CYFRA21-1 can predict the sensitivity to chemoradiotherapy of non-small-cell lung carcinoma

Background: The increasing panel of systemic therapies enables the individual management of lung cancer patients, even in advanced stages. However, predictive tools indicating the efficacy of chemoradiotherapy (CRT) are badly needed.

Aims: To determine the tumour markers for predicting the therapeutic effect in non-small-cell lung carcinoma (NSCLC) patients treated with CRT.

Methods: The serum levels of cytokeratin 19 fragment antigen 21-1 (CYFRA21-1), neurone-specific enolase (NSE) and carcinoembryonic antigen (CEA) were measured before CRT by enzyme-linked immunosorbent assays, while the tumour responses were assessed according to the World Health Organization (WHO) response criteria. The relationships between pretreatment expression of CYFRA21-1, NSE, CEA and the effectiveness of CRT were analysed.

Results: The complete response (CR) rate of the primary tumours estimated by computed tomography in patients with high levels of CYFRA21-1 was 2.9% (2/68) while in cases with low CYFRA21-1 it was 20.3% (12/59) (p?=?0.005). The effective rates (CR+PR) in CYFRA21-1 high and low groups were 52.9% (36/68) and 72.9% (43/59), respectively (p?=?0.022).

Conclusions: CYFRA21-1 may be a reliable surrogate marker of CRT efficacy in patients with NSCLC.     

Do poor-prognosis breast tumours express membrane cofactor proteins (CD46)?

CD46 or membrane cofactor protein (MCP) is a complement regulatory protein that has been identified on all nucleated cells and which protects them from attack by autologous complement. Breast carcinomas are reported to consistently express CD46.Aim and methods: Our previous immunohistochemical study showed that in breast carcinomas, loss of CD59 and CD55 correlated with poor survival. This study aimed to investigate the prognostic significance of CD46 on breast tumours using a rabbit polyclonal anti-CD46 antibody with a standard immunohistochemistry method. A total of 510 breast tissues from patients with primary operable breast cancer diagnosed between 1987 and 1992 had previously been included in tissue microarrays. They included patients 70 years of age or less (mean = 54 years) with a long-term follow-up (median = 82 months).Results: Immunohistochemical study revealed that 507/510 (99.4%) of breast tumours expressed CD46. Strong immunoreactivity was exhibited by 136/510 (27%) tumours, while moderate and weak staining was observed in 43% and 29% of tumours, respectively. Intensity of CD46 expression was significantly associated with tumour grade (p<0.05), histological type of tumour (p<0.001) and tumour recurrence (p<0.05). There was no correlation with lymph node stage or the presence of vascular invasion, nor with patient age or menopausal status. Interestingly, as most tumours expressed CD46, it would appear that poor-prognosis tumours that lose CD55 and CD59 still express CD46.Conclusion: Breast tumours express high levels of CD46 that correlates with tumour grade and recurrence. It is therefore likely that loss of CD55 and CD59 could be compensated by expression of CD46. However, loss of CD55 and CD59, even for tumours that still express CD46, is still associated with a poor prognosis. This may suggest that CD46 alone can protect from complement lysis but that loss of CD55 and CD59 are associated with other roles in immune regulation.     

Dickkopf‐1‐promoted vasculogenic mimicry in non‐small cell lung cancer is associated with EMT and development of a cancer stem‐like cell phenotype

To characterize the contributions of Dickkopf‐1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non‐small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial‐mesenchymal transition (EMT)‐related proteins (vimentin, Slug, and Twist), cancer stem‐like cell (CSC)‐related proteins (nestin and CD44), VM‐related proteins (MMP2, MMP9, and vascular endothelial‐cadherin), and β‐catenin‐nu were all elevated in VM‐positive and DKK1‐positive tumours, whereas the epithelial marker (E‐cadherin) was reduced in the VM‐positive and DKK1‐positive groups. Non‐small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC‐related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.     

