Biomarkers For Early Cancer Detection Research Articles

EP1.11-07 Development of a Chromosome Instabilities in Plasma Cell-Free DNA Assay for Early Lung Cancer Detection and Treatment Response Monitoring

Chromosomal instability (CIN) is one of the hallmarks of cancer. Tumor cells keep shedding DNA into blood stream which provides an opportunity for CIN analysis. Here we present a retrospective study to investigate the potential of CIN in plasma cell-free DNA (cfDNA) as a minimal-invasive biomarker for early cancer detection and cancer treatment responses monitoring. To characterization the baseline data of CIN in cfDNA, 171 plasma samples were collected since June 2017. 35 was from non-cancer individuals. 136 was from cancer patients. cfDNA was extracted and sent to low-coverage genome sequencing on the Illumina X10 platform, followed by chromosomal instability (CIN) analyses by a customized workflow Ultrasensitive Chromosomal Instability Detector (UCAD). Increased plasma cfDNA CIN was observed along with disease progression. In lung cancer, cfDNA CIN increased along with the development of lesions, from adenocarcinoma in-situ, minimal invasive adenocarcinoma, invasive adenocarcinoma (P=0.034) to relapsed cancer (P<0.01). The sensitivity of early lung cancer detection was 30.7%, 37.5%, 45.5%, 50.0% and 98.1% for AIS, MIA, IAC, SCC and relapsed lung cancer, at a specificity of 75%. In primary lung cancer, cfDNA CIN decreased after curative therapies. And cfDNA CIN significantly increased after disease relapsed (P<0.01). And cfDNA levels did not show statistical differences regarding metastases sites. cfDNA CIN can help early cancer detection and treatment response monitoring.

Multi-threshold and multi-input DNA logic design style for profiling the microRNA biomarkers of real cancers.

Early detection of cancer is very critical because it can reduce the treatment risk and cost. MicroRNAs (miRNAs) have been introduced in recent years as an efficient class of biomarkers for cancer early detection. Now, real-time polymerase chain reaction has been used to profile the miRNA expression, which is costly, time consuming and low accuracy. Most recently, DNA logic gates are used to detect the miRNA expression level that is more accurate and faster than previous methods. The DNA-based logic gates face with serious challenges such as the large complexity and low scalability. In this study, the authors proposed a methodology to design multi-threshold and multi-input DNA-based logic gates in response to specific miRNA inputs in live mammalian cells. The proposed design style can simultaneously recognise multiple miRNAs with different rising and falling thresholds. The design style has been evaluated on the lung cancer biomarkers and the experimental results show the efficiency of the proposed method in terms of accuracy, efficiency and speed.

Development of a plasma cell-free DNA chromosome instabilities assay for early cancer detection and treatment response monitoring of multiple tumor types.

e13072 Background: Tumor cells keep shedding DNA into blood stream. Here we present a retrospective study to investigate the potential of CIN in plasma cell-free DNA (cfDNA) as a minimal-invasive biomarker for early cancer detection and cancer treatment responses monitoring. Methods: To characterization cfDNA CIN, 160 plasma samples from non-cancer individuals and 569 from cancer patients, including tumors from the brain, respiratory tract, gastrointestinal tract, urinary tract, liver, gallbladder, female reproductive system, prostate, breast and sarcoma. cfDNA was extracted and sent to low-coverage whole genome sequencing by the Illumina X10, followed by CIN analyses by a customized workflow Ultrasensitive Chromosomal Instability Detector (UCAD). Results: In non-cancer individuals, increased CIN in cfDNA was found associated with active EBV infections(P&lt;0.01) and HBV infection (P=0.042). No statistical significances were found for the other parameters, including age, hypertension, diabetes, chronic kidney diseases, family history of cancer and etc. cfDNA CIN increased along with the development of lung cancer lesions, from adenocarcinoma in-situ, minimal invasive adenocarcinoma, invasive adenocarcinoma (P=0.034) to relapsed cancer (P&lt;0.01). The sensitivity of early lung cancer detection was 30.7%, 37.5%, 45.5%, 50.0% and 98.1% for AIS, MIA, IAC, SCC and relapsed lung cancer, at a specificity of 75%. cfDNA CIN levels did not show statistical differences regarding metastases sites. cfDNA CIN were further increased in relapsed breast cancer (P&lt;0.01). The sensitivity of relapsed breast cancer detection was 73.3%, 94.4%, 89.6% and 80.0% for HER2+, Luminal A, Luminal B and triple negative breast cancer, at a specificity of 90%. In primary liver cancer, cfDNA CIN decreased after curative therapies, including R0 resections and liver transplant. R0 resections showed similar performance as compared to liver transplant (P=0.35) in terms of cfDNA CIN decreasing. Furthermore, cfDNA CIN was higher in patients after R0 resections (P=0.003) and liver transplant (P=0.03) than that of HBV-positive cancer-free patients, indicating the potential risk of disease relapses. For advanced stage patients, continuously increasing cfDNA CIN level was found associated with worse survival, and a decreasing trend predicting better prognosis vice versa. Conclusions: Plasma cfDNA CIN analysis might be a useful tool for cancer management.

