Lung Cancer Biopsies Research Articles

MicroRNA-935 is reduced in non-small cell lung cancer tissue, is linked to poor outcome, and acts on signal transduction mediator E2F7 and the AKT pathway

ABSTRACTBackground: A potential role for microRNA-935 (miR-935) has been identified in several cancers but not in non-small cell lung cancer (NSCLC). We hypothesised changes in miR-935 in NSCLC, and proposed mechanisms that may further explain its role in carcinogenesis.Methods: NSCLC tissue and nearby normal tissue was obtained from 101 patients and was probed by qRT-PCR for miR-935 expression. The role of miR-935 and a potential target (signal transduction factor E2F7) was determined in cell lines by a dual luciferase assay. The function of miR-935 was investigated through metabolic activity (MTT) and transwell migration assays. Western blot and immunocytochemical assays examined protein expression level. Growth of miR-935 transfected or untransfected cells was measured via xenograft tumour formation.Results: miR-935 was reduced in cancer tissue and was related to lymph node metastases, tumour node metastasis status and poor prognosis (all p < 0.02). In vitro, miR-935 suppressed cell proliferation, migration and invasion in NSCLC cells through targeting E2F7. Furthermore, E2F7 was upregulated in NSCLC tissue associated with poor prognosis (p = 0.0203) of NSCLC patients. miR-935 suppressed the epithelial-mesenchymal transition and AKT pathways in NSCLC and inhibited the tumour growth in vivo.Conclusion: Altered miR-935 in lung cancer biopsy tissue may be a diagnostic tool and could direct treatment. Involvement in carcinogenesis is implied by its suppression of the development of NSCLC via targeting E2F7 and inhibiting AKT pathway.

Success of genomic profiling of non-small cell lung cancer biopsies obtained by trans-thoracic percutaneous needle biopsy.

Genomic profiling for personalized targeted therapy has become standard of care. We report the success of genomic profiling of non-small cell lung cancer (NSCLC) obtained by trans-thoracic needle biopsy (TTNB) in a single center experience. Patients with NSCLC who underwent TTNB for genomic were identified. Pathology specimens were evaluated for tumor adequacy and then analyzed for selected exons of epidermal growth factor receptor, KRAS, BRAF, PIK3CA, and ERBB2. ALK rearrangements were detected with fluorescence in situ hybridization and/or immunohistochemistry. Technical success was recorded and the factors affecting successful profiling were evaluated. Complications (pneumothorax, hemorrhage, and admission) were recorded. Comparison of yield and complications were done between the two groups (core biopsy and fine needle aspiration only group). Utility of PET-CT to guide the needle track for optimized yield was assessed in a subset of patients. Between December 6, 2009, and December 30, 2016, 765 patients with NSCLC underwent TTNB. Five-hundred and seventy-seven of 765 (75%) of all TTNB were profiled, for genomic analysis. Five-hundred and eight of 577 (88%) were successfully profiled. The number of samples obtained ranged from 1 to 10 (1 to 2 cm,18 to 20 G). Lesions biopsied ranged in size from 0.6 to 16 cm. No statistically significant difference was observed in the incidence of pneumothorax between two groups (P = 0.26). PET guidance was not found to be statistically significant ( P = 0.79) in the overall yield. Computed tomographic guided TTNB is a safe and efficacious technique for genomic profiling, enables the acquisition of sufficient tissue for genetic mutation analyses allowing for personalized therapy with an acceptable complication rate.

