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We have previously shown that Met activation through the hepatocyte growth factor (HGF) increases tumorogenesis, induces epithelial-to-mesenchymal transition (EMT) and chemoresistance in SCLC. We sought to evaluate circulating HGF levels in SCLC patients and assess correlation with outcome and EMT features in the tumor. Serum samples from patients with SCLC were prospectively obtained at diagnosis, response evaluation and progression. HGF serum (sHGF) was quantified by ELISA. EMT markers and p-Met/Met were assayed by immunohistochemistry in tumor samples. Clinical data were prospectively recorder. One-hundred twelve patients were included. High baseline levels of sHGF were associated with shorter overall survival (p=0.006) and remained independently associated with survival in the multivariate analysis (p=0.016). For stage IV patients, an increase of sHGF levels at response evaluation (p=0.042) and at progression (p=0.003) were associated with poor outcome. sHGF levels were associated (p<0.05) with a mesenchymal phenotype in the tumor. In conclusion, high sHGF at diagnosis and increases during the course of the disease predict for poor outcome in SCLC patients and associate with EMT in the tumor. These data provide novel evidence on a role of sHGF in the adverse clinical behavior of SCLC and supports testing Met inhibitors in patients with high sHGF.

Background: Although many DNA methylation (DNAm) alterations observed in peripheral whole blood/leukocytes and serum have been considered as potential diagnostic markers for cancer, their origin and their specificity for cancer (e.g., vs inflammatory diseases) remain unclear., Methods: From publicly available datasets, we identified changes in the methylation of blood-borne DNA for multiple cancers and inflammatory diseases. We compared the identified changes with DNAm difference between myeloid and lymphoid cells extracted from two datasets., Results: At least 94.7% of the differentially methylated DNA loci (DM loci) observed in peripheral whole blood/leukocytes and serum of cancer patients overlapped with DM loci that distinguish between myeloid and lymphoid cells and >99.9% of the overlapped DM loci had consistent alteration states (hyper- or hypomethylation) in cancer samples compared to normal controls with those in myeloid cells compared to lymphoid cells (binomial test, P-value <2.2 × 10(-16)). Similar results were observed for DM loci in peripheral whole blood/leukocytes in patients with rheumatoid arthritis or inflammatory bowel diseases. The direct comparison between DM loci observed in the peripheral whole blood/leukocytes of patients with inflammatory diseases and DM loci observed in the peripheral whole blood of patients with cancer showed that DM loci detected from cancer and inflammatory diseases also had significantly consistent alteration states (binomial test, P-value <2.2 × 10(-16))., Conclusions: DNAm changes observed in the peripheral whole blood/leukocytes and serum of cancer patients and in the peripheral whole blood/leukocytes of inflammatory disease patients are predominantly determined by the increase of myeloid cells and the decrease of lymphoid cells under the disease conditions, in the sense that their alteration states in disease samples compared to normal controls mainly reflect the DNAm difference between myeloid and lymphoid cells. These analyses highlight the importance of comparing cancer and inflammatory disease directly for the identification of cancer-specific diagnostic biomarkers.

A multiplex method for the determination of the small cell lung cancer (SCLC) markers progastrin releasing peptide (ProGRP) and neuron specific enolase (NSE) is presented, which involves coextraction by immunoaffinity (IA) beads and codetermination by selected reaction monitoring (SRM). The performance was compared with two IA SRM methods which were recently validated for individual marker determination. The multiplexing method reduces sample volume, handling time per sample, and reagent consumption and shows good linearity, recovery, quantitative measurements, and sensitivity with lower limit of detection (LLOD) values of 7.2 pM (=90 pg/mL) and 4.5 pM (=210 pg/mL) and lower limit of quantitation (LLOQ) values of 24 pM (=300 pg/mL) and 15 pM (=700 pg/mL), for total ProGRP and γ-NSE, respectively. The novel aspect of this approach is the multiplexing of ProGRP and NSE with the additional ability to perform fingerprinting by the selective determination of ProGRP isoform 1, ProGRP isoform 3, and total ProGRP, as well as the α- and the γ-subunit of NSE isoenzymes. Six serum samples from patients with SCLC were analyzed to demonstrate the methods feasibility to simultaneously differ between and individually quantify ProGRP, NSE, and their isoform and isoenzyme variants, respectively. Both the presence of and variation between all the isoforms and isoenzymes, as well as covarying results with the conventional immunometric assays for total ProGRP and γ-NSE, were seen in the analyses of patient serum samples.

We evaluated the expression and diagnostic value of human epididymis protein 4 (HE4) in small cell lung cancer (SCLC). Serum HE4 level was measured in serum samples from 30 healthy controls and 49 patients with SCLC using enzyme-linked immunosorbent assay. HE4 expression in tumor tissues was analyzed by immunohistochemical staining. The results show that serum HE4 level is significantly higher in SCLC patients than those in healthy controls, which is also the case in tumor tissues where strong intracytoplasmic staining is demonstrable. Using the optimal cutoff value of 84.19 pmol/l, serum HE4 level distinguishes SCLC patients from healthy controls with a sensitivity of 69.4 %, a specificity of 93.3 %, and the area under the receiver operating characteristic curve (AUC) of 0.85. Compared to other well-known tumor markers used in lung cancer diagnosis, HE4 shows the highest sensitivity (69.4 %, 34 of 49) and accuracy (78.5 %, 62 of 79) in diagnosing SCLC, and combinations with other tumor markers further increase the sensitivity and accuracy. These findings suggest that HE4 levels are greatly elevated in sera and tumor tissues of patients with SCLC and serum HE4 is a potential diagnostic marker for patients with SCLC.

Purpose: Amuvatinib is an oral multi-kinase inhibitor that suppresses RAD51, inhibits mutant c-KIT and platelet-derived growth factor receptor alpha, and has synergistic activity with DNA-damaging agents and topoisomerase inhibitors such as etoposide, doxorubicin, and topotecan. We conducted a phase 1B study to estimate the maximum tolerated dose (MTD) levels of amuvatinib with standard chemotherapy regimens and to define the safety profiles of specific amuvatinib + standard regimens., Methods: Five therapies each co-administered with amuvatinib 100-800 mg/day every 21 days were evaluated in treatment-naïve or moderately pre-treated subjects: paclitaxel IV followed by carboplatin IV; carboplatin IV followed by etoposide; topotecan IV; docetaxel IV; and erlotinib by mouth., Results: Among 97 treated subjects, no treatment arm reached the MTD. Dose-limiting toxicities included febrile neutropenia and diarrhea. No pharmacokinetic interactions of amuvatinib with any cancer regimens occurred. Of 12/97 (12 %) partial responses overall, 11 were seen in the amuvatinib and paclitaxel/carboplatin or carboplatin/etoposide arms and most commonly in the neuroendocrine (NE), non-small cell lung cancer (NSCLC), and small cell lung cancer (SCLC) tumors. Forty-four subjects (45 %) had stable disease. Adverse events reflected combination treatment and were primarily non-hematologic (fatigue, alopecia, diarrhea, nausea, anorexia) and hematologic (neutropenia, anemia, thrombocytopenia, leukopenia). Pharmacodynamic effects as measured by decreased levels of RAD51 and increased residual DNA damage (53BP1 foci) were seen in skin punch biopsies., Conclusion: Amuvatinib was well tolerated, modulated RAD51, and showed antitumor activity when combined with paclitaxel/carboplatin and carboplatin/etoposide in NE, NSCLC, and SCLC tumors.

