Autopsy findings in cancer patients infected with SARS-CoV-2 show a milder presentation of COVID-19 compared to non-cancer patients.

COVID-19, caused by SARS-CoV-2, manifests with differing severity across distinct patient subgroups, with outcomes influenced by underlying comorbidities such as cancer, which may cause functional and compositional alterations of the immune system during tumor progression. We aimed to investigate the association of SARS-CoV-2 infection and its complications with cancer in a large autopsy series and the role of COVID-19 in the fatal sequence leading to death. A total of 2641 adult autopsies were investigated, 539 of these were positive for SARS-CoV-2. Among the total number of patients analyzed, 829 had active cancer. Overall, the cohort included 100 patients who simultaneously had cancer and SARS-CoV-2 infection. The course of COVID-19 was less severe in cancer patients, including a significantly lower incidence of viral and bacterial pneumonia, occurring more frequently as a contributory disease or coexisting morbidity, or as SARS-CoV-2 positivity without viral disease. SARS-CoV-2 positivity was more frequent among non-metastatic than metastatic cancer cases, and in specific tumor types including hematologic malignancies. COVID-19 was more frequently found to be directly involved in the fatal sequence in patients undergoing active anticancer therapy, but less frequently in perioperative status, suggesting that the underlying malignancy and consequent surgery are more important factors leading to death perioperatively than viral disease. The course of COVID-19 in cancer patients was milder and balanced during the pandemic. This may be due to relative immunosuppressed status, and the fact that even early/mild viral infections can easily upset their condition, leading to death from their underlying cancer or its complications.

Dual spatially resolved transcriptomics for human host-pathogen colocalization studies in FFPE tissue sections.

Technologies to study localized host-pathogen interactions are urgently needed. Here, we present a spatial transcriptomics approach to simultaneously capture host and pathogen transcriptome-wide spatial gene expression information from human formalin-fixed paraffin-embedded (FFPE) tissue sections at a near single-cell resolution. We demonstrate this methodology in lung samples from COVID-19 patients and validate our spatial detection of SARS-CoV-2 against RNAScope and in situ sequencing. Host-pathogen colocalization analysis identified putative modulators of SARS-CoV-2 infection in human lung cells. Our approach provides new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes in FFPE samples.

Liver alterations and detection of SARS-CoV-2 RNA and proteins in COVID-19 autopsies.

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope®) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.

A COVID-19-association-dependent categorization of death causes in 100 autopsy cases.

From March through December 2020, 100 autopsies were performed (Semmelweis University, Budapest, Hungary), with chart review, of patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection demonstrated by real-time reverse-transcription polymerase chain reaction testing (mean age, 74.73 years, range 40-102 years; 50 males, mean age 71.96 years, and 50 females, mean age 77.5 years). Classified by the date of death, 21 cases were from the pandemic's "first wave" (March through July) and 79 from the "second wave" (August through December). Three mortality categories were defined by relevance of SARS-CoV-2 infection: (1) "strong" association (n=57), in which COVID-19 was primary responsible for death; (2) "contributive" association (n=27), in which a pre-existing condition independent of COVID-19 was primary responsible for death, albeit with substantial COVID-19 co-morbidity; (3) "weak" association (n=16), in which COVID-19 was minimally or not at all responsible for death. Distributions among categories differed between the first wave, in which the "contributive" association cases dominated (strong: 24%, contributive: 48%, weak: 28%), and the second wave, in which the "strong" association cases dominated (strong: 66%, contributive: 21%, weak: 13%). Charted co-morbidities included hypertension (85 %), cardiovascular diseases (71 %), diabetes (40 %), cerebrovascular diseases (31 %), chronic respiratory diseases (30 %), malignant tumors (20 %), renal diseases (19 %), diseases of the central nervous system (15 %), and liver diseases (6 %). Autopsy evaluation analyzed alterations on macroscopy as well as findings on microscopy of scanned and scored sections of formalin-fixed, paraffin-embedded tissue samples (50-80 blocks/case). Severity of histological abnormalities in the lung differed significantly between "strong" and "contributive" (p<0.0001) and between "strong" and "weak" categories (p<0.0001). Abnormalities included diffuse alveolar damage, macrophage infiltration, and vascular and alveolar fibrin aggregates (lung), with macro- and microvascular thrombi and thromboemboli (lung, kidney, liver). In conclusion, autopsies clarified in what extent COVID-19 was responsible for death, demonstrated the pathological background of clinical signs and symptoms, and identified organ alterations that led to the death. Clinicopathologic correlation, with conference discussions of severity of co-morbidities and of direct pathological signs of disease, permitted accurate categorization of cause of death and COVID-19 association as "strong," "contributive," or "weak." Lung involvement, with reduced ventilatory capacity, was the primary cause of death in the "strong" and "contributive" categories. Shifts in distribution among categories, with "strong" association between COVID-19 and death dominating in the second wave, may reflect improved clinical management of COVID-19 as expertise grew.

