Clinical validation of a blood-based classifier for diagnostic evaluation of asymptomatic individuals with pulmonary nodules.

BACKGROUND: The number of pulmonary nodules detected in the US is expected to increase substantially following recent recommendations for nationwide CT-based lung cancer screening. Given the low specificity of CT screening, non-invasive adjuvant methods are needed to differentiate cancerous lesions from benign nodules to help avoid unnecessary invasive procedures in the asymptomatic population. We have constructed a serum-based multi-biomarker panel and assessed its clinical accuracy in a retrospective analysis of samples collected from participants with suspicious radiographic findings in the Prostate, Lung, Chest and Ovarian (PLCO) cancer screening trial. METHODS: Starting with a set of 9 candidate biomarkers, we identified 8 that exhibited limited pre-analytical variability with increasing clotting time, a key pre-analytical variable associated with the collection of serum. These 8 biomarkers were evaluated in a training study consisting of 95 stage I NSCLC patients and 186 smoker controls where a 5-biomarker pulmonary nodule classifier (PNC) was selected. The clinical accuracy of the PNC was determined in a blinded study of asymptomatic individuals comprising 119 confirmed malignant nodule cases and 119 benign nodule controls selected from the PLCO screening trial. RESULTS: A PNC comprising 5 biomarkers: CEA, CYFRA 21-1, OPN, SCC, and TFPI, was selected in the training study. In an independent validation study, the PNC resolved lung cancer cases from benign nodule controls with an AUC of 0.653 (p < 0.0001). CEA and CYFRA 21-1, two of the markers included in the PNC, also accurately distinguished malignant lesions from benign controls. CONCLUSIONS: A 5-biomarker blood test has been developed for the diagnostic evaluation of asymptomatic individuals with solitary pulmonary nodules.

Blood-based lung cancer biomarkers identified through proteomic discovery in cancer tissues, cell lines and conditioned medium.

BACKGROUND: Support for early detection of lung cancer has emerged from the National Lung Screening Trial (NLST), in which low-dose computed tomography (LDCT) screening reduced lung cancer mortality by 20 % relative to chest x-ray. The US Preventive Services Task Force (USPSTF) recently recommended annual screening for the high-risk population, concluding that the benefits (life years gained) outweighed harms (false positive findings, abortive biopsy/surgery, radiation exposure). In making their recommendation, the USPSTF noted that the moderate net benefit of screening was dependent on the resolution of most false-positive results without invasive procedures. Circulating biomarkers may serve as a valuable adjunctive tool to imaging. RESULTS: We developed a broad-based proteomics discovery program, integrating liquid chromatography/mass spectrometry (LC/MS) analyses of freshly resected lung tumor specimens (n = 13), lung cancer cell lines (n = 17), and conditioned media collected from tumor cell lines (n = 7). To enrich for biomarkers likely to be found at elevated levels in the peripheral circulation of lung cancer patients, proteins were prioritized based on predicted subcellular localization (secreted, cell-membrane associated) and differential expression in disease samples. 179 candidate biomarkers were identified. Several markers selected for further validation showed elevated levels in serum collected from subjects with stage I NSCLC (n = 94), relative to healthy smoker controls (n = 189). An 8-marker model was developed (TFPI, MDK, OPN, MMP2, TIMP1, CEA, CYFRA 21-1, SCC) which accurately distinguished subjects with lung cancer (n = 50) from high risk smokers (n = 50) in an independent validation study (AUC = 0.775). CONCLUSIONS: Integrating biomarker discovery from multiple sample types (fresh tissue, cell lines and conditioned medium) has resulted in a diverse repertoire of candidate biomarkers. This unique collection of biomarkers may have clinical utility in lung cancer detection and diagnoses.

Conformation-dependent oligomers in cerebrospinal fluid of presymptomatic familial Alzheimer's disease mutation carriers.

