Mixed responses to first-line alectinib in non-small cell lung cancer patients with rare ALK gene fusions: A case series and literature review

Anaplastic lymphoma kinase (ALK) fusion is a well-defined biomarker for ALK tyrosine kinase inhibitors (TKIs) treatment in non-small cell lung cancer (NSCLC). Alectinib, a second-generation ALK-TKI, has been shown to have significantly longer progression-free survival (PFS) than first-generation ALK inhibitors in untreated ALK-rearranged NSCLC patients. However, its clinical efficacy on rare ALK fusions remains unclear. Herein, two advanced NSCLC patients received first-line alectinib treatment, given their positive ALK fusion status as determined by immunohistochemistry (IHC) testing results. Patients showed limited clinical response (PFS: 4 months) and primary resistance to alectinib respectively. Molecular profiling using next-generation sequencing (NGS) further revealed a striatin (STRN)-ALK fusion in the first patient accompanied by MET amplification, and a LIM domain only protein 7 (LMO7)-ALK fusion in another patient without any other known oncogenic alterations. Both patients demonstrated improved survival after they switched to second-line crizotinib (PFS: 11 months) and ensartinib (PFS: 18 months), respectively, up till the last follow-up assessment. In conclusion, the clinical efficacy of ALK-TKIs including alectinib for lung cancer with uncommon ALK gene fusions is still under evaluation. This study and literature review results showed mixed responses to alectinib in NSCLC patients who harboured rare ALK fusions. Comprehensive molecular profiling of tumour is thus strongly warranted for precise treatment strategies.     

Molecular Characteristics and Clinical Outcomes of EGFR Exon 19 C-Helix Deletion in Non–Small Cell Lung Cancer and Response to EGFR TKIs

Epidermal growth factor receptor (EGFR) exon 19 deletion (E19del) is the most common activating mutation in advanced non–small cell lung cancer (NSCLC) and associates with the sensitivity of EGFR tyrosine kinase inhibitors (TKIs) treatment. However, not all mutant patterns of E19del have been well studied for the limited coverage of regular EGFR mutation testing. Here, we performed a retrospective cohort study of the C-helix E19del in advanced NSCLC patients based on the screening data by the next-generation sequencing (NGS) platform. From May 2012 to December 2019, clinical information and specimen from 7544 consecutive advanced (IIIB/IV) NSCLC patients were collected and screened for EGFR gene mutations by NGS from multicenters in China. The molecular characteristics and responsiveness to first-line EGFR TKIs therapy in NSCLC patients with C-helix E19del were analyzed. The clinical characteristics were also compared between patients with classical E19del and C-helix E19del. Thirty-eight (2.6%) patients with C-helix E19del and 1400 (97.4%) patients with classical E19dels were identified from 1438 patients with E19del. No significant difference in clinical characteristics was observed between the C-helix E19del and classical E19del groups (P > .05), except for histology (P < .001). All 22 patients with C-helix E19del as p.S752_I759del, p.A750_E758del, p.A750_E758delinsP, p.T751_A755delinsNY, p.T751_I759delinsG, p.T751_I759delinsLD, p.T751_I759delinsN, p.T751_L760delinsNL, and p.T751_D761delinsLY reached the best response as partial response rate (72.7%), and the progression-free survival (PFS) was 12.0 months. The PFS after EGFR TKIs in patients with C-helix E19del tended to be longer than patients with classical E19del but has no statistical significance (12.0 months vs 8.5 months, P = .06). The C-helix E19del could be a positive biomarker for predicting response to EGFR TKIs in advanced NSCLC patients. NGS should be the appropriate platform to identify this rare population, especially when patients harbor no actionable driver mutation initially and are reluctant to accept chemotherapy as first-line therapy.     

Elevated Hsp90-beta contributes to differential diagnosis of pleural effusion caused by lung cancer and correlates with malignant biological behavior of lung cancer

Background

Hsp90-beta has been investigated to be correlated with the occurrence and development of tumor. The intention of this research was to test the level of Hsp90-beta in malignant pleural effusion (MPE) of patients with lung cancer and disclose the clinical significance of Hsp90-beta as a potential tumor marker for differential diagnosis of pleural effusion caused by lung cancer.

Methods

The level of Hsp90-beta was determined using enzyme-linked immunosorbent assay. Calculations of the Hsp90-beta threshold, the sensitivity and specificity for distinguishing MPE from benign pleural effusion were performed using receiver operator characteristic curve.

