Development and validation of a supervised deep learning algorithm for automated whole-slide programmed death-ligand 1 tumour proportion score assessment in non-small cell lung cancer.

AIMS: Immunohistochemical programmed death-ligand 1 (PD-L1) staining to predict responsiveness to immunotherapy in patients with advanced non-small cell lung cancer (NSCLC) has several drawbacks: a robust gold standard is lacking, and there is substantial interobserver and intraobserver variance, with up to 20% discordance around cutoff points. The aim of this study was to develop a new deep learning-based PD-L1 tumour proportion score (TPS) algorithm, trained and validated on a routine diagnostic dataset of digitised PD-L1 (22C3, laboratory-developed test)-stained samples. METHODS AND RESULTS: We designed a fully supervised deep learning algorithm for whole-slide PD-L1 assessment, consisting of four sequential convolutional neural networks (CNNs), using aiforia create software. We included 199 whole slide images (WSIs) of 'routine diagnostic' histology samples from stage IV NSCLC patients, and trained the algorithm by using a training set of 60 representative cases. We validated the algorithm by comparing the algorithm TPS with the reference score in a held-out validation set. The algorithm had similar concordance with the reference score (79%) as the pathologists had with one another (75%). The intraclass coefficient was 0.96 and Cohen's κ coefficient was 0.69 for the algorithm. Around the 1% and 50% cutoff points, concordance was also similar between pathologists and the algorithm. CONCLUSIONS: We designed a new, deep learning-based PD-L1 TPS algorithm that is similarly able to assess PD-L1 expression in daily routine diagnostic cases as pathologists. Successful validation on routine diagnostic WSIs and detailed visual feedback show that this algorithm meets the requirements for functioning as a 'scoring assistant'.

Does biomarker use in oncology improve clinical trial failure risk? A large-scale analysis.

PURPOSE: To date there has not been an extensive analysis of the outcomes of biomarker use in oncology. METHODS: Data were pooled across four indications in oncology drawing upon trial outcomes from www.clinicaltrials.gov: breast cancer, non-small cell lung cancer (NSCLC), melanoma and colorectal cancer from 1998 to 2017. We compared the likelihood drugs would progress through the stages of clinical trial testing to approval based on biomarker status. This was done with multi-state Markov models, tools that describe the stochastic process in which subjects move among a finite number of states. RESULTS: Over 10000 trials were screened, which yielded 745 drugs. The inclusion of biomarker status as a covariate significantly improved the fit of the Markov model in describing the drug trajectories through clinical trial testing stages. Hazard ratios based on the Markov models revealed the likelihood of drug approval with biomarkers having nearly a fivefold increase for all indications combined. A 12, 8 and 7-fold hazard ratio was observed for breast cancer, melanoma and NSCLC, respectively. Markov models with exploratory biomarkers outperformed Markov models with no biomarkers. CONCLUSION: This is the first systematic statistical evidence that biomarkers clearly increase clinical trial success rates in three different indications in oncology. Also, exploratory biomarkers, long before they are properly validated, appear to improve success rates in oncology. This supports early and aggressive adoption of biomarkers in oncology clinical trials.

Cost-effectiveness of pembrolizumab versus chemotherapy as first-line treatment in PD-L1-positive advanced non-small-cell lung cancer in the USA.

Aim: This analysis aimed to evaluate the cost-effectiveness of pembrolizumab monotherapy as first-line treatment in advanced non-small-cell lung cancer patients with a programmed death ligand 1 (PD-L1) tumor proportion score ≥1% from a US payer perspective. Materials & methods: A partitioned survival model was developed using efficacy and safety data from the KEYNOTE-042 trial and projected over 20 years. Costs accounted for treatment, toxicity and disease management. Quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios were reported. Results: Pembrolizumab resulted in an expected gain of 0.60 life years and 0.49 QALYs compared with platinum-based chemotherapy. The incremental cost-effectiveness ratio was US$130,155/QALY. Conclusion: Pembrolizumab is projected to be cost-effective compared with platinum-based chemotherapy as first-line treatment for advanced non-small-cell lung cancer with PD-L1 tumor proportion score ≥1%.