Despite the increasing incidence of combined small-cell lung cancer (C-SCLC) in recent years, there have not been many data on clinical prognostic factors predicting prognosis of C-SCLC patients. In present study, we sought pretreatment features especially basic laboratory parameters predicting survival of C-SCLC. We analyzed 613 small-cell lung cancer (SCLC) patients at our institution between January 2005 and December 2010. We identified 114 patients with C-SCLC. The pathologic and clinical characteristics of these patients were reviewed. Data of laboratory parameters obtained during regular examinations at diagnosis of these patients were examined. The Kaplan-Meier method was used to calculate the survival rate and depict the survival curves. The Cox regression model was used to analyze the independent factors affecting the overall survival (OS). These data were compared with the results obtained from our 499 pure SCLC patients who presented during the same time period. Of the 613 SCLC patients analyzed, 18.6 % of the patients presented with C-SCLC. No difference in OS was observed in patients with C-SCLC and patients with pure SCLC (P = 0.995). The Kaplan-Meier survival curves revealed that poor ECOG-PS (P < 0.001), extensive disease (P < 0.001), pathologic subtype of SC/LC (P < 0.001), not receiving surgery (P = 0.001), elevated serum lactate dehydrogenase (LDH) (P = 0.005), elevated NSE (P = 0.043), and elevated neutrophile-lymphocyte ratio (NLR) (P = 0.018) were associated with adverse prognosis of patients with C-SCLC. By multivariate analysis, OS was affected by ECOG-PS (hazard ratio 2.001, P = 0.012), disease extent (hazard ratio 3.406, P < 0.001), and NLR (hazard ratio 1.704, P = 0.030) in C-SCLC patients, while the risk factors that influenced the prognosis of the patients with pure SCLC were ECOG-PS (hazard ratio 2.132, P < 0.001), disease extent (hazard ratio 1.482, P < 0.001), and LDH (hazard ratio 1.811, P < 0.001). Patients with C-SCLC carry a similar prognosis than those with pure small-cell variety. Easily accessible pretreatment parameters such as NLR should be considered in defining the prognosis of C-SCLC patients besides disease extent and performance status.

Hyponatremia is a common electrolyte disorder occurring in patients with malignancy. Typically, it runs in the form of the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Among malignant diseases it is often ascertained from small cell lung cancer. In the form of paraneoplastic syndrome it may precede clinical and radiological symptoms malignant disease. Hyponatremia requires special attention because of the neurological consequences and the risk of death. We present a case of a patient in whom the occurrence of hyponatremia preceded the appearance of clinical symptoms of lung cancer and has been the reason to start the diagnosis. The normalization of serum sodium was the first signal response to chemotherapy. In contrast, a statement confirmed the recurrence of hyponatremia progression of the disease in the form of metastases to the central nervous system. Speeches hyponatremia refractory symptomatic treatment should be a cause of further investigation into the neoplastic process. Recurrent hyponatremia during or after treatment may suggest its progression. Therefore, monitoring the sodium level is required not only during treatment, but also after the oncological treatment.

Background: Patients with recurrent small-cell lung cancer (SCLC) have dismal outcomes. The failure of salvage therapy is due to the possible development of resistance mechanisms, such as the upregulation of the anti-apoptosis protein, Bcl-2. We conducted a phase II study to evaluate if modulation of Bcl-2 with 13-cis-retinoic acid (13-CRA) and interferon alpha could improve response rates when combined with paclitaxel in patients with recurrent SCLC., Methods: Patients with recurrent SCLC and measurable disease were treated with interferon alpha at 6 million units/m² subcutaneously and 13-CRA 1 mg/kg orally on days 1 and 2 and paclitaxel 75 mg/m² intravenously on day 2 of each week for 6 weeks of an 8-week treatment cycle. Treatment was continued until disease progression, development of unacceptable toxicity, or withdrawal of consent. The primary endpoint was response rate with secondary endpoints of progression-free survival (PFS) and overall survival (OS). Bcl-2 levels were assessed in peripheral blood mononuclear cells (PBMCs)., Results: Thirty-seven patients were enrolled; 34 were included in the intention-to-treat analysis as 3 patients were ineligible for the study. There were 3 partial responses (9 %), and 5 patients had stable disease (15 %) as best response. The median PFS was 2 months, and median OS was 6.2 months. Although mean Bcl-2 protein levels decreased with therapy in PBMCs, there was no association between Bcl-2 levels and response rate or survival., Conclusion: Despite sound pre-clinical evidence, the addition of 13-CRA and interferon alpha to paclitaxel did not improve outcomes for recurrent SCLC.

This study was to explore the association between the serum YKL-40 level and the clinical characteristics, the response to chemotherapy and prognosis in small cell lung cancer (SCLC). Serum YKL-40 levels were detected and compared in 120 patients with SCLC pre- and post-chemotherapy, and in 40 healthy controls. Receiver operating characteristics (ROC) curves were adopted for diagnosis and calculation of area under ROC curve in SCLC. The Kaplan-Meier method, univariate and multivariate Cox regression analysis were used to analyze the correlation between pre-chemotherapy serum YKL-40 levels and progression-free survival (PFS) and overall survival (OS). The pre-chemotherapy serum YKL-40 levels were significantly higher than those of the controls (p<0.001). The post-chemotherapy serum YKL-40 levels in the SCLC cases were lower than pre-chemotherapy serum YKL-40 levels in these cases (p = 0.026). The patients with high serum YKL-40 showed a poorer response to chemotherapy than those patients with low serumYKL-40 (p = 0.031). Univariate analysis revealed that SCLC patients with high serum YKL-40 had a shorter PFS and OS than those with low serum YKL-40 (HR of 1.74, p = 0.033; HR of 1.33, p = 0.001). Cox multivariate analysis indicated that YKL-40 was an independent prognostic indicator of PFS and OS (HR of 1.12, p = 0.029; HR of 1.84, p = 0.025). Kaplan-Meier survival curves further confirmed that patients with low serum YKL-40 have longer PFS and OS (p = 0.016 and p = 0.041, respectively). These results suggest that YKL-40 is a potential prognostic marker of chemotherapy response in SCLC.

Objective: To determine the expression of neuron-specific enolase (NSE) in patients with multiple myeloma (MM) and to evaluate its clinical value as a tumor marker and, an indicator of disease progression and treatment efficacy., Methods: Using electrochemiluminescence immunoassay (ECLIA), we measured the serum levels of NSE in 47 healthy subjects (control group), 25 patients with small cell lung cancer (lung cancer group), and 52 patients with MM (MM group). For the MM group, serum NSE levels were measured and other disease indicators and related symptoms were monitored before and after chemotherapy. The relationship between NSE expression and other MM-related factors was analyzed. In addition, immunohistochemical staining was performed on bone marrow biopsy specimens from patients with MM., Results: In the control group, serum NSE levels were within the normal range as previously reported, while the lung cancer group and the untreated MM group exhibited NSE levels that were significantly higher relative to the control group (P<0.05). The difference in NSE expression between the lung cancer group and untreated MM group was statistically significant (P<0.05). NSE levels were significantly decreased in MM patients after chemotherapy and were positively correlated with an MM disease index [beta-2 microglobulin (β2-MG)]. Changes in NSE were not related to the response rate to chemotherapy but rather were correlated with progression-free survival., Conclusions: Patients with MM may have increased serum NSE levels, and changes in NSE may provide insight into treatment efficacy of chemotherapy and disease progression. Perhaps NSE expression is a viable biomarker for MM and can be a useful reference for the design and adjustment of clinical MM treatment programs.

Aim: Detection of novel tumor biomarker will aid in diagnosis of early-stage small cell lung cancer (SCLC). The purpose of this study was to identify novel tumor biomarker in serum from patients with SCLC using a proteomics-based approach., Methods: Sera were analyzed before the initiation of chemotherapy. Serum proteins of SCLC patients and healthy controls were collected and separated by 2-D fluorescence differential gel electrophoresis (2-D DIGE). Positive spots were analyzed by LC-MS/MS. Different expression of identified biomarker was verified by immunohistochemical method in wax specimen from 40 patients., Results: A total of 86 proteins were shown to be differentially abundant between the serum of SCLC patients and normal subjects by 2-D DIGE. Fifteen proteins were identified by LC-MS/MS. According to the bioinformatic analysis, these proteins are mainly involved in development and carcinogenesis. Some of them have been previously demonstrated to be important prognostic factors. Differential expression of 5 proteins between the normal tissue and cancerious tissue was confirmed by immunochemistry of SCLC patients., Conclusion: We have identified different serum proteins between SCLC patients and healthy controls. These proteins may be potential serum biomarkers for early detection of SCLC and play a role in the development and metastasis of SCLC.