IGFBP-1 As a Serum Protein Biomarker for Early Detection of Gastrointestinal Cancer

Background: Early detection would significantly improve cancer prognosis. We aimed to identify serum protein biomarker for early cancer detection and assess the diagnostic value in various types of cancers. Methods: We performed a three-stage study, and recruited participants of normal controls and seven different types of cancer patients (oesophageal squamous cell carcinoma (ESCC), oesophagogastric junction adenocarcinoma (EJA), stomach cancer, colorectal cancer, lung cancer, nasopharyngeal cancer and breast cancer) from two medical centres. We applied two different antibody arrays to screen candidate serum protein which were increased in 20 ESCC patients compared with 20 matched normal controls. In biomarker assessment stage, the diagnostic value of the selected protein measured by ELISA was evaluated in seven cancer types, and was validated in two independent cohorts of ESCC patients and controls (cohorts 1 and 2). Receiver operating characteristics (ROCs) with the sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) were used to calculate diagnostic accuracy. Findings: We recruited 1628 participants to biomarker assessment stage: 287 with ESCC, 237 with EJA, 207 with stomach cancer, 138 with colorectal cancer, 126 with lung cancer, 150 with nasopharyngeal cancer, 150 with breast cancer and 333 normal controls, and 472 and 452 participants comprised of ESCC patients and controls to the two independent validation cohorts. Serum IGFBP-1 showed elevated level for ESCC in both antibody arrays. A significant increase in the serum IGFBP-1 level on all cancer types except breast cancer was confirmed by ELISA, compared with normal controls. Serum IGFBP-1 provided a high diagnostic accuracy of gastrointestinal tumours (AUC 0·880-0·938, sensitivity 65·22-77·22%, and specificity 90·39%). Similar results were noted for early-stage gastrointestinal tumours (0·858-0·936, 60·29-83·93%, and 90·39%). In two validation cohorts, serum IGFBP-1 maintained diagnostic performance for early stage ESCC (0·849, 68·04%, and 89·09% in cohort 1, and 0·911, 72·34% and 87·37% in cohort 2). Furthermore, when compared with CEA, Cyfra21-1 and SCCA tested alone or together, serum IGFBP-1 had significantly improved diagnostic accuracy for ESCC and early stage ESCC. Interpretation: Serum IGFBP-1 is a potential biomarker with high diagnostic value for the early detection of gastrointestinal cancer. Funding Statement: This work was supported by funding from the Natural Science Foundation of China (31600632, 81772532 and 81872372), the Population-based prospective cohort study of esophageal cancer and precancerous lesions in high risk areas (2016YFC0901404), the National Cohort of Esophageal Cancer of China (2016YFC0901400), and the Guangdong Esophageal Cancer Institute Science and Technology Program (M201713), the Natural Science Foundation of Guangdong Province (2018A030307079) and the Shantou University Medical College Clinical Research Enhancement Initiative (201428). Declaration of Interests: We declare that we have no conflicts of interest. Ethics Approval Statement: The study was approved by the Institutional Review Boards for Human Research at each hospital, and informed consent was obtained from all participants.