Results

The level of Hsp90-beta in MPE of lung cancer patients was higher than that in control individuals (P <?0.05) and increased MPE Hsp90-beta was correlated with the pathological differentiation, tumor size and lymphatic metastasis (P <?0.05). The cutoff value of Hsp90-beta produced by receiver operator characteristic curve for distinguishing lung cancer from control individuals were 1.659?ng/mL and the sensitivity and specificity were 93.46 and 79%.

Conclusions

Increased Hsp90-beta in MPE was correlated with malignant biological behavior of lung cancer patients, indicating that the level of Hsp90-beta could be a tool of referential value for differential diagnosis of pleural effusion caused by lung cancer.

     

DNA damage of tumor-associated lymphocytes and total antioxidant capacity in cancerous patients

The purpose of this study was to assess DNA damage of tumor-associated lymphocytes (TALs) in malignant pleural effusion (MPE), the total antioxidant capacity (TAC) of plasma and MPE from patients with carcinoma, and DNA repair effect of melatonin. TAC of plasma was measured in 28 cancer patients with MPE and in 33 healthy persons, and also TAC of MPE supernatant was measured in these patients. DNA damages of peripheral blood mononuclear cells (PBMCs) and of TALs were assessed using comet assay. The TAC of plasma was remarkably lower in cancer patients (8.41+/-1.78 U/ml) than that in healthy persons (10.52+/-1.64 U/ml, P<0.001). The TAC of MPE supernatant (6.34+/-1.57 U/ml) was significantly lower than that of plasma in cancer patients (8.41+/-1.78 U/ml, P<0.001). The comet percentage of PBMCs was higher in cancer patients (16.8+/-7.9) than that in healthy persons (10.4+/-4.9, P<0.01). Within cancer patients, the comet percentage of TALs (41.9+/-11.7) was significantly higher than that of PBMCs (16.8+/-7.9, P<0.001). A negative correlation was observed between the TAC of MPE supernatant and the comet percentage of TALs in patients (r=-0.538, P<0.01). After treatment with melatonin, comet percentage of TALs declined significantly from 42.6+/-12.8 to 27.1+/-9.9 (P<0.001). These data show that lower TAC of MPE supernatant may be related to higher degree of DNA damage of TALs and that melatonin may facilitate the repair of the damaged DNA.     

Cell-Free MicroRNA Expression Profiles in Malignant Effusion Associated with Patient Survival in Non-Small Cell Lung Cancer

Objective

MicroRNAs (miRNAs) expression is altered in cancer cells, and miRNAs could serve as diagnostic and prognostic biomarker for cancer patients. This study was designed to analyze circulating miRNAs expression in the malignant pleural effusion (MPE) and their association with patient survival in non-small cell lung cancer (NSCLC).

Methods

Pleural effusion from 184 patients with NSCLC and MPE were collected. MiRNA microarray and bioinformatics interpretation were used to evaluate miRNA expression profiles in 10 NSCLC patients with different survival prognosis. Associations were validated in 184 patients (randomly classified into training and validation set with equal number in each group) using quantitative RT-PCR. Risk scores were formulated based on the expression signature of miRNAs. Clinical data, such as patient survival, were collected for correlation analysis.

Results

Thirty-three miRNAs were found to be altered more than two-fold by microarray in malignant effusions between longer-survival and shorter-survival groups, and levels of five miRNAs (miRNA-93, miRNA-100, miRNA-134, miRNA-151 and miRNA-345) were significantly associated with overall survival. High expression of miR-100 and low expression of miRNA-93, miRNA-134, miRNA-151 and miRNA-345 were associated with poor survival in both the training and validation cohort. Patients with high risk scores had overall poor survival compared to the patients with low risk scores. Risk score was an independent predictor of patient survival.

Conclusions

Expression patterns of miRNAs are systematically altered in MPE of patient with NSCLC. The five miRNA signature from the effusion may serve as a predictor for the overall survival of patients with lung cancers.     

The anti-myeloma activity of a novel purine scaffold HSP90 inhibitor PU-H71 is via inhibition of both HSP90A and HSP90B1

Background

The ribonucleotide reductase M1 (RRM1) gene encodes the regulatory subunit of ribonucleotide reductase, the molecular target of gemcitabine. The overexpression of RRM1 mRNA in tumor tissues is reported to be associated with gemcitabine resistance. Thus, single nucleotide polymorphisms (SNPs) of the RRM1 gene are potential biomarkers of the response to gemcitabine chemotherapy. We investigated whether RRM1 expression in peripheral blood mononuclear cells (PBMCs) or SNPs were associated with clinical outcome after gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients.