Cancer testis antigen PRAME: An anti-cancer target with immunomodulatory potential

PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen with restricted expression in somatic tissues and re-expression in poor prognostic solid tumours. PRAME has been extensively investigated as a target for immunotherapy, however, its role in modulating the anti-tumour immune response remains largely unknown. Here, we show that PRAME tumour expression is associated with worse survival in the TCGA breast cancer cohort, particularly in immune-unfavourable tumours. Using direct and indirect co-culture models, we found that PRAME overexpressing MDA-MB-468 breast cancer cells inhibit T cell activation and cytolytic potential, which could be partly restored by silencing of PRAME. Furthermore, silencing of PRAME reduced expression of several immune checkpoints and their ligands, including PD-1, LAG3, PD-L1, CD86, Gal-9 and VISTA. Interestingly, silencing of PRAME induced cancer cell killing to levels similar to anti-PD-L1 atezolizumab treatment. Comprehensive analysis of soluble inflammatory mediators and cancer cell expression of immune-related genes showed that PRAME tumour expression can suppress the expression and secretion of multiple pro-inflammatory cytokines, and mediators of T cell activation, differentiation and cytolysis. Together, our data indicate that targeting of PRAME offers a potential, novel dual therapeutic approach to specifically target tumour cells and regulate immune activation in the tumour microenvironment.     

非小细胞肺癌CD166+干细胞的分离和鉴定

目的:分选人非小细胞肺癌中的CD166+细胞,并验证其肿瘤干细胞的特性。方法:收集手术切除的15例非小细胞癌组织标本以及患者的临床资料,并通过流式细胞术对组织中CD166+的表达水平进行检测,对CD166+细胞进行原代培养,对比CD166+细胞和CD166-细胞的成球能力和集落形成能力;并将CD166+细胞和CD166-细胞注射到小鼠体内,比较其体内肿瘤形成能力。结果:在15例临床样本中均检测到CD166+细胞,CD166阳性表达率最高为12.1%,最低为0.3%。CD166的高表达与肿瘤TNM分期、肿瘤转移、以及肿瘤分型密切相关(P均0.05)。CD166+细胞的成球形成能力和集落形成能力显著的高于CD166-细胞,且CD166+细胞在裸鼠体内肿瘤形成能力显著高于CD166-细胞(P均0.05)。结论:非小细胞肺癌CD166+细胞具有肿瘤干细胞的特征,CD166在非小细胞肺癌的发生发展中可能具有重要的作用。     

Comparison of K-ras gene mutations in tumour and sputum DNA of patients with lung cancer

Mutations in the K-ras gene are frequently found in lung tumours and are implicated in the development of lung cancer. In order to investigate the clinical usefulness of these mutations in lung cancer, we applied a sensitive method to compare mutations in codon 12 of the K-ras gene in DNA extracted from lung tumours and the matched sputum samples obtained from 22 lung cancer patients. K-ras mutations were identified in the lung tumours of 12 patients (54.5%) and in the sputum samples of 10 patients (45.5%). Nine patients showed an identical mutation in both the tumour and the matched sputum samples. There was a significant association between the presence of a K-ras mutation in a lung tumour and the detection of an identical mutation in the matched sputum sample of the lung cancer patient (κ = 0.64, 95% confidence interval 0.32-0.95, p <0.01). K-ras mutations were detected in sputum samples from cancer patients with all lung tumour grades, and both in the presence and the absence of lymph node metastasis. Therefore, K-ras mutations may provide useful diagnostic markers for lung cancer.     

The expression of IL-8 and IL-8 receptors in pancreatic adenocarcinomas and pancreatic neuroendocrine tumours

