Post-mortem studies can provide important information for understanding new diseases and small autopsy case series have already reported different findings in COVID-19 patients.
We evaluated whether ...some specific post-mortem features are observed in these patients and if these changes are related to the presence of the virus in different organs. Complete macroscopic and microscopic autopsies were performed on different organs in 17 COVID-19 non-survivors. Presence of SARS-CoV-2 was evaluated with immunohistochemistry (IHC) in lung samples and with real-time reverse-transcription polymerase chain reaction (RT-PCR) test in the lung and other organs.
Pulmonary findings revealed early-stage diffuse alveolar damage (DAD) in 15 out of 17 patients and microthrombi in small lung arteries in 11 patients. Late-stage DAD, atypical pneumocytes, and/or acute pneumonia were also observed. Four lung infarcts, two acute myocardial infarctions, and one ischemic enteritis were observed. There was no evidence of myocarditis, hepatitis, or encephalitis. Kidney evaluation revealed the presence of hemosiderin in tubules or pigmented casts in most patients. Spongiosis and vascular congestion were the most frequently encountered brain lesions. No specific SARS-CoV-2 lesions were observed in any organ. IHC revealed positive cells with a heterogeneous distribution in the lungs of 11 of the 17 (65%) patients; RT-PCR yielded a wide distribution of SARS-CoV-2 in different tissues, with 8 patients showing viral presence in all tested organs (i.e., lung, heart, spleen, liver, colon, kidney, and brain).
In conclusion, autopsies revealed a great heterogeneity of COVID-19-associated organ injury and the remarkable absence of any specific viral lesions, even when RT-PCR identified the presence of the virus in many organs.
Twist1 promotes epithelial-to-mesenchymal transition (EMT), invasion, metastasis, and cancer stem cell (CSC) properties. However, it remains unclear whether Twist1 is also required for tumor ...initiation and whether Twist1-induced cancer stemness and EMT are functionally linked. Using a conditional deletion of Twist1 at different stages of skin carcinogenesis, we show that Twist1 is required for skin tumor initiation and progression in a gene-dosage-dependent manner. Moreover, conditional ablation of Twist1 in benign tumors leads to increased apoptosis, reduced cell proliferation, and defective tumor maintenance and propagation independently of its EMT-inducing abilities. Concomitant deletion of Twist1 and p53 rescues the apoptotic response, but not the cell proliferation and propagation defects. These results reveal that Twist1 is required for tumor initiation and maintenance in a p53-dependent and -independent manner. Importantly, our findings also indicate that tumor stemness and EMT can be regulated by distinct mechanisms.
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•Twist1 is required for skin tumor initiation•Twist1 controls tumor initiation and progression in a gene-dosage-dependent manner•Twist1 controls apoptosis and propagation in a p53-dependent and -independent manner•Twist1 controls tumor maintenance and stemness independently of its EMT function
Twist1 promotes tumor stemness and EMT. It remains unclear whether Twist1 also controls tumor initiation and whether the different Twist1 functions are linked. Using Twist1 deletion at different stages of skin tumorigenesis, Beck et al. show that Twist1 is required for tumor initiation, stemness, and progression in a gene-dosage-dependent manner.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Accumulating evidence suggests that extracellular galectin-1 and galectin-3 promote angiogenesis. Increased expression of galectin-1 and/or galectin-3 has been reported to be associated with tumour ...progression. Thus, it is critical to identify their influence on angiogenesis.
We examined the individual and combined effects of galectin-1 and galectin-3 on endothelial cell (EC) growth and tube formation using two EC lines, EA.hy926 and HUVEC. The activation of vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) was determined by ELISA and Western blots. We evaluated the VEGFR1 and VEGFR2 levels in endosomes by proximity ligation assay.
We observed different responses to exogenous galectins depending on the EC line. An enhanced effect on EA.hy926 cell growth and tube formation was observed when both galectins were added together. Focusing on this enhanced effect, we observed that together galectins induced the phosphorylation of both VEGFR1 and VEGFR2, whereas galectin-1 and -3 alone induced VEGFR2 phosphorylation only. In the same way, the addition of a blocking VEGFR1 antibody completely abolished the increase in tube formation induced by the combined addition of both galectins. In contrast, the addition of a blocking VEGFR2 antibody only partially inhibited this effect. Finally, the addition of both galectins induced a decrease in the VEGFR1 and VEGFR2 endocytic pools, with a significantly enhanced effect on the VEGFR1 endocytic pool. These results suggest that the combined action of galectin-1 and galectin-3 has an enhanced effect on angiogenesis via VEGFR1 activation, which could be related to a decrease in receptor endocytosis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recently, Next Generation Sequencing (NGS) has begun to supplant other technologies for gene mutation testing that is now required for targeted therapies. However, transfer of NGS technology to ...clinical daily practice requires validation.
We validated the Ion Torrent AmpliSeq Colon and Lung cancer panel interrogating 1850 hotspots in 22 genes using the Ion Torrent Personal Genome Machine. First, we used commercial reference standards that carry mutations at defined allelic frequency (AF). Then, 51 colorectal adenocarcinomas (CRC) and 39 non small cell lung carcinomas (NSCLC) were retrospectively analyzed.
