CL-K1 Promotes Complement Activation and Regulates Opsonophagocytosis of Macrophages with CD93 Interaction in a Primitive Vertebrate.

Collectin is a crucial component of the innate immune system and plays a vital role in the initial line of defense against pathogen infection. In mammals, collectin kidney 1 (CL-K1) is a soluble collectin that has recently been identified to have significant functions in host defense. However, the evolutionary origins of immune defense of CL-K1 and its mechanism in clearance of pathogenic microorganisms remain unclear, especially in early vertebrates. In this study, the Oreochromis niloticus CL-K1 (OnCL-K1) protein was purified and identified, which was capable of binding to two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila. Interestingly, OnCL-K1 exhibited direct bactericidal activity by binding to lipoteichoic acid or LPS on cell walls, disrupting the permeability and integrity of the bacterial membrane in vitro. Upon bacterial challenge, OnCL-K1 significantly inhibited the proliferation of pathogenic bacteria, reduced the inflammatory response, and improved the survival of tilapia. Further research revealed that OnCL-K1 could associate with OnMASPs to initiate and regulate the lectin complement pathway. Additionally, OnCD93 reduced the complement-mediated hemolysis by competing with OnMASPs for binding to OnCL-K1. More importantly, OnCL-K1 could facilitate phagocytosis by collaborating with cell surface CD93 in a lectin pathway-independent manner. Moreover, OnCL-K1 also promoted the formation of phagolysosomes, which degraded and killed ingested bacteria. Therefore, this study reveals the antibacterial response mechanism of CL-K1 in primitive vertebrates, including promoting complement activation, enhancing opsonophagocytosis, and killing of macrophages, as well as its internal links, all of which provide (to our knowledge) new insights into the understanding of the evolutionary origins and regulatory roles of the collectins in innate immunity.

Patients with ASPSCR1-TFE3 fusion achieve better response to ICI based combination therapy among TFE3-rearranged renal cell carcinoma.

BACKGROUND: TFE3-rearranged renal cell carcinoma (TFE3-rRCC) is a rare but highly heterogeneous renal cell carcinoma (RCC) entity, of which the clinical treatment landscape is largely undefined. This study aims to evaluate and compare the efficacy of different systemic treatments and further explore the molecular correlates. METHODS: Thirty-eight patients with metastatic TFE3-rRCC were enrolled. Main outcomes included progression-free survival (PFS), overall survival, objective response rate (ORR) and disease control rate. RNA sequencing was performed on 32 tumors. RESULTS: Patients receiving first-line immune checkpoint inhibitor (ICI) based combination therapy achieved longer PFS than those treated without ICI (median PFS: 11.5 vs. 5.1 months, P = 0.098). After stratification of fusion partners, the superior efficacy of first-line ICI based combination therapy was predominantly observed in ASPSCR1-TFE3 rRCC (median PFS: not reached vs. 6.5 months, P = 0.01; ORR: 67.5% vs. 10.0%, P = 0.019), but almost not in non-ASPSCR1-TFE3 rRCC. Transcriptomic data revealed enrichment of ECM and collagen-related signaling in ASPSCR1-TFE3 rRCC, which might interfere with the potential efficacy of anti-angiogenic monotherapy. Whereas angiogenesis and immune activities were exclusively enriched in ASPSCR1-TFE3 rRCC and promised the better clinical outcomes with ICI plus tyrosine kinase inhibitor combination therapy. CONCLUSIONS: The current study represents the largest cohort comparing treatment outcomes and investigating molecular correlates of metastatic TFE3-rRCC based on fusion partner stratification. ICI based combination therapy could serve as an effective first-line treatment option for metastatic ASPSCR1-TFE3 rRCC patients. Regarding with other fusion subtypes, further investigations should be performed to explore the molecular mechanisms to propose pointed therapeutic strategy accordingly.

Affinity-Driven Site-Specific High Mannose Modification Determines the Structural Polymerization and Function of Tetrameric IgM in a Primitive Vertebrate.