Lung cancer is the most common cancer, and small-cell lung cancer (SCLC) accounts for around 20 % of lung cancers. SCLC has a neuroendocrine cellular origin, and the tumor cells usually express neuroendocrine markers. There have been major recent advances in the management of SCLC, and multimodal approaches are now the norm. An improved knowledge of the prognostic variables would assist in defining which patients were better candidates to receive these newer intensive therapies. This single-center retrospective study of 97 previously untreated and histologically proven SCLC patients analysed the circulating neuroendocrine markers chromogranin A (CGA), pro-gastrin-releasing peptide (ProGRP), and neuron-specific enolase (NSE) in addition to the other more classical variables. Fifty patients had limited-stage disease and 47 had extensive disease. Sixty patients had an ECOG performance status (PS) of 0-1 and 37 had PS 2-4. Median survival for the whole study population was 13 months. Univariate analysis and univariate Cox regression modeling found a statistically significant association between survival and PS, disease stage, and CGA, ProGRP, and NSE levels. Age and sex were not prognostic. A shorter survival time was found in patients with a PS equal to or >2, extensive stage disease, a serum CGA level >56 ng/ml, a serum ProGRP level >58 pg/ml, and a serum NSE level >19 ng/ml. This study has found that there is a potential role for ProGRP, NSE, and CGA in both staging and prognosing survival in SCLC patients.

Objectives: To study possibilities of immunological and electrophysiological methods for the diagnosis of paraneoplastic polyneuropathy in cancer., Methods: We studied 88 cancer patients using electromyography and immunological assay (serum neuronal antibodies)., Results: A symmetrical, distal, sensory-motor, axonal-demyelinating form of polyneuropathy can develop in breast cancer and small cell lung cancer. Onconeural antibodies were detected in the serum of more than half of study participants as well as in some healthy donors. Symptoms of polyneuropathy appeared earlier than the diagnosed tumor., Conclusion: The diagnostic value of the methods used for the early diagnosis of breast cancer and small cell lung cancer is emphasized.

In diagnosis of lung cancer, rapid distinction between small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) tumors is very important. Serum markers, including lactate dehydrogenase (LDH), C-reactive protein (CRP), carcino-embryonic antigen (CEA), neurone specific enolase (NSE) and Cyfra21-1, are reported to reflect lung cancer characteristics. In this study classification of lung tumors was made based on biomarkers (measured in 120 NSCLC and 60 SCLC patients) by setting up optimal biomarker joint models with a powerful computerized tool - gene expression programming (GEP). GEP is a learning algorithm that combines the advantages of genetic programming (GP) and genetic algorithms (GA). It specifically focuses on relationships between variables in sets of data and then builds models to explain these relationships, and has been successfully used in formula finding and function mining. As a basis for defining a GEP environment for SCLC and NSCLC prediction, three explicit predictive models were constructed. CEA and NSE are frequently- used lung cancer markers in clinical trials, CRP, LDH and Cyfra21-1 have significant meaning in lung cancer, basis on CEA and NSE we set up three GEP models-GEP 1(CEA, NSE, Cyfra21-1), GEP2 (CEA, NSE, LDH), GEP3 (CEA, NSE, CRP). The best classification result of GEP gained when CEA, NSE and Cyfra21-1 were combined: 128 of 135 subjects in the training set and 40 of 45 subjects in the test set were classified correctly, the accuracy rate is 94.8% in training set; on collection of samples for testing, the accuracy rate is 88.9%. With GEP2, the accuracy was significantly decreased by 1.5% and 6.6% in training set and test set, in GEP3 was 0.82% and 4.45% respectively. Serum Cyfra21-1 is a useful and sensitive serum biomarker in discriminating between NSCLC and SCLC. GEP modeling is a promising and excellent tool in diagnosis of lung cancer.

Purpose: To investigate the clinical value in lung cancer of a combination of four serum tumor markers, haptoglobin (Hp), carcinoembryonic antigen (CEA), neuron specific enolase (NSE) as well as the cytokeratin 19 fragment (CYFRA21-1)., Materials and Methods: Serum Hp (with immune-turbidimetric method), CEA, NSE, CYFRA21-1 (with chemiluminescence method) level were assessed in 193 patients with lung cancer, 87 patients with benign lung disease and 150 healthy controls. Differences of expression were compared among groups, and joint effects of these tumor markers for the diagnosis of lung cancer were analyzed., Results: Serum tumor marker levels in patients with lung cancer were obviously higher than those with benign lung disease and normal controls (p<0.01). The sensitivities of Hp, CEA, NSE and CYFRA21-1 were 43.5%, 40.9%, 23.3% and 41.5%, with specificities of 90.7%, 99.2%, 97.9% and 97.9%. Four tumor markers combined together could produce a positive detection rate of 85.0%, significantly higher than that of any single test. With squamous carcinomas, the positive detection rates with Hp and CYFRA21-1 were higher than that of other markers. In the adenocarcinoma case , the positive detection rate of CEA was higher than that of other markers. For small cell carcinomas, the positive detection rate of NSE was highest. The area under receiver operating characteristic curve (AUCROC) of Hp in squamous carcinoma (0.805) was higher than in adenocarcinoma (0.664) and small cell carcinoma (0.665)., Conclusions: Hp can be used as a new serum tumor marker for lung cancer. Combination detection of Hp, CEA, NSE and CYFRA21-1 could significantly improve the sensitivity and specificity in diagnosis of lung cancer, and could be useful for pathological typing.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer, and the detection of SCLCs at an early stage is necessary for successful therapy and for improving cancer survival rates. Fucosylation is one of the most common glycosylation-based modifications. Increased levels of fucosylation have been reported in a number of pathological conditions, including cancers. In this study, we aimed to identify and validate the aberrant and selective fucosylated glycoproteins in the sera of patients with SCLC. Fucosylated glycoproteins were enriched by the Aleuria aurantia lectin column after serum albumin and IgG depletion. In a narrowed down and comparative data analysis of both label-free proteomics and isobaric peptide-tagging chemistry iTRAQ approaches, the fucosylated glycoproteins were identified as up- or down-regulated in the sera of limited disease and extensive disease stage patients with SCLC. Verification was performed by multiple reaction monitoring-mass spectrometry to select reliable markers. Four fucosylated proteins, APCS, C9, SERPINA4, and PON1, were selected and subsequently validated by hybrid A. aurantia lectin ELISA (HLE) and Western blotting. Compared with Western blotting, the HLE analysis of these four proteins produced more optimal diagnostic values for SCLC. The PON1 protein levels were significantly reduced in the sera of patients with SCLC, whereas the fucosylation levels of PON1 were significantly increased. Fucosylated PON1 exhibited an area under curve of 0.91 for the extensive disease stage by HLE, whereas the PON1 protein levels produced an area under curve of 0.82 by Western blot. The glycan structural analysis of PON1 by MS/MS identified a biantennary fucosylated glycan modification consisting of a core + 2HexNAc + 1Fuc at increased levels in the sera of patients with SCLC. In addition, the PON1 levels were decreased in the sera of the Lewis lung carcinoma lung cancer mouse model that we examined. Our data suggest that fucosylated protein biomarkers, such as PON1, and their fucosylation levels and patterns can serve as diagnostic and prognostic serological markers for SCLC.

Background: According to the cancer stem cell theory, tumors originate from a subset of cells known as cancer stem cells (CSCs) that are responsible for tumor initiation, resistance and relapse. CD44 is a cell adhesion molecule that can aid in the identification of CSCs in various malignancies., Objective: The purpose of the current study is to evaluate the expression level and clinical significance of CD44 in lung cancer samples., Methods: One hundred and ninety-five lung tumor samples including 74 (38%) squamous cell carcinomas (SCC), 61 (31%) adenocarcinomas (ADC), 23 (12%) large cell carcinoma (LCC) in non-small cell lung cancer (NSCLC) group and 37 (19%) small cell lung cancer (SCLC) samples were examined for the expression of CD44 using immunohistochemistry method. The correlation of CD44 expression with clinicopathological parameters as well as Ki-67 status was also assessed., Results: Univariate analysis demonstrated that CD44 expression was significantly higher in NSCLC compared to SCLC (P < 0.001). Among NSCLC, higher level of CD44 expression was found in SCC compared to ADC (P< 0.001) and LCC (P=0.046). Increased expression of CD44 was significantly correlated with higher grade tumors which correspond to poor prognosis in SCC (P=0.012) and the lower level of CD44 expression was more often found in well differentiated ADC tumors (P=0.03). In addition, high expression of CD44 was significantly associated with decreased level of proliferative marker Ki-67 (P=0.04)., Conclusions: CD44 could be a valuable tool for the study of lung CSCs and provide a novel therapeutic target for treatment of the patients with lung cancer in combination with conventional therapy.