Detection of miRNA cancer biomarkers using light activated Molecular Beacons.

Early detection of cancer biomarkers can reduce cancer mortality rate. miRNAs are small non-coding RNAs whose expression changes upon the onset of various types of cancer. Biosensors that specifically detect such biomarkers can be engineered and integrated into point-of-care devices (POC) using label-free detection, high sensibility and compactness. In this paper, a new engineered Molecular Beacon (MB) construct used to detect miRNAs is presented. Such a construct is immobilized onto biosensor surfaces in a covalent and spatially oriented way using the photonic technology Light Assisted Molecular Immobilization (LAMI). The construct consists of a Cy3 labelled MB covalently attached to a light-switchable peptide. One MB construct contains a poly-A sequence in its loop region while the other contains a sequence complementary to the cancer biomarker miRNA-21. The constructs have been characterized by UV-Vis spectroscopy, mass spectrometry and HPLC. LAMI led to the successful immobilization of the engineered constructs onto thiol functionalized optically flat quartz slides and Silicon on Insulator (SOI) sensor surfaces. The immobilized Cy3 labelled MB construct has been imaged using confocal fluorescence microscopy (CFM). The bioavailability of the immobilized engineered MB biosensors was confirmed through specific hybridization with the Cy5 labelled complementary sequence and imaged by CFM and FRET. Hybridization kinetics have been monitored using steady state fluorescence spectroscopy. The label-free detection of miRNA-21 was also achieved by using integrated photonic sensing structures. The engineered light sensitive constructs can be immobilized onto thiol reactive surfaces and are currently being integrated in a POC device for the detection of cancer biomarkers.

Identification and Verification of a Biomarker Panel for Early Diagnosis of Lung Cancer Patients

Background: Lung cancer is the most common cause of cancer related mortality worldwide, characterised by late clinical presentation (49-53% of patients are diagnosed at stage IV) and consequently poor outcomes. One challenge in identifying biomarkers of early disease is the collection of samples from patients prior to symptomatic presentation. An additional complication in determining differential biomarkers arises from protein expression change from non-specific systemic responses and co-morbidities such as Chronic Obstructive Pulmonary Disease (COPD). Methods: We employed isobaric tagging, relative quantification mass spectrometry to analyse protein markers that differed in blood entering and egressing from the tumour bearing lobe of the diseased lung immediately prior to lung resection surgery. These potential biomarkers were assessed as lung cancer early detection biomarkers using the orthogonal mass spectrometry (MS) technique Sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS which we showed had equivalent quantification value to a selected reaction monitoring (SRM) approach. Findings: In a second cohort, SWATH-MS was used to measure 974 proteins in serum samples taken <50 months prior to and at lung cancer diagnosis. Two proteins found to increase in expression egressing from a tumour had value as biomarkers. SWATH-MS profiling followed by machine learning generated a predictive 13 protein panel resulted in a mean AUC of 0.80, an accuracy of 0.78, a sensitivity of 0.79 and a specificity of 0.78. Interpretation: Thus, whilst tumour egressing proteins did not contribute significantly to a MS early detection biomarker signature, SWATH-MS and machine learning have great potential value for early detection biomarker algorithms. Funding Statement: Mass spectrometry was supported with equipment grants from Bloodwise and Medical Research Council. This work was supported by the CRUK Manchester Centre award (C5759/A25254). CD and ADW are supported by the NIHR Manchester Biomedical Research Centre. CD is also supported via core funding to the CRUK Manchester Institute and via the CRUK Lung Cancer Centre of Excellence (C5759/A20465). The pulmonary vein study was funded by Roy Castle Lung Cancer Foundation (PC) and the North West Lung Centre Charity (PC). UKCTOCS was funded by the Medical Research Council (G9901012 and G0801228), Cancer Research UK (C1479/A2884), the Department of Health and with additional support from The Eve Appeal. Senior investigators at UCL supported by the NIHR University College London Hospitals (UCLH) Biomedical Research Centre. Declaration of Interests: The authors have no financial conflicts of interest. Ethics Approval Statement: UKCTOCS was approved by the UK North West Multicentre Research Ethics Committee (North West MREC 00/8/34) with site specific approval from the local regional ethics committees and the Caldicott guardians (data controllers) of the primary care trusts. All women gave written consent for use of samples and data in ethically approved secondary studies. The subset of samples used for the present study has been approved by the Yorkshire & The Humber - Sheffield Research Ethics Committee (REC Ref 15/YH/0044).