Methods

PBMC samples were obtained from 62 stage IIIB and IV patients treated with gemcitabine-based chemotherapy. RRM1 mRNA expression levels were assessed by real-time PCR. Three RRM1 SNPs, -37C→A, 2455A→G and 2464G→A, were assessed by direct sequencing.

Results

RRM1 expression was detectable in 57 PBMC samples, and SNPs were sequenced in 56 samples. The overall response rate to gemcitabine was 18%; there was no significant association between RRM1 mRNA expression and response rate (P = 0.560). The median progression-free survival (PFS) was 23.3 weeks in the lower expression group and 26.9 weeks in the higher expression group (P = 0.659). For the -37C→A polymorphism, the median PFS was 30.7 weeks in the C(-)37A group, 24.7 weeks in the A(-)37A group, and 23.3 weeks in the C(-)37C group (P = 0.043). No significant difference in PFS was observed for the SNP 2455A→G or 2464G→A.

Conclusions

The RRM1 polymorphism -37C→A correlated with PFS in NSCLC patients treated with gemcitabine-based chemotherapy. No significant correlation was found between PBMC RRM1 mRNA expression and the efficacy of gemcitabine.     

A Combined test using both cell sediment and supernatant cell‐free DNA in pleural effusion shows increased sensitivity in detecting activating EGFR mutation in lung cancer patients

Introduction

The aim of this study was to examine whether a combined test using both cell sediment and supernatant cytology cell‐free DNA (ccfDNA) is more useful in detecting EGFR mutation than using cell sediment DNA or supernatant ccfDNA alone in pleural effusion of lung cancer patients.

Methods

A total of 74 lung adenocarcinoma patients with paired samples between primary tumour and corresponding metastatic tumour with both cell sediment and supernatant ccfDNA of pleural effusion cytology were enrolled in this study. Cell sediment and supernatant ccfDNA were analysed separately for EGFR mutations by polymerase chain reaction.

Results

Out of 45 patients with mutant EGFR in primary tumours, EGFR mutations were detected in 23 cell sediments of corresponding metastases (sensitivity; 51.1%) and 20 supernatant ccfDNA corresponding metastases (sensitivity; 44.4%). By contrast, the combined test detected EGFR mutations in 27 corresponding metastases (sensitivity; 60.0%), and had a higher sensitivity than the cell sediment or the supernatant ccfDNA alone (P < .05). Out of 45 patients with mutant EGFR, 24, three and 18 were cytologically diagnosed as positive, atypical or negative, respectively. The detection rate in the combined test was highest (95.8%) in the positive group, and mutant EGFR was also detected in four of 18 samples (22.2%) in the negative group.

Conclusions

A combined test using both cell sediment DNA and supernatant ccfDNA samples increases the concordance rate of EGFR mutations between primary tumour and corresponding metastases. Our findings indicate that supernatant ccfDNA is useful even in cases where the cytological diagnosis is negative.     

Prognostic value of neuron-specific enolase in patients with advanced and metastatic non-neuroendocrine non-small cell lung cancer

Background: Increased serum neuron-specific enolase (NSE) level was found in a substantial proportion (30–69%) of patients with non-small-cell lung cancer (NSCLC), but little was known about the clinical properties of NSE in NSCLC.Objective: We aimed to assess the level of serum NSE to predict prognosis and treatment response in patients with advanced or metastatic non-neuroendocrine NSCLC.Methods: We retrospectively analyzed 363 patients with advanced and metastatic NSCLC between January 2011 and October 2016. The serum NSE level was measured before initiation of treatment.Results: Patients with high NSE level (≥26.1 ng/ml) showed significantly shorter progression-free survival (PFS) (5.69 vs 8.09 months; P=0.02) and significantly shorter overall survival (OS) than patients with low NSE level (11.41 vs 24.31 months; P=0.01).NSE level was an independent prognostic factor for short PFS (univariate analysis, hazard ratio [HR] = 2.40 (1.71–3.38), P<0.001; multivariate analysis, [HR] = 1.81 (1.28–2.56), P=0.001) and OS (univariate analysis, [HR] = 2.40 (1.71–3.37), P<0.001; multivariate analysis, [HR] = 1.76 (1.24–2.50), P=0.002).Conclusion: The survival of NSCLC patients with high serum NSE level was shorter than that of NSCLC patients with low serum NSE levels. Serum NSE level was a predictor of treatment response and an independent prognostic factor.     

