Novel intergenic KIF5B-MET fusion variant in a patient with gastric cancer: A case report

BACKGROUND MET fusion is a key driver mutation, but it is rare in gastric cancer (GC). Several MET (hepatocyte growth factor receptor) inhibitors have been approved for the treatment of MET-positive patients, but the tumor response is heterogeneous. With the development of next-generation sequencing, diverse MET fusion partner genes have been identified. We herein report a fusion variant involving KIF5B-MET in GC.CASE SUMMARYAfter thoracoscopic inferior lobectomy plus lymph node dissection under general anesthesia, a “tumor within a tumor” was found in the lung tumor tissue of a 64-year-old non-smoking male patient. Combining the medical history and the results of enzyme labeling, the focal area was considered to be GC. To seek potential therapeutic regimens, an intergenic region between KIF5B and MET fusion was identified. This fusion contains a MET kinase domain and coil-coiled domains encoded by KIF5B exons 1-25, which might drive the oncogenesis. CONCLUSIONOur finding could extend the spectrum and genomic landscape of MET fusions in GC and favor the development of personalized therapy.     

Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR-mutant lung cancer

Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor–mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.     

Digital PCR detection of EGFR somatic mutations in non-small-cell lung cancer formalin fixed paraffin embedded samples

BackgroundDigital PCR (dPCR) is proposed to replace real time PCR and Sanger sequencing for detection and quantification of rare mutations, frequently unnoticed in the mass of tumoral cells. Screening of endothelial growth factor receptor (EGFR) mutations is mandatory before treatment with EGFR-targeted therapy with small-molecule tyrosine kinase inhibitors, which has been approved for the treatment of advanced non-small-cell lung cancer (NSCLC).ObjectiveIn order to establish a cost-effective method for detection of mutations, we optimized dPCR identification of EGFR mutations in exons 18–21, and determined dPCR sensitivity, limits of detection (LoD) and quantification (LoQ).MethodsFor clinical validation, we compared the performance of dPCR and castPCR in 57 NSCL formalin fixed paraffin embedded samples and 10 lung cancer-free formalin fixed paraffin embedded samples.ResultsEGFR mutations DEL19, p.L858R, p.G719X, p.L861Q and p.T790 M were detected by dPCR in 27 samples versus 11 detected by castPCR (p = 0.014). LoD was determined as 100 molecules of DNA/uL and LoQ as 1%. Most of the samples (87%) identified by competitive Allele-Specific TaqMan (castPCR) as wild-type and by dPCR as mutated, presented less than 10% mutated DNA molecules (mean 4.57%). Accuracy of dPCR was 94.44%, as measured with the assay recommended by the College of American Pathologists.ConclusionThese results indicated higher sensibility and specificity of dPCR for screening EGFR mutations in NSCLC biopsies, compared to castPCR.     

Profile of the therascreen® EGFR RGQ PCR kit as a companion diagnostic for gefitinib in non-small cell lung cancer

Introduction: Gefitinib is recently approved by the US Food and Drug Administration as a first-line treatment for non-small cell lung cancer (NSCLC) patients harboring EGFR mutations. The therascreen® EGFR RGQ PCR Kit is approved as a companion diagnostic to select patients with EGFR exon 19 deletions and L858R mutation for treatment with gefitinib.

Areas covered: This article reviews the methods for detecting EGFR mutations, the technology and indication of the therascreen® kit, and the clinical utility of the assay in phase 3 and phase 4 clinical trials. Studies that compared the performance of the therascreen® kit with other assays and assessed the kit’s application in non-tissue samples are also discussed.

Expert commentary: The therascreen® kit is a highly sensitive real-time polymerase chain reaction test that provides standardised testing and automated interpretation of EGFR mutation status in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Although not indicated for these applications, the test has also shown utility in detecting uncommon sensitizing EGFR mutations as well as mutations in non-tissue samples.     

Assessment of the clinical application of detecting EGFR,KRAS, PIK3CA and BRAF mutations in patients with non-small cell lung cancer using next-generation sequencing

Background: Next-generation sequencing (NGS) has been widely applied in clinical research, while its application in routine clinical molecular testing requires careful validation. The aim of our study was to assess the clinical usefulness of the NextDaySeq Lung panel on Ion Torrent? PGM in mutation detection of actionable genes in lung cancer.

Methods: The NextDaySeq assay was evaluated by blinded comparisons to Quantitative Real-Time PCR (qPCR) assays with 188 consecutive samples from Chinese patients with non-small cell lung cancer (NSCLC) to detect mutations in EGFR, KRAS, PIK3CA and BRAF. Discordant variants were further validated by Sanger sequencing and independent qPCR and NGS assays.