BackgroundInflammatory mediators influence tumour progression. IL-8 has been shown to have pro-angiogenic, mitogenic and motogenic effects and several studies have demonstrated the expression of IL-8 by various human pancreatic cancer cell lines. Methods: The expression of IL-8 and IL-8 receptors was studied in 52 pancreatic adenocarcinomas and 52 pancreatic neuroendocrine tumours using immunohistochemistry. The expression of IL-8 and IL-8 receptors was also assessed in eight pancreatic adenocarcinomas and seven neuroendocrine tumours in comparison to normal pancreatic tissue using real time quantitative PCR (qRT-PCR). Results: Immunohistochemical analysis of the expression of IL-8, IL-8RA and IL-8RB in 52 pancreatic adenocarcinomas demonstrated expression in 25%, 75% and 79% of pancreatic adenocarcinomas, respectively. There was no statistically significant correlation between expression and tumour grade and stage for any of the three antigens. IL-8, IL-8RA and IL-8RB expression was detected in 21%, 63% and 92% of 52 pancreatic neuroendocrine tumours. There was no statistically significant correlation between expression and tumour grade for any of the three antigens. Using qRT-PCR, the expression of each of IL-8, IL-8RA and IL-8RB mRNA was increased in 75% of pancreatic adenocarcinomas. IL-8, IL-8RA and IL-8RB mRNA expression was also increased in 57%, 43% and 29% of pancreatic neuroendocrine tumours. Quantitatively, there was a significant increase in expression level of IL-8 in tumours of both types in comparison to normal pancreatic tissue (38.5-fold in adenocarcinomas and 43.9-fold in neuroendocrine tumours). There was also increased expression of IL-8RA in both tumour types, with higher levels in adenocarcinomas, 2.7-fold and neuroendocrine tumours, 1.7-fold. IL-8RB was slightly increased in adenocarcinomas in comparison to normal pancreas (1.4-fold), but the expression was decreased in neuroendocrine tumours compared with normal pancreas (0.9-fold). Conclusion: This is the first study to show that IL-8 and IL-8 receptors are upregulated in both pancreatic adenocarcinomas and neuroendocrine tumours, and indicate this signalling pathway may modulate tumour behaviour through autocrine and/or paracrine loops.     

Phospholipase C ε‐1 inhibits p53 expression in lung cancer

The pathogenesis of lung cancer is to be further investigated. Recent reports indicate that phospholipase C ε‐1 (PLCE1) is a critical molecule involved in tumour growth. This study aims to investigate the role of PLCE1 in the regulation of apoptosis in lung cancer cells. In this study, the surgically removed non‐small‐cell lung cancer (NSCLC) tissue was collected from 36 patients. Single NSCLC cells were prepared from the tissue, in which immune cells of CD3⁺, CD11c⁺, CD19⁺, CD68⁺ and CD14⁺ were eliminated by magnetic cell sorting. The expression of PLCE1 and p53 was assessed by quantitative real‐time polymerase chain reaction and Western blotting. Apoptosis of NSCLC cells was analysed by flow cytometry. The results showed that, in cultured NSCLC cells, high levels of PLCE1 and low levels p53 were detected; the two molecules showed a negative correlation (p < 0.01). The addition of anti‐PLCE1 antibody increased the expression of p53 in NSCLC cells, which increased the frequency of apoptotic NSCLC cells. We conclude that NSCLC cells express high levels of PLCE1, which suppresses the expression of p53 in NSCLC cells. PLCE1 can be a therapeutic target of NSCLC. Copyright © 2013 John Wiley & Sons, Ltd.     

3-O-methylfunicone, from Penicillium pinophilum, is a selective inhibitor of breast cancer stem cells

Objectives: Cancer stem cells make up a subpopulation of cells within tumours that drive tumour initiation, growth and recurrence. They are resistant to many current types of cancer treatment, causing failure of such therapeutic approaches, including chemotherapy and radiotherapy. In the study described here, anti‐proliferative effects of 3‐O‐methylfunicone (OMF), a metabolite from Penicillium pinophilum, were investigated on human breast cancer MCF‐7 cells and cancer stem cells selected as mammospheres derived from MCF‐7s. Materials and methods: Stemness markers were analysed on isolated mammospheres showing positive expression of CD24, CD29, CD44, CD133, CD184 and CD338. Cell proliferation and apoptosis were analysed by flow cytometry and RT‐PCR. Cell colony formation assays were performed to evaluate colony formation of mammospheres. Results and conclusion: OMF treatment affected both MCF‐7 and mammosphere growth, inducing apoptosis. In addition, OMF strongly reduced stemness markers and survivin, hTERT and Nanog‐1 gene expression. Growth of colonies in soft‐agar was significantly affected by OMF treatment, too. Lastly, we tested ability of MCF‐7 cells to form mammospheres after treatment with OMF or cisplatin, demonstrating that OMF treatment resulted in drastic reduction in number of mammospheres. These results introduce OMF as an effective molecule in suppressing breast cancer stem cells.     