Percutaneous Fine-Needle 5% Ethanol-Cisplatin Intratumoral Injection Combined with Second-Line Chemotherapy Improves On the Standard of Care in Patients with Platinum-Pretreated Stage IV Non–Small Cell Lung Cancer

BACKGROUND: Efficacy of second-line chemotherapy in platinum-pretreated non–small cell lung cancer (NSCLC) is poor. This study investigated efficacy of computed tomography–guided percutaneous fine-needle 5% ethanol-cisplatin intratumoral injection (CT-PFNECII) combined with second-line chemotherapy in patients with platinum-pretreated stage IV NSCLC. PATIENTS: Between October 2011 and July 2013, 34 eligible patients were randomly assigned to receive either CT-PFNECII combined with second-line chemotherapy (combination group, n = 17) or second-line chemotherapy alone (chemotherapy group, n = 17). The primary end points were the proportions of patients who achieved an overall response rate (ORR) and disease control rate (DCR). Secondary end points were median survival and progression-free survival (PFS). RESULTS: The ORR and DCR in the combination group were significantly higher than in the chemotherapy group (23.53% vs 11.76% for ORR, P < .01; and 58.82% vs 35.29% for DCR, P < .01). Compared with patients in the chemotherapy group, patients in the combination group had significantly longer PFS (5.4 months vs 3.0 months, P < .01) and median survival (9.5 months vs 5.3 months, P < .01). CONCLUSIONS: CT-PFNECII combined with second-line chemotherapy provided a higher response rate and improved survival than second-line chemotherapy for patients with platinum-pretreated stage IV NSCLC.     

Relative expressions of miR-205-5p, miR-205-3p, and miR-21 in tissues and serum of non-small cell lung cancer patients

Objective was to assess and compare the relative expressions of miR-205-5p, miR-205-3p, and miR-21-3p in tissues and serum among non-small cell lung carcinoma (NSCLC) patients, benign pulmonary conditions patients, and healthy volunteers. Serum samples were obtained between October 2011 and September 2012 from 20 NSCLC patients undergoing surgical treatment, 20 patients diagnosed with a benign lung disease (pulmonary tuberculosis, pneumonia, chronic obstructive pulmonary disease, or interstitial pneumonia) (lesion group), and 20 healthy volunteers (control group). NSCLC patients provided cancer tissues and cancer-adjacent normal tissues during surgery (paired specimens). Quantitative RT-PCR was used to assess miR-205-5p, miR-205-3p, and miR-21-3p expressions in serum and tissue samples. The relative expressions of miR-205-5p and miR-205-3p were significantly higher in NSCLC tissues compared with cancer-adjacent paired specimens (both P < 0.001). In the serum, significantly higher miR-205-5p, miR-205-3p, and miR-21-3p relative expressions were observed in the NSCLC group compared with the two other groups (all P < 0.001). The relative expressions of miR-205-5p and miR-21-3p in NSCLC tissues and serum were significantly correlated (r = 0.553, P = 0.011; and r = ?0.541, P = 0.014, respectively), while no significant correlation was observed for miR-205-3P (P = 0.120). Expressions of miR-205-5p and miR-205-3P in squamous cell carcinoma specimens were significantly higher than in lung adenocarcinoma specimens (both P = 0.001). Similarly, higher serum miR-205-5p and miR-205-3p levels were observed in squamous cell carcinoma patients (P = 0.033 and P = 0.002, respectively). In this preliminary and novel study, miR-205-5p was more useful as a marker for NSCLC than miR-205-3p or miR-21, indicating a potential for future applications in NSCLC diagnosis and prognosis.     

Prognosis of ALK-rearranged non-small-cell lung cancer patients carrying TP53 mutations