We hereby describe the rare case of a 59-year-old patient presenting with marked hyperamylasaemia mimicking acute pancreatitis upon admission. Investigation of co-existent hypokalemia revealed the presence of ectopic adrenocorticotropic hormone secretion, leading to the final diagnosis of small cell lung cancer, exhibiting dual paraneoplastic syndromes including Cushing Syndrome and hyperamylasaemia. Although, paraneoplastic syndromes occur commonly, paraneoplastic hyperamylasaemia especially in the context of dual paraneoplastic syndromes occurring simultaneously, is extremely rare. Such misleading manifestations require a high index of suspicion on behalf of the physician, so that an underlying malignancy is not missed, and a final diagnosis combining all clinical and laboratory findings is reached. In turn, in rare cases common biochemical markers such as amylase can be used as a useful follow up index driving further management.

Objective: To analyze the expression of co-stimulatory molecules PD-1/PD-L1 in peripheral blood mononuclear cells in lung cancer patients, and to explore its biological significance., Methods: One hundred and thirty-three lung cancer patients, 25 lung infection patients and 23 healthy donors were enrolled in this study. 100 µl of whole blood from these subjects were collected. Multi-color immunofluorescence staining and flow cytometry were used to detect PD-1/PD-L1 expression. The results were statistically analyzed., Results: The expression level of CD3⁺CD8⁺ T cells in the lung cancer patients was (38.83 ± 1.74)%, significantly lower than that in the control group [(43.25 ± 3.35)%, P < 0.05]. CD8⁺CD28⁺ T cell subset in the peripheral blood of lung cancer patients was (17.73 ± 1.21)% significantly lower than that of the healthy donors [(27.96 ± 2.72)%, P < 0.01]. The CD8⁺CD28⁻ T cell subset was (21.19 ± 1.92)% in the lung cancer patients, significantly higher than that of the healthy control group [(15.18 ± 2.93)%, P < 0.05]. The expression level of PD-1 on the surface of CD8⁺CD28⁺ T cells was (10.67 ± 1.12)% in the group of lung cancer patients, significantly higher than that of the control group [(5.32 ± 1.58)%, P < 0.01]. It was also found that the expression of PD-1 on CD8⁺CD28⁻ T cells was up-regulated in the group of lung cancer patients (7.46 ± 1.25)%, significantly higher than that of the healthy control group [(2.68+1.07)%, P < 0.01]. The expression level of PD-L1 on CD68⁺ cells in the lung cancer patients was (16.03 ± 2.06)%, significantly higher than that of the healthy control group [(9.32 ± 2.00)%, P < 0.05]., Conclusion: Up-regulation of PD-1/PD-L1 on peripheral blood cells in lung cancer patients negatively regulates the lymphocytes, inhibits the immune response for killing tumor cells, and promotes tumor development and immune escape.

Small-cell lung caner (SCLC) is the most aggressive form of lung cancer. The aim of this study was to investigate whether the presence of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTCs) predicts treatment response, progression-free survival (PFS), and overall survival (OS) in SCLC patients who received standard therapy. Fifty-five SCLC patients were enrolled in this single-center prospective study. CK-19 mRNA-positive CTCs in blood samples were detected using real-time quantitative-PCR assay before the initiation of chemotherapy (B0) and after one chemotherapy cycle (B1) and three chemotherapy cycles (B3). The association with known prognostic factors and the effect of CK-19 mRNA-positive CTCs on patients' prognosis were analyzed. Patients with positivity for CK-19 mRNA-positive CTCs at B0, B1, and B3 time points had shorter PFS and OS compared with patients without (P = 0.014 and P = 0.01, respectively, at B0; P = 0.008 and P = 0.002, respectively, at B1; P = 0.003 and P = 0.001, respectively, at B3). Conversion of initial positivity for CK-19 mRNA-positive CTCs to negativity at B1 and B3 time points was associated with longer PFS and OS compared with patients with persistent positivity at three time points (P = 0.008 and P = 0.010, respectively). Multivariate analysis demonstrated that the presence of CK-19 mRNA-positive CTCs at B0, B1, and B3 time points remained strong predictors of PFS and OS after adjustment for clinically significant factors. In conclusion, detection of CK-19 mRNA-positive CTCs before and during chemotherapy is an accurate indication of subsequent disease progression and mortality for SCLC patients.

Lung cancer is one of the leading causes of death from malignancy worldwide. In particular small cell lung cancers, which comprise about 15-20% of all lung cancers, are extremely aggressive and cure rates are extremely low. Therefore, new treatment modalities are needed and detection at an early stage of disease, as well as adequate monitoring of treatment response is essential in order to improve outcome. In this respect, the use of non-invasive tools for screening and monitoring has gained increasing interest and the clinical applicability of reliable, tumor-related substances that can be detected as tumor markers in easily accessible body fluids is subject of intense investigation. Some of these indicators, such as high LDH levels in serum as a reflection of the disease, have been in use for a long time as a general tumor marker. To allow for improved monitoring of the efficacy of new therapeutic modalities and for accurate subtyping, there is a strong need for specific and sensitive markers that are more directly related to the biology and behavior of small cell lung cancer. In this review the current status of these potential markers, like CEA, NSE, ProGRP, CK-BB, SCC, CgA, NCAM and several cytokeratins will be critically analyzed with respect to their performance in blood based assays. Based on known cleavage sites for cytoplasmic and extracellular proteases, a prediction of stable fragments can be obtained and used for optimal test design. Furthermore, insight into the synthesis of specific splice variants and neo-epitopes resulting from protein modification and cleavage, offers further opportunities for improvement of tumor assays. Finally, we discuss the possibility that detection of SCLC related autoantibodies in paraneoplastic disease can be used as a very early indicator of SCLC., (Copyright © 2013. Published by Elsevier B.V.)

Objective: To report the clinical features of 20 newly diagnosed patients with GABAB receptor (GABABR) antibodies and determine the frequency of associated tumors and concurrent neuronal autoantibodies., Methods: Clinical data were retrospectively obtained and evaluated. Serum and CSF samples were examined for additional antibodies using methods previously reported., Results: Seventeen patients presented with seizures, memory loss, and confusion, compatible with limbic encephalitis (LE), one patient presented with ataxia, one patient presented with status epilepticus, and one patient presented with opsoclonus-myoclonus syndrome (OMS). Nineteen (95%) patients eventually developed LE during the course of the disease. Small-cell lung cancer (SCLC) was identified in 10 (50%) patients, all with LE. Treatment and outcome was available from 19 patients: 15 showed complete (n = 7) or partial (n = 8) neurologic improvement after steroids, IV immunoglobulins, or plasma exchange and oncologic treatment when indicated; 1 patient died of tumor progression shortly after the first cycle of immunotherapy, and 3 were not treated. Five patients with SCLC had additional onconeuronal antibodies (Ri, amphiphysin, or SOX1), and 2 without tumor had GAD65 and NMDAR antibodies, respectively. GABABR antibodies were not detected in serum of 116 patients with SCLC without neurologic symptoms., Conclusion: Our study confirms GABABR as an autoantigen of paraneoplastic and nonparaneoplastic LE and expands the phenotype of GABABR antibodies to ataxia, OMS, and status epilepticus. The long-term prognosis is dictated by the presence of a tumor. Recognition of syndromes associated with GABABR antibodies is important because they usually respond to treatment.