Molecular Genetic Testing and Liquid Biopsy in Lung Cancer: Present and Future

Molecular Genetic Testing and Liquid Biopsy in Lung Cancer: Present and Future

9 Liquid biopsy in small cell lung cancer (SCLC)

IntroductionAnalysis of circulating cell-free DNA (cfDNA) and circulating tumour cells (CTCs) obtained from the peripheral blood of patients with cancer is a powerful and minimally invasive approach that allows the reconstruction of tumour genomes, with potential as a companion diagnostic for both patient stratification and monitoring. Here, we use cfDNA next-generation sequencing (NGS) analysis to assess and compare circulating tumour DNA (ctDNA) profiles in patients with SCLC.Material and methodsNGS libraries were prepared from cfDNA isolated from the pre-treatment peripheral blood samples from 69 patients and 32 cancer-free controls. Libraries were subjected to wholegenome sequencing (WGS) to establish genome wide copy number aberrations (CNA) as well as targeted mutation analysis of 110 genes frequently altered in SCLC. In addition CellSearch, an epitope dependent enrichment platform was used to enumerate CTCs from a parallel blood sample.Results and discussionsCNA patterns revealed SCLC associated changes including losses on chromosomes 3 p, 5q and 17 p and gains on chromosome 3q and 5 p as well as amplification of MYC in 21/69 (30%) and SOX2 in 36/69 (52%) as well losses on FHIT in 40/69 (58%), RASSFI in 38/69 (55%) and RB1 in 24/69 (35%) patients. CNA metrics enabled detection of tumour associated changes in 58/64 (84%) patients, Targeted NGS detected tumour associated mutations in 59/63 (94%) with TP53 mutations detected in 50 patients (85% of patients with any detectable ctDNA). CNA and somatic mutations were detected in both limited stage (LS-SCLC, confined to 1 hemithorax) and extensive stage (ES-SCLC, with distant metastases) with statistically significant differences seen for CNA metrics and variant allele frequencies of mutations consistent with higher levels of ctDNA in ES-SCLC. However, no significant differences between ES and LS were observed in the mutation patterns with respect to DNA damage repair, RAS and PI3K and transcriptional regulation pathways between the two stages of SCLC. There was considerable overlap between the detection of ctDNA and CTC counts, with cfDNA readouts detecting tumour related changes in 93% and CTCs detected only in 78% of patients.ConclusionWe have established sensitive methods for detecting ctDNA in SCLC and combined with CTC enumeration we have an effective liquid biopsy applicable to 98% of patients in this cohort. Future work will involve utilising this optimised NGS approach in an independent cohort of patients to correlate cfDNA metrics with clinical outcome.

PO-429 Identification of the right immunostimulatory chemotherapeutic partner for anti-CD70 immunotherapy in non-small cell lung cancer

IntroductionCD70 has emerged as a promising immunotherapeutic target in various haematological and solid malignancies. Its overexpression on tumour cells is associated with immunosuppression in the tumour microenvironment. We have previously demonstrated constitutive CD70 overexpression on tumour cells in a subset of primary non-small cell lung cancer (NSCLC) biopsies. More importantly, preliminary findings from our group demonstrated the ability of cisplatin to induce CD70 overexpression on NSCLC cells, which broadens the therapeutic window of anti-CD70 immunotherapy. On the other hand, certain chemotherapeutic agents can elicit immunostimulatory changes in the tumour microenvironment by triggering immunogenic cell death and enabling tumor-specific cytotoxic T cell responses. In this regard, strategically combining such a chemotherapeutic agent with anti-CD70 immunotherapy has the potential to achieve enhanced anti-tumour effects.Material and methodsThe immunostimulatory potential of different chemotherapeutics (cisplatin, oxaliplatin, cyclophosphamide, docetaxel and carboplatin) was evaluated on several NSCLC cell lines, varying in genetic aberrations, by assessing the most important hallmarks of immunogenic cell death: IFNα and HMGB1 production (ELISA), membrane calreticulin expression (flow cytometry) and ATP secretion (bioluminescence). Furthermore, the potential of these different chemotherapeutics to induce CD70 overexpression on NSCLC cells was explored (flow cytometry).Results and discussionsCytotoxicity experiments demonstrated different mechanisms of action for all chemotherapeutics with divergent sensitivity to NSCLC cell lines. At least three out of five chemotherapeutic agents demonstrated ATP secretion and ecto-calreticulin induction on various NSCLC cell lines. In addition, initial experiments showed the highest induction of CD70 expression on NSCLC cells upon low doses of cisplatin and carboplatin. In vitro data on the immunogenic effects of clinically relevant combination strategies in different cell lines are currently being analysed.ConclusionThese in vitro results demonstrate the immunostimulatory effects of clinically relevant chemotherapeutics, making it worthwhile to further investigate the potential of a novel combination strategy of chemotherapy and anti-CD70 immunotherapy in NSCLC.