Non-small-cell lung cancer (NSCLC) is the primary cause of cancer-related death. Gene rearrangements involving the anaplastic lymphoma kinase (ALK) tyrosine kinase identify a clinical and molecular subset of NSCLC patients, who benefit from the monotherapy with ALK tyrosine kinase inhibitors. Nonetheless, responsiveness to TKIs and prognosis of these patients are influenced by several factors, including resistance mechanisms and mutations affecting genes involved in key molecular pathways of cancer cells. In a cohort of 98 NSCLC patients with ALK gene rearrangements, we investigated the role of Tumor Protein (TP53) gene mutations in predicting patients prognosis. TP53 mutations were evaluated in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS).Results: In patients with available clinical and TP53 mutation information, we found that 13 patients (20.3%) were affected by TP53 mutations. Considered together, even though showing a trend, TP53 mutations were not associated with PFS and OS. Considering the different TP53 mutations by functionality in terms of disruptive and non-disruptive mutations, we observed that TP53 non-disruptive mutations were able to predict worse OS in the overall case series. Moreover, a worse PFS was seen in the subgroup of patients with TP53 non-disruptive mutation, in first-, second-, and third line of treatment. Our results show that mutations affecting TP53 gene, especially non-disruptive mutations, are able to affect prognosis of ALK-rearranged NSCLC patients.     

Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide. Liquid biopsy by a blood draw to detect circulating tumor cells (CTCs) is a tool for molecular profiling of cancer using single-cell and next-generation sequencing (NGS) technologies. The aim of the study was to identify somatic variants in single CTCs isolated from NSCLC patients by targeted NGS. Thirty-one subjects (20 NSCLC patients, 11 smokers without cancer) were enrolled for blood draws (7.5 mL). CTCs were identified by immunofluorescence, individually retrieved, and DNA-extracted. Targeted NGS was performed to detect somatic variants (single-nucleotide variants (SNVs) and insertions/deletions (Indels)) across 65 oncogenes and tumor suppressor genes. Cancer-associated variants were classified using OncoKB database. NSCLC patients had significantly higher CTC counts than control smokers (p = 0.0132; Mann–Whitney test). Analyzing 23 CTCs and 13 white blood cells across seven patients revealed a total of 644 somatic variants that occurred in all CTCs within the same subject, ranging from 1 to 137 per patient. The highest number of variants detected in ≥1 CTC within a patient was 441. A total of 18/65 (27.7%) genes were highly mutated. Mutations with oncogenic impact were identified in functional domains of seven oncogenes/tumor suppressor genes (NF1, PTCH1, TP53, SMARCB1, SMAD4, KRAS, and ERBB2). Single CTC-targeted NGS detects heterogeneous and shared mutational signatures within and between NSCLC patients. CTC single-cell genomics have potential for integration in NSCLC precision oncology.     

KIT (CD117) Expression in a Subset of Non-Small Cell Lung Carcinoma (NSCLC) Patients

We have previously described the expression of CD44, CD90, CD117 and CD133 in NSCLC tumors, adjacent normal lung, and malignant pleural effusions (MPE). Here we describe the unique subset of tumors expressing CD117 (KIT), a potential therapeutic target. Tumor and adjacent tissue were collected from 58 patients. Six MPE were obtained before therapy. Tissue was paraffin embedded for immunofluorescent microscopy, disaggregated and stained for flow cytometry or cryopreserved for later culture. The effect of imatinib on CD117^high/KIT+ tumors was determined on first passage cells; absolute cell counts and flow cytometry were readouts for drug sensitivity of cell subsets. Primary tumors divided into KIT^neg and KIT⁺ by immunofluorescence. By more sensitive flow cytometric analysis, CD117+ cytokeratin+ cells were detected in all tissues (1.1% of cytokeratin+ cells in normal lung, 1.29% in KIT “negative” tumors, 40.7% in KIT⁺ tumors, and 0.4% in MPE). In KIT⁺/CD117^high, but not KIT⁺/CD117^low tumors, CD117 was overexpressed 3.1-fold compared to normal lung. Primary cultures of CD117^high tumors were sensitive to imatinib (5 µM) in short term culture. We conclude that NSCLC tumors divide into CD117^low and CD117^high. Overexpression of CD117 in CD117^high NSCLC supports exploring KIT as a therapeutic target in this subset of patients.     

In-depth Proteomic Analysis of Six Types of Exudative Pleural Effusions for Nonsmall Cell Lung Cancer Biomarker Discovery