Results: Our results showed 93.3% concordance of reportable variants mutually covered in both NGS and qPCR assays, with a clinical sensitivity of 89.9%, specificity of 97.5%. Through the comparison, the NGS assays demonstrated its advantages in offering more clinical relevant information, such as detecting non-hotspot mutations and providing mutation allele frequencies (MAF) and accurate mutation sequences. The analytical sensitivity of NGS to detect mutations with low MAF needs further improvement.

Conclusions: The NextDaySeq Lung panel exhibited good clinical performance, strongly supporting the implementation of the NGS assay in routine clinical use to facilitate therapeutic decision-making for lung cancer patients.     

Up-regulated Golgi phosphoprotein 2 (GOLPH2) expression in lung adenocarcinoma tissue

ObjectivesThe aim of the study was to evaluate Golgi Phosphoprotein 2 (GOLPH2) expression in lung cancer.Design and methodsGOLPH2 expression was analyzed from 178 lung cancer tumor tissue samples using immunohistochemistry. Levels of 133 lung cancer patients' serum GOLPH2 (sGOLPH2) were also quantitatively compared with 70 healthy individuals by a sandwich enzyme-linked immunosorbent assay (ELISA).ResultsAll adenocarcinoma (ADC) tissue samples (100%) displayed strong staining, 83.6% of which displayed cytoplasmic diffused staining. However, for squamous cell carcinoma samples, only 16.3% displayed strong staining intensity. Statistically, GOLPH2 expression in tissues of ADC was significantly higher than in other types of lung cancer (P < 0.001). Levels of sGOLPH2 were also detected. The mean value of sGOLPH2 concentration is 69 ng/ml in lung cancer patients and 53 ng/ml in healthy individuals.ConclusionsSignificantly elevated GOLPH2 expression was observed in ADC tumor tissues. The levels of sGOLPH2 were about 30% higher in lung cancer patients compared with healthy individuals.     

microRNA regulation of cell viability and drug sensitivity in lung cancer

Introduction: microRNAs (miRNAs) are 19 – 23 nucleotide long RNAs found in multiple organisms that regulate gene expression and have been shown to play important roles in tumorigenesis. In the context of lung cancer, numerous studies have shown that tumor suppressor genes and oncogenes that play crucial roles in lung tumor development and progression are targets of miRNA regulation. Manipulation of miRNA levels that modulate lung cancer cell survival and drug sensitivity can therefore provide novel therapeutic targets and agents.

Areas covered: Here, the authors review the published in vitro, in vivo and preclinical studies on the functional role of miRNAs in modulating lung cancer cell viability and drug response, and discuss the limitations and promise of translating current findings into miRNA-based therapeutic and diagnostic strategies.

Expert opinion: Although many miRNAs have been identified as potent regulators of cell viability and drug sensitivity in lung cancer, most of them have not been characterized for potential clinical application. Further study is warranted to evaluate translation of the current findings to the clinic to improve the diagnosis and treatment of lung cancer. In addition, most studies have focused on non-small cell lung cancer (NSCLC). It is therefore important to raise interest in investigating miRNAs in small cell lung cancer (SCLC) as well as in comparative studies of miRNA expression and function in different histological subtypes of lung cancer.     

A profile on cobas® EGFR Mutation Test v2 as companion diagnostic for first-line treatment of patients with non-small cell lung cancer

ABSTRACT

Introduction

Among non-small cell lung cancer (NSCLC) patients, there is one molecularly defined subgroup harboring activating mutations in the epidermal growth factor receptor gene (EGFR), which results in constitutive activation of its intrinsic kinase activity. Consistent data have demonstrated that these patients have a better outcome when treated with specific tyrosine-kinase inhibitors (EGFR-TKIs). Therefore, analysis of EGFR mutational status for treatment guidance is mandatory in this context.     

Tracking of Tumor Cell–Derived Extracellular Vesicles In Vivo Reveals a Specific Distribution Pattern with Consecutive Biological Effects on Target Sites of Metastasis

Purpose

Extracellular vesicles, small vesicles carrying inter alia proteins, miRNA and RNA, are important mediators of intercellular communication. The purpose of this study was to assess the distribution of extracellular vesicles from highly malignant breast cancer and their subsequent effect on the immune cell infiltrate in target organs of metastasis.

Procedures

Extracellular vesicles were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was verified by electron microscopy and western blotting. Extracellular vesicles were additionally subjected to proteome analysis. After labeling with the fluorescent dye DiR, extracellular vesicles were injected into healthy BALB/c mice and their in vivo distribution was assessed using fluorescence reflectance imaging (FRI). Following ex vivo imaging of the organs, lung tissue samples were analyzed for extracellular vesicle-mediated changes of myeloid cells and T cell numbers, using flow cytometry. Proteome analysis revealed major differences in the cargo of tumor cell–derived versus extracellular vesicles from healthy serum.