A Systematic Review of Economic Evaluations Assessing the Cost-Effectiveness of Licensed Drugs Used for Previously Treated Epidermal Growth Factor Receptor (EGFR) and Anaplastic Lymphoma Kinase (ALK) Negative Advanced/Metastatic Non-Small Cell Lung Cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most commonly diagnosed cancers. There are many published studies of cost-effectiveness analyses of licensed treatments, but no study has compared these studies or their approaches simultaneously. OBJECTIVE: To investigate the methodology used in published economic analyses of licensed interventions for previously treated advanced/metastatic NSCLC in patients without anaplastic lymphoma kinase or epidermal growth factor receptor expression. METHODS: A systematic review was performed, including a systematic search of key databases (e.g. MEDLINE, EMBASE, Web of Knowledge, Cost-effectiveness Registry) limited to the period from 01 January 2001 to 26 July 2019. Two reviewers independently screened, extracted data and quality appraised identified studies. The reporting quality of the studies was assessed by using the Consolidated Health Economic Evaluation Reporting Standards and the Philips' checklists. RESULTS: Thirty-one published records met the inclusion criteria, which corresponded to 30 individual cost-effectiveness analyses. Analytical approaches included partitioned survival models (n = 14), state-transition models (n = 7) and retrospective analyses of new or published data (n = 8). Model structure was generally consistent, with pre-progression, post-progression and death health states used most commonly. Other characteristics varied more widely, including the perspective of analysis, discounting, time horizon, usually to align with the country that the analysis was set in. CONCLUSIONS: There are a wide range of approaches in the modelling of treatments for advanced NSCLC; however, the model structures are consistent. There is variation in the exploration of sensitivity analyses, with considerable uncertainty remaining in most evaluations. Improved reporting is necessary to ensure transparency in future analyses.

Evaluating the Application of Tissue-Specific Dose Kernels Instead of Water Dose Kernels in Internal Dosimetry: A Monte Carlo Study.

PURPOSE: The aim of this work is to evaluate the application of tissue-specific dose kernels instead of water dose kernels to improve the accuracy of patient-specific dosimetry by taking tissue heterogeneities into consideration. MATERIALS AND METHODS: Tissue-specific dose point kernels (DPKs) and dose voxel kernels (DVKs) for yttrium-90 (90Y), lutetium-177 (177Lu), and phosphorus-32 (32P) are calculated using the Monte Carlo (MC) simulation code GATE (version 7). The calculated DPKs for bone, lung, adipose, breast, heart, intestine, kidney, liver, and spleen are compared with those of water. The dose distribution in normal and tumorous tissues in lung, liver, and bone of a Zubal phantom is calculated using tissue-specific DVKs instead of those of water in conventional methods. For a tumor defined in a heterogeneous region in the Zubal phantom, the absorbed dose is calculated using a proposed algorithm, taking tissue heterogeneity into account. The algorithm is validated against full MC simulations. RESULTS: The simulation results indicate that the highest differences between water and other tissue DPKs occur in bone for 90Y (12.2% ± 0.6%), 32P (18.8% ± 1.3%), and 177Lu (16.9% ± 1.3%). The second highest discrepancy corresponds to the lung for 90Y (6.3% ± 0.2%), 32P (8.9% ± 0.4%), and 177Lu (7.7% ± 0.3%). For 90Y, the mean absorbed dose in tumorous and normal tissues is calculated using tissue-specific DVKs in lung, liver, and bone. The results are compared with doses calculated considering the Zubal phantom water equivalent and the relative differences are 4.50%, 0.73%, and 12.23%, respectively. For the tumor in the heterogeneous region of the Zubal phantom that includes lung, liver, and bone, the relative difference between mean calculated dose in tumorous and normal tissues based on the proposed algorithm and the values obtained from full MC dosimetry is 5.18%. CONCLUSIONS: A novel technique is proposed considering tissue-specific dose kernels in the dose calculation algorithm. This algorithm potentially enables patient-specific dosimetry and improves estimation of the average absorbed dose of 90Y in a tumor located in lung, bone, and soft tissue interface by 6.98% compared with the conventional methods.

Hormonal receptors in lung adenocarcinoma: expression and difference in outcome by sex.