Objectives: The aim of the study was to investigate the clinical significance of serum mitochondrial DNA (mtDNA) in lung cancer., Design and Methods: Serum mtDNA from 65 lung cancer patients, 20 patients with benign lung diseases and 55 healthy individuals was quantified using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). Data were analyzed using statistical software SPSS 13.0., Results: Serum mtDNA levels in lung cancer patients were significantly higher, compared to those in patients with benign lung diseases and healthy individuals (u=108, p=0.000; u=293, p=0.000), and closely associated with TNM stage (p=0.01). The use of serum mtDNA facilitated detection of lung cancer at a cutoff value of 0.74×10⁴ copies/μL with a sensitivity of 86.2% and specificity of 80.7%. However, serum mtDNA levels were not associated with patient age, gender, histological type, and lymph node metastasis (p>0.05)., Conclusions: Quantification of serum mtDNA using FQ-PCR potentially serves as a novel complementary tool to improve the clinical screening and detection of lung cancer., (Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

This study aims to evaluate the association of serum neuron-specific enolase (NSE) levels with the prognosis of small cell lung cancer (SCLC). Literature retrieval, trials selection and assessment, data collection, and statistical analysis were performed according to the Revman 5.0 guidelines. Literature-based searching was guided to gather data and either fixed-effect or random-effect model was used to pool the hazard ratio (HR) according to the test of heterogeneity. A total of 11 eligible studies that included 3,497 SCLC patients and 3,344 control subjects were analyzed. About 68.6 % of patients had high serum levels of NSE, according to the cutoff value defined by the authors. The HR of high levels of NSE for overall survival (OS) was 1.74 times that of low levels of NSE in SCLC patients (95 % CI, 1.14 to 2.65; P = 0.01). Patients with high levels of NSE appear to have a poorer OS compared with those with low levels of NSE.

Introduction: The aim of this study was to develop a multiplex polymerase chain reaction (PCR)-based method for detection of circulating tumor cells in peripheral blood of lung cancer (LC) patients., Patients and Methods: Peripheral blood was collected from 71 healthy donors and 125 LC patients at different pathological stages. Samples were analyzed using multiplex PCR, and specific primers for CK19, PTHrP, and LUNX mRNA. The sensitivity of our method was set at 10 LC cells (A549 cells) in 3 mL of peripheral blood of healthy donors using spiking experiments., Results: The detection rates in LC patients for CK19, PTHrP, and LUNX were 45.6%, 64.8%, and 28%, and in healthy individuals were 7%, 7%, and 5.6%, respectively. Overall, our method produced 77.8% positive detections for at least 1 molecular marker. Twenty-eight (22.2%) were negative for expression of all markers, 39 (31.2%) were positive for expression of 1 marker, 42 (33.6%) were positive for expression of 2 markers, and 17 (13.6%) were positive for expression of all 3 markers. Detection of CK19 mRNA expression positively correlated with LC stage and distant metastases. PTHrP mRNA detection correlated positively with LC stage, presence of bone metastasis, and squamous cell carcinoma, and LUNX mRNA detection correlated with lymph node involvement. Combined detection of 2 or 3 markers was significantly correlated with metastatic disease, and negative detection of all 3 molecular markers was correlated with early stage nonmetastatic disease., Conclusion: Multiple PCR-based detection of CK19, PTHrP, and LUNX mRNA expression provides useful information for disease stage and dissemination in LC patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: Cancer-related carbohydrate epitopes, which are regarded as potential diagnostic and prognostic biomarkers, are carried on the main acute phase proteins. It is not clear, however, if the glycosylation profile is similar in different glycoproteins, or it is protein specific to some extent. The aim of the study was to compare fucosylation, α2,3 sialylation and expression of sialyl-Lewisx epitopes (sLe(x)) in the serum as a whole, AGP and haptoglobin of small cell (SCLC) and non-small cell lung cancer (NSCLC) patients with respect to healthy subjects as well as the cancer stage and its histological type., Material and Methods: Thirty-three NSCLC, 13 SCLC patients and 20 healthy volunteers were included in the study. Carbohydrate epitopes were detected by means of their reactivity with specific lectins and monoclonal anti-sLe(x) antibodies in direct or dual-ligand ELISA tests., Results: Significantly increased fucosylation was found in total serum in both cancer groups and in NSCLC haptoglobin. No difference was observed in SCLC haptoglobin or α₁-acid glycoprotein in both cancer groups. Also α2,3 sialylation was elevated in total serum, but not in α₁-acid glycoprotein. This type of sialylation was undetectable in haptoglobin by means of MAA reactivity, in both healthy and cancer subjects. Complete sLe(x) antigens were overexpressed in total NSCLC serum and SCLC AGP, and their level was considerably lowered in cancer haptoglobin., Discussion: Typical acute phase proteins, haptoglobin and AGP, exhibit different glycosylation profiles in lung cancer. Alterations observed in haptoglobin reflected the disease process better than those in AGP. Comparison of haptoglobin and AGP glycosylation to that observed in total serum suggests that some efficient carriers of disease-altered glycoproteins still remain unidentified.

We report the case of an 80-year-old male with relapsed EGFR exon 19 deletion lung adenocarcinoma treated with EGFR-tyrosine kinase inhibitor (TKI), but with poor response and rapid increase of serum neuron specific enolase (NSE). Repeat biopsy identified pathological transformation to small cell lung cancers (SCLC) retaining the same EGFR mutation. This case highlights routine serological testing of NSE may benefit for the lung adenocarcinoma patients resistant to TKIs., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Purpose: To determine the dose-limiting toxicities (DLTs) and the maximum tolerated dose (MTD) of oral topotecan administered weekly in patients with relapsed small cell lung cancer (SCLC)., Patients and Methods: Patients were treated with oral topotecan on days 1, 8, and 15, every 28 days. The dose was escalated by 0.5 mg/m² increments from the starting dose of 3 mg/m² until the MTD was reached. DLTs were defined as grade 4 neutropenia, febrile neutropenia, grade 4 thrombocytopenia, non-hematologic toxicity ≥grade 3, any toxicity precluding the treatment on days 8 or 15 of the first cycle, or delay of the second cycle for more than 7 days., Results: Eighteen patients were enrolled. Thirteen patients received oral topotecan as second-line and five as third- or further-line treatment. The DLT level was reached at 4.5 mg/m², and the MTD was determined to be 4 mg/m². DLTs consisted of grade 2/3 neutropenia and grade 2 thrombocytopenia precluding treatment on day 15 of the first cycle or on day 1 of the second cycle. The most frequent toxicities were grade 2-3 neutropenia (27.8 % of patients), grade 2-3 anemia (33.3 %), grade 2 thrombocytopenia (16.7 %), and grade 2-3 fatigue (44.4 %). The response rate was 11.1 %, the median progression-free survival 2.3 months, and the median overall survival 5.1 months., Conclusion: The recommended phase II dose of weekly oral topotecan in pretreated patients with SCLC is 4 mg/m² on days 1, 8, and 15 every 28 days.

Introduction: Soluble mesothelin-related peptides (SMRP) have been reported as potential markers for the diagnosis of malignant pleural mesothelioma (MPM). We wondered, whether a combination with a carcinoembryonic antigen (CEA) test might improve the relatively low diagnostic yield of the SMRP test., Methods: In a retrospective study, SMRP (mesothelin) and CEA serum concentrations were measured, using commercially available kits, in 93 previously untreated MPM patients, 75 patients with benign asbestos disease, and 139 patients suffering from lung cancer (LC)., Results: The differentiation between MPM, LC, and benign asbestos disease could be improved by applying the ratio mesothelin/CEA. Whereas CEA expression was found to be low in MPM, most LC patients had elevated CEA serum levels. The area under curve (AUC) of the receiver operator characteristics curve for mesothelin alone was found to be only 0.708. For mesothelin/CEA the AUC of the receiver operator characteristics curve increased to 0.978. The sensitivity was 93% (69%) at 95% (100%) specificity for the differentiation between MPM and LC. Comparison of MPM and benign asbestos disease showed that the AUC was 0.887 and the sensitivity 56% (47%) at 95% (100%) specificity. In contrast, the AUC for the mesothelin test alone was only 0.715, and for the CEA test alone it was 0.16. An average increment in sensitivity of 38% (range, 16%-63%) could be achieved by the quotient mesothelin/CEA compared with the sensitivity of mesothelin alone., Conclusion: The diagnostic yield of the mesothelin test can be considerably improved when combined with a CEA test with regard to the differential diagnosis between MPM and LC and between MPM and benign asbestos disease.