Teleost tetramer IgM is the predominant Ig in the immune system and plays essential roles in host defense against microbial infection. Due to variable disulfide polymerization of the monomeric subunits, tetrameric IgM possesses considerable structural diversity. Previous work indicated that the teleost IgM H chain was fully occupied with complex-type N-glycans. However, after challenge with trinitrophenyl (TNP) Ag, the complex N-glycans in the Asn-509 site of Oreochromis niloticus IgM H chain transformed into high mannose. This study, therefore, was conducted to examine the functional roles of the affinity-related high-mannose modification in tilapia IgM. The TNP-specific IgM Ab affinity maturation was revealed in tilapia over the response. A positive correlation between TNP-specific IgM affinity and its disulfide polymerization level of isomeric structure was demonstrated. Mass spectrometric analysis indicated that the relationship between IgM affinity and disulfide polymerization was associated with the Asn-509 site-specific high-mannose modification. Furthermore, the increase of high mannose content promoted the combination of IgM and mannose receptor (MR) on the surface of phagocytes. Moreover, the increased interaction of IgM and MR amplified the phagocytic ability of phagocytes to Streptococcus agalactiae. To our knowledge, this study demonstrates that site-specific high-mannose modification associates with IgM Ab affinity and its structural disulfide polymerization and amplifies the phagocytosis of phagocytes by the combination of IgM and MR. The present study provides evidence for understanding the association of IgM structure and function during the evolution of the immune system.

The tumor-repressing effect of CYP27A1 on renal cell carcinoma by 27-HC arising from cholesterol metabolism.

As a key approach to mediate cholesterol metabolism, the role of the CYP27A1/27-HC axis in renal cell carcinoma (RCC) remains unclear. Analysis of CYP27A1 expression from public databases and metastatic cases in our center suggested that CYP27A1 was obviously downregulated in RCC tissues, and survival analysis further showed its correlation with favorable clinicopathological features and prognosis. In vitro, up and downregulation of CYP27A1 expression in RCC cell lines could definitely illustrate its anticipation involving apoptosis, proliferation, invasion, migration, and clonality. This could be achieved through upregulation of 27-HC concentration, which mediates the activation of signaling pathways of apoptosis and cell cycle arrest. Further, recovery of CYP27A1 expression could definitely inhibit the proliferation of RCC tumors in vivo. This is the first study to explore the role of the CYP27A1/27-HC axis in RCC. Attempts to maintain the normal function of the axis may be a potential strategy in the treatment of RCC, and the predictive value of CYP27A1 detection on the efficacy of targeted therapy in metastatic RCC is also worthy of attention.

The roles of mutated SPINK1 gene in prostate cancer cells.

SPINK1-positive prostate cancer (PCa) has been identified as an aggressive PCa subtype. However, there is a lack of definite studies to elucidate the underlying mechanism of the loss of SPINK1 expression in most PCa cells except 22Rv1 cells, which are derived from a human prostatic carcinoma xenograft, CWR22R. The aim of this study was to investigate the mechanisms of SPINK1 protein positive/negative expression and its biological roles in PCa cell lines. SPINK1 mRNA was highly expressed in 22Rv1 cells compared with LNCaP, C4-2B, DU145, and PC-3 cells, and the protein was only detected in 22Rv1 cells. Among these cell lines, the wild-type SPINK1 coding sequence was only found in 22Rv1 cells, and two mutation sites, the c.194G>A missense mutation and the c.210T>C synonymous mutation, were found in other cell lines. Our further research showed that the mutations were associated with a reduction in SPINK1 mRNA and protein levels. Functional experiments indicated that SPINK1 promoted PC-3 cell proliferation, migration, and invasion, while knockdown of SPINK1 attenuated 22Rv1 cell proliferation, migration, and invasion. The wild-type SPINK1 gene can promote the malignant behaviors of cells more than the mutated ones. Cell cycle analysis by flow cytometry showed that SPINK1 decreased the percentage of cells in the G0/G1 phase and increased the percentage of S phase cells. We demonstrated that the c.194G>A and c.210T>C mutations in the SPINK1 gene decreased the mRNA and protein levels. The wild-type SPINK1 gene is related to aggressive biological behaviors of PCa cells and may be a potential therapeutic target for PCa.

Circulating tumour DNA reveals genetic traits of patients with intraductal carcinoma of the prostate.