BACKGROUND/AIMS: Oligomerization of amyloid beta (Aß) is a hypothesized step in the formation of plaques in Alzheimer's disease (AD) but has been difficult to demonstrate in vivo in humans. As persons destined to develop familial AD (FAD) due to fully penetrant autosomal dominant mutations are essentially certain to develop the disease, they provide the opportunity to identify oligomers during the presymptomatic stage of the disease. METHODS: We measured levels of Aß(42) using a conventional immunoassay and prefibrillar, fibrillar, and annular protofibrillar oligomers using polyclonal conformation-dependent antibodies in the cerebrospinal fluid (CSF) of 7 persons at risk for inheriting FAD mutations. Levels of oligomers were compared between FAD mutation carriers and noncarriers. RESULTS: Compared to 2 noncarriers, annular protofibrillar oligomers were elevated, prefibrillar and fibrillar oligomers trended towards elevation and Aß(42) monomer trended towards being decreased in 5 FAD mutation carriers. CONCLUSION: Our data provide evidence for an identifiable elevation of CSF oligomers during the presymptomatic phase of FAD.

Amyloid-beta peptide and oligomers in the brain and cerebrospinal fluid of aged canines.

The study of Alzheimer's disease (AD) pathogenesis requires the use of animal models that develop some amount of amyloid pathology in the brain. Aged canines (beagles) naturally accumulate human-type amyloid-beta peptide (Abeta) and develop parallel declines in cognitive function. However, the type and quantity of biochemically extracted Abeta in brain and cerebrospinal fluid (CSF), its link to aging, and similarity to human aging has not been examined systematically. Thirty beagles, aged 4.5-15.7 years, were studied. Abeta40 and Abeta42 were measured in CSF by ELISA, and from SDS and formic acid extracted prefrontal cortex. A sample of the contralateral hemisphere, used to assess immunohistochemical amyloid load, was used for comparison. In the brain, increases in Abeta42 were detected at a younger age, prior to increases in Abeta40, and were correlated with an increased amyloid load. In the CSF, Abeta42 decreased with age while Abeta40 levels remained constant. The CSF Abeta42/40 ratio was also a good predictor of the amount of Abeta in the brain. The amount of soluble oligomers in CSF was inversely related to brain extractable Abeta, whereas oligomers in the brain were correlated with SDS soluble Abeta42. These findings indicate that the Abeta in the brain of the aged canine exhibits patterns that mirror Abeta deposited in the human brain. These parallels support the idea that the aged canine is a useful intermediate between transgenic mice and humans for studying the development of amyloid pathology and is a potentially useful model for the refinement of therapeutic interventions.

Soluble fibrillar oligomer levels are elevated in Alzheimer's disease brain and correlate with cognitive dysfunction.

Recent evidence has suggested a role for soluble oligomeric Abeta species in the pathology of Alzheimer's disease (AD). Fibrillar plaque deposits are present in non-demented individuals and levels of soluble Abeta correlate better with cognitive dysfunction in AD and transgenic mouse models. We have previously reported that there are at least two conformationally distinct types of Abeta oligomers: prefibrillar oligomers that are kinetic intermediates in fibril assembly reactions and are specifically recognized by A11 antibody and fibrillar oligomers that may represent fibril seeds or small pieces of fibrils and are recognized by a fibril specific antibody, OC. We have examined the levels of these two types of oligomers in the PBS soluble fraction of brain tissue from control cases, cases with senile degenerative changes (SDC) and AD patients. We found that the levels of soluble fibrillar oligomers detected by OC antibody are significantly elevated in multiple brain regions of AD patients. The elevated fibrillar oligomer levels were found not to be an artifact of tissue homogenization, nor a result of increased Abeta or APP levels. The concentration of fibrillar oligomers in adjacent brain regions of the same patient can vary widely and were not detected in post-mortem cerebrospinal fluid. In contrast, the level of prefibrillar oligomers are variable in both AD and age matched controls, indicating that they are not correlated with cognitive dysfunction and suggesting that they precede dementia in AD. Significant correlations were found between the levels of fibrillar oligomers and cognitive decline (MMSE scores) as well as the neuropathological hallmarks of AD. These results indicate that fibrillar oligomers may play a key role in the pathology of AD and may be a new target for diagnostic and therapeutic development.