Changes of placental syndecan-1 expression in preeclampsia and HELLP syndrome.

Preeclampsia is characterized by maternal systemic anti-angiogenic and pro-inflammatory states. Syndecan-1 is a cell surface proteoglycan expressed by the syncytiotrophoblast, which plays an important role in angiogenesis and resolution of inflammation. Our aim was to examine placental syndecan-1 expression in preeclampsia with or without hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome. Placentas were obtained from women in the following groups: (1) late-onset preeclampsia (n = 8); (2) early-onset preeclampsia without (n = 7) and (3) with HELLP syndrome (n = 8); (4) preterm controls (n = 5); and (5) term controls (n = 9). Tissue microarrays (TMAs) were constructed from paraffin-embedded placentas. TMA slides were immunostained for syndecan-1 and evaluated using microscopy, virtual microscopy, and semi-automated image analysis. Maternal sera from patients with preeclampsia (n = 49) and controls (n = 32) were immunoassayed for syndecan-1. BeWo cells were treated with Forskolin or Latrunculin B or kept in ischemic conditions. SDC1 expression and syndecan-1 production were investigated with qRT-PCR, confocal microscopy, and immunoassays. Syndecan-1 was localized to the syncytiotrophoblast apical membrane in normal placentas. Syndecan-1 immunoscores were higher in late-onset preeclampsia (p = 0.0001) and early-onset preeclampsia with or without HELLP syndrome (p = 0.02 for both) than in controls. Maternal serum syndecan-1 concentration was lower in preeclampsia (median, 673 ng/ml; interquartile range, 459-1,161 ng/ml) than in controls (1,158 ng/ml; 622-1,480 ng/ml). SDC1 expression and syndecan-1 immunostainings in BeWo cells and syndecan-1 concentrations in supernatants increased during cell differentiation. Disruption of the actin cytoskeleton with Latrunculin B decreased syndecan-1 release, while ischemic conditions increased it. Syncytiotrophoblastic syndecan-1 expression depends on the differentiation of villous trophoblasts, and trophoblastic syndecan-1 release is decreased in preeclampsia and HELLP syndrome. This phenomenon may be related to the disturbed syncytiotrophoblastic cortical actin cytoskeleton and associated with maternal anti-angiogenic and pro-inflammatory states in these syndromes.

Placental protein 13 (PP13/galectin-13) undergoes lipid raft-associated subcellular redistribution in the syncytiotrophoblast in preterm preeclampsia and HELLP syndrome.

OBJECTIVE: To investigate placental protein 13 (PP13) localization in relation to cytoskeleton and lipid rafts in preeclampsia and HELLP syndrome. STUDY DESIGN: Placental cryosections from patients with preeclampsia and HELLP, and controls were stained for PP13, actin, PLAP (lipid raft marker), and CD71 (nonraft marker). BeWo cells exposed to stress conditions were stained for PP13 and actin. Protein localizations were investigated by confocal microscopy, PP13 concentrations by ELISA. RESULTS: PP13-actin colocalization was increased in syncytiotrophoblast juxtamembrane regions in term/preterm preeclampsia and HELLP. PP13-CD71 colocalization was decreased and PP13-PLAP proximity was increased in preterm but not term preeclampsia and HELLP. PP13-release from BeWo cells was inhibited by cytoskeleton disruption, and augmented by Ca2+-influx and ischemic stress. CONCLUSION: The actin cytoskeleton, probably in connection with lipid rafts, controls trophoblastic "nonclassical" PP13 export. PP13 is released from the syncytiotrophoblast in preterm preeclampsia and HELLP, mimicked in BeWo cells by ischemic stress, suggesting PP13 is a placental alarmin.