Sensitivity and accuracy for detecting variants at an AF >4% was 100% for commercial reference standards. Among the 90 cases, 89 (98.9%) were successfully sequenced. Among the 86 samples for which NGS and the reference test were both informative, 83 showed concordant results between NGS and the reference test; i.e. KRAS and BRAF for CRC and EGFR for NSCLC, with the 3 discordant cases each characterized by an AF <10%.
Overall, the AmpliSeq colon/lung cancer panel was specific and sensitive for mutation analysis of gene panels and can be incorporated into clinical daily practice.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The tumour micro-environment (TME) plays a crucial role in the onset and progression of prostate cancer (PCa). Here we studied the potential of a selected panel of TME-markers to predict clinical ...recurrence (CLR) in PCa. Patient cohorts were matched for the presence or absence of CLR 5 years post-prostatectomy. Tissue micro-arrays (TMA) were composed with both prostate non-tumour (PNT) and PCa tissue and subsequently processed for immunohistochemistry (IHC). The IHC panel included markers for cancer activated fibroblasts (CAFs), blood vessels and steroid hormone receptors ((SHR): androgen receptor (AR), progesterone receptor (PR) and estrogen receptor (ER)). Stained slides were digitalised, selectively annotated and analysed for percentage of marker expression with standardized and validated image analysis algorithms. A univariable analysis identified several TME markers with significant impact on CR: expression of CD31 (vascular marker) in PNT stroma, expression of alpha smooth muscle actin (αSMA) in PCa stroma, and PR expression ratio between PCa stroma and PNT stroma. A multivariable model, which included CD31 expression (vascular marker) in PNT stroma and PR expression ratio between PCa stroma and PNT stroma, could significantly stratify patients for CLR, with the identification of a low risk and high-risk subgroup. If validated and confirmed in an independent prospective series, this subgroup might have clinical potential for PCa patient stratification.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
Fibroblast activation protein-α (FAPα) is a marker of activated fibroblasts that can be selectively targeted by an inhibitor (FAPI) and visualised by PET/CT imaging. We evaluated ...whether the measurement of FAPα in bronchoalveolar lavage fluids (BALF) and the uptake of FAPI by PET/CT could be used as biomarkers of fibrogenesis.
Methods
The dynamics of lung uptake of
18
F-labeled FAPI (
18
FFAPI-74) was assessed in the bleomycin mouse model at various time points and using different concentrations of bleomycin by PET/CT. FAPα was measured in BALFs from these bleomycin-treated and control mice. FAPα levels were also assessed in BALFs from controls and patients with idiopathic pulmonary fibrosis (IPF).
Results
Bleomycin-treated mice presented a significantly higher uptake of
18
FFAPI-74 during lung fibrinogenesis (days 10 and 16 after instillation) compared to control mice. No significant difference was observed at initial inflammatory phase (3 days) and when fibrosis was already established (28 days).
18
FFAPI-74 tracer was unable to show a dose-response to bleomycin treatment. On the other hand, BALF FAPα levels were steeply higher in bleomycin-treated mice at day 10 and a significant dose-response effect was observed. Moreover, FAPα levels were strongly correlated with lung fibrosis as measured by the modified Aschroft histological analysis, hydroxyproline and the percentage of weight loss. Importantly, higher levels of FAPα were observed in IPF patients where the disease was progressing as compared to stable patients and controls. Moreover, patients with FAPα BALF levels higher than 192.5 pg/mL presented a higher risk of progression, transplantation or death compared to patients with lower levels.
Conclusions
Our preclinical data highlight a specific increase of
18
FFAPI-74 lung uptake during the fibrotic phase of the bleomycin murine model. The measurement of FAPα in BALF appears to be a promising marker of the fibrotic activity in preclinical models of lung fibrosis and in IPF patients. Further studies are required to confirm the role of FAPα in BALF as biomarker of IPF activity and assess the relationship between FAPα levels in BALF and
18
FFAPI-74 uptake on PET/CT in patients with fibrotic lung disease.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pancreatic ductal adenocarcinoma (PDAC) is reported to be amongst the cancers with the lowest survival rate at 5 years. In the present study we aimed to validate a targeted next-generation sequencing ...(tNGS) panel to use in clinical routine, investigating genes important for PDAC diagnostic, prognostic and potential theragnostic aspect. In this NGS panel we also designed target regions to inquire about loss of heterozygosity (LOH) of chromosome 18 that has been described to be possibly linked to a worse disease progression. Copy number alteration has also been explored for a subset of genes. The last two methods are not commonly used for routine diagnostic with tNGS panels and we investigated their possible contribution to better characterize PDAC. A series of 140 formalin-fixed paraffin-embedded (FFPE) PDAC samples from 140 patients was characterized using this panel. Ninety-two % of patients showed alterations in at least one of the investigated genes (most frequent KRAS, TP53, SMAD4, CDKN2A and RNF43). Regarding LOH evaluation, we were able to detect chr18 LOH starting at 20% cell tumor percentage. The presence of LOH on chr18 is associated with a worse disease- and metastasis-free survival, in uni- and multivariate analyses. The present study validates the use of a tNGS panel for PDAC characterization, also evaluating chr18 LOH status for prognostic stratification.