Platelets in cancer development and diagnosis.

Platelets are involved in the development and progression of cancer through several mechanisms. Platelet activation at the site of tissue damage contributes to the initiation of a cascade of events which promote tumorigenesis. In fact, platelets release a wide array of proteins, including growth and angiogenic factors, lipids and extracellular vesicles rich in genetic material, which can mediate the induction of phenotypic changes in target cells, such as immune, stromal and tumor cells, and promote carcinogenesis and metastasis formation. Importantly, the role of platelets in tumor immune escape has been described. These lines of evidence open the way to novel strategies to fight cancer based on the use of antiplatelet agents. In addition to their ability to release factors, platelets are able of up-taking proteins and genetic material present in the bloodstream. Platelets are like 'sentinels' of the disease state. The evaluation of proteomics and transcriptomics signature of platelets and platelet-derived microparticles could represent a new strategy for the development of biomarkers for early cancer detection and/or therapeutic drug monitoring in cancer chemotherapy. Owing to the ability of platelets to interact with cancer cells and to deliver their cargo, platelets have been proposed as a 'biomimetic drug delivery system' for anti-tumor drugs to prevent the occurrence of off-target adverse events associated with the use of traditional chemotherapy.

Highly sensitive detection of DNA hypermethylation in melanoma cancer cells

Aberrant hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a promising biomarker for early cancer detection. This methylation status is reflected in the methylation pattern of ctDNA shed from the primary tumor; however, to realize the full clinical utility of ctDNA methylation detection via liquid biopsy for early cancer diagnosis, improvements in the sensitivity and multiplexability of existing technologies must be improved. Additionally, the assay must be cheap and easy to perform in a clinical setting. We report the integration of methylation specific PCR (MSP) to melt curve analysis on giant magnetoresistive (GMR) biosensors to greatly enhance the sensitivity of our DNA hybridization assay for methylation detection. Our GMR sensor is functionalized with synthetic DNA probes that target methylated or unmethylated CpG sites in the MSP amplicon, and measures the difference in melting temperature (Tm) between the two probes (ΔTm), giving an analytical limit of detection down to 0.1% methylated DNA in solution. Additionally, linear regression of ΔTm's for serial dilutions of methylated:unmethylated mixtures allows for quantification of methylation percentage, which could have diagnostic and prognostic utility. Lastly, we performed multiplexed MSP on two different genes, and show the ability of our GMR assay to resolve this mixture, despite their amplicons’ overlapping Tm's in standard EvaGreen melt analysis. The multiplexing ability of our assay and its enhanced sensitivity, without necessitating deep sequencing, represent important steps toward realizing an assay for the detection of methylated ctDNA in plasma for early cancer detection in a clinical setting.

Creating Models to Identify New Therapeutic Options for Aggressive African Breast Cancers