Pleural effusion (PE), a tumor-proximal body fluid, may be a promising source for biomarker discovery in human cancers. Because a variety of pathological conditions can lead to PE, characterization of the relative PE proteomic profiles from different types of PEs would accelerate discovery of potential PE biomarkers specifically used to diagnose pulmonary disorders. Using quantitative proteomic approaches, we identified 772 nonredundant proteins from six types of exudative PEs, including three malignant PEs (MPE, from lung, breast, and gastric cancers), one lung cancer paramalignant PE, and two benign diseases (tuberculosis and pneumonia). Spectral counting was utilized to semiquantify PE protein levels. Principal component analysis, hierarchical clustering, and Gene Ontology of cellular process analyses revealed differential levels and functional profiling of proteins in each type of PE. We identified 30 candidate proteins with twofold higher levels (q<0.05) in lung cancer MPEs than in the two benign PEs. Three potential markers, MET, DPP4, and PTPRF, were further verified by ELISA using 345 PE samples. The protein levels of these potential biomarkers were significantly higher in lung cancer MPE than in benign diseases or lung cancer paramalignant PE. The area under the receiver-operator characteristic curve for three combined biomarkers in discriminating lung cancer MPE from benign diseases was 0.903. We also observed that the PE protein levels were more clearly discriminated in effusions in which the cytological examination was positive and that they would be useful in rescuing the false negative of cytological examination in diagnosis of nonsmall cell lung cancer-MPE. Western blotting analysis further demonstrated that MET overexpression in lung cancer cells would contribute to the elevation of soluble MET in MPE. Our results collectively demonstrate the utility of label-free quantitative proteomic approaches in establishing differential PE proteomes and provide a new database of proteins that can be used to facilitate identification of pulmonary disorder-related biomarkers.The lungs are covered by parietal and visceral pleural membranes, including a small amount of fluid (10–20 ml) in the pleural cavity that helps the lungs expand and contract smoothly. Pleural effusions (PE)¹, an accumulation of pleural fluid, contain proteins originating from the plasma filtrate and are released by inflammatory or epithelial cells. PE is triggered by a variety of etiologies, including malignancies and benign diseases such as pneumonia (PN), tuberculosis (TB), pulmonary embolism, heart failure, renal dysfunction, and autoimmune disease (1). Based on their biochemical characteristics, PEs are classified as transudative or exudative; determination of the PE type is a crucial step in the differential diagnosis and management of PEs. Transudative effusions, generally caused by systemic diseases, can be effectively distinguished from exudative PEs using the established modified Light''s criteria (2, 3). However, further discrimination among different exudate types such as malignant and nonmalignant effusions (e.g. paramalignancies or acute and chronic inflammatory diseases) is sometimes diagnostically challenging because of similar biochemical and/or cellular profiles. For example, neutrophil-rich fluid is generally observed in patients with bacterial PN whereas lymphocytic effusions are generally observed in cancer or chronic inflammatory diseases such as TB (4).PEs caused by cancer are generally divided into two categories, malignant (MPE) and paramalignant (PMPE). MPEs result when cancer cells metastasize to the pleural cavity (stage IV), wherein exfoliated malignant cells are observed in pleural fluid by cytological examination or detected in percutaneous pleural biopsy, thoracoscopy, thoracotomy, or at autopsy (5). PMPE occurs in cancer patients with no evidence of tumor invasion in the pleural space and may be caused by airway obstruction with lung collapse, lymphatic obstruction, or the systemic effects of cancer treatment (5). A high percentage of MPEs (>75%) arise from lung, breast, and ovarian cancer or lymphoma/leukemia. Lung cancer is a major etiology underlying MPE (6); however, only ∼40–87% patients with MPE can be accurately diagnosed upon initial examination (7). Inaccurate diagnosis of MPE and PMPE underestimates or overestimates the disease stage and leads to inappropriate therapy. Thus, it is important to identify a specific and powerful biomarker to distinguish MPE from benign diseases and PMPE.Notably, tumor-proximal body fluids are promising sources for biomarker discovery because they represent a reservoir of in vivo tumor-secreted proteins without a large dynamic range or complexity of plasma or serum (8). Tumor-proximal fluids include PEs, nipple aspirate, stool, saliva, lavage, and ascites fluid. Previously, we utilized the powerful analytical capability of high-abundance protein depletion followed by one-dimensional SDS-PAGE combined with nano-LC-MS/MS (GeLC-MS/MS) for biomarker discovery to generate a comprehensive MPE proteome data set from 13 pooled nonsmall cell lung cancer (NSCLC) patients (9). Because a variety of pathological conditions can lead to exudative effusions, generating different PE proteomic profiles would accelerate discovery of potential PE biomarkers that can be used to discriminate between malignant and nonmalignant pulmonary disorders. The aim of this study is to establish differential PE proteomes from six types of exudative PEs, including three MPEs (from NSCLC, breast, and gastric cancers), one PMPE from NSCLC, and two benign diseases (TB and PN), using a label-free semiquantitative proteomics approach. Our results were verified by clinical validation of three potential biomarkers using an enzyme-linked immunosorbent assay (ELISA; Fig. 1).Open in a separate windowFig. 1.Biomarker discovery strategy for identifying differentially expressed proteins from six pleural effusion (PE) types. The strategy comprised prefractionation by removal of high-abundance proteins, GeLC-MS/MS, comparative analysis of the six PE proteomes based on spectral counts, proteome clustering, functional classification of differentially expressed proteins, and selection and validation of biomarker candidates by ELISA.     