Serum circulating cell free DNA as potential diagnostic and prognostic biomarker in non small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) is leading cause of cancer related death and the survival rate for patients with NSCLC remain poor so early diagnosis of NSCLC represents the best opportunity for cure. Cell-free DNA (cf-DNA) is extracellular nucleic acids found in cell-free plasma/serum of humans, given the recent approval of a liquid biopsy in lung cancer, the use of circulating tumor DNA as a novel non-invasive diagnostic and prognostic biomarker is promising. ObjectivesStudying whether the concentrations of circulating Cell Free DNA in serum can be used as a diagnostic and prognostic biomarker for NSCLC patients. MethodThis study was carried out on 140 subjects included 60 patients with non small cell lung cancer,40 patients with Chronic Obstructive Pulmonary Disease (COPD) and 40 healthy controls. Quantitative analysis of serum circulating cf-DNA was done b y AlU-based quantitative real time PCR. Serum level of CEA was measured by ELISA. ResultsNSCLC patients demonstrated significantly higher values of each of ALU 215, ALU 247, and DNA integrity than both COPD patients and controls. On ROC curve analysis, the total accuracy of ALU 247, ALU 115, DNA integrity (92.1%, 83.6%, 56.4%) at cutoff points (325, 565 & 0.48) respectively. On combining both DNA integrity and CEA, improved sensitivity to 93.3% was noted. For NSCLC patients, ALU 115 & ALU 247 increased significantly with more advanced stage and highest level was noticed in metastatic patients. Regarding survival there was better overall survival among patients with low DNA integrity. ConclusionSerum cf-DNA concentrations and integrity index may be valuable tool in early diagnosis of NSCLC and prediction of prognosis of those patients.

Clinical Implications of Transbronchial Biopsy for Surgically-resected Non-small Cell Lung Cancer.

Lung biopsies might cause metastasis and/or dissemination. The aim of this study was to review our institutional experience and analyze the outcomes of resection for non-small cell lung cancer (NSCLC), in patients who had received preoperative transbronchial biopsy using fiberoptic bronchoscopy with fluoroscopic imaging (BFS). The medical records of consecutive patients between 2010 and 2015 were retrospectively reviewed. Patients were divided into two groups (BFS and Non-BFS). Overall survival (OS) curves and recurrence-free survival (RFS) curves were plotted using the Kaplan-Meier method. Cox regression analyses were used to evaluate the hazard ratio (HR) with the endpoint OS or RFS. We studied the medical records of 531 patients. The 5-year OS rate was 91.8% and 79.8%, in the BFS group and in the Non-BFS group, respectively (p<0.001). BFS was an independent factor associated with RFS HR=2.164 (95%CI=1.399-2.346). Preoperative BFS is a prognostic factor in patients receiving surgery for NSCLC.

PMD7 - Biopsy Procedures in Patients with Primary Lung Cancer

Biopsy is critical in diagnosis and staging of lung cancer. The study aims to assess the use of biopsy procedures, including the approaches, the frequency, and the associated costs, in the care pathway of diagnosis or monitoring of lung cancer. This study used Truven MarketScan® Database from 2013 to 2015. The 2014 data was used to identify baseline patients with primary diagnosis of lung cancer. We included patients who were ≥18 years old, had continuous enrollment of 12 months before and after the first recorded primary lung cancer diagnosis dated in 2014, and had ≥2 primary lung cancer diagnoses within two months. CPT, ICD-9-CM, and ICD-10-PCS codes were used to capture lung cancer diagnosis and biopsy procedures. The radiologic procedures (e.g. ultrasound, fluoroscopic, CT, or MR) for needle placement were excluded from biopsy enumeration. We identified three major biopsy approaches: surgical, percutaneous, and bronchoscopic. Descriptive statistics of frequency, duration and costs were performed for biopsies within a year before and after the primary diagnosis. Among 22,161 patients included in 2014, 14,280 (64.44%) patients had ≥1 biopsies within 1 year before or after the primary diagnosis; 17.59% surgical, 41.57% percutaneous, and 33.93% bronchoscopic. Specifically, 2,905 (13.11%) patients had 1 biopsy with an average procedure cost of $5,489 (standard deviation (SD): $22,593); 4,625 (20.87%) patients had 2 biopsies, with an average of 15.7 days from the first biopsy and an average procedure cost of $7,729 (SD: $4,664); and 6,750 (30.46%) patients had ≥3 biopsies with an average of 22.7 days between biopsies and an average procedure cost of $11,899 (SD: $15,438). All costs were significantly different (p<0.0001). More than 50% of patients with primary lung cancer had multiple biopsies with high costs in short time frame. Further research is needed to identify ways to reduce unnecessary biopsies.