OBJECTIVES: To investigate the genetic alterations of patients with prostate cancer (PCa) with and without intraductal carcinoma of the prostate (IDC-P). PATIENTS AND METHODS: We performed targeted sequencing of plasma cell-free DNA on 161 patients with prostate adenocarcinoma (PAC) with IDC-P and 84 without IDC-P. Genomic alterations were compared between these two groups. The association between genetic alterations and patients' survival outcomes was also explored. RESULTS: We identified that 29.8% (48/161) and 21.4% (18/84) of patients with and without IDC-P harboured genomic alterations in DNA repair pathways, respectively (P = 0.210). Pathogenic germline DNA repair alterations were frequently detected in IDC-P carriers compared to IDC-P non-carriers (11.8% [19/161] vs 2.4% [two of 84], P = 0.024). Germline BReast CAncer type 2 susceptibility protein (BRCA2) and somatic cyclin-dependent kinase 12 (CDK12) defects were specifically identified in IDC-P carriers relative to PAC (BRCA2: 8.7% [14/161] vs 0% and CDK12: 6.8% [11/161] vs 1.2% [one of 84]). Patients with IDC-P had a distinct androgen receptor (AR) pathway alteration, characterised by an enrichment of nuclear receptor corepressor 2 (NCOR2) mutations compared with patients with pure PAC (21.1% [34/161] vs 6.0% [five of 84], P = 0.004). Increased AR alterations were detected in patients harbouring tumours with an IDC-P proportion of ≥10% vs those with an IDC-P proportion of <10% (6.4% [five of 78] vs 18.1% [15/83], P = 0.045). For IDC-P carriers, tumour protein p53 (TP53) mutation was associated with shorter castration-resistant-free survival (median 10.9 vs 28.9 months, P = 0.026), and BRCA2 alteration was related to rapid prostate-specific antigen progression for those receiving abiraterone treatment (median 9.1 vs 11.9 months, P = 0.036). CONCLUSION: Our findings provide genomic evidence explaining the aggressive phenotype of tumours with IDC-P, highlighting the potential therapeutic strategies for this patient population.

Survival in patients with Parkinson's disease: a ten-year follow-up study in northern China.

BACKGROUND: A thorough understanding of the factors that influence patient survival in Parkinson's disease (PD) will aid in prognosis prediction and provide a new direction for disease modification treatment. Currently, there are no standardized mortality ratio (SMR) data for PD patients in the northern Chinese mainland. The main focus of this study was to determine which factors in the prospectively collected baseline characteristics can affect the survival of PD patients. In addition, for the first time, we investigated the SMR of PD patients in northern China. METHODS: Between 2009 and 2012, 218 PD patients were continuously recruited from the movement disorder clinic of the First Affiliated Hospital of Dalian Medical University and followed up until death or May 31, 2021. The prespecified prognostic variables were demographics, clinical features, lifestyle factors, and drug dose prospectively collected at baseline. To determine the independent predictors of survival during follow-up, the Cox proportional hazards model was used. Kaplan-Meier analysis was applied to estimate the overall survival curve and to compare survival between layers based on statistically significant predictors. The SMR of this northern Chinese mainland PD cohort was calculated. RESULTS: After a mean follow-up of 9.58 ± 2.27 years, 50 patients (22.90%) died. Factors that could individually predict shortened survival during follow-up included older age at onset (hazard ratio [HR] 1.10, 95% confidence interval [CI] 1.06-1.15), Hoehn and Yahr (H&Y) stage ≥ 3 (HR 9.36, 95% CI 2.82-31.03) and severe cognitive impairment (HR 6.18, 95% CI 2.75-13.88). Univariate Cox regression revealed that a certain amount of physical activity was associated with better survival (HR 0.41, 95% CI 0.22-0.74), while fatigue was associated with an increased risk of death (HR 2.54, 95% CI 1.37-4.70). The overall SMR was 1.32 (95% CI 0.98-1.74). CONCLUSIONS: Older age at onset, higher baseline H&Y stage, and severe cognitive impairment have a negative impact on survival. The 10-year survival of PD patients is not significantly different from that of the general population in China.

Mannose-Binding Lectin Possesses Agglutination Activity and Promotes Opsonophagocytosis of Macrophages with Calreticulin Interaction in an Early Vertebrate.

The innate immune system is an ancient defense system in the process of biological evolution, which can quickly and efficiently resist pathogen infection. In mammals, mannose-binding lectin (MBL) is a key molecule in the innate immune and plays an essential role in the first line of host defense against pathogenic bacteria. However, the evolutionary origins and ancient roles of immune defense of MBL and its mechanism in clearance of microbial pathogens are still unclear, especially in early vertebrates. In this study, Oreochromis niloticus MBL (OnMBL) was successfully isolated and purified from the serum of Nile tilapia (O. niloticus). The OnMBL was able to bind and agglutinate with two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila Interestingly, the OnMBL was able to significantly inhibit the proliferation of pathogenic bacteria and reduce the inflammatory response. Upon bacterial challenge, the downregulation of OnMBL expression by RNA interference could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by reinjection of the OnMBL, which restored the healthy status of the knockdown tilapia. Moreover, a mechanistic analysis revealed that the OnMBL could clear pathogenic bacteria by collaborating with cell-surface calreticulin to facilitate phagocytosis in a complement activation-independent manner. To our knowledge, these results provide the first evidence on the antibacterial response mechanism of MBL performing evolutionary conserved function to promote opsonophagocytosis of macrophages in early vertebrates and reveals new insights into the understanding of the evolutionary origins and ancient roles basis of the C-type lectins in the innate immune defense.