Leptin receptor (LEPR) SNP polymorphisms in HELLP syndrome patients determined by quantitative real-time PCR and melting curve analysis.

BACKGROUND: Several studies have shown overexpression of leptin in microarray experiments in pre-eclampsia (PE) and in hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. We decided to study four leptin receptor (LEPR) SNP polymorphisms in HELLP syndrome patients by using quantitative real-time PCR and melting curve analysis. METHODS: DNA was isolated from blood samples from 83 normotensive pregnant women and 75 HELLP syndrome patients. Four SNPs, LEPR c.326A>G (K109), LEPR c.668A>G (Q223R), LEPR c.1968G>C (K656N) and LEPR c.3024A>G (S1008) were determined by quantitative real-time PCR and melting curve analysis. Investigators were blinded to clinical outcomes. RESULTS: LEPR c.326A>G, LEPR c.668A>G, LEPR c.1968G>C and LEPR c.3024A>G allele, genotype and haplotype polymorphisms were not different in HELLP syndrome patients and normotensive healthy pregnants. There were strong linkage disequilibrium (LD) between loci c.326A>G and c.6687A>G (D' = 0.974), and c.668A>G and c.1968G>C (D' = 0.934), and c.326A>G and c.1968G>C (D' = 0.885), and c.1968G>C and c.3024A>G (D' = 1.0). However, linkages of c.3024A>G with c.668A>G (D' = 0.111) and c.326A>G (D' = 0.398) were weak. The Hardy-Weinberg equilibrium was observed for all polymorphisms. However the LEPR c.326A>G AG genotype was twice more frequent and the (AG AG GG AG) haplotype was three times more frequent in HELLP syndrome patients. The introduced quantitative real-time PCR combined with melting curve analysis is a fast and reliable method for the determination of LEPR SNPs. CONCLUSION: Although certain LEPR haplotypes are more frequent in HELLP syndrome, we conclude that there is no compelling evidence that the four studied LEPR SNP polymorphisms associated with the development of HELLP syndrome.

Leptin gene (TTTC)(n) microsatellite polymorphism in pre-eclampsia and HELLP syndrome.

BACKGROUND: Leptin plays an important role in energy homeostasis. There is polymorphism on the leptin (LEP) gene. Our aim was to compare the tetranucleotide repeat (TTTC)(n) polymorphism in the 3'-flanking region in the LEP gene on DNA samples from patients with pre-eclampsia (PE), hemolysis, elevated liver enzymes, and low platelet (HELLP) syndrome and healthy pregnant controls. METHODS: Blood samples were collected from healthy pregnant women (n=88), patients with PE (n=79) and HELLP (n=77) syndrome. Fluorescent PCR and DNA fragment analysis was performed from the isolated DNA for the detection of (TTTC) repeats. The electrophoretograms were evaluated and patients were assigned to two groups; class I low (<190 bp) or class II high (> or =190 bp) PCR fragments. RESULTS: We observed similar distributions of the class I and class II (TTTC) alleles in the groups studied (class I allele: healthy pregnant 58.5%; severe pre-eclamptic 58.3%; HELLP syndrome 52.6%). We detected a higher frequency of the II/II genotype in HELLP syndrome patients (32.4%) compared to healthy controls (22.7%). However, the difference was not statistically significant. CONCLUSIONS: In an ethnically homogenous population, the LEP gene (TTTC) microsatellite polymorphism in the 3'-flanking region does not show a significant difference in the allele and genotype distribution in healthy pregnant, pre-eclamptic and HELLP syndrome patients. Furthermore, we recommend a new classification of the class I and class II alleles based on the distribution of the (TTTC) microsatellites.

Association between tumor necrosis factor (TNF)-alpha G-308A gene polymorphism and preeclampsia complicated by severe fetal growth restriction.

