Stillbirth due to SARS-CoV-2 placentitis without evidence of intrauterine transmission to fetus: association with maternal risk factors.

OBJECTIVES: To describe the placental pathology, fetal autopsy findings and clinical characteristics of pregnancies that resulted in stillbirth owing to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) placentitis, and to identify potential risk factors. METHODS: This was a prospective multicenter study of non-vaccinated pregnant women affected by coronavirus disease 2019 (COVID-19) in Greece from April 2020 to August 2021. A total of 165 placentas were examined histologically and six cases of stillbirth associated with SARS-CoV-2 placentitis were retrieved. Complete fetal autopsy was performed in three of these cases. Gross, histopathological, immunohistochemical, molecular and electron microscopy examinations were carried out in the stillbirth placentas and fetal organs. The histological findings of cases with SARS-CoV-2 placentitis were compared with those in 159 cases with maternal COVID-19 which resulted in a live birth. Regression analysis was used to identify predisposing risk factors for SARS-CoV-2 placentitis. RESULTS: The placentas of all six stillborn cases showed severe and extensive histological changes typical of SARS-CoV-2 placentitis, characterized by a combination of marked intervillositis with a mixed inflammatory infiltrate and massive perivillous fibrinoid deposition with trophoblast damage, associated with intensely positive immunostaining for SARS-CoV-2 spike protein, the presence of virions on electron microscopy and positive reverse-transcription polymerase chain reaction test of placental tissues. The histological lesions obliterated over 75% of the maternal intervillous space, accounting for intrauterine fetal death. Similar histological lesions affecting less than 25% of the placenta were observed in seven liveborn neonates, while the remaining 152 placentas of COVID-19-affected pregnancies with a live birth did not show these findings. Complete fetal autopsy showed evidence of an asphyctic mode of death without evidence of viral transmission to the fetus. The mothers had mild clinical symptoms or were asymptomatic, and the interval between maternal COVID-19 diagnosis and fetal death ranged from 3 to 15 days. Statistically significant predisposing factors for SARS-CoV-2 placentitis included thrombophilia and prenatally diagnosed fetal growth restriction (FGR). Multiple sclerosis was seen in one case. CONCLUSIONS: SARS-CoV-2 placentitis occurred uncommonly in COVID-19-affected pregnancies of non-vaccinated mothers and, when extensive, caused fetal demise, with no evidence of transplacental fetal infection. Thrombophilia and prenatally detected FGR emerged as independent predisposing factors for the potentially lethal SARS-CoV-2 placentitis. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

Age-associated inflammation connects RAS-induced senescence to stem cell dysfunction and epidermal malignancy.

Aging is the single biggest risk factor for malignant transformation. Among the most common age-associated malignancies are non-melanoma skin cancers, comprising the most common types of human cancer. Here we show that mutant H-Ras activation in mouse epidermis, a frequent event in cutaneous squamous cell carcinoma (SCC), elicits a differential outcome in aged versus young mice. Whereas H-Ras activation in the young skin results in hyperplasia that is mainly accompanied by rapid hair growth, H-Ras activation in the aged skin results in more dysplasia and gradual progression to in situ SCC. Progression is associated with increased inflammation, pronounced accumulation of immune cells including T cells, macrophages and mast cells as well as excessive cell senescence. We found not only an age-dependent increase in expression of several pro-inflammatory mediators, but also activation of a strong anti-inflammatory response involving enhanced IL4/IL10 expression and immune skewing toward a Th2 response. In addition, we observed an age-dependent increase in the expression of Pdl1, encoding an immune suppressive ligand that promotes cancer immune evasion. Moreover, upon switching off oncogenic H-Ras activity, young but not aged skin regenerates successfully, suggesting a failure of the aged epidermal stem cells to repair damaged tissue. Our findings support an age-dependent link between accumulation of senescent cells, immune infiltration and cancer progression, which may contribute to the increased cancer risk associated with old age.

Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation.