Background: Many cancers metastasize to bone, which may cause an increase in bone resorption because of the direct effects of the tumor itself or osteoclastic activation., Patients and Methods: Levels of urinary cross-linked N-telopeptide of type I collagen (uNTx) and serum cross-linked N-telopeptide of type I collagen (sNTx) were measured in 100 patients with lung cancer and 50 patients with benign respiratory disease using the Osteomark NTx urine and serum assays (Osteomark, Princeton, NJ). Bone metastasis was diagnosed by bone scintigraphy. Receiver operating characteristic (ROC) analysis was used to evaluate the detection of bone metastasis. Sensitivity and specificity to detect bone metastasis were calculated when cutoff points were set to 64 nmol bone collagen equivalents (BCE)/mmol Cr for uNTx and 22 nmol BCE/L for sNTx., Results: Patients with lung cancer and bone metastasis had significantly higher levels of both uNTx and sNTx (uNTx median [range], 61.3 [22.7-593.1] nmol BCE/mmol creatinine [Cr]; sNTx median [range], 19.7 [10.7-97.1] nmol BCE/L) than did patients with lung cancer without bone metastasis (uNTx median [range], 45.2 [19.8-153.0] nmol BCE/mmol Cr; sNTx median [range], 16.7 [11.0-28.4] nmol BCE/L), or patients with benign respiratory diseases (uNTx median [range], 40.6 [15.2-155.9] nmol BCE/mmol Cr; sNTx median [range], 14.8 [9.5-55.5] nmol BCE/L.). There was good correlation between uNTx and sNTx (R = 0.807). Area under the curve (AUC) for ROC was 0.743 for uNTx and 0.712 for sNTx. The sensitivity and specificity for the diagnosis of bone metastasis were 48.0% and 86.0%, respectively, using uNTx, and 40.0% and 87.0%, respectively, using sNTx., Conclusion: This prospective study indicates equivalency between sNTx and uNTx in sensitivity and specificity to detect bone metastasis, and both uNTx and sNTx may have value as aids in the diagnosis of bone metastasis in patients with lung cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: Vascular endothelial growth factor (VEGF) is considered as the best-validated key regulator of angiogenesis, while the prognostic role of circulating VEGF in lung cancer remains controversial. We conducted a meta-analysis to evaluate the prognostic role of circulating VEGF., Methods: Nineteen studies with a total number of 2,890 patients were analyzed in our meta-analysis. Hazard ratios (HRs) and their 95 % confidence intervals (CIs) were used to quantify the predictive ability of circulating VEGF on survival., Results: The pooled HR of all 17 studies evaluating overall survival (OS) was 1.29 (95 % CI 1.19-1.40, p < 0.001), indicating high circulating VEGF predicted poor OS. When grouped by disease stages, the pooled HRs were 0.97 (95 % CI 0.47-1.47, p < 0.001) for operable stage and 1.34 (95 % CI 1.18-1.49, p < 0.001) for inoperable stage. The pooled HRs were 1.28 (95 % CI 1.15-1.42, p < 0.001) for serum and 1.31 (95 % CI 1.13-1.49, p < 0.001) for plasma, when categorized by blood sample. Meta-analysis of circulating VEGF related to progression-free survival (PFS) was performed in 7 studies, and the pooled HR was 1.03 (95 % CI 0.96-1.09)., Conclusions: Our results indicate that high level of circulating VEGF predicts poor OS in lung cancer, yet it does not predict poor PFS.

Background: Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTC) has prognostic value in patients with solid tumors, such as advanced breast, colon, and prostate cancer. However, poor sensitivity has been reported for non-small cell lung cancer (NSCLC). To address this problem, we developed a microcavity array (MCA) system integrated with a miniaturized device for CTC isolation without relying on EpCAM expression. Here, we report the results of a clinical study on CTCs of advanced lung cancer patients in which we compared the MCA system with the CellSearch system, which employs the conventional EpCAM-based method., Methods: Paired peripheral blood samples were collected from 43 metastatic lung cancer patients to enumerate CTCs using the CellSearch system according to the manufacturer's protocol and the MCA system by immunolabeling and cytomorphological analysis. The presence of CTCs was assessed blindly and independently by both systems., Results: CTCs were detected in 17 of 22 NSCLC patients using the MCA system versus 7 of 22 patients using the CellSearch system. On the other hand, CTCs were detected in 20 of 21 small cell lung cancer (SCLC) patients using the MCA system versus 12 of 21 patients using the CellSearch system. Significantly more CTCs in NSCLC patients were detected by the MCA system (median 13, range 0-291 cells/7.5 mL) than by the CellSearch system (median 0, range 0-37 cells/7.5 ml) demonstrating statistical superiority (p = 0.0015). Statistical significance was not reached in SCLC though the trend favoring the MCA system over the CellSearch system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch system., Conclusions: The MCA system has a potential to isolate significantly more CTCs and CTC clusters in advanced lung cancer patients compared to the CellSearch system.

In this study, we present a method for efficient enrichment of small-sized circulating tumor cells (CTCs) such as those found in the blood of small-cell lung cancer (SCLC) patients using a microcavity array (MCA) system. To enrich CTCs from whole blood, a microfabricated nickel filter with a rectangular MCA (10(4) cavities/filter) was integrated with a miniaturized device, allowing for the isolation of tumor cells based on differences in size and deformability between tumor and blood cells. The shape and porosity of the MCA were optimized to efficiently capture small tumor cells on the microcavities under low flow resistance conditions, while allowing other blood cells to effectively pass through. Under optimized conditions, approximately 80% of SCLC (NCI-H69 and NCI-H82) cells spiked in 1 mL of whole blood were successfully recovered. In clinical samples, CTCs were detectable in 16 of 16 SCLC patients. In addition, the number of leukocytes captured on the rectangular MCA was significantly lower than that on the circular MCA (p < 0.001), suggesting that the use of the rectangular MCA diminishes a considerable number of carryover leukocytes. Therefore, our system has potential as a tool for the detection of CTCs in small cell-type tumors and detailed molecular analyses of CTCs.

Brain metastasis (BM) is a major cause of mortality in small-cell lung cancer (SCLC) patients; however, the molecular pathway of SCLC BM remains largely unknown because of a lack of investigation. Here we screen the levels of some candidate-soluble factors in the serum of SCLC patients and find that SCLC patients with high levels of placental growth factor (PLGF) are prone to BM. Using in vitro blood-brain barrier model, we show that PLGF derived from SCLC cells triggers vascular endothelial growth factor receptor-1-Rho-extracellular regulated protein kinase 1/2 signaling axis activation, results in disassembly of tight junction in brain endothelial cells and promotes SCLC cell transendothelial migration. Furthermore, the downregulation of PLGF suppresses SCLC cell metastasis to the brain in an experimental BM model. These data suggest that PLGF is a potential signature of SCLC BM and a prospective therapeutic target for SCLC BM.

Adipokines have a significant effect on metabolism, immunoinflammatory responses as well as on carcinogenesis; therefore, we aimed at evaluating their potential predictive and prognostic significance in lung cancer. Eighty patients--mean age 62.9 ± 9.2 years--with previously untreated lung cancer (61 NSCLC and 19 SCLC) of all stages and 40 healthy individuals were enrolled in this study. Serum levels of leptin, adiponectin and ghrelin were measured using human Radioimmunoassay kits. Serum leptin levels in lung cancer patients were lower compared to control (p < 0.0001), while adiponectin and ghrelin levels were significantly increased in patients (p = 0.0003 and p = 0.0043, respectively). Additionally, the leptin/adiponectin ratio was significantly lower in the patients group compared to controls (p < 0.0001]. There was no association between serum levels of adipokines and any of the patient clinicopathological characteristics or response to therapy. Nevertheless, patients with lower values of serum leptin had shorter overall survival (p = 0.014), whereas multivariate analysis revealed leptin levels as an independent prognostic factor for survival (p = 0.024, HR 0.452, CI 95 % 0.232-0.899). These results suggest that adipokines may play a role in the pathogenesis of lung cancer, while leptin serum levels might provide useful prognostic information.