BACKGROUND: Preeclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome are multifactorial disorders with genetic and environmental components. Given that the tumor necrosis factor (TNF)-alpha G-308A single nucleotide polymorphism (SNP) affects TNF-alpha gene transcription and that preeclampsia and HELLP syndrome are characterized by a shift towards a Th1-type maternal immune response with increased TNF-alpha production, the aim of the current study was to investigate whether this SNP is associated with preeclampsia and HELLP syndrome in a Caucasian population from Hungary. Additionally, we aimed to examine whether TNF-alpha G-308A polymorphism can influence the risk for fetal growth restriction in preeclamptic patients, which issue none of the earlier studies dealt with. METHODS: In a case-control study, we analyzed blood samples from 140 preeclamptic patients, 69 patients with HELLP syndrome and 144 normotensive, healthy pregnant women using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. We performed also a meta-analysis with our results and those of 8 previously published studies. RESULTS: There were no significant differences in the genotype and allele frequencies of the TNF-alpha G-308A polymorphism between preeclamptic patients and normotensive, healthy pregnant women. However, the mutant (TNF2 or A) allele occurred significantly more frequently in preeclamptic patients with IUGR than in those without IUGR (18.5% versus 7.1%, p=0.003). In addition, the frequency of the mutant allele carriers was significantly higher among preeclamptic patients with IUGR compared to those without IUGR (30.6% versus 12.8%, p=0.010). The mutant allele carriers were found to have an increased risk of severe IUGR-complicated preeclampsia, which was independent of maternal age, prepregnancy BMI and primiparity (odds ratio (OR): 2.89, 95% confidence interval (CI): 1.16-7.22, p=0.023; adjusted OR: 2.78, 95% CI: 1.04-7.45, p=0.042). Nevertheless, no significant differences were detected in the genotype and allele frequencies of the TNF-alpha G-308A polymorphism between patients with HELLP syndrome and control subjects. In the meta-analysis, no association was observed between this SNP and preeclampsia (summary OR: 0.956, 95% CI: 0.693-1.319). CONCLUSIONS: Although the meta-analysis demonstrated a lack of an overall association between TNF-alpha G-308A polymorphism and preeclampsia, our results suggest a role of this SNP in the risk of severe IUGR-complicated preeclampsia. However, further studies are required with a larger sample size to confirm our findings.

Vascular endothelial growth factor (VEGF) polymorphisms in HELLP syndrome patients determined by quantitative real-time PCR and melting curve analyses.

BACKGROUND: The vascular endothelial growth factor (VEGF) has a critical role in vasculogenesis and vascular permeability in several diseases including preeclampsia. There are at least 30 single nucleotide polymorphic (SNP) places on this gene. VEGF G+405C, C-2578A and C-460T SNPs are known to be related to VEGF production. VEGF polymorphisms were studied in preeclampsia, but not in HELLP syndrome. Therefore, we decided to determine the allele and genotype frequencies of VEGF G+405C, C-460T and C-2578A SNPs in healthy pregnant women and HELLP syndrome patients. METHODS: The authors introduced a quantitative real-time PCR method for the determination of the three VEGF SNPs. Blood samples were collected from 71 HELLP syndrome patients and 93 healthy controls. DNA was isolated by using silica adsorption method. The SNPs were determined by quantitative real-time PCR and melting curve analysis using LightCycler. RESULTS: There were significant differences in the allele and genotype frequencies of VEGF C-460T SNP between the two study groups. The T allele was present in 71.1% in the HELLP group, while in 53.8% in the controls (p=0.0014). The TT genotype occurred significantly more frequently in the HELLP group than in the control group (45.1% vs. 21.5%; p (for genotype frequencies)=0.0011). The TT genotype carriers had an increased risk of HELLP syndrome, which was independent of maternal age and primiparity (adjusted odds ratio (OR)=3.03, 95% confidence interval (CI)=1.51-6.08; p=0.002). Although the VEGF G+405C allele and genotype distributions did not differ significantly between the two groups, the CC genotype carriers were also found to have an increased risk for HELLP syndrome after adjustment for maternal age and primiparity (adjusted OR=3.67, 95% CI=1.05-12.75; p=0.041). The VEGF C-2578A SNP was not associated with HELLP syndrome. CONCLUSIONS: The quantitative real-time PCR combined with melting curve analyses is a fast and reliable method for the determination of VEGF SNPs. We found that the VEGF -460TT and +405CC genotype carriers have an increased risk of HELLP syndrome. As these two SNPs were previously observed to be related to production of the VEGF protein, we suppose that these VEGF polymorphisms -- interacting with other genetic and environmental factors - could play a role in the development of HELLP syndrome.