Oncogene-induced reactive oxygen species (ROS) have been proposed to be signaling molecules that mediate proliferative cues. However, ROS may also cause DNA damage and proliferative arrest. How these apparently opposite roles can be reconciled, especially in the context of oncogene-induced cellular senescence, which is associated both with aberrant mitogenic signaling and DNA damage response (DDR)-mediated arrest, is unclear. Here, we show that ROS are indeed mitogenic signaling molecules that fuel oncogene-driven aberrant cell proliferation. However, by their very same ability to mediate cell hyperproliferation, ROS eventually cause DDR activation. We also show that oncogenic Ras-induced ROS are produced in a Rac1 and NADPH oxidase (Nox4)-dependent manner. In addition, we show that Ras-induced ROS can be detected and modulated in a living transparent animal: the zebrafish. Finally, in cancer we show that Nox4 is increased in both human tumors and a mouse model of pancreatic cancer and specific Nox4 small-molecule inhibitors act synergistically with existing chemotherapic agents.

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues.

There is shortage of extensive clinicopathologic studies of cellular senescence because the most reliable senescence biomarker, the detection of Senescence-Associated-beta-galactosidase activity (SA-ß-gal), is inapplicable in archival material and requires snap-frozen tissues. We validated the histochemical Sudan-Black-B (SBB) specific stain of lipofuscin, an aggregate of oxidized proteins, lipids and metals, known to accumulate in aged tissues, as an additional reliable approach to detect senescent cells independently of sample preparation. We analyzed cellular systems in which senescence was triggered by replicative exhaustion or stressful stimuli, conditional knock-in mice producing precancerous lesions exhibiting senescence, and human preneoplastic lesions known to contain senescent cells. In the above settings we demonstrated co-localization of lipofuscin and SA-ß-gal in senescent cells in vitro and in vivo (cryo-preserved tissue), strongly supporting the candidacy of lipofuscin for a biomarker of cellular senescence. Furthermore, cryo-preserved tissues positive for SA-ß-gal were formalin-fixed, paraffin-embedded, and stained with SBB. The corresponding SA-ß-gal positive tissue areas stained specifically for lipofuscin by SBB, whereas tissues negative for SA-ß-gal were lipofuscin negative, validating the sensitivity and specificity of the SBB staining to visualize senescent cells in archival material. The latter unique property of SBB could be exploited in research on widely available retrospective tissue material.

The tumor suppressor gene ARF as a sensor of oxidative stress.

Oxidative stress as a result of either exogenous stimuli or cellular metabolism affects several cellular processes such as proliferation, apoptosis, cell death and senescence. Consequently, it is implicated in the pathogenesis of various human diseases like cancer, diabetes mellitus, atherosclerosis, neurodegenerative diseases and aging. Oxidative stress is implicated in carcinogenesis either by directly provoking DNA damage or through the regulation of intracellular signaling cascades. In both cases the cellular response to oxidative stress is determined by the cellular context. ARF, the alternative protein product of the CDKN2A locus has been recently recognized as a novel sensor of oxidative stress, in a ß-catenin and Hsp70-mediated manner. Since, improved understanding of cellular responses to oxidative stress may facilitate the design of novel antineoplastic regimens, we herein review the mechanisms by which oxidative stress promotes carcinogenesis, focusing on the role of ARF as a sensor of oxidative stress.

Targeting DNA damage and repair: embracing the pharmacological era for successful cancer therapy.

DNA is under constant assault from genotoxic agents which creates different kinds of DNA damage. The precise replication of the genome and the continuous surveillance of its integrity are critical for survival and the avoidance of carcinogenesis. Cells have evolved an arsenal of repair pathways and cell cycle checkpoints to detect and repair DNA damage. When repair fails, typically cell cycle progression is halted and apoptosis is initiated. Here, we review the different sources and types of DNA damage including DNA replication stress and oxidative stress, the repair pathways that cells utilize to repair damaged DNA, and discuss their biological significance, especially with reference to cancer induction and cancer therapy. We also describe the main methodologies currently used for the detection of DNA damage with their strengths and limitations. We conclude with an outline as to how this information can be used to identify novel pharmacological targets for DNA repair pathways or enhancers of DNA damage to develop improved treatment strategies that will benefit cancer patients.

Update of the cancer-associated molecular mechanisms in oral lichen planus, a disease with possible premalignant nature.