BACKGROUND: Lung cancer seems to have different epidemiological, biomolecular and clinical characteristics in females than in males, with a better prognosis for women. The aim of the study is to determine gender differences in lung adenocarcinoma in terms of androgen (AR), estrogen (ER)α and progesterone (PgR) receptors expression and their impact on outcome. RESULTS: Overall survival was significantly better in ERα and in PgR positive lung adenocarcinoma patients (median survival 45 vs. 19 months).Eight out of 62 patients showed positive expression of nuclear (n) AR and 18 of cytoplasmic (c) AR with a significantly better survival (49 vs. 19 and 45 vs. 19 months, respectively). There was a significant difference in survival between patients with vs. without c-AR expression (30 vs. 17 months). Finally, in the subgroup of women, median survival was greater in positive expression of c-AR than for women with negative c-AR (45 vs. 21 months). MATERIALS AND METHODS: We conducted an analysis on a cohort of 62 patients with advanced NSCLC treated at our institution. We investigated the immunohistochemical expression of n/c AR, ERα and PgR in 62 NSCLC and we correlated it with patients' clinic-pathologic characteristics and with prognosis. CONCLUSIONS: Our results showed that the positive expression of one hormonal receptor could represent a prognostic factor.Furthermore our study suggests that AR should become object of close examination in a larger series of lung adenocarcinoma patients, also for selection of the patients with best prognosis that can perform more chemotherapy lines.

Value of Ki-67 and computed tomography in the assessment of peripheral lung adenocarcinoma.

PURPOSE: This study was designed to determine whether proliferation antigen Ki-67 and/or a computed tomography (CT) value could be used to evaluate the clinical-pathological features of peripheral lung adenocarcinoma. MATERIALS AND METHODS: A total of 116 eligible lung cancer patients were enrolled. Nodule size, lymph node metastasis, differentiation, Ki-67 expression and CT findings were assessed. The relationship between clinic parameters and the CT feature was analysed statistically. RESULTS: The percentage of lesions that had ground-glass opacity or localised air bronchogram was significantly greater in low CT value group (<30, p < 0.05). No significant association was observed between CT value and size in the subgroup with CT value > 0 (p = 0.66). As a proliferative marker of lung cancer, Ki-67 was present in a total of 115 (99.9%) of the 116 evaluable primary lung cancers. There was a statistically significant correlation between the Ki-67 index and CT value (p < 0.05). Compared to CT value, Ki-67 index possessed higher sensitivity to predict the differentiation and lymph node metastasis of peripheral lung adenocarcinoma, adding of CT value would enhance its specificity. CONCLUSION: Combination of Ki-67 expression and CT value determination was useful for the classification of differentiation and metastatic or proliferative potential of peripheral lung adenocarcinoma.

[Issues and current limits for immunohistochemical assessment of PD-L1 status in bronchial biopsies]. / Enjeux et limites actuelles de l'évaluation du statut de PD-L1 par immunohistochimie sur des biopsies bronchiques.

Immunotherapy targeting the PD-L1/PD-1 axis has shown recently some promising results in metastatic lung cancer patients. This treatment seems to be more effective when a high expression of PD-L1 is detected by immunohistochemistry in bronchial biopsies. In this regard, this targeted therapy will be proposed soon in metastatic lung cancer patients. This immunotherapy could be dependent to the immunohistochemical (IHC) assessment of PD-L1 expression, thus considered as a companion diagnostic test. This near perspective poses challenges with regard to the positivity threshold value for PD-L1 expression before therapy administration, the positive cellular compartment (tumour cells and/or immune cells), the percentage of positive cells and the clone which is used. A couple of patients have a good response to treatment targeting the PD-1/PD-L1 axis despite the absence or a weak PD-L1 expression. However the assessment of PD-L1 expression by immunohistochemistry will be the only mandatory approach before therapeutic strategies targeting the PD1/PD-L1 axis for lung cancer patients. In this review, the main challenges of using PD-L1 immunohistochemistry as a potential companion diagnostic tool for metastatic lung cancer patient immunotherapy will be discussed.

Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non-Small-Cell Lung Cancer.

IMPORTANCE: Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non-small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression. OBJECTIVE: To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies. DESIGN, SETTING, AND PARTICIPANTS: PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non-small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined. MAIN OUTCOMES AND MEASURES: PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study. RESULTS: Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02). CONCLUSIONS AND RELEVANCE: Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.