Complement C3 Regulates Inflammatory Response and Monocyte/Macrophage Phagocytosis of Streptococcus agalactiae in a Teleost Fish.

The complement system is composed of a complex protein network and is pivotal to innate immunity. Complement 3 (C3) is a critical protein in the complement cascade and participates in complement activation and immune defense. In this study, C3 from Nile tilapia (Oreochromis niloticus) was cloned and its function in resisting pathogen infection was characterized. The full length of OnC3 open reading frame is 4974 bp, encoding 1657 aa, and the predicted protein mass weight is 185.93 kDa. The OnC3 amino acid sequence contains macroglobulin domains. The expression pattern of OnC3 mRNA in the tissues of healthy fish was detected, with the highest in the liver and the lowest in the muscle. After challenged with Streptococcus agalactiae and Aeromonas hydrophila, the expression of OnC3 mRNA was significantly up-regulated in the liver, spleen, and head kidney. Further, the recombinant OnC3 protein alleviated the inflammatory response and pathological damage of tissues after infected with S. agalactiae. Moreover, the OnC3 promoted the phagocytosis of monocytes/macrophages to S. agalactiae. The data obtained in this study provide a theoretical reference for in-depth understanding of C3 in host defense against bacterial infection and the immunomodulatory roles in teleost fish.

Functional Characterization of Serum Amyloid P Component (SAP) in Host Defense against Bacterial Infection in a Primary Vertebrate.

Serum amyloid P component (SAP), an ancient short pentraxin of the pentraxin family, plays an essential role in resistance to bacterial infection. In this study, the expression and functional characterization of SAP (OnSAP) in Nile tilapia (Oreochromis niloticus), a primary vertebrate, are investigated. The open reading frame of OnSAP is 645 bp of a nucleotide sequence encoding a polypeptide of 214 amino acids. As a calcium-binding protein, the structure and relative motif of OnSAP is highly similar to those of humans, containing amino acid residues Asn, Glu, Gln and Asp. In healthy fish, OnSAP mRNA is extensively distributed in all eleven tissues examined, with the highest level in spleen. The mRNA expression of OnSAP was significantly up-regulated after being challenged with gram-positive bacterium Streptococcus agalactiae and gram-negative bacterium Aeromonas hydrophila in vivo. In addition, recombinant OnSAP ((r)OnSAP) protein had capacities of binding S. agalactiae or A. hydrophila in the presence of Ca2+. Further, (r)OnSAP helped monocytes/macrophages to efficiently phagocytize bacteria. Moreover, the (r)OnSAP was able to enhance the complement-mediated lysis of the chicken red blood cells. Collectively, the evidence of SAP in tilapia, based on the results including its evolutionary conserved protein structure, bacterial binding and agglutination, opsonophagocytosis of macrophage and hemolysis enhancement, enriches a better understanding of the biological functions of the pentraxin family.

Identification and functional characterization of CD154 in T cell-dependent immune response in Nile tilapia (Oreochromis niloticus).

CD154, a member of the TNF superfamily, is a multifunctional molecule highly expressed in activated T cells, and plays important roles in T cell-dependent humoral immune response. In this study, CD154 of Nile tilapia (Oreochromis niloticus) was identified, and its functions in the T cell-dependent immune response were demonstrated. The open reading frame (ORF) of OnCD154 is 699 bp, encoding a protein of 232 amino acids with a 23 amino acid transmembrane region. Amino acid sequence of OnCD154 is highly homologous to that of other teleost fish, especially rainbow trout. Quantitative real-time PCR (qRT-PCR) demonstrated that mRNA of OnCD154 is highly expressed in immune organs, especially in spleen, thymus, gills, head kidney, etc. In addition, the anti-OnCD154 polyclonal antibody (anti-(r)OnCD154) was successfully prepared, and it can react with natural protein in head kidney leukocytes. Following two immunizations with keyhole limpet hemocyanin (KLH) in vivo, the significantly up-regulated expression level of OnCD154 mRNA appeared earlier (fifth day) and higher (42.9 folds) in the second challenge than the first on in head kidney. Further, after stimulation with KLH in vitro, the expressions of T cell-dependent immune response-related molecules (activated T cell specific surface molecules CD3ε and CD154) and B cell differentiation-related molecules (Blimp1 and sIgM) and CD40 were significantly up-regulated in head kidney leukocytes. Moreover, the up-regulated expressions of these molecules were blocked with the treatment of anti-(r)OnCD154 antibody. Taken together, these results indicate that OnCD154 might get involved in T cell-dependent immune response, and provide a new insight into the humoral immune response of teleost fish.