Background: Population-based incidence rates of breast cancer (BC) that does not express the estrogen receptor (ER), progesterone receptor (PR) or overexpress the human epidermal growth factor 2 HER2/ neu (triple negative breast cancer, TNBC) are higher among Africans compared with white women. However the underlying biologic and genetic differences among different ethnicities are poorly understood and there are currently very few ethnically diverse BC models available for identifying new therapeutic options. Aim: Establish an international collaboration to: i, characterize African breast tumors ii, create models for studying these tumors and iii, identify biomarkers for early detection and treatment personalization. Methods: We have collected tumors from 154 white Americans WA, 76 African Americans, AA, 190 Ethiopians, Eth, and 286 Ghanaian (Gh) BC patients. We then established a unique resource of patient derived xenografts (PDX) from these tumors. The PDXs were then fully characterized using whole exome and RNA sequencing for the primary tumor, matched normal DNA, and corresponding low passage PDXs. Using immunohistochemistry, we evaluated the ER, PR, HER2/ neu, androgen receptor (AR), and ALDH1 (cancer stem cell marker) expression among these tumors. Based on biomarker expression the PDXs were then tested against a panel of IND drugs, either alone or in combinations, in an ex vivo organoid culture system to discover potential new therapeutic options. Results: Mean age at BC diagnosis was 43; 49; 60; and 57 years for the Eth; Gh; AA; and WA patients, respectively. The proportion of TNBC was significantly higher for the AA and Gh patients (41% and 54%, respectively) compared with the WA and Eth patients (23% and 15%, respectively); P &lt; 0.001. Significant differences were observed for distribution of AR positivity, which was 71%; 55%; 42% and 50% for the WA; AA; Gh; and Eth cases, respectively ( P = 0.008). The Gh breast tumors exhibited the highest number of loss of function and missense mutations that are likely to impact therapy with a high frequency of P53, APC, and FGFR mutations. These mutations were maintained in the corresponding PDXs that were developed, and were thus used as biomarkers for drug screening. These tumors exhibited a gene expression signature based on the ethnicity of the patients with 2385 genes differentially expressed between Gh and AA, 1573 between AA and CA and 1317 between GH and CA. Results from our ongoing drug screening and biomarker identification will be available soon. Conclusions: Establishing the molecular and genetic platform of aggressive breast cancers occurring in women with African ancestry will help in identifying biomarkers for early cancer detection and targeted treatment stratification for optimum patient outcome. The availability of tumor models based on tumors from diverse African populations is the important missing pieces that have to be incorporated into current drug discovery efforts.

The multifaceted role of glutathione S-transferases in cancer

Glutathione S-transferases (GSTs) are phase II detoxifying enzymes involved in the maintenance of cell integrity, oxidative stress and protection against DNA damage by catalyzing the conjugation of glutathione to a wide variety of electrophilic substrates. Though enzymes of the glutathione synthesis and salvage pathways have been well characterized in the past, there is still a lack of comprehensive understanding of their independent and coordinate regulatory mechanisms in carcinogenesis. The present review discusses implication of GST in cancer development and progression, gene polymorphism, drug resistance, signaling and epigenetic regulation involving their role in cancer. It is anticipated that GST especially the GSTP1 class can be developed as a biomarker either used alone or in combination with other biomarkers for early cancer detection and/or diagnosis as well as for future targeted preventive and therapeutic interventions with dietary agents.

Abstract A24: Bone marrow hematopoietic adaptation as a sensor of early, pre-invasive, epithelial malignancy

Abstract Tumor development and progression is in part dependent on the ability of bystander cells, mostly of bone marrow (BM) origin, to establish a pro-tumorigenic microenvironment. We hypothesized that signs of the cross-talk between elements of the tumor microenvironment and the BM can be identified in the very early phases of cancer development, being finalized to the instruction of a tumor-promoting hematopoiesis. By integrating in situ BM histopathological and immunophenotypical analyses with flow cytometry and gene expression profiling of hematopoietic populations in a spontaneous mouse model of breast carcinogenesis (MMTV/NeuT) we investigated the occurrence and quality of modifications in the hematopoietic and stromal BM components and their correlation with the pathological stage of the mammary lesions. At the invasive carcinoma stage, histophathology and immunophenotyping of the BM hematopoietic parenchyma revealed a clear-cut switch towards a preferential granulo-monocytic fate, which was paralleled by a significant contraction in B-cell lymphoid populations and associated with the remodeling of the BM mesenchymal niches. At this stage, gene expression profiling of BM cells identified a differential gene signature on unsupervised analyses that allowed distinguishing between transgenic and control mice on the basis of inflammatory and innate immune response programs. The investigation at earlier stages of cancer development of such modifications in BM microarchitecture, hematopoietic composition, and gene profile demonstrated that their induction was already detectable at the high-grade dysplasia/in situ cancer stage, which provided the first evidence of the BM acting as a very early sensor of peripheral transformation. We hypothesized that the variations of the BM transcriptional profile could be paralleled by a reprogramming of the miRNA circulating in the peripheral blood. To text this hypothesis, we profiled plasmatic miRNAs at late and early stages of tumor progression and found, already at early time points, differentially expressed miRNAs, which could potentially be used as early biomarkers of cancerogenesis. In conclusion, our data lay a first demonstration that BM hematopoietic adaptation to cancer is not confined to the engendering of a general immunosuppressive state associated with advanced cancers, rather it represents an early process co-evolving with malignant clone expansion. These results, besides casting light on the identification of potential new biomarkers for early cancer detection, offer a whole new prospect on investigating the significance of early cancer-adapted hematopoiesis. Citation Format: Claudia Chiodoni, Andrea Massimiliano Tomirotti, Tiziana Ada Renzi, Matteo Dugo, Valeria Cancila, Claudio Tripodo, Mario Paolo Colombo. Bone marrow hematopoietic adaptation as a sensor of early, pre-invasive, epithelial malignancy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A24.