Total Synthesis and Biological Assessment of Mandelalide†A.

A new convergent total synthesis of the marine macrolide mandelalide A (1) has been developed that is based on macrocyclic ring closure by a Shiina-type macrolactonization and the construction of the requisite precursor seco acid by a highly efficient Sonogashira cross-coupling reaction between two fragments of comparable complexity. Key steps in the elaboration of the acid building block were the enantioselective, catalytic addition of a protected acetylene to crotonaldehyde and the construction of the tetrahydropyran unit that is embedded in the macrocycle by means of an acid-catalyzed Prins reaction. The synthesis of the alcohol fragment features the formation of the trisubstituted tetrahydrofuran ring through an acetal cleavage/epoxide opening cascade reaction and a rarely used radical alkynylation of a primary alkyl iodide. Intriguingly, the dihydroxylation of a terminal double bond as part of the synthesis of this building block gave the same major product for both the α- and ß-AD-mix reagents, albeit with moderate or low selectivity. Synthetic mandelalide A (1) was a potent proliferation inhibitor of A549, HT460, and H1299 human lung cancer cells in vitro, but not of SK-N-SH neuroblastoma cells. However, in no case did we observe complete cell kill even at the highest compound concentration tested (5 µm).

A novel device based on a fluorescent cross-responsive sensor array for detecting lung cancer related volatile organic compounds.

In this paper, a novel, simple, rapid, and low-cost detection device for lung cancer related Volatile Organic Compounds (VOCs) was constructed. For this task, a sensor array based on cross-responsive mechanism was designed. A special gas chamber was made to insure sensor array exposed to VOCs sufficiently and evenly, and FLUENT software was used to simulate the performance of the gas chamber. The data collection and processing system was used to detect fluorescent changes of the sensor arrays before and after reaction, and to extract unique patterns of the tested VOCs. Four selected VOCs, p-xylene, styrene, isoprene, and hexanal, were detected by the proposed device. Unsupervised pattern recognition methods, hierarchical cluster analysis and principal component analysis, were used to analyze data. The results showed that the methods could 100% discriminate the four VOCs. What is more, combined with artificial neural network, the correct rate of quantitative detection was up to 100%, and the device obtained responses at concentrations below 50 ppb. In conclusion, the proposed detection device showed excellent selectivity and discrimination ability for the VOCs related to lung cancer. Furthermore, our preliminary study demonstrated that the proposed detection device has brilliant potential application for early clinical diagnosis of lung cancer.

A fine decision tree consisted of CK5/6, IMP3 and TTF1 for cytological diagnosis among reactive mesothelial cells, metastatic adenocarcinoma of lung and non-lung origin in pleural effusion.

The utility of combination with CK5/6, IMP3 and TTF1 to differentiate among reactive mesothelial cells (RMs), metastatic adenocarcinoma of lung (LAC) and non-lung (NLAC) origin was investigated by using immunocytochemistry (ICC) and conventional PCR (C-PCR) in pleural effusion. A total of 108 cell blocks (32 RMs, 51 LAC and 25 NLAC were evaluated by ICC for CK5/6, IMP3 and TTF1 protein expression. In addition, we further performed C-PCR for amplification of CK5/6, IMP3 and TTF1 DNA from 28 specimens (9 MAC and 7 RMs, 6 LAC and 6 NLAC) for molecular diagnosis. CK5/6 staining was observed in the majority of reactive specimens (78.1%) and was rare in adenocarcinoma cells (14.5%), whereas the opposite was true for IMP3 and TTF1. We found a high frequency of TTF1 positivity (76.5%) in LAC, but not in NLAC (4.0%); while there was no significant difference of IMP3 expression in LAC (88.2%) and NLAC (88.0%). The 487 bp DNA fragments of IMP3 was expected to be amplified in 6/9 of adenocarcinoma cases showed negative in ICC; and the 394 bp DNA fragments of CK5/6 was also expected to be amplified in 4/7 of RMs cases showed negative in ICC. Consistent with ICC results, there was significant difference of TTF1 expression in the LAC and NLAC compared with IMP3 expression. The combination with CK5/6, IMP3 and TTF1 immunostaining appears to be useful to improve the accuracy of cytological diagnoses between RMs, metastatic adenocarcinoma of lung and non-lung origin in pleural effusion. In addition, C-PCR may act as a useful supplemental approach for ICC, especially negative cases in ICC for differential cytological diagnosis.