Functional characterization of TNF-α in pufferfish (Takifugu obscurus) in immune response and apoptosis against Aeromonas hydrophila.

Tumour necrosis factor-α (TNF-α) is a multifunctional cytokine involved in immune system homeostasis, antimicrobial defence, regulation of apoptosis, cell proliferation and differentiation. Although the pro-inflammatory property of TNF-α has been made new progress, detailed research on host defence against bacterial infection and inducing apoptosis remains to be revealed in early vertebrates. Here, we reported the TNF-α homologue (ToTNF-α) from pufferfish (Takifugu obscurus). The open reading frame (ORF) of ToTNF-α was 753 bp, encoding a protein of 250 aa contained the TNF family signature and conserved cysteine residues. The mRNA expression of ToTNF-α had a wide range of tested tissues, with the highest expression in the skin. After Aeromonas hydrophila infection, the mRNA expression of ToTNF-α was significantly up-regulated both in vivo and in vitro experiments. After stimulation by recombinant protein of ToTNF-α ((r)ToTNF-α), the relative expressions of endogenous TNF-α, caspase 8, caspase 3, p53, and Bax inhibitor-1 in head kidney leucocytes were all notably up-regulated. These results showed that ToTNF-α might induce apoptosis depend on pro- and anti-apoptotic proteins at mRNA level. Moreover, flow cytometry analysis indicated that the (r)ToTNF-α can induce apoptosis of head kidney leucocytes. Taken together, these characteristics suggest that ToTNF-α can participate in immune response against A. hydrophila and induce apoptosis at mRNA and cellular level, which will help to understand the mechanism of apoptosis and immune response in teleost fish.

Expression and functional analysis of Nile tilapia transferrin receptors (TfRs) in host resistance to pathogenic bacteria and iron ion metabolism.

Transferrin receptors (TfRs) play an essential role in iron-withholding strategy, and are involved in immune response against bacterial infection. In this study, the transferrin receptor 1 (OnTfR1) and transferrin receptor 2 (OnTfR2) genes are identified and characterized in Nile tilapia (Oreochromis niloticus). The open reading frames of OnTfR1 and OnTfR2 are 2220 and 2343 bp of nucleotide sequence, encoding 739 and 780 amino acids, respectively. The deduced proteins of OnTfR1 and OnTfR2 are highly homologous to those of other species, containing three conserved TfR superfamily domains (PA TfR domain, M28 TfR domain and TfR dimer domain). Expression analyses of OnTfRs in the healthy tilapia reveal that the OnTfR1 and OnTfR2 transcripts are the most abundant in the liver. The in vivo studies show that the expressions of OnTfRs are significantly up-regulate in liver and spleen, following infections of Streptococcus agalactiae and Aeromonas hydrophila. In addition, the in vitro studies reveal that the up-regulations of OnTfR expressions are also significant in monocytes/macrophages and hepatocytes upon the stimulations of S. agalactiae and A. hydrophila. Moreover, the iron ion (Fe3+) could significantly increase the expressions of OnTfRs in monocytes/macrophages and hepatocytes. Taken together, the present study indicates that OnTfRs may be involved in host defense against bacterial infection and possess the function of combining or transporting iron ions in Nile tilapia.

A microfibril-associated glycoprotein 4 (MFAP4) from Nile tilapia (Oreochromis niloticus) possesses agglutination and opsonization ability to bacterial pathogens.

Microfibril-associated glycoprotein 4 (MFAP4), a pattern recognition-like molecule with a fibrinogen-like domain (FBG), has the ability to combine and agglutinate pathogens, playing an essential role in the first line of innate immune defense. In this study, the sequence of Nile tilapia (Oreochromis niloticus) microfibril-associated glycoprotein 4 (OnMFAP4) open reading frame (ORF) was amplified and identified. The ORF of OnMFAP4 is 720 bp of nucleotides and codes for 239 amino acids. Spatial mRNA encoding analysis indicated that OnMFAP4 was highly produced in liver, intestine and head kidney in healthy tilapia, and with the lowest expression in muscle. After challenges with Streptococcus agalactiae (S. agalactiae) and Aeromonas hydrophila (A. hydrophila), the expression of OnMFAP4 mRNA was prominently produced in the liver, spleen and head kidney. The up-regulation of OnMFAP4 expression was also presented in head kidney monocytes/macrophages (MO/MΦ) and hepatocytes. Recombinant OnMFAP4 ((r)OnMFAP4) could bind and agglutinate both bacterial pathogens. Moreover, (r)OnMFAP4 could take part in the modulation of inflammation and phagocytosis. In conclusion, this study revealed that OnMFAP4 might take effect in host defense against bacterial infection in Nile tilapia, with agglutination and opsonization capability to bacterial pathogens.