A liquid biopsy in primary lung cancer.

A tissue biopsy is the "golden standard" for molecular profiling that is essential in decision-making regarding treatment for malignant tumors, including primary lung cancer. However, tumor biopsies are associated with several limitations, including invasiveness and difficulty in achieving access. Liquid biopsies have several potential advantages over tissue biopsies, and recent advances in molecular technologies have enabled liquid biopsies to be introduced into daily clinical practice. Cell-free blood-based liquid biopsies to detect mutations in the epidermal growth factor receptor (EGFR) gene in the plasma have been approved and may be useful in selecting patients for treatment with tyrosine kinase inhibitors of EGFR. We herein describe blood-based liquid biopsies and review the current status and future perspectives of plasma genotyping in primary lung cancer.

Liquid biopsy for early stage lung cancer.

Liquid biopsy, which analyzes biological fluids especially blood specimen to detect and quantify circulating cancer biomarkers, have been rapidly introduced and represents a promising potency in clinical practice of lung cancer diagnosis and prognosis. Unlike conventional tissue biopsy, liquid biopsy is non-invasive, safe, simple in procedure, and is not influenced by manipulators' skills. Notably, some circulating cancer biomarkers are already detectable in disease with low-burden, making liquid biopsy feasible in detecting early stage lung cancer. In this review, we described a landscape of different liquid biopsy methods by highlighting the rationale and advantages, accessing the value of various circulating biomarkers and discussing their possible future development in the detection of early lung cancer.

Liquid Biopsy in Lung Cancer: Clinical Applications of Circulating Biomarkers (CTCs and ctDNA).

Lung cancer is by far the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Recent advances in the understanding of the biology of tumors and in highly sensitive detection technologies for molecular analysis offer targeted therapies, such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. However, our understanding of an individual patient’s lung cancer is often limited by tumor accessibility because of the high risk and invasive nature of current tissue biopsy procedures. “Liquid biopsy”, the analysis of circulating biomarkers from peripheral blood, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), offers a new source of cancer-derived materials that may reflect the status of the disease better and thereby contribute to more personalized treatment. In this review, we examined the clinical significance and uniqueness of CTCs and ctDNA from NSCLC patients, isolation and detection methods developed to analyze each type of circulating biomarker, and examples of clinical studies of potential applications for early diagnosis, prognosis, treatment monitoring, and prediction of resistance to therapy. We also discuss challenges that remain to be addressed before such tools are implemented for routine use in clinical settings.

Conditionally reprogrammed cells (CRC) methodology does not allow the in vitro expansion of patient-derived primary and metastatic lung cancer cells.

Availability of tumor and non-tumor patient-derived models would promote the development of more effective therapeutics for non-small cell lung cancer (NSCLC). Recently, conditionally reprogrammed cells (CRC) methodology demonstrated exceptional potential for the expansion of epithelial cells from patient tissues. However, the possibility to expand patient-derived lung cancer cells using CRC protocols is controversial. Here, we used CRC approach to expand cells from non-tumoral and tumor biopsies of patients with primary or metastatic NSCLC as well as pulmonary metastases of colorectal or breast cancers. CRC cultures were obtained from both tumor and non-malignant tissues with extraordinary high efficiency. Tumor cells were tracked in vitro through tumorigenicity assay, monitoring of tumor-specific genetic alterations and marker expression. Cultures were composed of EpCAM+ lung epithelial cells lacking tumorigenic potential. NSCLC biopsies-derived cultures rapidly lost patient-specific genetic mutations or tumor antigens. Similarly, pulmonary metastases of colon or breast cancer generated CRC cultures of lung epithelial cells. All CRC cultures examined displayed epithelial lung stem cell phenotype and function. In contrast, brain metastatic lung cancer biopsies failed to generate CRC cultures. In conclusion, patient-derived primary and metastatic lung cancer cells were negatively selected under CRC conditions, limiting the expansion to non-malignant lung epithelial stem cells from either tumor or non-tumor tissue sources. Thus, CRC approach cannot be applied for direct therapeutic testing of patient lung tumor cells, as the tumor-derived CRC cultures are composed of (non-tumoral) airway basal cells.