Expression Profile of Three Splicing Factors in Pleural Cells Based on the Underlying Etiology and Its Clinical Values in Patients with Pleural Effusion

Splicing factors (SFs) are involved in oncogenesis or immune modulation, the common underlying processes giving rise to pleural effusion (PE). The expression profiles of three SFs (HNRNPA1, SRSF1, and SRSF3) and their clinical values have never been assessed in PE. The three SFs (in pellets of PE) and conventional tumor markers were analyzed using PE samples in patients with PE (N = 336). The sum of higher–molecular weight (Mw) forms of HNRNPA1 (Sum-HMws-HNRNPA1) and SRSF1 (Sum-HMws-SRSF1) and SRSF3 levels were upregulated in malignant PE (MPE) compared to benign PE (BPE); they were highest in cytology-positive MPE, followed by tuberculous PE and parapneumonic PE. Meanwhile, the lowest-Mw HNRNPA1 (LMw-HNRNPA1) and SRSF1 (LMw-SRSF1) levels were not upregulated in MPE. Sum-HMws-HNRNPA1, Sum-HMws-SRSF1, and SRSF3, but neither LMw-HNRNPA1 nor LMw-SRSF1, showed positive correlations with cancer cell percentages in MPE. The detection accuracy for MPE was high in the order of carcinoembryonic antigen (CEA, 85%), Sum-HMws-HNRNPA1 (76%), Sum-HMws-SRSF1 (68%), SRSF3, cytokeratin-19 fragments (CYFRA 21-1), LMw-HNRNPA1, and LMw-SRSF1. Sum-HMws-HNRNPA1 detected more than half of the MPE cases that were undetected by cytology and CEA. Sum-HMws-HNRNPA1, but not other SFs or conventional tumor markers, showed an association with longer overall survival among patients with MPE receiving chemotherapy. Our results demonstrated different levels of the three SFs with their Mw-specific profiles depending on the etiology of PE. We suggest that Sum-HMws-HNRNPA1 is a supplementary diagnostic marker for MPE and a favorable prognostic indicator for patients with MPE receiving chemotherapy.     

Peripheral Blood for Epidermal Growth Factor Receptor Mutation Detection in Non-Small Cell Lung Cancer Patients

OBJECTIVE: It is important to analyze and track Epidermal Growth Factor Receptor (EGFR) mutation status for predicting efficacy and monitoring resistance throughout EGFR-tyrosine kinase inhibitors (TKIs) treatment in non-small cell lung cancer (NSCLC) patients. The objective of this study was to determine the feasibility and predictive utility of EGFR mutation detection in peripheral blood. METHODS: Plasma, serum and tumor tissue samples from 164 NSCLC patients were assessed for EGFR mutations using Amplification Refractory Mutation System (ARMS). RESULTS: Compared with matched tumor tissue, the concordance rate of EGFR mutation status in plasma and serum was 73.6% and 66.3%, respectively. ARMS for EGFR mutation detection in blood showed low sensitivity (plasma, 48.2%; serum, 39.6%) but high specificity (plasma, 95.4%; serum, 95.5%). Treated with EGFR-TKIs, patients with EGFR mutations in blood had significantly higher objective response rate (ORR) and insignificantly longer progression-free survival (PFS) than those without mutations (ORR: plasma, 68.4% versus 38.9%, P = 0.037; serum, 75.0% versus 39.5%, P = 0.017; PFS: plasma, 7.9 months versus 6.1 months, P = 0.953; serum, 7.9 months versus 5.7 months, P = 0.889). In patients with mutant tumors, those without EGFR mutations in blood tended to have prolonged PFS than patients with mutations (19.7 months versus 11.0 months, P = 0.102). CONCLUSIONS: EGFR mutations detected in blood may be highly predictive of identical mutations in corresponding tumor, as well as showing correlations with tumor response and survival benefit from EGFR-TKIs. Therefore, blood for EGFR mutation detection may allow NSCLC patients with unavailable or insufficient tumor tissue the opportunity to benefit from personalized treatment. However, due to the high false negative rate in blood samples, analysis for EGFR mutations in tumor tissue remains the gold standard.     