Identification and functional characterization of three caspases in Takifugu obscurus in response to bacterial infection.

Caspases are evolutionarily conserved proteases, which are inextricably linked with the apoptosis and immune system in mammals. However, the expression pattern and function of some caspases remain largely unknown in pufferfish. In this study, three different pufferfish caspases (caspase-2 (Pfcasp-2), caspase-3 (Pfcasp-3), and caspase-8 (Pfcasp-8)) were characterized, and their expression patterns and functions were determined following Aeromonas hydrophila infection. The open reading frames of Pfcasp-2, -3, and -8 are 1,320, 846, and 1455 bp, respectively. Analyses of sequence alignment and phylogenetic tree showed that casp-2, -3, and -8 share 52%-65%, 33%-40%, 63%-78% overall sequence identities with those of other vertebrates, respectively. 3D structures of Pfcasp-2, -3, and -8 enjoy conservation in core area together, while each owns a distinctive profile. Comparisons of deduced amino acid sequences indicated that Pfcaspases possessed the caspase domain and conserved active sites like 'HG' and 'QACXG' (X for R or G). qRT-PCR results revealed that Pfcasp-2, -3, and -8 were expressed constitutively in a wide range of organs, especially in immune-related organs including whole blood and kidney. In vitro, the expressions of the three caspases (Pfcasp-2, 3, and -8) and immune-related genes (IgM and IL-8) were significantly up-regulated in kidney leukocytes after A. Hydrophila challenge and inhibitors treatment. The expressions of Pfcasp-2 and Pfcasp-3 were successfully inhibited in the kidney leukocytes by Ac-DEVD-CHO (an inhibitor to caspase-3), but the expression of Pfcasp-8 was not affected. Cellular localization analysis showed that the distribution of Pfcasp-2, -3, and -8 was in cytoplasm. Further, overexpression of Pfcasp-2, -3, or -8 was found to cause DNA damage and apoptosis, suggesting that three caspases may be related to apoptosis and mediate different apoptosis pathways in pufferfish. Moreover, the expressions of these caspases were also up-regulated in whole blood and kidney after A. hydrophila challenge, indicating their possible involvement in the immune response against A. hydrophia stimulation. Taken together, the results of this study suggest that the caspase-2,-3, and -8 may play an important role in the apoptosis and immune response in pufferfish.

The influence of dynamic changes in lipid metabolism on survival outcomes in patients with metastatic renal cell carcinoma treated with tyrosine kinase inhibitors.

BACKGROUND: The role of lipid metabolic status in tyrosine kinase inhibitors-treated patients with metastatic renal cell carcinoma is insufficient. OBJECTIVE: To analyse the influence of dynamic changes of lipid metabolism on survival outcomes in tyrosine kinase inhibitors-treated metastatic renal cell carcinoma. PATIENTS AND METHODS: Serum levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and non-high-density lipoprotein cholesterol were collected, both before tyrosine kinase inhibitors therapy and at different time points of tyrosine kinase inhibitors treatment duration. Other clinicopathological and survival data were retrospectively reviewed. The clinical outcomes, including tumour response, progression-free survival and overall survival, were analysed. Kaplan-Meier survival curves were plotted and the log-rank test was used to analyse statistical significance. RESULTS: A total of 127 patients with metastatic renal cell carcinoma, initially treated with tyrosine kinase inhibitors as first-line systemic therapy, were included. In the whole cohort, the serum levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and non-high-density lipoprotein cholesterol fluctuated but gradually increased during tyrosine kinase inhibitors treatment. In survival analysis, the higher serum level of lipid metabolism, the longer progression-free survival was observed. In terms of overall survival, all post-treatment lipid metabolism, including the percentages of increasing change, were correlated with better survival. Further multivariate analysis showed that patients with five components of treatment-related dysfunction of lipid metabolism had superior survival to those with less than five components. However, lipid metabolism was not correlated with tumour response. CONCLUSION: Increasing parameters of lipid metabolism indicated improvement of survival in tyrosine kinase inhibitors-treated metastatic renal cell carcinoma, especially the increasing percentages.

The expression profile and heterogeneity analysis of ERG in 633 consecutive prostate cancers from a single center.