Lung cancer biopsy: Can diagnosis be changed after immunohistochemistry when the H&E-Based morphology corresponds to a specific tumor subtype?

OBJECTIVES:Advancements in non-small cell lung cancer treatment based on targeted therapies have made the differentiation between adenocarcinoma and squamous cell carcinoma increasingly important. Pathologists are challenged to make the correct diagnosis in small specimens. We studied the accuracy of an immunohistochemical panel in subclassifying non-small cell lung cancer in routine small biopsies and compared the results with the diagnosis from resected lung specimens, autopsy samples or biopsied/resected metastases.METHODS:In total, 340 lung cancer biopsies were investigated for the expression of CK5, TTF1, p63 and surfactant.RESULTS:We characterized 166 adenocarcinomas and 124 squamous cell carcinomas. Overall, 85% of cases displayed binary staining (TTF1 positive/p63 negative, and vice versa). The diagnoses of ten cases with a morphology that indicated a specific tumor subtype were changed after immunohistochemistry (IHC). A second specimen was available for 71 patients, and the first diagnosis at biopsy was confirmed in 95% of these cases. Most non-small cell lung cancer cases present a binary immunohistochemical profile in small biopsies, contributing to good diagnostic accuracy with routine markers. In a small proportion of cases, the diagnosis can be changed after IHC even when the morphological aspects indicate one specific tumor subtype.CONCLUSIONS:We recommend that routine small biopsies of lung cancer without classic morphology should be subjected to a minimum immunohistochemical panel to differentiate adenocarcinoma from squamous cell carcinoma.

Association of pulmonary tuberculosis with lung carcinoma: An epidemiological study

Introduction: Tuberculosis (TB) is a serious public health problem in developing countries, with India bearing highest burden. Lung cancer (LC), especially adenocarcinoma, is also increasing all over the world, with India having highest mortality in males due to lung cancer. Both diseases do co-exists producing diagnostic dilemma and treatment controversies. We intended to explore the incidence of both diseases at a tertiary cancer centre. Materials and Methods: This longitudinal study was conducted between 2014 to 2017. Diagnosis and treatment of TB were according to DOTS protocol. For lung cancer biopsy was mandatory for diagnosis. Results: Our study was conducted on 42 patients at the Department of Radiotherapy, R.G. Kar Medical College. In 19 of the 42 patients, the diagnosis of TB and LC occurred simultaneously, whereas, in 23, the occurrence was sequential. Fifteen (35.7%) patients reported having had TB twice. TB treatment was given as Category 1 of DOTS in 21 patients (50%) and Category 2 in 15 (35.7%). In 6 (14.3%) patients, the data on treatment regimen were either not provided or unavailable. Regarding carcinoma lung management, 50% of patients were purely treated as palliative intent. Twenty percent of patients received chemotherapy with platinum doublet. Thirty percent of patients were given best supportive care. Conclusion: The symptoms of TB and lung carcinoma overlap with each other. If we get any patient with lung cancer not much improving after anticancer treatment, diagnosis of TB should be kept in mind. Similarly, in any diagnosed case of pulmonary TB, development of lung cancer should be considered if not improved with anti-tubercular treatment.

Contrast-enhanced ultrasound for needle biopsy of central lung cancer with atelectasis.

Contrast-enhanced ultrasound (CEUS) can distinguish between central lung cancer and atelectatic lung tissue. The aim of this study was to explore the clinical value of CEUS for biopsy in patients with central lung cancer with obstructive atelectasis. One hundred and twelve patients were selected and CEUS was performed to display central lung cancer and atelectatic lung tissue. The front edge of central lung cancer was punctured with a needle, avoiding the necrotic area, under the guidance of CEUS. All of the 112 lesions were diagnosed with a clear central lung cancer mass and atelectatic lung tissue. In 104 cases, the central lung cancer mass presented with a "slow-in and fast-out" pattern compared to atelectatic lung tissue. In eight cases, the central lung cancer mass presented with a "fast-in and fast-out" pattern compared to atelectatic lung tissue. The mean number of punctures was 2.6, and the success rate of puncture biopsy was 98%. Of the 112 patients, six cases had hemoptysis during the procedure and 10 patients had bloody sputum in the postoperative period. No complications were found in the other cases. CEUS has important clinical value for needle biopsy of central lung cancer with atelectasis.