Cost effectiveness of EML4-ALK fusion testing and first-line crizotinib treatment for patients with advanced ALK-positive non-small-cell lung cancer.

PURPOSE: ALK-targeted therapy with crizotinib offers significant improvement in clinical outcomes for the treatment of EML4-ALK fusion-positive non-small-cell lung cancer (NSCLC). We estimated the cost effectiveness of EML4-ALK fusion testing in combination with targeted first-line crizotinib treatment in Ontario. PATIENTS AND METHODS: A cost-effectiveness analysis was conducted using a Markov model from the Canadian Public health (Ontario) perspective and a lifetime horizon in patients with stage IV NSCLC with nonsquamous histology. Transition probabilities and mortality rates were calculated from the Ontario Cancer Registry and Cancer Care Ontario New Drug Funding Program (CCO NDFP). Costs were obtained from the Ontario Case Costing Initiative, CCO NDFP, University Health Network, and literature. RESULTS: Molecular testing with first-line targeted crizotinib treatment in the population with advanced nonsquamous NSCLC resulted in a gain of 0.011 quality-adjusted life-years (QALYs) compared with standard care. The incremental cost was Canadian $2,725 per patient, and the incremental cost-effectiveness ratio (ICER) was $255,970 per QALY gained. Among patients with known EML4-ALK-positive advanced NSCLC, first-line crizotinib therapy provided 0.379 additional QALYs, cost an additional $95,043 compared with standard care, and produced an ICER of $250,632 per QALY gained. The major driver of cost effectiveness was drug price. CONCLUSION: EML4-ALK fusion testing in stage IV nonsquamous NSCLC with crizotinib treatment for ALK-positive patients is not cost effective in the setting of high drug costs and a low biomarker frequency in the population.

Longitudinal assessment of TUBB3 expression in non-small cell lung cancer patients.

INTRODUCTION: Class-III-beta-tubulin (TUBB3) expression may be a potential predictive factor for treatment with microtubule interfering cytotoxic drugs in non-small cell lung cancer (NSCLC). Potential changes in TUBB3 expression during chemotherapy may be of interest if future choice of chemotherapy is to be based on TUBB3 expression. If the biomarker expression changes during chemotherapy, biopsies before initiation of chemotherapy beyond first line may be needed if treatment decision is to be based on TUBB3 expression. Thus, the aim was to explore TUBB3 expression heterogeneity and changes during chemotherapy. MATERIALS AND METHODS: TUBB3 expression was investigated by immunohistochemistry performed on diagnostic biopsies and on available subsequent resection specimens in 65 NSCLC patients stage T1-3N0-2 who received neoadjuvant carboplatin and paclitaxel (NAC-group). Another group of 53 NSCLC patients stage T1-4N0-1 was treated with surgery alone without preceding chemotherapy (OP-group). Paired repeated samples were compared in order to evaluate for changes in TUBB3 expression. RESULTS: No statistically significant change in TUBB3 expression was observed between initial diagnostic biopsies and subsequent surgical resections of primary tumors in either the OP-group (p = 0.124) or the NAC-group (p = 0.414). When dichotomized into high and low TUBB3 expression, discordance between diagnostic biopsies and resection specimens of the primary tumors occurred in 22 % and 40 % in the OP-group and NAC-group, respectively (p = 0.169). Significantly more patients having low TUBB3 expression experienced down-staging during neoadjuvant chemotherapy compared to patients having high TUBB3 expression (p = 0.022). CONCLUSION: A high degree of discordance of TUBB3 expression between paired repeated tumor samples was observed, which likely reflects intratumoral heterogeneity. This emphasizes a need for optimal tumor tissue samples in order to stratify patients based on TUBB3 expression. No significant changes in TUBB3 expression after neoadjuvant carboplatin and paclitaxel chemotherapy occurred, suggesting no need for rebiopsy in case second-line chemotherapy with microtubule interfering cytotoxic treatments is necessary.

Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.

BACKGROUND: Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA. METHODS: Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues. RESULTS: One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants. CONCLUSIONS: The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.