New p53-based anti-cancer therapeutic strategies

Thep53 gene is frequently mutated in human tumours and therefore an important target for therapeutic intervention. Several p53-based strategies for treatment of cancer are currently under development.p53 gene therapy has resulted in tumour regression in patients with lung cancer. A mutant adenovirus can obliterate tumour cells carrying mutant p53 or lacking p53, but is unable to replicate in normal cells. Furthermore, current studies suggest that reactivation of mutant p53 proteins in tumours using small p53-activating molecules may initiate p53-dependent apoptosis and thus eliminate the tumour.     

Enhanced expression of the complement regulatory protein CD55 predicts a poor prognosis in colorectal cancer patients

This study prospectively correlated the level of expression of CD55 on tumours with 7-year survival in 136 colorectal cancer patients. Patients with tumours expressing high levels of CD55 had a significantly worse survival (24%) than patients with low CD55 levels (50%, p<0.02). A similar difference was seen for patients (Duke's B or C) with a high risk of recurrence (29% vs 58%, p<0.05). Furthermore, there was a progressive deterioration in prognosis with increasing antigen expression (p=0.01). It remains unclear if CD55 is overexpressed by tumours to protect them from complement or if it is related to the recent observation that CD55 is a ligand for the T-cell activation antigen CD97. However, it is a marker of aggression, as colorectal cancer patients whose tumours overexpress CD55 have a significantly reduced 7-year survival.     

Alterations of the levels of primary antioxidant enzymes in different grades of human astrocytoma tissues

Malignant astrocytoma is the most commonly occurring brain tumour in humans. Oxidative stress is implicated in the development of cancers. Superoxide dismutase 2 (SOD2) was found to exert tumour suppressive effect in basic research, but increased SOD2 protein level was associated with higher aggressiveness of human astrocytomas. However, studies reporting alterations of antioxidant enzymes in human astrocytomas often employed less accurate methods or included different types of tumours. Here we analysed the mRNA levels, activities, and protein levels of primary antioxidant enzymes in control brain tissues and various grades of astrocytomas obtained from 40 patients. SOD1 expression, SOD1 activity, and SOD1 protein level were lower in Grade IV astrocytomas. SOD2 expression was lower in low-grade (Grades I and II) and Grade III astrocytomas than in controls, but SOD2 expression and SOD2 protein level were higher in Grade IV astrocytomas than in Grade III astrocytomas. Although there was no change in SOD2 activity and a lower activity of citrate synthase (CS), the MnSOD:CS ratio increased in Grade IV astrocytomas compared with controls and low-grade astrocytomas. Furthermore, SOD1 activity, CS activity, SOD1 expression, GPX4 expression, and GPX4 protein level were inversely correlated with the malignancy, whereas catalase activity, catalase protein, SOD2 protein level, and the SOD2:CS ratio were positively correlated with the degree of malignancy. Lower SOD2:CS ratio was associated with poor outcomes for Grade IV astrocytomas. This is the first study to quantify changes of various primary antioxidant enzymes in different grades of astrocytomas at different levels concurrently in human astrocytomas.     