Distribution and Clinical Significance of CTLA-4, PD-1 and PD-L1 in Peripheral Blood of Patients with Small Cell Lung Cancer

背景与目的本研究旨在探索细胞毒性T淋巴细胞相关抗原（cytotoxic T lymphocyte associated antigen-4, CTLA-4）、程序坏死因子（programmed death 1, PD-1）和程序坏死因子配体（programmed death ligand 1, PD-L1）在小细胞肺癌（small cell lung cancer, SCLC）患者外周血中的分布情况，探索其免疫作用机制并评估其作为生物标志物的临床价值。方法招募290例SCLC患者及60例健康志愿者，收集治疗前和治疗2nd周期末SCLC患者EDTA抗凝血2 mL。应用流式细胞仪检测CTLA-4、PD-1和PD-L1在外周血CD3、CD4、CD8及CD25的分布，分析其与临床病理特征的相关性；采用细胞免疫化学法和流式细胞法检测PD-L1在SCLC细胞系H446中的表达。结果SCLC患者外周血中CTLA-4+细胞和PD-1+细胞水平分别为（1.56±1.24）%和（8.07±3.97）%、CTLA-4在CD3细胞和CD4细胞中的表达水平无明显差异，分别为（4.87±5.18）%和（3.85±2.60）%，均低于PD-1在CD3+或CD4+细胞中的表达（26.63±9.04）%和（20.79±9.41）%，与健康对照组相比，SCLC中CD4+CD25+CTLA-4+细胞水平明显升高（1.91±1.27）% vs（7.09±5.09）%，P < 0.001；PD-1+（CD8+）细胞表达水平明显降低，分别为（22.56±4.21）% vs（11.47±5.85）%，P < 0.001。CD4+CD25+CTLA-4+细胞或CD8+PD-1+细胞水平与患者的年龄、性别、吸烟状况、临床分期以及是否转移等因素无关（P > 0.05）。化疗两周期末CD4+CD25+CTLA-4+和CD8+PD-1+细胞的水平对比化疗前明显下降，分别为（5.11±2.60）% vs（6.94±4.91）%；（8.74±3.39）% vs（11.48±5.91）%，P值均 < 0.000, 1，但与无疾病进展生存和总生存无显著相关性。PD-L1高表达于SCLC细胞系H446中并定位在细胞膜和细胞浆，但在外周血中未见表达。结论本研究首次证实SCLC外周血中CTLA4高表达于调节性T细胞中，而PD-1低表达于效应性T细胞，该结果为揭示SCLC免疫监测点免疫逃逸机制提供了理论依据，可能作为一种新的无创性且可实时监测的生物标志物。

MiR-99a reveals two novel oncogenic proteins E2F2 and EMR2 and represses stemness in lung cancer

Lung cancer is one of the most aggressive tumours with very low life expectancy. Altered microRNA expression is found in human tumours because it is involved in tumour growth, progression and metastasis. In this study, we analysed microRNA expression in 47 lung cancer biopsies. Among the most downregulated microRNAs we focussed on the miR-99a characterisation. In vitro experiments showed that miR-99a expression decreases the proliferation of H1650, H1975 and H1299 lung cancer cells causing cell cycle arrest and apoptosis. We identified two novel proteins, E2F2 (E2F transcription factor 2) and EMR2 (EGF-like module-containing, mucin-like, hormone receptor-like 2), downregulated by miR-99a by its direct binding to their 3′-UTR. Moreover, miR-99a expression prevented cancer cell epithelial-to-mesenchymal transition (EMT) and repressed the tumourigenic potential of the cancer stem cell (CSC) population in both these cell lines and mice tumours originated from H1975 cells. The expression of E2F2 and EMR2 at protein level was studied in 119 lung cancer biopsies. E2F2 and EMR2 are preferentially expressed in adenocarcinomas subtypes versus other tumour types (squamous and others). Interestingly, the expression of E2F2 correlates with the presence of vimentin and both E2F2 and EMR2 correlate with the presence of β-catenin. Moreover, miR-99a expression correlates inversely with E2F2 and directly with β-catenin expression in lung cancer biopsies. In conclusion, miR-99a reveals two novel targets E2F2 and EMR2 that play a key role in lung tumourigenesis. By inhibiting E2F2 and EMR2, miR-99a represses in vivo the transition of epithelial cells through an EMT process concomitantly with the inhibition of stemness features and consequently decreasing the CSC population.