BACKGROUND: Overexpression of ERG protein resulting from TMPRSS2:ERG rearrangement is highly specific for prostate cancer (PCa). However, the biological function of this fusion protein and its relationship with clinicopathological features still remain controversial. METHOD: In this study, we evaluated ERG protein expression/gene rearrangement and heterogeneity by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in a cohort of 633 consecutive PCa initially diagnosed by core-needle biopsy in the West China Hospital. RESULT: Overall, ERG protein expression was detected in 16.7% (106 of 633) cases, and frequently observed in PCa patients less than 60 years of age (31.9% vs 15.5%, P = 0.004) and in PCa with Gleason score less than 8 (20.0% vs 13.4%, P = 0.027), but infrequently observed in cases with intraductal carcinoma of the prostate (IDC-P) (10.0% vs 18.6%, P = 0.012). Follow-up analysis found that patients who progressed to castration-resistant prostate cancer (CRPC) have a lower frequency of ERG protein expression at initial biopsies compared to androgen deprivation therapy (ADT)-sensitive cases (14.1% vs 23.5%, P = 0.042), but Kaplan-Meier curve showed that ERG protein expression was not an independent prognostic marker. Of all the 106 ERG-positive cases, eight cases (7.5%) exhibited heterogeneous expression of ERG protein, in which ERG was only positive in tumors with Gleason pattern 3, but negative in Gleason pattern 4. The FISH analysis was consistent with IHC in six of these cases. In the other two cases, ERG rearrangement was detected in tumors with both Gleason pattern 3 and 4 by FISH, despite the negative protein expression in Gleason pattern 4. In case 1, a repeated biopsy was performed when the disease progressed to CRPC, and no ERG-positive cells were identified neither by IHC nor FISH. CONCLUSION: This was by far the largest series of ERG expression and heterogeneity analysis in Chinese PCa. The ERG rearrangement seemed to be frequently expressed in patients with relatively younger age and lower Gleason score and infrequently expressed in PCa with the IDC-P. PCa with positive ERG were less frequently to progress to CRPC, but there was no prognostic significance of ERG expression. In heterogeneous cases, ERG protein was detectable only in tumors with Gleason pattern 3 but not in pattern 4. Tumor cells with positive ERG expression/rearrangement seemed easily response to ADT.

AKR1C3 expression in primary lesion rebiopsy at the time of metastatic castration-resistant prostate cancer is strongly associated with poor efficacy of abiraterone as a first-line therapy.

BACKGROUND: Previous studies had demonstrated that aldo-keto reductase family 1 member C3 (AKR1C3), a crucial enzyme in the steroidogenic pathway, played an important role in abiraterone (ABI)-resistance in metastatic castration-resistant prostate cancer (mCRPC) by increasing intratumoral androgen synthesis. However, its value in predicting treatment response in patients with mCRPC is unknown. METHOD AND MATERIALS: Data of 163 patients with metastatic prostate cancer between 2016 and 2018 were retrospectively analyzed. All patients received androgen deprivation therapy plus bicalutamide after initial diagnosis. After mCRPC, either ABI or docetaxel (DOC) treatment was used. No patient had the experience of therapy to the primary tumor. AKR1C3 protein was detected by immunohistochemical staining from rebiopsy (re-Bx) of primary prostate lesions at mCRPC. Kaplan-Meier curves and Cox regression were used to analyze the association between AKR1C3 and treatment outcomes. RESULTS: AKR1C3 was positive in 58 of 163 (35.6%) cases. AKR1C3 was associated with significantly shorter median prostate-specific antigen progression-free survival (mPSA-PFS, 5.6 mo vs 10.7 mo; P < .001), median radiographic progression-free survival (mrPFS, 11.1 mo vs 18.0 mo; P = .018), and numerically shorter median overall survival (mOS, 20.4 mo vs 26.4 mo; P = .157). Notably, AKR1C3-positive patients treated with ABI, but not DOC, had shorter mPSA-PFS and mrPFS compared with AKR1C3-negative men, (mPSA-PFS, 5.7 mo vs. 11.2 mo; P < .001; mrPFS, 12.4 mo vs 23.3 mo; P = .048). However, AKR1C3 expression had no correlation to PSA response or OS. Multivariate Cox regression indicated that AKR1C3 was independently accompanied with rapid PSA progression (hazard ratio [HR], 3.64; 95% confidence interval [CI], 2.10-6.31; P < 0.001) and radiological progression (HR, 2.08; 95% CI, 1.05-4.11; P = .036) in the ABI-treated subgroup. CONCLUSION: This study demonstrated that AKR1C3 detection in tissues from prostate re-Bx at mCRPC was associated with early resistance to ABI but not DOC. These results will help to make optimal personalized treatment decisions for patients with mCRPC, facilitate physicians predicting the effectiveness of ABI.