Objective: To establish a quantitative method to detect circulating tumor cells (CTC) in patients with small cell lung cancer, and analyze its sensitivity and stability., Methods: A specific primer and probe for prepro-gastrin-releasing peptide (preproGRP) was designed and a quantitative RT-PCR method was established to detect preproGRP mRNA. Cell incorporation method was used to evaluate the sensitivity. Magnetic cell sorting (MACS) was used to isolate and purify CTC from peripheral blood, and the MACS in combination with morphological diagnosis were used for cell counting., Results: The isolation rate of CTC by MACS was 30% and the lower detection limit was 5 cells per ml blood. The sensitivity of quantitative RT-PCR in detection of preproGRP mRNA in CTC was 0.64 cells per reaction, and the lower detection limit was 50 cells per ml blood, which was lower than that of MACS. However, the cell numbers calculated by Ct value was in greater accordance (about 80%) with actual cell numbers than that obtained by MACS., Conclusions: PreproGRP quantitative RT-PCR and MACS have both advantages and disadvantages in detecting CTC of SCLC patients. MACS has a higher sensitivity, and is more favorable when CTC count is below 50 per ml blood. Meanwhile, preproGRP mRNA quantitative RT-PCR is more reliable in calculating actual cell numbers.

Background: Phenotypic biomarkers of DNA damage repair may enhance cancer risk prediction. The γ-H2AX formed at the sites of double-strand break (DSB) after ionizing radiation is a specific marker of DNA damage., Methods: In an ongoing case-control study, the baseline and ionizing radiation-induced γ-H2AX levels in peripheral blood lymphocytes (PBL) from frequency-matched 306 untreated patients with lung cancer and 306 controls were measured by a laser scanning cytometer-based immunocytochemical method. The ratio of ionizing radiation-induced γ-H2AX level to the baseline was used to evaluate interindividual variation of DSB damage response and to assess the risk of lung cancer by using unconditional multivariable logistic regression with adjustment of age, sex, ethnicity, smoking status, family history of lung cancer, dust exposure, and emphysema., Results: The mean γ-H2AX ratio was significantly higher in cases than controls (1.46 ± 0.14 vs. 1.41 ± 0.12, P < 0.001). Dichotomized at the median in controls, high γ-H2AX ratio was significantly associated with increased risk of lung cancer [OR = 2.43; 95% confidence interval (CI): 1.66-3.56]. There was also a significant dose-response relationship between γ-H2AX ratio and lung cancer risk in quartile analysis. Analysis of joint effects with other epidemiologic risk factors revealed elevated risk with increasing number of risk factors., Conclusion: γ-H2AX activity as shown by measuring DSB damage in ionizing radiation-irradiated PBLs may be a novel phenotypic marker of lung cancer risk., Impact: γ-H2AX assay is a robust and quantifiable image-based cytometer method that measures mutagen-induced DSB response in PBLs as a potential biomarker in lung cancer risk assessment.

The prognosis of small cell lung cancer (SCLC) remains poor with a 5-year survival rate of 4-6%. In non-small cell lung cancer (NSCLC), high levels of intact and cleaved forms of the receptor for urokinase plasminogen activator (uPAR) are significantly associated with short overall survival. Our aim was therefore to determine the prognostic value of the different uPAR forms in blood from SCLC patients. Serum samples from 92 treatment naive SCLC patients were analysed. Intact uPAR, uPAR(I-III), intact and cleaved uPAR, uPAR(I-III) + uPAR(II-III) and the liberated domain I, uPAR(I) were measured using time-resolved fluorescence immunoassays (TR-FIA 1-3). Assessment of association of the uPAR forms to overall survival (OS) was done using Cox regression analysis adjusted for clinical covariates [age, gender, stage, lactate dehydrogenase (LDH), WHO performance status (PS)]. Multivariate survival analysis demonstrated that high levels of uPAR(I) were significantly (p = 0.009) associated with short overall survival (OS). Patients with uPAR(I) levels above the second tertile had a hazard ratio (HR) of 1.9 (95% confidence interval (CI): 1.1-3.3), compared to patients with levels below the first tertile. High serum uPAR(I) levels are associated with short OS in SCLC patient, independent of LDH and PS.

Introduction: Plasma concentrations of circulatory markers of hemostatic activation which may be associated with tumor growth and dissemination have been shown to predict prognosis in malignancy. The present study was designed to investigate the prognostic value of plasma D-dimer level in lung cancer., Materials and Methods: Plasma levels of the D-dimer in 138 lung cancer patients [98 non-small cell lung cancer (NSCLC), 40 small cell lung cancer (SCLC)] were measured before the initiation of any therapy and each chemotherapy., Results: There were 124 (89.9%) men and 14 (10.1%) women with a mean age of 62.8 years (range 38-84). There were no statistically significant differences among the histopathologic types for NSCLC patients. Stage IIIA NSCLC group had statistically significant higher D-dimer level than stages I-II and IV. D-dimer levels were increased significantly after 4 cycles of chemotherapy in progressive disease. The median survival times in NSCLC patients were 26.6 months (95% CI, 17.6-35.6) and 15.9 months (95% CI, 4.2-27.7; p= 0.037) respectively, for patients with a low D-dimer level (≤ 1.2 ng/L) and a high D-dimer level (> 1.2 ng/L).With the cox-regression analysis, the plasma level of D-dimer and tumour stage were identified as independent predictive factors of the survival., Conclusion: These results suggest that plasmalevel of D-dimer can act as a predictive factor of decreased survival and a poor response to the treatment in lung cancer.

Introduction: Lung cancer, the leading cause of cancer deaths, is divided into 2 main classes based on its biology, therapy and prognosis: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Many cases are at an advanced stage at diagnosis, which is a major obstacle to improving outcomes. It is important to define the high risk group patients for early diagnosis and chance of cure. Blood group antigens are chemical components on erythrocyte membranes but they are also expressed on a variety of epithelial cells. Links between ABO blood groups with benign or malignant diseases, such as gastric and pancreas cancers, have been observed for a long time. In this study, we aimed to investigate any possible relationship between lung cancer histological subtypes and ABO-Rh blood groups., Materials and Methods: The files of 307 pathologically confirmed lung cancer patients were were reviewed retrospectively. Cases with a serologically determined blood group and Rh factor were included and those with a history of another primary cancer were excluded, leaving a total of 221. The distribution of blood groups of the lung cancer patients were compared with the distribution of blood groups of healthy donors admitted to the Turkish Red Crescent Blood Service in our city in the year 2012., Results: There was no significant difference between patients with lung cancer of either type and the control group in terms of distribution of ABO blood groups and Rh factor (p: 0.073). There was also no relationship with non small cell cancer histological subtypes., Conclusions: In this study, we found no relationship between the ABO-Rhesus blood groups and NSCLC and SCLC groups. To our knowledge this is the first analysis of ABO blood groups in SCLC patients.

Lung cancer accounts for the highest number of cancer-related deaths worldwide. Early diagnosis significantly increases the disease-free survival rate and a large amount of effort has been expended in screening trials and the development of early molecular diagnostics. However, a gold standard diagnostic strategy is not yet available. Here, based on miRNA expression profile in lung cancer and using a novel in silico reverse-transcriptomics approach, followed by analysis of the interactome; we have identified potential transcription factor (TF) markers that would facilitate diagnosis of subtype specific lung cancer. A subset of seven TF markers has been used in a microarray screen and was then validated by blood-based qPCR using stage-II and IV non-small cell lung carcinomas (NSCLC). Our results suggest that overexpression of HMGA1, E2F6, IRF1, and TFDP1 and downregulation or no expression of SUV39H1, RBL1, and HNRPD in blood is suitable for diagnosis of lung adenocarcinoma and squamous cell carcinoma sub-types of NSCLC. Here, E2F6 was, for the first time, found to be upregulated in NSCLC blood samples. The miRNA-TF-miRNA interaction based molecular mechanisms of these seven markers in NSCLC revealed that HMGA1 and TFDP1 play vital roles in lung cancer tumorigenesis. The strategy developed in this work is applicable to any other cancer or disease and can assist in the identification of potential biomarkers.

Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule, member of the immunoglobulin gene superfamily that seems to participate in the evolution of the metastatic process. We investigated the significance of baseline soluble ICAM-1 levels on the outcome of patients with small-cell lung cancer and whether soluble ICAM-1 is a predictive marker for objective response during and after chemotherapy in patients with small-cell lung cancer. Fifty patients with recently diagnosed small-cell lung cancer, as well as 27 healthy smokers, were enrolled. Blood samples were collected at the time of diagnosis, during and at the end of chemotherapy. Data were correlated with the characteristics of the patients and survival as well as with ICAM-1 predictive role for objective response. Statistical significant values of baseline soluble ICAM between patients and controls (p < 0.001) were observed. Multivariate analysis revealed an elevated risk of death of 9 % in the first year after diagnosis for every 10 units of increased soluble ICAM-1 at the baseline (p = 0.046). Performance status and disease stage were also independent prognostic factors. Patients with extensive disease who achieved an objective response during chemotherapy showed a significant decrease (25.8 %) in their soluble ICAM-1 levels compared with baseline levels (p = 0.001). Alongside performance status and disease stage, baseline soluble ICAM-1 could be evaluated as an additional prognostic factor in patients with small-cell lung cancer. Also, a possible role for soluble ICAM-1 may exist as a predictive marker for objective response during chemotherapy for patients with extensive disease (p = 0.001).

