Abstract Background For the advancement of cancer research, the collection of tissue specimens from drug‐resistant tumors after targeted therapy is crucial. Although patients with lung cancer are often provided targeted therapy, post‐therapy specimens are not routinely collected due to the risks of collection, limiting the study of targeted therapy resistance mechanisms. Posthumous rapid tissue donation (RTD) is an expedient collection process that provides an opportunity to understand treatment‐resistant lung cancers. Methods Consent to participate in the thoracic RTD protocol was obtained during patient care. When death occurred, tumor and paired non‐tumor, cytology, and blood specimens were collected within 48 hours and preserved as formalin‐fixed and frozen specimens. Tissue sections were evaluated with hematoxylin and eosin staining and immunohistochemistry (IHC) against multiple biomarkers, including various programmed death ligand 1 (PD‐L1) clones. Next‐generation sequencing was performed on 13 specimens from 5 patients. Results Postmortem specimens (N = 180) were well preserved from 9 patients with lung cancer. PD‐L1 IHC revealed heterogeneity within and between tumors. An AGK‐BRAF fusion was newly identified in tumor from a donor with a known echinoderm microtubule‐associated protein‐like 4 to anaplastic lymphoma kinase ( EML4‐ALK ) fusion and history of anaplastic lymphoma kinase (ALK) inhibitor therapy. RNA expression analysis revealed a clonal genetic origin of metastatic cancer cells. Conclusions Post‐therapy specimens demonstrated PD‐L1 heterogeneity and an acyl glycerol kinase to B‐rapidly accelerated fibrosarcoma ( AGK‐BRAF ) fusion in a patient with an EML4‐ALK –positive lung adenocarcinoma as a potential resistance mechanism to ALK inhibitor therapy. Rapid tissue donation collection of postmortem tissue from lung cancer patients is a novel approach to cancer research that enables studies of molecular evolution and drug resistance.
Improvements offered by viability droplet digital PCR (v-ddPCR) include increased precision, specificity and decreased time to results making for an attractive alternative method to traditional plate count enumeration of probiotic products. A major hurdle faced in v-ddPCR, however, is distinguishing between live and dead cells. The objective of this study was to evaluate a combination of PMA and EMA (PE51) for viability treatment of freeze-dried probiotic powders. Lactobacillus acidophilus La-14 and Bifidobacterium animalis subsp. lactis Bi-07 were analyzed over a 2-log concentration gradient to investigate the efficiency of PE51 across genus and assay targets. Results suggest a need to optimize viability dye concentration based on the genera of the organism, but also the assay target, even when analyzing the same organism. When optimized for PE51 concentration, strain specific v-ddPCR assays for both La-14 and Bi-07 were demonstrated to agree with plate count enumeration results. In conclusion, while these v-ddPCR assays require highly specific optimization, they are better suited for the future of the probiotic industry and are suggested to be implemented in probiotic product testing.
Global data on the treatment rate with direct-acting antivirals (DAAs) for chronic hepatitis C (CHC) are sparse. We aimed to evaluate the CHC treatment rate and barriers to treatment in the DAA era.We searched PubMed, EMBASE and Cochrane from inception to 5 August 2021, for relevant articles. Patients treated with DAAs without interferon (IFN) therapy were categorized as IFN-free DAAs. Patients receiving DAA with IFN or unclear IFN status were categorized as DAA/IFN.We identified and analysed data from 146 studies (1 760 352 CHC patients). DAA/IFN treatment rate was 16.0% (95% CI: 9.9-23.3, 49 studies, 886 535 patients). IFN-free DAA treatment rate was 52.3% (95% CI: 46.2-58.4, 123 studies, 1 276 754 patients): 45.4% in North America, 64.2% in South America (1 study), 90.4% in Africa (most data from Egypt), 54.4% in Europe, 60.7% in Australia and 60.5% in Asia, (p < .0001); 49% with hepatitis B co-infection and 32.3% with hepatocellular carcinoma (HCC). Treatment was not a priority in 22.8% of patients in Europe and 16.7% in Australia, compared to only 4.8% in North America and 2.1% in Asia (p < .0001). Poor adherence to clinical follow-up was the cause of no treatment in 74.7% of patients in Australia, 37.0% in North America, 7.9% in Europe and 14.3% in Asia (p < .0001).Though a marked improvement from IFN/DAA, the treatment rate with IFN-free DAA remains suboptimal (52.3% overall, 32.3% in HCC patients). Non-adherence to clinical follow-up and lack of disease awareness were treatment barriers.
Abstract Background Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest. Results Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed. Conclusions We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them.
Probiotics are typically enumerated by agar plate counting (PC) techniques. PC has several limitations including poor specificity, high variability, inability to enumerate dead cells, viable but non-culturable cells and cells in complex matrices. Viability droplet digital polymerase chain reaction (v-ddPCR) is an emerging enumeration technique with improved specificity, precision, and the ability to enumerate cells in varying states of culturability or in complex matrices. Good correlation and agreement between v-ddPCR and PC is well documented, but not much research has been published on the comparison when enumerating freeze-dried (FD) probiotics during storage. In this study, v-ddPCR utilizing PE51 (PE51-ddPCR), a combination of propidium monoazide (PMA) and ethidium monoazide (EMA), was evaluated as alternative enumeration technique to PC on blends of four FD probiotic strains over the course of a 3-month storage study with accelerated conditions. When PMA and EMA are combined (PE51), this study demonstrates agreement (bias = 7.63e+9, LOA = 4.38e+10 to 5.9e+10) and association ( r = 0.762) between PC and v-ddPCR, at or above levels of an accepted alternative method. Additionally, v-ddPCR with individual dyes PMA and EMA provide insight into how they individually contribute to the viable counts obtained by PE51-ddPCR and provide a more specific physiological understanding of how probiotics cope with or experience damage during storage.
Simian virus 40 (SV40) is significantly associated with some human cancers. However, the frequency of tumor-associated virus detection differs by geographic regions, so it is important to understand the status of SV40 infections in different populations. Poliovaccines potentially containing live SV40 were used in well-documented nationwide vaccination programs in Hungary and the Czech Republic that are reported here. We analyzed serum samples from periodic surveillance programs in those two countries for antibodies to SV40 using a specific plaque reduction neutralization assay. The prevalence of antibodies was between 1.3 and 8.7% in Hungary and from 1.0 to 4.0% in the Czech Republic. Females had a higher rate of antibodies than males, reaching in certain age groups 15.6% in Hungary and 8.3% in the Czech Republic. Antibodies to SV40 were found in similar proportions in both countries among persons not directly exposed to poliovaccines and subjects vaccinated in the era of SV40-free vaccines. Complexities and limitations of current serological approaches to epidemiological studies of SV40 in humans are discussed. These data suggest that SV40 may be present in these populations and emphasize the importance of follow-up studies to determine the pathogenesis of infections by this emerging human agent.
