Electronic field effect detection of SARS-CoV-2 N-protein before the onset of symptoms.

As part of the efforts to contain the pandemic, researchers around the world have raced to develop testing platforms to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the Coronavirus disease 2019 (COVID-19). Within the different detection platforms studied, the field effect transistor (FET) is a promising device due to its high sensitivity and fast detection capabilities. In this work, a graphene-based FET which uses a boron and nitrogen co-doped graphene oxide gel (BN-GO gel) transducer functionalized with nucleoprotein antibodies, has been investigated for the detection of SARS-CoV-2 nucleocapsid (N)-protein in buffer. This biosensor was able to detect the viral protein in less than 4 min, with a limit of detection (LOD) as low as 10 ag/mL and a wide linear detection range stretching over 11 orders of magnitude from 10 ag/mL-1 µg/mL. This represents the lowest LOD and widest detection range of any COVID-19 sensor and thus can potentially enable the detection of infected individuals before they become contagious. In addition to its potential use in the COVID-19 pandemic, our device serves as a proof-of-concept of the ability of functionalized BN-GO gel FETs to be used for ultrasensitive yet robust biosensors.

Serum LAMC2 enhances the prognostic value of a multi-parametric panel in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) lacks reliable serological biomarkers for predicting patients' survival and response to treatment. The present study examined the capability of serum LAMC2 and four known tumour markers for disease prognosis and patients' risk stratification. METHODS: LAMC2, CA 125, CEA, CYFRA 21-1 and SCC levels were retrospectively measured in sera obtained from 127 patients diagnosed with NSCLC by commercial immunoassays. Prognostic performance of the markers was compared with established clinical parameters and multivariate models were constructed to assess the prognostic complementarity of variables. RESULTS: LAMC2 showed significant prognostic ability for overall survival (hazards ratio: 1.607, 95% confidence interval: 1.268-2.037, P<0.0001) in the full cohort. LAMC2 and CYFRA 21-1 combination enhanced prognostic models based on common clinical parameters (c-index: 0.81 vs 0.72, P=0.00018), further enabling stratification of patients into clear risk groups. A bootstrap-based cross-validation analysis was supportive of our findings. Combination of LAMC2 and CA 125 showed similar performance. CONCLUSIONS: Our preliminary study proposes LAMC2 as a novel NSCLC prognostic factor. LAMC2 combined with CA 125 and CYFRA 21-1 could aid in clinical prediction of NSCLC patients' overall survival and inform clinical practice. Larger studies are necessary to unravel LAMC2's full potential as a new NSCLC biomarker.

Clinical utility of kallikrein-related peptidases (KLK) in urogenital malignancies.

Kallikrein-related peptidases (KLK), which represent a major tissue-associated proteolytic system, stand for a rich source of biomarkers that may allow molecular classification, early diagnosis and prognosis of human malignancies as well as prediction of response or failure to cancer-directed drugs. International research points to an important role of certain KLKs in female and male urogenital tract malignancies, in addition to cancers of the lung, brain, skin, head and neck, and the gastrointestinal tract. Regarding the female/male urogenital tract, remarkably, all of the KLKs are expressed in the normal prostate, testis, and kidney whereas the uterus, the ovary, and the urinary bladder are expressing a limited number of KLKs only. Most of the information regarding KLK expression in tumour-affected organs is available for ovarian cancer; all of the 12 KLKs tested so far were found to be elevated in the malignant state, depicting them as valuable biomarkers to distinguish between the normal and the cancerous phenotype. In contrast, for kidney cancer, a series of KLKs was found to be downregulated, while other KLKs were not expressed. Evidently, depending on the type of cancer or cancer stage, individual KLKs may show characteristics of a Janus-faced behaviour, by either expanding or inhibiting cancer progression and metastasis.

Folate-receptor 1 (FOLR1) protein is elevated in the serum of ovarian cancer patients.