A Panel of Novel Detection and Prognostic Methylated DNA Markers in Primary Non-Small Cell Lung Cancer and Serum DNA.

Purpose: To establish a novel panel of cancer-specific methylated genes for cancer detection and prognostic stratification of early-stage non-small cell lung cancer (NSCLC).Experimental Design: Identification of differentially methylated regions (DMR) was performed with bumphunter on "The Cancer Genome Atlas (TCGA)" dataset, and clinical utility was assessed using quantitative methylation-specific PCR assay in multiple sets of primary NSCLC and body fluids that included serum, pleural effusion, and ascites samples.Results: A methylation panel of 6 genes (CDO1, HOXA9, AJAP1, PTGDR, UNCX, and MARCH11) was selected from TCGA dataset. Promoter methylation of the gene panel was detected in 92.2% (83/90) of the training cohort with a specificity of 72.0% (18/25) and in 93.0% (40/43) of an independent cohort of stage IA primary NSCLC. In serum samples from the later 43 stage IA subjects and population-matched 42 control subjects, the gene panel yielded a sensitivity of 72.1% (31/41) and specificity of 71.4% (30/42). Similar diagnostic accuracy was observed in pleural effusion and ascites samples. A prognostic risk category based on the methylation status of CDO1, HOXA9, PTGDR, and AJAP1 refined the risk stratification for outcomes as an independent prognostic factor for an early-stage disease. Moreover, the paralog group for HOXA9, predominantly overexpressed in subjects with HOXA9 methylation, showed poor outcomes.Conclusions: Promoter methylation of a panel of 6 genes has potential for use as a biomarker for early cancer detection and to predict prognosis at the time of diagnosis. Clin Cancer Res; 23(22); 7141-52. ©2017 AACR.

Screening for Multiple Autoantibodies in Plasma of Patients with Breast Cancer.

Autoantibodies have potential as circulating biomarkers for early cancer detection. This study aimed to screen for known autoantibodies in human plasma using an Autoantibody Profiling System (APS) and quantify the levels in plasma of donors with/without breast cancer. Plasma from nine female donors diagnosed with breast cancer (test group) and nine matched donors with no personal history of cancer (reference group) were screened with an APS containing probes for 30 autoantibodies. Autoantibody levels ≥1.5 times the mean concentration of the group were considered elevated, and test/reference ratios ≥1.3 were considered higher in the test group compared to the reference group. Twenty percent of the probes detected elevated levels of autoantibodies against proteins involved in different cancer mechanisms. Amongst these, the levels of autoantibodies against interleukin 29 (IL29), osteoprotegerin (OPG), survivin (SUR), growth hormone (GRH) and resistin (RES) were significantly higher in the cancer group compared to the reference group (p<0.05), whereas the level of autoantibody against cytotoxic T-lymphocyte associated antigen-4 (CTLA4) was not significantly different between the two groups (p=0.38). Disease-relevant autoantibodies were detected in the plasma of patients with breast cancer and donors without breast cancer. This means that identifying the type and level of autoantibodies in samples will be important in determining their significance in the disease process. A microtiter plate-based array system could be a fast and inexpensive screening method for identifying and quantifying autoantibodies in human plasma.