Metabolic signatures of lung cancer in biofluids: NMR-based metabonomics of blood plasma.

In this work, the variations in the metabolic profile of blood plasma from lung cancer patients and healthy controls were investigated through NMR-based metabonomics, to assess the potential of this approach for lung cancer screening and diagnosis. PLS-DA modeling of CPMG spectra from plasma, subjected to Monte Carlo Cross Validation, allowed cancer patients to be discriminated from controls with sensitivity and specificity levels of about 90%. Relatively lower HDL and higher VLDL + LDL in the patients' plasma, together with increased lactate and pyruvate and decreased levels of glucose, citrate, formate, acetate, several amino acids (alanine, glutamine, histidine, tyrosine, valine), and methanol, could be detected. These changes were found to be present at initial disease stages and could be related to known cancer biochemical hallmarks, such as enhanced glycolysis, glutaminolysis, and gluconeogenesis, together with suppressed Krebs cycle and reduced lipid catabolism, thus supporting the hypothesis of a systemic metabolic signature for lung cancer. Despite the possible confounding influence of age, smoking habits, and other uncontrolled factors, these results indicate that NMR-based metabonomics of blood plasma can be useful as a screening tool to identify suspicious cases for subsequent, more specific radiological tests, thus contributing to improved disease management.

Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry.

Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and ß-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.

Pathologic assessment of radiofrequency ablation of pulmonary metastases.

PURPOSE: To evaluate pathologically the effectiveness of radiofrequency (RF) ablation in the treatment of pulmonary metastases. MATERIALS AND METHODS: Patients with multiple pulmonary metastases scheduled for surgical resection were prospectively enrolled. Patients underwent RF ablation of one percutaneously accessible tumor and within 2-4 weeks underwent surgical resection of the ablated tumor and any additional lesions. Resected tumors all were assessed by routine light microscopy, and selected tumors were assessed by immunohistochemistry with MIB1 and proliferative cell nuclear antigen (PCNA). Relationship of ablation zone to the tumor and viability of the ablated tumors were assessed. RESULTS: Nine patients (four men and five women) 46-76 years of age were included in the study. Four patients had metastatic colorectal carcinoma, and five patients had metastases from soft tissue sarcomas. Ablated tumors ranged from 1.0-3.0 cm in diameter. Each target lesion was completely encompassed by the ablation zone. All tumor tissue within the ablation zone showed characteristic changes of coagulative necrosis with hematoxylin and eosin staining. Tumors showed preservation of MIB1 staining but loss of PCNA protein staining. RF ablation resulted in complete coagulative necrosis of all the pulmonary metastases treated in the study. CONCLUSIONS: Although this series is small, it provides histologic support for RF ablation as an effective treatment for selected pulmonary metastases.

Usefulness of tissue microarrays for assessment of protein expression, gene copy number and mutational status of EGFR in lung adenocarcinoma.

Specific inhibitors targeting the epidermal growth factor receptor (EGFR) can increase survival rates in certain lung adenocarcinoma patients with mutations in the EGFR gene. Although such EGFR-targeted therapies have been approved for use, there is no general consensus among surgical pathologists on how the EGFR status should be tested in lung adenocarcinoma tissues and whether the results of immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and mutational analysis by molecular methods correlate. We evaluated the EGFR status in 61 lung adenocarcinomas by IHC (using total and mutant-specific antibodies against EGFR), by FISH analysis on tissue microarrays (TMAs), and by direct sequencing. The results of each method were compared using χ² and κappa statistics. The sensitivity and negative predictive value estimating the presence of abnormal EGFR for each test was calculated. The results show that, with respect to expression patterns and clinicopathological parameters, the total and mutant-specific EGFR detected by immunohistochemistry and FISH analysis on TMAs are valid and are equivalent to conventional methods performed on whole-tissue sections. Abnormal EGFR was detected in 52.4% of patients by IHC, FISH, and sequencing. The best sensitivity (100%) and negative predictive value (100%) was determined by evaluating the EGFR status with all methods. Testing for molecular changes in EGFR using a single test is likely to underestimate the presence of EGFR abnormalities. Taken together, these results demonstrate the high potential of TMAs to test for the major mechanisms of EGFR activation in patients with lung adenocarcinoma.