The aim of this study was to identify serum protein fingerprints of small cell lung cancer (SCLC) and potential biomarkers related to chemotherapy resistance of SCLC with surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS). A total of 60 SCLC patients and 48 age- and sex-matched healthy individuals were enrolled. The chemotherapy regimen was cisplatin plus etoposide. All patients received two cycles of chemotherapy. Serum protein profiles were detected using SELDI-TOF MS and the spectra were analyzed with support vector machines (SVMs). Western blotting was performed to verify the results of SELDI-TOF MS. Three top scored peaks, at m/z of 6269, 9043 and 13124 Da, were finally selected as potential biomarkers for detection of SCLC. The SVM classifier separated the SCLC from the healthy samples in the blind test, with a sensitivity of 92.4% and a specificity of 92.5%. For the 56 eligible chemotherapy patients, 4 had a complete response (7.14%), 39 patients had a partial response (69.6%), 9 patients had a stable disease (16.1%) and 4 patients had a progressive disease (7.14%). The model constructed using two protein peaks with m/z of 8830 and 10468 Da separated the chemotherapy-resistant group from the chemotherapy-sensitive group with a sensitivity of 80.0% and a specificity of 80.0%. Initial protein database searching identified 10468 Da as S100-A9 which was confirmed by western blotting. The present results suggest that the combination of SELDI-TOF MS with SVM may provide a useful means in the search for serum biomarkers for predicting chemotherapy resistance in patients with SCLC.

Glycosylation is a key post-translational protein modification which appears important in malignant transformation and tumor metastasis. Abnormal glycosylation of different proteins can often be measured in the blood serum. In this study, we extend our serum-based structural investigations to samples provided by patients diagnosed with lung cancer, paying particular attention to the effects of smoking on the serum glycomic traces. Following a battery of glycomic tests, we find that several fucosylated tetra-antennary structures with varying degrees of sialylation are increased in their abundances in control samples provided by the former smokers, with further elevations in the lung cancer patients who were former smokers. Further detailed investigations demonstrated that the level of outer-arm fucosylation was elevated in the control samples of the former smokers and again in the lung cancer samples provided by the former smokers. This trend was particularly noticeable for the tri- and tetra-antennary structures. Different ratios of sialylation linkages were also observed that could be correlated with the different states of health and smoking status. Decreases in the abundance levels of isomers with two and three α2,3-linked sialic acids and an increased abundance of an isomer with two α2,6-linked sialic acids were noted for a fucosylated tri-sialylated tri-antennary glycan. These results demonstrate the long-term effects of smoking on glycomic profiles and that this factor needs to be considered in these and other serum-based analyses.

We evaluated the diagnostic and prognostic efficacy of human epididymis protein 4 (HE4) for lung cancer patients by using our novel enzyme-linked immunosorbent assay (ELISA) system. We measured serum HE4 levels of cancer patients including 49 lung cancer and 18 ovarian cancer patients. Furthermore, we evaluated the relationship between serum HE4 levels and overall survival after chemotherapy of 24 lung cancer patients. Serum HE4 levels were significantly higher for non-small, small cell lung cancer and ovarian cancer patients than for healthy controls. The area under the receiver operating characteristic curve (AUC) was calculated for differentiation of lung cancer patients and healthy controls. AUC for serum HE4 was 0.988 for differentiating lung cancer patients from healthy controls, with a cutoff value of 6.56 ng/ml (sensitivity = 89.8%, specificity = 100%). Serum HE4 levels were elevated in 36/40 (90.0%) non-small cell lung cancer patients, 8/9 (88.9%) small cell lung cancer patients and 8/18 (44.4%) ovarian cancer patients. High levels of serum HE4 (>15 ng/ml) after chemotherapy were significantly correlated with worse overall survival after the treatment. These findings suggest that serum HE4 is a potential diagnostic and prognostic marker for lung cancer patients.

Introduction: We aim to develop a simple and sensitive array-based method for the detection of epidermal growth factor receptor (EGFR) gene mutations in the plasma of non-small-cell lung cancer patients and determine its use in the follow-up of those on tyrosine-kinase inhibitor (TKI) therapy., Method: DNA from 100 μl of plasma was amplified in the presence of peptide nucleic acid clamp to provide single-stranded template for the allele-specific arrayed primer extension reaction, incorporating cyanine-5-deoxycytidine triphosphate in the newly synthesized strands. The fluorescent product was visualized by laser at 670 nm., Results: Eleven different types of EGFR TKI drug-sensitive mutants (SM) were identified in plasma-DNA from 46 of 51 patients. Five patients carried only wild-type sequence. Plasma-DNA finding was concordant in 36 of 37 cases with tumor-sequencing data. This method could detect as little as 62.5 copies of mutant L858R; 125 copies of E709K + G719A or 625 copies of del 746-750 in the presence of 100,000 copies of wild-type EGFR. In 21 patients on longitudinal follow-up for up to 18 months, SM was found in all initial plasma samples, except for three samples collected after recent chemotherapy. Nine of 16 patients (56%) who responded to TKI had undetectable plasma EGFR mutant. SM was present concurrently with drug-resistant mutant in 44% of patients with disease progression while on TKI, the remaining 56% might have other mechanisms of resistance., Conclusion: The EGFR array provides a sensitive, inexpensive, and robust method for monitoring non-small-cell lung cancer patients' response to TKI, and obviates the need of repeated lung biopsy.

Background and Objectives: We investigated various tumor markers in patients with surgically treated small cell lung cancer (SCLC) to identify the markers closely correlated to pathological staging and to predict survival by retrospective analyses., Methods: Reviewing database records between 1990 and 2007 revealed 36 patients with SCLC, that were grouped according to clinical and pathological stages. Receiver operating characteristic (ROC) curves were calculated for serum levels of various tumor makers to predict the pathological stage. The cut-off value was calculated from the ROC curve of the significant marker. Survival in patient groups divided by the new cut-off value was calculated., Results: Serum levels of various tumor makers were not significantly different between the pathological stage groups, except for serum sialyl Lewis X (SLX). ROC curve of SLX was significantly correlated to pathological stages (P = 0.0136). The calculated SLX cut-off value was 25.1 U/ml, with 80% sensitivity and 70% specificity. Five-year survival of patients selected by this new cut-off was 82.5%, whereas that with the standard cut-off (38.0 U/ml) was 55.9%., Conclusions: Serum SLX values were associated with pathological stage and survival after surgery in SCLC patients., (Copyright © 2011 Wiley Periodicals, Inc.)

Introduction: To investigate whether decrease in the serum levels of pro-gastrin releasing peptide (ProGRP) and neuron-specific enolase (NSE) were correlated with the radiological response in patients with small-cell lung cancer (SCLC)., Methods: Of the 196 patients, we retrospectively reviewed 118 patients elevated baseline levels of ProGRP and NSE prior to the initial therapy (IT) who survived for more than 1 month. The radiological response was assessed by Response Evaluation Criteria in Solid Tumors (RECIST 1.1)., Results: Decrease in the serum ProGRP was strongly correlated with the decrease of the sum of the tumor diameters (SOD) before the third course (ρ=0.50) and after the fourth course (ρ=0.42) of IT. Decrease in the serum NSE was weakly correlated with the decrease of the SOD after the fourth course (ρ=0.27), but not before the third courses (ρ=0.22). In the receiver operating characteristic (ROC) curves predicting 1-year survivors, the area under the curve (AUC) for percent changes in serum ProGRP before the third course were significantly larger than those for NSE (0.714 vs. 0.527, p=0.004)., Conclusions: Percent changes in serum ProGRP showed better correlation to SOD and prognostic impact than that of NSE., (Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.)