The era of a "liquid biopsy" may be on the horizon in non-small cell lung cancer.

As an integral part of precision medicine, the role of liquid biopsy in non-small cell lung cancer (NSCLC) is highlighted in this special issue of Journal of Thoracic Disease with a selection of articles written by a panel of eminent experts.

Critical issues in the clinical application of liquid biopsy in non-small cell lung cancer.

Current therapeutic options for non-small cell lung cancer (NSCLC) patients are chemotherapy and targeted therapy directed mainly against epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements. Targeted therapy relies on the availability of tumor biopsies for molecular profiling at diagnosis and to longitudinally monitor treatment response and resistance development. Unfortunately, tumor biopsy might be invasive, recover poor material of suboptimal quality, and cause sample bias due to tumor heterogeneity. Many studies have illustrated the potential of liquid biopsy as minimal invasive approach to respond to the urgent need for real time monitoring, stratification, and personalized optimized treatment in NSCLC patients. In principle, the liquid biopsy could provide the genetic landscape of primary and metastatic cancerous lesions, detecting "druggable" genomic alterations or associated with treatment resistance. Moreover, it would guarantee the prognostic/predictive biomarkers evaluation in patients for whom biopsies are inaccessible or difficult to repeat. At this regard, the prognostic value of circulating tumor cells (CTCs) in NSCLC patients has been largely investigated, but still their clinical utility as tumor biomarker is hampered by the lack of a consensus on the criteria necessary and sufficient to define them and on the standard operating procedures (SOPs) for their assessment. This review will summarize current developments on liquid biopsy in NSCLC, addressing the technology issues that contribute to the poor ability to track CTCs in the blood of NSCLC patients, thus limiting their extensive use in the clinical practice, and analyzing the solutions adopted to overcome such limits, on the road towards the clinical validation.

Quantitative Proteomic Analysis of Optimal Cutting Temperature (OCT) Embedded Core-Needle Biopsy of Lung Cancer.

With recent advances in understanding the genomic underpinnings and oncogenic drivers of pathogenesis in different subtypes, it is increasingly clear that proper pretreatment diagnostics are essential for the choice of appropriate treatment options for non-small cell lung cancer (NSCLC). Tumor tissue preservation in optimal cutting temperature (OCT) compound is commonly used in the surgical suite. However, proteins recovered from OCT-embedded specimens pose a challenge for LC-MS/MS experiments, due to the large amounts of polymers present in OCT. Here we present a simple workflow for whole proteome analysis of OCT-embedded NSCLC tissue samples, which involves a simple trichloroacetic acid precipitation step. Comparisons of protein recovery between frozen versus OCT-embedded tissue showed excellent consistency with more than 9200 proteins identified. Using an isobaric labeling strategy, we quantified more than 5400 proteins in tumor versus normal OCT-embedded core needle biopsy samples. Gene ontology analysis indicated that a number of proliferative as well as squamous cell carcinoma (SqCC) marker proteins were overexpressed in the tumor, consistent with the patient's pathology based diagnosis of "poorly differentiated SqCC". Among the most downregulated proteins in the tumor sample, we noted a number of proteins with potential immunomodulatory functions. Finally, interrogation of the aberrantly expressed proteins using a candidate approach and cross-referencing with publicly available databases led to the identification of potential druggable targets in DNA replication and DNA damage repair pathways. We conclude that our approach allows LC-MS/MS proteomic analyses on OCT-embedded lung cancer specimens, opening the way to bring powerful proteomics into the clinic. Graphical Abstract ᅟ.