Oral lichen planus (OLP) is a relatively common inflammatory disease. Several reports of oral squamous cell carcinomas (OSCC) developing in the ground of previous OLP lesions exist in the current medical literature. Hence, there is a debate concerning the possible premalignant nature of OLP. The studies that examined the malignant potential of OLP for many years were mainly observational and were seeking to detect the percentage of OLP patients that developed OSCC. The results of these studies varied significantly with reported percents of malignant transformation of OLP ranging from 0 to 12.5%. In recent years the number of OLP studies that investigate molecular biomarkers identified in cancer is on the rise. This article is an update of the molecular pathways identified in OLP that could be suggestive of a malignant potential of this condition.

Oncogene-induced replication stress preferentially targets common fragile sites in preneoplastic lesions. A genome-wide study.

Common fragile sites (CFSs) are regions of the genome prone to breakage by replication inhibitors (extrinsic replication stress). Recently, we and others observed that oncogene-induced replication stress (RS) induces DNA damage from the earliest stages of cancer. Our aim was to perform a genome-wide analysis in precancerous and cancerous experimental models to examine whether allelic imbalance occurs within CFSs. Subsequently, CFSs sequence characteristics were assessed. We used a growth-factor-induced human skin hyperplasia and a H-ras-induced mouse hyperplastic urothelium as preneoplastic models, along with an inducible U2OS-CDT1(Tet-ON) cancer cell line model, all bearing established oncogene-induced RS stimuli. Human DNA was analysed with Affymetrix SNP microarrays, while mouse DNA was analysed with Nimblegen array CGH. We studied 56 aphidicolin-type CFSs and 1914 regions of control, nonfragile DNA. Our theoretical in silico analysis spanned 2.16 billion nonoverlapping bases on human chromosomes 1-22. Our results provide direct experimental evidence indicating that genomic alterations were more common within CFSs in epidermal and urothelial preneoplastic lesions as well as in cancer. CFSs were on average less flexible than nonfragile regions, contained more guanine-cytosine (GC) and Alu sequences. Importantly, regions with loss-of-heterozygosity were also less flexible and had a higher Alu percentage.

E2F-1 overexpression correlates with decreased proliferation and better prognosis in adenocarcinomas of Barrett oesophagus.

BACKGROUND: E2F-1 expression is positively associated with tumour growth in oesophageal squamous-cell carcinomas (OSCC), while it exhibits oncosuppressive features in colonic adenocarcinomas (AC). To date there are no data regarding E2F-1 expression and its relationship with tumour kinetics (proliferation, apoptosis) in adenocarcinomas that develop on Barrett oesophagus. AIM: As oesophageal adenocarcinomas occur almost exclusively in the metaplastic Barrett epithelium and the opposing E2F-1 behaviour seems to be cell and tissue-type dependent, we examined the manner in which E2F-1 acts in ACs of Barrett oesophagus. METHODS: We estimated the immunohistochemical expression of E2F-1, Ki-67, caspase-3 and p53 immunohistochemical status in 35 Barrett oesophagus ACs. RESULTS: E2F-1 immunopositivity correlated inversely with Ki-67, by semi-serial section and statistical analysis (p = 0.023, Spearman correlation). Semi-serial section analysis revealed a direct association between E2F-1 and caspase-3 staining. No correlation was found with p53 status. Cases with higher E2F-1 immunoexpression exhibited longer survival (p = 0.047, Cox-regression). CONCLUSIONS: E2F-1 expression was negatively related to tumour proliferation in ACs of Barrett oesophagus. Additionally, E2F-1 immunohistochemical status correlated positively with patient survival. These findings are opposite from those seen in OSCCs, suggesting that the tumour-suppressing E2F-1 behaviour in oesophageal adenocarcinomas is possibly due to the intestinal-type nature of the metaplastic Barrett mucosa.

Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer.