The Exosome Total Isolation Chip.

Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4-1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the device's broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10-100 μL) of blood.

Assessing biological and technological variability in protein levels measured in pre-diagnostic plasma samples of women with breast cancer

BackgroundQuantitative proteomics allows for the discovery and functional investigation of blood-based pre-diagnostic biomarkers for early cancer detection. However, a major limitation of proteomic investigations in biomarker studies remains the biological and technical variability in the analysis of complex clinical samples. Moreover, unlike ‘omics analogues such as genomics and transcriptomics, proteomics has yet to achieve reproducibility and long-term stability on a unified technological platform. Few studies have thoroughly investigated protein variability in pre-diagnostic samples of cancer patients across multiple platforms.MethodsWe obtained ten blood plasma “case” samples collected up to 2 years prior to breast cancer diagnosis. Each case sample was paired with a matched control plasma from a full biological sister without breast cancer. We measured protein levels using both mass-spectrometry and antibody-based technologies to: (1) assess the technical considerations in different protein assays when analyzing limited clinical samples, and (2) evaluate the statistical power of potential diagnostic analytes.ResultsAlthough we found inherent technical variation in the three assays used, we detected protein dependent biological signal from the limited samples. The three assay types yielded 32 proteins with statistically significantly (p < 1E-01) altered expression levels between cases and controls, with no proteins retaining statistical significance after false discovery correction.ConclusionsTechnical, practical, and study design considerations are essential to maximize information obtained in limited pre-diagnostic samples of cancer patients. This study provides a framework that estimates biological effect sizes critical for consideration in designing studies for pre-diagnostic blood-based biomarker detection.

Optomechanical devices for deep plasma cancer proteomics.

Most of the cancer deaths could be avoided by early detection of the tumor when it is confined to its primary site and it has not metastasized. To this aim, one of the most promising strategies is the discovery and detection of protein biomarkers shed by the young tumor to the bloodstream. Proteomic technologies, mainly mass spectrometry and multiplexed immunoassays, have rapidly developed during last years with improved limits of detection and multiplexing capability. Unfortunately, these developments together major investments and large international efforts have not resulted into new useful protein biomarkers. Here, we analyze the potential and limitations of current proteomic technologies for detecting protein biomarkers released into circulation by the tumor. We find that these technologies can hardly probe the deepest region of the plasma proteome, at concentrations below the pg/mL level, where protein biomarkers for early cancer detection may exist. This clearly indicates the need of incorporating novel ultrasensitive techniques to the proteomic tool-box that can cover the inaccessible regions of the plasma proteome. We here propose biological detectors based on nanomechanical systems for discovery and detection of cancer protein biomarkers in plasma. We review the modes of operation of these devices, putting our focus on recent developments on nanomechanical sandwich immunoassays and nanomechanical spectrometry. The first technique enables reproducible immunodetection of proteins at concentrations well below the pg/mL level, with a limit of detection on the verge of 10 ag/mL. This technology can potentially detect low abundance tumor-associated proteins in plasma at the very early stages of the tumor. The second technique enables the identification of individual intact proteins by two physical coordinates, the mass and stiffness, instead of the mass-to-charge ratio of the protein constituents. This technology enormously simplifies the identification of proteins and it can provide useful information on interactions and posttranslational modifications, that otherwise is lost in mass spectrometry.

Label-free Detection for a DNA Methylation Assay Using Raman Spectroscopy.