miR-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为研究

目的:探讨mi R-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为。方法:qRT-PCR法检测并比较肺癌组织、癌旁正常组织、肺癌细胞、正常肺上皮细胞中的mi R-199a-3p m RNA相对表达量。比较远处转移肺癌组织、未转移肺癌组织中mi R-199a-3p m RNA相对表达量。qRT-PCR法、Western Blot法检测并比较肺癌组织、癌旁正常组织中的CBX7 m RNA及蛋白的表达水平。荧光素酶活性法检测mi R-199a-3p与靶基因CBX7的结合。比较mi R-199a-3p模拟物转染组与阴性对照组的肺癌细胞中的CBX7 m RNA相对表达量及CBX7蛋白表达水平。CCK8实验检测mi R-199a-3p对肺癌细胞增殖的促进作用。Tranwell实验检测mi R-199a-3p对肺癌细胞侵袭与迁移能力的影响。结果:肺癌组织中mi R-199a-3p明显高于癌旁正常组织,发生远处转移的肺癌组织中mi R-199a-3p m RNA的表达量明显高于未发生转移的肺癌组织,差异有统计学意义(P<0.001)。肺癌组织中CBX7m RNA、CBX7蛋白表达水平均明显低于癌旁正常组织,差异有统计学意义(P<0.001)。荧光素酶活性法证实mi R-199a-3p可与靶基因CBX7结合抑制CBX7的表达。肺癌细胞中mi R-199a-3p m RNA的相对表达量明显高于正常肺上皮细胞,CBX7 m RNA相对表达量明显低于正常肺上皮细胞(P<0.05)。对于肺癌细胞,mi R-199a-3p模拟物转染组的CBX7 m RNA相对表达量及CBX7蛋白表达水平均明显低于阴性对照组(P<0.001)。CCK8实验证实mi R-199a-3p能够促进肺癌细胞的增殖,Tranwell实验证实mi R-199a-3p对肺癌细胞侵袭与迁移具有积极的促进作用。结论:mi R-199a-3p在肺癌的发生发展过程中发挥重要作用,能够通过抑制CBX7基因的表达,促进肺癌细胞的增殖、侵袭和转移。     

An investigation into the potential use of serum Hsp70 as a novel tumour biomarker for Hsp90 inhibitors

Hsp90 inhibitors are under investigation in multiple human clinical trials for the treatment of cancers, including myeloma, breast cancer, prostate, lung, melanoma, gastrointestinal stromal tumour and acute myeloid leukaemia. The pharmacodynamic activity of Hsp90 inhibitors in the clinic is currently assessed by Hsp70 induction in peripheral blood mononuclear cells using Western blot analysis, a method that is laborious, semiquantitative and difficult to implement in the clinic. Since Hsp70 was reported to be secreted by tumour cells and elevated in sera of cancer patients, serum Hsp70 has been evaluated as a potentially more robust, easily and reproducibly measured biomarker of Hsp90 inhibition as an alternative to cytosolic Hsp70. A highly sensitive and specific electrochemiluminescent ELISA was developed to measure serum Hsp70 and employed to evaluate Hsp70 levels in both ex vivo and xenograft samples. In ex vivo studies, maximal secretion of Hsp70 by tumour cells was observed between 48 and 72?h after exposure to Hsp90 inhibitors. In in vivo studies a 3–4-fold increase in serum Hsp70 was observed following treatment with BIIB021 in tumour-bearing mice. Strikingly, secreted Hsp70 was detectable in mice transplanted with human tumours but not in naive mice indicating a direct origination from the transplanted tumours. Analysis of clinical samples revealed low baseline levels (2–15?ng ml^?1) of Hsp70 in the serum of cancer patients and normal donors. Together these findings in laboratory studies and archived cancer patient sera suggest that serum Hsp70 could be a novel biomarker to assess reliably the pharmacological effects of Hsp90 inhibitors in clinical trials, especially under conditions where collection of tumour biopsies is not feasible.     

Effects of cannabinoid receptor 1 (brain) on lipid accumulation by transcriptional control of CPT1A and CPT1B