Early detection of decreased soluble HLA-G levels in the maternal circulation predicts the occurrence of preeclampsia and intrauterine growth retardation during further course of pregnancy.

PROBLEM: Soluble (s) HLA-G1/G5 molecules may potentially affect immune homeostasis during pregnancy. The aim of this study was to determine changes of sHLA-G1/G5 plasma levels throughout normal pregnancy and to assess its predictive value for the occurrence of characteristic gestation-associated diseases during further course of pregnancy. METHOD OF STUDY: sHLA-G1/G5 levels were estimated in plasma samples of 40 non-pregnant women, 291 women throughout normal pregnancy and 236 women affected by different complications. RESULTS: In comparison with non-pregnant women sHLA-G1/G5 levels strongly increased during the first trimenon and then decreased continuously toward term. Non-parametric discriminant analysis showed that women with significantly decreased sHLA-G1/G5 levels in the second trimenon had an increased risk of developing preeclampsia and/or intrauterine growth retardation (IUGR) during further course of pregnancy. However, in the third trimenon, sHLA-G1/G5 levels in affected women did not deviate significantly from those of non-affected women. Surprisingly, significantly increased sHLA-G1/G5 levels were detected in third trimenon women with uncontrollable preterm labor, but not in women with prolonged preterm rupture of fetal membranes. CONCLUSION: For the identification of women with an increased risk of IUGR and/or preeclampsia, measurement of sHLA-G1/G5 plasma levels may be a powerful new tool in prenatal diagnostics.

[Assessment of prognostic factors in common ovarian tumors of varying malignancy]. / Különbözô malignitású petefészek-daganattal kezelt betegek prognosztikai faktorainak vizsgálata.

OBJECTIVE: The outcome and prognosis of apparently similar cases of epithelial ovarian cancers with the same histology and stage is highly variable. The objective was to compare survival and prognostic factors of patients treated at the 1st Department of Obstetrics and Gynecology of Semmelweis University between 1993-2003 with the similar data of the 25th Annual Report on the Results of Treatment in Gynecological Cancer of FIGO. In addition, the aim was to assess the prognostic value of MMP activities and fibronectin concentration in ovarian tumour patients. METHODS: The 25th Annual Report of FIGO included 5694 patients with ovarian tumours from 32 countries diagnosed and treated between 1995 and 1998. Hungary did not participate in this report. Between 1993 and 2003, 180 patients with common epithelial ovarian tumours had been treated at the 1st Department of Obstetrics and Gynecology of Semmelweis University. Treatment and survival data derived from medical record review and from the Population Register Office. In order to compare different prognostic factors, a multivariate Cox proportional regression analysis was performed. The authors measured MMP activities in 33 surgically removed ovarian tumours, serum and ascites by applying zymographic technique. Fibronectin content was determined by Western blot analysis and quantitated by densitometry. RESULTS: The 5-year survival was 90.0% and 30.9% in the case of ovarian tumours with low malignant potential and of epithelial ovarian cancers, respectively. Multivariate analysis identified adverse prognostic factors including advanced age (>60 years) and stage, high grade and suboptimal operation with residual macroscopic disease and the presence of ascites. However, the histological type was not identified to be an adverse prognostic factor in this study. No correlation could be seen between the histology of the ovarian tumours and the elevation of MMP-2/9 activity. More interestingly, however, MMP-9 expression and fibronectin concentration were significantly elevated and the activated forms of both MMP-9 and MMP-2 were more frequent in ovarian cancer patients who developed recurrent disease. CONCLUSION: A great deal of effort should be devoted to identification of further prognostic factors to improve treatment of ovarian cancer. These prognostic factors might help to identify those ovarian cancer patients at the time of diagnosis whose disease will have unfavourable outcome. Our data support the notion that high expression of MMP-9 and fibronectin indicates poor prognosis of ovarian cancer patients.