Background:DNA methylation has been suggested as a biomarker for early cancer detection and treatment. Varieties of technologies for detecting DNA methylation have been developed, but they are not sufficiently sensitive for use in diagnostic devices. The aim of this study was to determine the suitability of Raman spectroscopy for label-free detection of methylated DNA.Methods:The methylated promoter regions of cancer-related genes cadherin 1 (CDH1) and retinoic acid receptor beta (RARB) served as target DNA sequences. Based on bisulfite conversion, oligonucleotides of methylated or nonmethylated probes and targets were synthesized for the DNA methylation assay. Principal component analysis with linear discriminant analysis (PCA-DA) was used to discriminate the hybridization between probes and targets (methylated probe and methylated target or nonmethylated probe and nonmethylated target) of CDH1 and RARB from nonhybridization between the probe and targets (methylated probe and nonmethylated target or nonmethylated probe and methylated target).Results:This study revealed that the CDH1 and RARB oligo sets and their hybridization data could be classified using PCA-DA. The classification results for CDH1 methylated probe + CDH1 methylated target versus CDH1 methylated probe + CDH1 unmethylated target showed sensitivity, specificity, and error rates of 92%, 100%, and 8%, respectively. The classification results for the RARB methylated probe + RARB methylated target versus RARB methylated probe + RARB unmethylated target showed sensitivity, specificity, and error rates of 92%, 93%, and 11%, respectively.Conclusions:Label-free detection of DNA methylation could be achieved using Raman spectroscopy with discriminant analysis.

Nanotechnology-Enhanced No-Wash Biosensors for in Vitro Diagnostics of Cancer.

In vitro biosensors have been an integral component for early diagnosis of cancer in the clinic. Among them, no-wash biosensors, which only depend on the simple mixing of the signal generating probes and the sample solution without additional washing and separation steps, have been found to be particularly attractive. The outstanding advantages of facile, convenient, and rapid response of no-wash biosensors are especially suitable for point-of-care testing (POCT). One fast-growing field of no-wash biosensor design involves the usage of nanomaterials as signal amplification carriers or direct signal generating elements. The analytical capacity of no-wash biosensors with respect to sensitivity or limit of detection, specificity, stability, and multiplexing detection capacity is largely improved because of their large surface area, excellent optical, electrical, catalytic, and magnetic properties. This review provides a comprehensive overview of various nanomaterial-enhanced no-wash biosensing technologies and focuses on the analysis of the underlying mechanism of these technologies applied for the early detection of cancer biomarkers ranging from small molecules to proteins, and even whole cancerous cells. Representative examples are selected to demonstrate the proof-of-concept with promising applications for in vitro diagnostics of cancer. Finally, a brief discussion of common unresolved issues and a perspective outlook on the field are provided.

Reprimo tissue-specific expression pattern is conserved between zebrafish and human.

Reprimo (RPRM), a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRM and RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb), RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH), we demonstrate that rprm (rprma/rprmb) and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb) and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS). We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC) staining, respectively. Strikingly, tissue-specific expression patterns of the RPRM transcripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.

Identification of potential serum biomarkers for breast cancer using a functional proteomics technology

BackgroundCancer is a genetic disease; its development and metastasis depend on the function of many proteins. Human serum contains thousands of proteins; it is a window for the homeostasis of individual’s health. Many of the proteins found in the human serum could be potential biomarkers for cancer early detection and drug efficacy evaluation.MethodsIn this study, a functional proteomics technology was used to systematically monitor metabolic enzyme and protease activities from resolved serum proteins produced by a modified 2-D gel separation and subsequent Protein Elution Plate, a method collectively called PEP. All the experiments were repeated at least twice to ensure the validity of the findings.ResultsFor the first time, significant differences were found between breast cancer patient serum and normal serum in two families of enzymes known to be involved in cancer development and metastasis: metabolic enzymes and proteases. Multiple enzyme species were identified in the serum assayed directly or after enrichment. Both qualitative and quantitative differences in the metabolic enzyme and protease activity were detected between breast cancer patient and control group, providing excellent biomarker candidates for breast cancer diagnosis and drug development.ConclusionsThis study identified several potential functional protein biomarkers from breast cancer patient serum. It also demonstrated that the functional proteomics technology, PEP, can be applied to the analysis of any functional proteins in human serum which contains thousands of proteins. The study indicated that the functional domain of the human serum could be unlocked with the PEP technology, pointing to a novel alternative for the development of diagnosis biomarkers for breast cancer and other diseases.