•PDAC molecular profiling is used for diagnostic, prognostic and theranostic purposes.•Targeted NGS (tNGS) is an effective approach in daily practice for TAT and cost.•This study focuses on the validation of a tNGS panel integrating evaluation of LOH status.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Right ventricular (RV) dysfunction remains a major problem after heart transplantation and may be associated with brain death (BD) in a donor. A calcineurin inhibitor tacrolimus was recently found to ...have beneficial effects on heart function. Here, we examined whether tacrolimus might prevent BD-induced RV dysfunction and the associated pathobiological changes.
After randomized tacrolimus (
= 8; 0.05 mg·kg
·day
) or placebo (
= 9) pretreatment, pigs were assigned to a BD procedure and hemodynamically investigated 1, 3, 5, and 7 h after the Cushing reflex. After euthanasia, myocardial tissue was sampled for pathobiological evaluation. Seven pigs were used as controls.
Calcineurin inhibition prevented increases in pulmonary vascular resistance and RV-arterial decoupling induced by BD. BD was associated with an increased RV pro-apoptotic Bax-to-Bcl2 ratio and RV and LV apoptotic rates, which were prevented by tacrolimus. BD induced increased expression of the pro-inflammatory IL-6-to-IL-10 ratio, their related receptors, and vascular cell adhesion molecule-1 in both the RV and LV. These changes were prevented by tacrolimus. RV and LV neutrophil infiltration induced by BD was partly prevented by tacrolimus. BD was associated with decreased RV expression of the β-1 adrenergic receptor and sarcomere (myosin heavy chain MYH7-to-MYH6 ratio) components, while β-3 adrenergic receptor, nitric oxide-synthase 3, and glucose transporter 1 expression increased. These changes were prevented by tacrolimus.
Brain death was associated with isolated RV dysfunction. Tacrolimus prevented RV dysfunction induced by BD through the inhibition of apoptosis and inflammation activation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cyclin D‐CDK4/6 are the first CDK complexes to be activated in the G1 phase in response to oncogenic pathways. The specific CDK4/6 inhibitor PD0332991 (palbociclib) was recently approved by the FDA ...and EMA for treatment of advanced ER‐positive breast tumors. Unfortunately, no reliable predictive tools are available for identifying potentially responsive or insensitive tumors. We had shown that the activating T172 phosphorylation of CDK4 is the central rate‐limiting event that initiates the cell cycle decision and signals the presence of active CDK4. Here, we report that the profile of post‐translational modification including T172 phosphorylation of CDK4 differs among breast tumors and associates with their subtypes and risk. A gene expression signature faithfully predicted CDK4 modification profiles in tumors and cell lines. Moreover, in breast cancer cell lines, the CDK4 T172 phosphorylation best correlated with sensitivity to PD0332991. This gene expression signature identifies tumors that are unlikely to respond to CDK4/6 inhibitors and could help to select a subset of patients with HER2‐positive and basal‐like tumors for clinical studies on this class of drugs.
Synopsis
Biomarkers able to predict breast cancer sensitivity to the newly FDA/EMA‐approved CDK4/6 inhibitor palbociclib are still lacking. Based on the T172‐phosphorylation of CDK4 as critical to the kinase activation, an 11‐gene classifier helps to segregate cancer subtypes and predict palbociclib sensitivity.
In frozen breast cancers samples (n = 56), three distinct CDK4 modification profiles are observed using 2D‐gel electrophoresis: profile H, preponderant presence of T172‐phosphorylated CDK4 (most luminal B and HER2‐positive cancers); profile L, minor presence of phosphorylated CDK4 (90% of lower risk luminal A tumors); profile A, no phosphorylated CDK4 despite high proliferation index (60% of triple‐negative cancers).
An 11‐gene classifier that correctly predicts the three CDK4 modification profiles was generated and optimized using these breast tumors.
In 4,000 breast cancer samples, the CDK4 modification profiles as predicted by the 11‐gene classifier are associated with subtype, grade, relapse status and risk profile.
The 11‐gene classifier correctly predicts the observed presence or absence of CDK4 phosphorylation in 24 out of 25 breast cancer cell lines and the observed or reported sensitivity to palbociclib in 50 out of 52 breast cancer cell lines.
Once transposed to a qPCR assay compatible with formalin‐fixed breast tumor samples, this classifier should predict the potential sensitivity or resistance to CDK4 inhibitors and the risk and grade of tumors.
Biomarkers able to predict breast cancer sensitivity to the newly FDA/EMA‐approved CDK4/6 inhibitor palbociclib are still lacking. Based on the T172‐phosphorylation of CDK4 as critical to the kinase activation, an 11‐gene classifier helps to segregate cancer subtypes and predict palbociclib sensitivity.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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