ZBTB7A (Pokemon) is a member of the POK family of transcriptional repressors. Its main function is the suppression of the p14ARF tumour suppressor gene. Although ZBTB7A expression has been found to be increased in various types of lymphoma, there are no reports dealing with its expression in solid tumours. Given that p14(ARF) inhibits MDM2, the main negative regulator of p53, we hypothesized that overexpression of ZBTB7A could lead indirectly to p53 inactivation. To this end, we examined the status of ZBTB7A and its relationship with tumour kinetics (proliferation and apoptosis) and nodal members of the p53 network in a panel of 83 non-small cell lung carcinomas (NSCLCs). We observed, in the majority of the samples, prominent expression of ZBTB7A in the cancerous areas compared to negligible presence in the adjacent normal tissue elements. Gene amplification (two- to five-fold) was found in 27.7% of the cases, denoting its significance as a mechanism driving ZBTB7A overproduction in NSCLCs. In the remaining non-amplified group of carcinomas, analysis of the mRNA and protein expression patterns suggested that deregulation at the transcriptional and post-translational level accounts for ZBTB7A overexpression. Proliferation was associated with ZBTB7A expression (p = 0.033) but not apoptosis. The association with proliferation was reflected in the positive correlation between ZBTB7A expression and tumour size (p = 0.018). The overexpression of ZBTB7A in both p53 mutant and p53 wild-type cases, implies either a synergistic effect or that ZBTB7A exerts its oncogenic properties independently of the p14(ARF)-MDM2-p53 axis. The concomitant expression of ZBTB7A with p14(ARF) (p = 0.039), instead of the anticipated inverse relation, supports the latter notion. In conclusion, regardless of the pathway followed, the distinct expression of ZBTB7A in cancerous areas and the association with proliferation and tumour size pinpoints a role for this novel cell cycle regulator in the pathogenesis of lung cancer.

The prophylactic effect of L-arginine in acute ischaemic colitis in a rat model of ischaemia/reperfusion injury.

BACKGROUND/AIMS: The decreased synthesis of nitric oxide (NO) during ischaemia/reperfusion (I/R) has been implicated as the major underlying mechanism for the pathogenesis of acute ischaemic colitis (A.I.C.). The aim of this study was to investigate the prophylactic effect of L-arginine, a NO donor, on tissue injury during intestinal I/R, and compare its efficacy with that of exogenous vasodilators (molsidomine) and inert nitrogen-containing molecules (casein). MATERIAL AND METHODS: One hundred forty four Wistar rats underwent occlusion of the superior mesentery artery for 30, 60 and 90 min for induction of intestinal ischaemia, followed by 90 min of reperfusion. The rats were randomly assigned to receive L-arginine, molsidomine, or casein hydrolysate. In all groups, apart of the histological study, we determined the levels of serum malondialdehyde (MDA), a reliable marker indicating the degree of the tissue damage after intestinal I/R. RESULTS: Serum MDA levels were significantly lower in the L-arginine group compared to the untreated animals or those that had received molsidomine or casein, after a period of ischaemia of 90 minutes (p < 0.0005), as well as after a period of ischaemia of 60 or 90 minutes followed by a 90 minutes reperfusion (p = 0.011, and p < 0.0005, respectively). In addition, lesser histopathological damage was noted after the use of L-arginine compared to that caused by the administration of molsidomine and casein. CONCLUSION: These findings support a prophylactic effect of L-arginine in experimentally induced intestinal ischaemia. In short, L-arginine attenuates the degree of tissue damage in intestinal ischaemia and promotes healing of intestinal mucosa.

Advances in the biology of oral cancer.

The incidence of oral cancer remains high and is associated with many deaths in both Western and Asian countries. Several risk factors for the development of oral cancer are now well known, including smoking, drinking and consumption of smokeless tobacco products. Genetic predisposition to oral cancer has been found in certain cases but its components are not yet entirely clear. In accordance with the multi-step theory of carcinogenesis, the natural history of oral cancer seems to gradually evolve through transitional precursor lesions from normal epithelium to a full-blown metastatic phenotype. A number of genomic lesions accompany this transformation and a wealth of related results has appeared in recent literature and is being summarized here. Furthermore, several key genes have been implicated, especially well-known tumor suppressors like the cyclin-dependent kinase inhibitors, TP53 and RB1 and oncogenes like the cyclin family, EGFR and ras. Viral infections, particularly with oncogenic HPV subtypes and EBV, can have a tumorigenic effect on oral epithelia and their role is discussed, along with potential therapeutic interventions. A brief explanatory theoretical model of oral carcinogenesis is provided and potential avenues for further research are highlighted.

Evaluation of claspin as a proliferation marker in human cancer and normal tissues.