Classification of Epidermal Growth Factor Receptor Gene Mutation Status Using Serum Proteomic Profiling Predicts Tumor Response in Patients with Stage IIIB or IV Non-Small-Cell Lung Cancer

Objectives

Epidermal growth factor receptor (EGFR) gene mutations in tumors predict tumor response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). However, obtaining tumor tissue for mutation analysis is challenging. Here, we aimed to detect serum peptides/proteins associated with EGFR gene mutation status, and test whether a classification algorithm based on serum proteomic profiling could be developed to analyze EGFR gene mutation status to aid therapeutic decision-making.

Patients and Methods

Serum collected from 223 stage IIIB or IV NSCLC patients with known EGFR gene mutation status in their tumors prior to therapy was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ClinProTools software. Differences in serum peptides/proteins between patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes were detected in a training group of 100 patients; based on this analysis, a serum proteomic classification algorithm was developed to classify EGFR gene mutation status and tested in an independent validation group of 123 patients. The correlation between EGFR gene mutation status, as identified with the serum proteomic classifier and response to EGFR-TKIs was analyzed.

Results

Nine peptide/protein peaks were significantly different between NSCLC patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes in the training group. A genetic algorithm model consisting of five peptides/proteins (m/z 4092.4, 4585.05, 1365.1, 4643.49 and 4438.43) was developed from the training group to separate patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes. The classifier exhibited a sensitivity of 84.6% and a specificity of 77.5% in the validation group. In the 81 patients from the validation group treated with EGFR-TKIs, 28 (59.6%) of 47 patients whose matched samples were labeled as “mutant” by the classifier and 3 (8.8%) of 34 patients whose matched samples were labeled as “wild” achieved an objective response (p<0.0001). Patients whose matched samples were labeled as “mutant” by the classifier had a significantly longer progression-free survival (PFS) than patients whose matched samples were labeled as “wild” (p=0.001).

Conclusion

Peptides/proteins related to EGFR gene mutation status were found in the serum. Classification of EGFR gene mutation status using the serum proteomic classifier established in the present study in patients with stage IIIB or IV NSCLC is feasible and may predict tumor response to EGFR-TKIs.     

Effectiveness and Safety of Chemotherapy Combined with Dendritic Cells Co-Cultured with Cytokine-Induced Killer Cells in the Treatment of Advanced Non-Small-Cell Lung Cancer: A Systematic Review and Meta-Analysis

Background

Lung cancer, particularly non-small-cell lung cancer (NSCLC) is the leading cause of cancer mortality. Chemotherapy combined dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK) immunotherapy has been applied in advanced NSCLC patients'' treatment, but couldn''t provide consistent beneficial results. Therefore, it is necessary to evaluate the efficiency and safety of combination therapy to promote the application.

Methods

A literature search for randomized controlled trials of NSCLC was conducted in PubMed database. Before meta-analysis was performed, studies were evaluated heterogeneity. Pooled risk ratios (RRs) were estimated and 95% confidence intervals (CIs) were calculated using a fixed-effect model. Sensitivity analysis was also performed.

Results

Six eligible trials were enrolled. Efficiency and safety of chemotherapy followed by DC-CIK immunotherapy (experimental group) and chemotherapy alone (control group) were compared. 1-year overall survival (OS) (P = 0.02) and progression free survival (PFS) (P = 0.005) in the experimental group were significantly increased compared with the control. Disease control rate (DCR) (P = 0.006) rose significantly in experimental group. However, no significant differences between the two groups were observed in 2-year OS (P = 0.21), 2-year PFS (P = 0.10), overall response rate (ORR) (P = 0.76) and partial response (PR) (P = 0.22). Temporary fever, anemia, leukopenia and nausea were the four major adverse events (AEs) treated by chemotherapy. The incidence of anemia, leukopenia and nausea in the experimental group was obviously lower than the control group. Temporary fever rate was higher in experimental group than that in the control, but could be alleviated by taking sufficient rest.

Conclusions

Chemotherapy combined with DC-CIK immunotherapy showed superiority in DCR, 1-year OS and PFS, and no more AEs appeared, however, there was no significant improvement in ORR, PR, 2-year OS and PFS. As a whole, the combination therapy is safer but modest in efficacy for advanced NSCLC patients.