Results

In contrast to control extracellular vesicles, DiR-labeled extracellular vesicles from tumor cells preferentially accumulated in lung, liver, and spine. Subsequent flow cytometry of the immune cell composition of lung tissue samples revealed an increase of cytotoxic CD8+ T cells and a decrease of CD4+ T-helper cells as well as an increase in mature macrophages in response to tumor cell EV.

Conclusions

In conclusion, distribution of tumor cell–derived extracellular vesicles follows a specific pattern and can be monitored, using dedicated imaging. Extracellular vesicles alter the immune cell composition in target organs of metastasis, using a specific proteome cargo.

     

Efficacy of afatinib in a patient with rare EGFR (G724S/R776H) mutations and amplification in lung adenocarcinoma: A case report

BACKGROUNDThe most common EGFR mutations are in-frame deletions in exon 19 and point mutations in exon 21. Cases with classical EGFR mutations show a good response to EGFR tyrosine kinase inhibitors (TKIs), the standard first-line treatment. With the development of next generation sequencing, some uncommon genomic mutations have been detected. However, the effect of TKIs on such uncommon EGFR mutations remains unclear.CASE SUMMARYHere, we report a case of rare EGFR co-mutation in non-small cell lung cancer and the efficacy of afatinib on this EGFR co-mutation. A 64-year-old woman was diagnosed with thoracolumbar and bilateral local rib bone metastases, bilateral pulmonary nodules, and pericardial and left pleural effusion. The pathological diagnosis was lung adenocarcinoma. To seek potential therapeutic regimens, rare co-mutation comprising rare EGFR G724S/R776H mutations and amplification were identified. The patient experienced a significant clinical response with a progression-free survival of 17 mo. CONCLUSIONA case of non-small cell lung cancer with rare EGFR G724S/R776H mutations and EGFR amplification responds well to TKI treatment.     

B1场校正T1 mapping测量肺癌初始T1值的可重复性及其与表观弥散系数和Ki-67表达的相关性

目的 观察B1场校正MR纵向弛豫时间成像(T1 mapping)测量肺癌初始T1值的可重复性及其与弥散加权成像(DWI)定量参数表观弥散系数(ADC)及Ki-67表达的相关性。方法 收集36例经穿刺活检或手术病理确诊的肺癌患者,包括33例单发及3例多发共39个病灶;采集胸部B1场校正T1 mapping及DWI,以免疫组织化学检测方法评估病灶组织Ki-67表达水平。由2名影像科医师(观察者A、B)独立测量病灶初始T1值和ADC,评估观察者内及观察者间测量T1值的一致性及其差异,分析肺癌初始T1值、ADC及Ki-67表达的相关性。结果 观察者A所测肺癌初始T1值为(1 436.38±222.26)ms及(1 449.58±229.98)ms,差异无统计学意义(t=-0.960,P=0.343);观察者B所测肺癌初始T1值为(1 461.30±236.44)ms,与观察者A差异无统计学意义(t=-1.532,P=0.134);观察者内[组内相关系数(ICC)=0.963,95%CI(0.928,0.980)]与观察者间[ICC=0.948,95%CI(0.901,0.973)]测量肺癌初始...     

Low‐level gastrokine 2 promoted progress of NSCLC and as a potential biomarker

BackgroundGastrokine 2 (GKN2) is significantly downregulated in non‐small cell lung cancer (NSCLC) tissues than in normal tissues (NT), as assessed by mRNA microassay; however, the mechanism and clinical value of GKN2 is unknown in NSCLC.MethodsA total of 60 NSCLC samples and corresponding NT samples were prospectively collected GKN2 expression in NSCLC tissues was estimated. Also, the expression level of GKN2 promoter methylation and correlation with clinical data in NSCLC patients from public databases were analyzed. Cytology experiments were also carried out.ResultsThe GKN2 mRNA and protein expression level in NSCLC was significantly lower than that in the NT, and the GKN2 expression level in large tumors NSCLC was significantly lower than that in the small tumor group. Public data showed that expression of GKN2 in LUAD with P53 mutation group was lower than that of the P53 non‐mutation group, and GKN2 promoter methylation level of LUAD was significantly higher than its NT and close to age and clinical stage. Cell migration, invasion, and proliferation ability of GKN2 overexpressed were lower in A549 and PC9 groups than those in GKN2 overexpressed A549 and PC9 negative control groups, while the percentage of apoptotic cells increased in the GKN2 overexpressed A549 and PC9 groups. The DNMT3B mRNA expression levels were higher in PC9 and A549 cells than BEAS‐2B cells.ConclusionThe overexpression of GKN2 significantly inhibited cell proliferation, migration, and invasion and promoted apoptosis. Low‐level GKN2 promoted the progression of NSCLC via DNMT3B and is expected to be a biomarker for NSCLC.     