Differential expression of TIM-3 between primary and metastatic sites in renal cell carcinoma.

BACKGROUND: Due to the significant heterogeneity of renal cell carcinoma (RCC), immune checkpoints may express differently between primary and metastatic tumor. We aimed to evaluate the differential expression of TIM-3 between the primary and metastatic sites of RCC. METHODS: Cases of RCC with metastases resected or biopsied at West China Hospital between January 2009 and November 2016 were included. Clinicopathological parameters were retrospectively extracted. SPPS 22.0, GraphPad Prism 6 and R statistical software were applied for data analysis. RESULTS: A total of 163 cases were included. Immunohistochemical results showed that the overall detection rate of TIM-3 was 56.4% (92/163). The detection rate of TIM-3 in the primary (53.0%, 44/83) was numerically higher than that of the metastasis (42.6%,79/174). Although the concordance rate of TIM-3 between the primary and metastasis was as high as 66.3% (55/83) in the paired cohort, a significant statistically difference of TIM-3 expression between the primary and metastasis was observed (χ2 = 4.664, p = 0.002), with a poor consistency (Kappa = 0.331, p = 0.002). Subsequent survival analysis suggested that TIM-3 expression either in the primary or metastatic tumor was associated with longer progression-free survival (PFS) (HR: 0.67, 95% CI 0.45-0.99, P = 0.02) and overall survival (OS) (HR: 0.52, 95% CI 0.33-0.82, P < 0.001). The expressions of TIM-3 in the primary, metastatic tumors and patients treated with targeted agents all played as favorable factors for PFS and OS. Further multivariate analysis showed that, in the whole cohort, TIM-3 expression in metastatic tumor increased the predicted accuracy (PA) of the whole model of PFS from 74.7 to 75.6% (P = 0.02). For OS, the PA of whole model was increased from 78.1 to 81.1% by adding TIM-3 expression in the metastasis (P = 0.005). The same trends were also observed in paired patients and patients treated with targeted agents. In conclusion, the expression difference between the primary and metastatic tumor of TIM-3 was significant. Biopsy or resection of the metastases may provide a more accurate biological information for clinician's decision-making and the patient's prognosis. What's more, the role of TIM-3 in the RCC still remains controversy, further study are needed to verify the conclusion.

Differential expressions of PD-1, PD-L1 and PD-L2 between primary and metastatic sites in renal cell carcinoma.

BACKGROUND: In clinical practice, the detection of biomarkers is mostly based on primary tumors for its convenience in acquisition. However, immune checkpoints may express differently between primary and metastatic tumor. Therefore, we aimed to compare the differential expressions of PD-1, PD-L1 and PD-L2 between the primary and metastatic sites of renal cell carcinoma (RCC). METHODS: Patients diagnosed with RCC by resection or fine needle aspiration of metastasis were included. Immunohistochemistry (IHC) was applied to detect PD-1, PD-L1 and PD-L2 expressions. SPSS 22.0 was applied to conduct Chi-square, consistency tests and Cox's proportional hazards regression models. GraphPad Prism 6 was used to plot survival curves and R software was used to calculate Predictive accuracy (PA). RESULTS: In the whole cohort (N = 163), IHC results suggested a higher detection rate of PD-L1 in the metastasis than that of the primary site (χ2 = 4.66, p = 0.03), with a low consistent rate of 32.5%. Among different metastatic tumors, PD-1 was highly expressed in the lung/lymph node (65.3%) and poorly expressed in the brain (10.5%) and visceral metastases (12.5%). PD-L1 was highly expressed in lung/lymph node (37.5%) and the bone metastases (12.2%) on the contrary. In terms of survival analysis, patients with PD-1 expression either in the primary or metastasis had a shorter overall survival (OS) (HR: 1.59, 95% CI 1.08-2.36, p = 0.02). Also, PD-L1 expression in the primary was associated with a shorter OS (HR 2.55, 95% CI 1.06-6.15, p = 0.04). In the multivariate analysis, the predictive accuracy of the whole model for PFS was increased from 0.683 to 0.699 after adding PD-1. CONCLUSION: PD-1, PD-L1 and PD-L2 were differentially expressed between primary and metastatic tumors. Histopathological examination of these immune check points in metastatic lesions of mRCC should be noticed, and its accurate diagnosis may be one of the effective ways to realize the individualized treatment.