Claspin is a nuclear protein involved in DNA replication and the DNA damage response. Its structural and functional properties suggest that it may represent a potentially useful proliferation marker. To this end, a monoclonal antibody was generated and the expression of claspin was investigated in normal fibroblasts and various cancer cell lines, as well as in tumour and normal tissues from patients with primary epithelial carcinomas. Immunoblotting analysis confirmed the specificity of the antibody, while immunohistochemistry demonstrated its applicability in archival material. In normal cells and tissues, claspin expression was weak, whereas increased levels were observed in cancer cell lines and tumour specimens. Claspin staining correlated strongly with Ki67 staining in both normal (p < 0.001) and tumour tissues (p < 0.001). However, the labelling index (LI) of claspin was consistently lower than that of Ki67, suggesting that claspin expression may be limited to a narrower part of the cell cycle. Co-localization assays with cyclin A and cell synchronization experiments indicated that claspin expression coincides with the S phase. Interestingly, the relative increase of the claspin LI in tumour samples compared with normal tissues was significantly higher (14-fold) than that of the Ki67 LI (five-fold), suggesting that claspin may be a more sensitive marker of aberrant proliferation.

Heregulin, cysteine rich-61 and matrix metalloproteinase 9 expression in human carotid atherosclerotic plaques: relationship with clinical data.

OBJECTIVES: Heregulins (HRGs) are known to induce expression of angiogenic factors such as cysteine rich-61 (CYR61) and collectively to promote neoangiogenesis. Along with extracellular matrix remodelling, mediated by matrix metalloproteinases (MMPs), these factors are important in atherogenesis. The aim of the present study was to investigate HRG, CYR61 and MMP-9 expression and their relationship with clinical and histopathological findings in carotid occlusive disease. MATERIALS AND METHODS: Specimens of human carotid atherosclerotic plaque (n=90) were obtained by endarterectomy. Expression of HRG, CYR61 and MMP-9 was assessed by immunohistochemical and Western blot analysis. Associations between protein expression and degree of carotid stenosis, presence of symptoms, presence of an infarct in CT scan and carotid plaque histopathology were investigated. RESULTS: An increase in HRG, CYR61 and MMP-9 expression was found, particularly in neovascularized regions of the plaques. High HRG expression was associated with the degree of carotid stenosis (p=0.028) and plaque histopathology (p=0.002). More than half of specimens from plaques with >90% stenosis had intense expression of CYR61 (p=0.047). Increased expression of MMP-9 was associated with degree of stenosis and presence of cerebral infarct on CT scan (p=0.05). CONCLUSION: HRG, CYR61 and MMP-9 are highly expressed in human atherosclerotic carotid plaques. The association with the degree of stenosis and/or plaque histopathology implies an involvement in lesion progression.

Centrosome abnormalities are frequently observed in non-small-cell lung cancer and are associated with aneuploidy and cyclin E overexpression.

Centrosome abnormalities are observed in human cancers and have been associated with aneuploidy, a driving force in tumour progression. However, the exact pathways that tend to cause centrosome abnormalities have not been fully elucidated in human tumours. Using a series of 68 non-small-cell lung carcinomas and an array of in vitro experiments, the relationship between centrosome abnormalities, aneuploidy, and the status of key G1 to S-phase transition cell-cycle molecules, involved in the regulation of centrosome duplication, was investigated. Centrosome amplification and structural abnormalities were common (53%), were strongly related to aneuploidy, and, surprisingly, were even seen in adjacent hyperplastic regions, suggesting the possibility that these are early lesions in lung carcinogenesis. Cyclin E and E2F1 overexpression, but not p53 mutation, was observed to correlate with centrosome abnormalities in vivo (p = 0.029 and p = 0.015, respectively). This was further strengthened by the observation that cyclin E was specifically present in the nucleus and/or cytoplasm of the cells that contained centrosome aberrations. The cytoplasmic cyclin E signal may be attributed, in part, to the presence of truncated low-molecular-weight isoforms of cyclin E. In order to isolate the effect of cyclin E on the appearance of centrosome abnormalities, a U2OS tetracycline-repressible cyclin E cell line that has a normal centrosome profile by default was used. With this system, it was confirmed in vitro that persistent cyclin E overexpression is sufficient to cause the appearance of centrosome abnormalities.

Involvement of E2F transcription factor family in cancer.

The E2F family of transcription factors is a central modulator of important cellular events, including cell cycle progression, apoptosis and DNA damage response. The role of E2F family members in various human malignancies is yet unclear and may provide vital clues to the diagnosis, prognosis and therapy of cancer patients. In this review we provide a brief but concise overview of E2F function and its putative role in the most common human tumour types.