CB1 (also known as CNR1), a main receptor for cannabinoids acting at PPARs, can enhance fat deposition. Carnitine palmitoyltransferase‐1 (CPT1), an enzyme responsible for the transport of long‐chain fatty acids for β‐oxidation, is closely related to fat deposition. Whether CB1 can regulate intramuscular adipocytes lipid accumulation through regulation of CPT1 is unclear. Based on the investigation of tissue‐ and breed‐specific CPT1A and CPT1B mRNA expression levels in Jinhua and Landrace pigs, we studied the effects of CB1 on lipid accumulation and CPT1B expression by treating porcine intramuscular adipocytes with CB1 antagonist Δ9‐THC and antagonist SR141716. Results showed that muscle CPT1 mRNA was expressed at higher levels in the longissimus dorsi and subcutaneous fat. Liver CPT1A mRNA expression levels were higher in the pancreas, duodenum and liver. Compared with Landrace pigs, CPT1A and CPT1B in the longissimus dorsi of Jinhua pigs were significantly higher and positively correlated with intramuscular fat content. However, for subcutaneous fat, CPT1 levels were significantly lower and negatively correlated with body fat percentage. Δ9‐THC significantly increased CB1 mRNA levels and lipid accumulation but decreased CPT1A and CPT1B mRNA levels. Conversely, SR141716 reduced CB1 mRNA levels but increased CPT1A and CPT1B mRNA levels, resulting in decreased lipid accumulation. The CPT1 antagonist etomoxir did not affect CB1 expression, suggesting that CB1 is likely upstream of CPT1A and CPT1B. Meanwhile, PPARA expression was greatly decreased when CPT1A and CPT1B were inhibited and enhanced when CPT1A and CPT1B were activated. Taken together, these data indicate that CB1 can affect intramuscular fat deposition by regulating both CPT1A and CPT1B mRNA expression, with the PPARA signal pathway likely playing a major role in this process.     

Demonstration of a Melanoma-Specific CD44 Alternative Splicing Pattern That Remains Qualitatively Stable,but Shows Quantitative Changes during Tumour Progression

The role of CD44 in the progression of human melanoma has mostly been characterised by qualitative changes in expression of its individual variable exons. These exons however, may be expressed to form a number of molecules, the alternative splice variants of CD44, which may be structurally and functionally different. Using real-time PCR measurements with variable exon specific primers we have determined that all are expressed in human melanoma. To permit comparison between different tumours we identified a stable CD44 variable exon (CD44v) expression pattern, or CD44 ‘fingerprint’. This was found to remain unchanged in melanoma cell lines cultured in different matrix environments. To evaluate evolution of this fingerprint during tumour progression we established a scid mouse model, in which the pure expression pattern of metastatic primary tumours, circulating cells and metastases, non-metastatic primary tumours and lung colonies could be studied. Our analyses demonstrated, that although the melanoma CD44 fingerprint is qualitatively stable, quantitative changes are observed suggesting a possible role in tumour progression.     

肺癌患者外周血淋巴细胞亚群水平的表达及临床意义

目的:探讨肺癌患者外周血淋巴细胞亚群水平的表达及临床意义。方法:选择2016年3月~2017年3月期间我院收治的88例肺癌患者作为研究组,选择同期于我院进行健康体检的88例受检者作为对照组。两组研究对象均通过流式细胞仪检测外周血T淋巴细胞亚群水平。观察对比两组研究对象外周血T淋巴细胞亚群的表达水平,以及研究组不同临床病理特征患者外周血T淋巴细胞亚群的表达水平。结果:研究组CD4~+/CD8~+、CD4~+、CD3~+表达水平均低于对照组,CD8~+表达水平高于对照组,差异具有统计学意义(P0.05)。研究组Ⅲ期与Ⅳ期患者CD4~+/CD8~+、CD4~+、CD3~+表达水平均低于Ⅰ期与Ⅱ期,Ⅲ期与Ⅳ期患者CD8~+表达水平高于Ⅰ期与Ⅱ期(P0.05);小细胞肺癌患者CD4~+/CD8~+、CD4~+、CD3~+表达水平均低于非小细胞肺癌患者,CD8~+表达水平高于非小细胞肺癌患者,差异具有统计学意义(P0.05)。不同性别、年龄、肿瘤分化程度肺癌患者CD4~+/CD8~+、CD8~+、CD4~+、CD3~+水平无统计学差异(P0.05)。结论:肺癌患者外周血淋巴细胞亚群水平的表达呈现异常状态,且表达水平与疾病的分期和病理分型有关。