non-squamous histology or between 1 versus >1 prior line of therapy. Four patients (7%) were off treatment due to toxicity, none were grade 4 or 5., Conclusion: Nivolumab treatment outside clinical trials seems to perform as expected.

We report an improved single-step synthesis to generate the membrane-permeant acetoxymethyl esters (AM-esters) of cGMP and three cGMP-analogues. These bioactivatable compounds were found to induce cell death in rat IPC-81 cells, a model system for acute myelocytic leukemia, in micromolar doses, while the corresponding non-modified cGMP-analogues were inactive.

Microinjection of cytochrome c induced apoptosis in all the cell types we tested (IPC-81, Swiss 3T3, Clone 8 fibroblasts, NRK, H295, Y1, HEK 293). The apoptotic phenotype induced by injected cytochrome c was characterized by externalization of phosphatidyl serine, cell detachment from substratum and from neighbor cells, and had the classic ultrastructural features of membrane budding, chromatin condensation and cell shrinkage. Depending on the cell type and concentration of cytochrome c, the induction of apoptosis was remarkably rapid. The development of apoptosis was prevented by the caspase inhibitor Z-VAD.fmk. Four of the cell types (Clone 8, Swiss 3T3, NRK, Y1) were transfected with bcl-2 and these all showed a markedly decreased sensitivity towards injected cytochrome c. Our data suggest that extramitochondrial cytochrome c is a general apoptogen in cells with a functioning caspase system. They also indicate that, in preventing apoptosis, Bcl-2 acts not only at the level of regulation of cytochrome c release from mitochondria, but can also interfere with caspase activation induced by cytochrome c microinjected directly into the cytoplasm.

The effect of interleukin 10 (IL-10) on proliferation and cytokine secretion by acute myelogenous leukemia (AML) blast cells was investigated in vitro. IL-10 inhibited spontaneous AML blast proliferation for a majority of patients, whereas in the presence of exogenous growth factors (granulocyte-stimulating factor, G-CSF; granulocyte-macrophage colony-stimulating factor, GM-CSF; interleukin 3) the IL-10 effect on blast proliferation showed a wide variation depending on the individual AML patient. IL-10 seemed to cause an irreversible inhibitory effect on AML blasts, as inhibition could also be demonstrated when IL-10 was present only during the initial preincubation of the leukemia cells. IL-10 also inhibited AML blast colony formation. However, independent of the effect on AML blast proliferation, IL-10 decrease cytokine secretion from AML blast cells for all patients, as demonstrated for IL-1 alpha, IL-1 beta, tumor necrosis factor-alpha, GM-CSF and interleukin 6. IL-10 did not inhibit development of apoptosis in AML blasts cultured in vitro. Expression of complement receptors and capability to adhere and internalize bacteria by AML blasts were not altered by IL-10.

Bacterial beta-galactosidase, coded by lacZ, is a widely used reporter for studies of transcriptional activity of eukaryotic promoters at the single cell level. Unfortunately, current detection methods, like X-gal cytochemistry, are slow, have suboptimal sensitivity, and are incompatible with cell survival. By a novel approach based on microinjection into cells of the fluorogenic substrate 5-chloromethylfluorescein di-beta-D-galactopyranoside lacZ gene expression was detected without affecting cell viability or proliferative capacity. The method was far more sensitive than the conventional X-gal cytochemistry in all cell systems tested (primary hepatocytes, fibroblasts, and glioma cells). Results were obtained within seconds to minutes after injection, and cells remained fluorescent for hours.