IL-6 has no acute effect on the regulation of urea synthesis in vivo in rats

Abstract

Background. Clinical or experimentally induced, active inflammation up-regulates the in vivo capacity of urea synthesis (CUNS), which promotes nitrogen removal from the body and metabolic catabolism. We have shown that tumor necrosis factor α (TNF-α) up-regulates CUNS and increases interleukin 6 expression (IL-6) within hours of administration. The described effect of TNF-α on nitrogen homeostasis may, therefore, depend on IL-6. Methods. Three hours after the i.v. injection of 125 μg.kg^?1 of IL-6 or placebo, we evaluated the CUNS, hepatocyte urea cycle enzyme protein levels and the mRNA levels of the urea cycle enzyme genes in rats. The prevailing rat serum acute phase proteins and their liver mRNA levels were also measured. Results. IL-6 did not change CUNS or hepatocyte urea cycle enzyme protein levels, whereas urea cycle enzyme mRNA levels, except for ornithine transcarbamylase (OTC), decreased by approximately 20%. The liver mRNA levels of α2MG, haptoglobin and α1AGP all increased by 1.5- to 2-fold (p < 0.001). In serum, only the α2MG concentration slightly increased (p < 0.001), whereas the levels of the other circulating acute phase proteins remained unchanged. Conclusion. IL-6 is not the mediator of the in vivo CUNS up-regulation observed 3 h after TNF-α administration, but it may be involved in the down-regulation of urea cycle genes. IL-6 may also mediate TNF-α effects on acute phase protein gene expression. Thus, IL-6 did not contribute to the in vivo hepatic component of inflammation-associated catabolism.     

肿瘤微环境促进子宫内膜癌细胞分泌雌激素

目的:筛选子宫内膜癌微环境中促进子宫内膜癌细胞雌激素分泌的细胞因子,并探讨相关机制。方法:提取子宫内膜癌间质细胞,用双重免疫荧光法进行鉴定。检测子宫内膜癌细胞(RL95-2、HEC-1A和HEC-1B)与间质细胞共培养和单独培养模式下,培养液上清雌激素浓度和肿瘤细胞芳香化酶的表达情况。用细胞因子芯片筛选2种模式下差异性表达的细胞因子,并用real-time PCR进行验证。结果:成功提取肿瘤间质细胞(波形蛋白为阳性)。共培养组上清雌激素浓度和肿瘤细胞中芳香化酶mRNA表达量均高于单独培养组(P<0.01)。共培养组上清中生长分化因子-15(GDF-15)的表达高于单独培养组,10 ng/mL的GDF-15可显著上调子宫内膜癌细胞中芳香化酶的表达(P<0.01)。结论:子宫内膜癌细胞与间质细胞共培养情况下,子宫内膜癌细胞中芳香化酶的表达,进而促进间质细胞雌激素浓度的合成,其中子宫内膜癌细胞中GDF-15可能发挥重要作用。     

体素内不相干运动成像在体检测人类结肠癌SW480裸鼠耐药性

目的 探讨体素内不相干运动（IVIM）成像活体评价人类结肠癌SW480裸鼠耐药性及其可行性。方法 对耐药组（n=5）和不耐药组（n=5）人类结肠癌SW480荷瘤裸鼠于肿瘤最长径大于1.50 cm后分别行IVIM DWI检查,测量肿瘤真扩散系数（D）、假扩散系数（D^*）及灌注分数（f）。处死荷瘤鼠,检测肿瘤坏死（HE染色）、凋亡（TUNEL法）及P-糖蛋白（P-gp）、多药耐药相关蛋白1（MRP1）、蛋白激酶C（PKC）蛋白表达（Western Blot）,对IVIM参数和肿瘤蛋白表达情况进行相关分析。结果 不耐药组D较耐药组增高（P<0.05）,2组D^*和f差异无统计学意义（P均>0.05）。2组肿瘤组织坏死区域范围相似;耐药组细胞核较不耐药组增大,且细胞间排列更紧密,细胞密度较大。2组肿瘤细胞凋亡指数差异无统计学意义（P>0.05）。耐药组PKC、P-gp、MRP1蛋白表达均较不耐药组增加（P均<0.05）。肿瘤D值与P-gp、MRP1、PKC表达均呈负相关（P均<0.05）。结论 IVIM成像参数D值可能成为评估小鼠人类结肠癌SW480耐药性的指标。