OBJECTIVES: Ovarian cancer is the most lethal gynecological malignancy in North America. Although survival rates are high when the disease is diagnosed at an early stage, this decreases exponentially in late-stage diagnoses. As such, there is a need for novel early detection biomarkers. Through an integrated approach to ovarian cancer biomarker discovery that combines proteomics with transcriptomics and bioinformatics, our laboratory has identified folate-receptor 1 (FOLR1) and Dickkopf-related protein 3 (Dkk-3) as putative biomarkers. The objective of this study was to measure the levels of FOLR1 and Dkk-3 in the serum of patients with ovarian cancer, benign gynecological conditions and healthy women. DESIGN AND METHODS: FOLR1 and Dkk-3 were analyzed in serum of 100 ovarian cancer patients, 100 patients with benign gynecological conditions, and 100 healthy women using enzyme-linked immunosorbent assays (ELISAs). All specimens were analyzed in triplicate. RESULTS: FOLR1 was significantly elevated in the serum of ovarian cancer patients compared to serum of both healthy controls (P<0.0001) and patients with benign gynecological conditions (P<0.0001). Furthermore, FOLR1 was strongly correlated with CA125 as both were elevated in the serous histotype and in late-stage disease. FOLR1 did not outperform CA125 in receiver operating characteristic curve analysis and there was no significant complementarity between the two markers. Dkk-3 was not significantly different between the three serum cohorts and was not correlated with CA125. CONCLUSIONS: FOLR1 is a new biomarker for ovarian cancer which correlates closely with CA125. The role of FOLR1 in the pathogenesis of ovarian cancer warrants further investigation.

CUB and zona pellucida-like domain-containing protein 1 (CUZD1): a novel serological biomarker for ovarian cancer.

OBJECTIVES: To measure the levels of serum CUB and zona pellucida-like domain-containing protein 1 (CUZD1) in patients with ovarian cancer (OvCa), benign gynecological conditions and healthy women and in a number of other cancer types (breast, colorectal, lung, prostate and testicular). DESIGN AND METHODS: Serum CUZD1 levels were measured with a commercial enzyme-linked immunosorbent assay (ELISA). All specimens were analyzed in duplicate. Preliminary verification was performed in serum using 9 healthy women and 20 late stage (III-IV) OvCa patients. An independent cohort of serum samples was used to validate the verification results (18 late stage OvCa, 8 benign gynecological conditions and 8 healthy controls). The following specimens were used for the other cancer types of unknown stage-breast (n=11), colorectal (n=10), lung (n=10), prostate (n=15) and testicular (n=10). RESULTS: Serum CUZD1 was significantly elevated in ovarian cancer patients (range 95-668 µg/L) as compared to healthy controls (range 0.7-2.5 µg/L). The independent cohort of OvCa samples confirmed the preliminary verification results. CUZD1 was also elevated in breast and lung cancer specimens and not in colorectal, prostate and testicular cancer specimens. CONCLUSIONS: CUZD1 appears to be a highly promising novel serum biomarker for OvCa diagnosis. Its performance in the 2 independent cohorts examined, and in lung and breast cancer patients warrants further investigation.

Circulating biomarker tissue kallikrein-related peptidase KLK5 impacts ovarian cancer patients' survival.

BACKGROUND: Effective cancer biomarkers for early detection, prognosis, or therapy response prediction are urgently needed in ovarian cancer. Kallikrein-related peptidases, including KLK5, have been reported to play an important role in the course of the disease. PATIENTS AND METHODS: KLK5 antigen content was determined by enzyme-linked immunosorbent assay in ovarian cancer patients' [FIGO (International Federation of Gynecology and Obstetrics) stages I-IV, n = 52] serum as well as ascitic fluid and compared with KLK5 content in serum of patients with benign ovarian tumors (n = 45). RESULTS: KLK5 antigen content was significantly elevated in the serum of ovarian cancer patients compared with the serum of patients with benign ovarian tumors. Forty-two of 52 ovarian cancer serum samples, 42 of 43 benign ovarian tumor serum samples, and all 41 ascitic fluid samples were KLK5 positive. Elevated KLK5 antigen in serum and ascitic fluid of ovarian cancer patients was a prognostic factor for progression-free survival. CONCLUSIONS: Our data support the finding that ovarian cancer patients release significant amounts of KLK5 into serum and ascitic fluid but KLK5 antigen is low in serum of patients with benign ovarian tumors. Increased serum and ascitic fluid KLK5 levels are associated with poor patient outcome, thus underlining the importance of KLK5 as a biomarker for early detection as well as for disease management in ovarian cancer.

Platform for establishing interlaboratory reproducibility of selected reaction monitoring-based mass spectrometry peptide assays.

Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Four independent laboratories in North America, using identical triple-quadrupole mass spectrometers (Quantum Ultra, Thermo), were provided with standard protocols and instrumentation settings to analyze unknown samples and internal standards in a digested plasma matrix to quantify 51 peptides from 39 human proteins using a multiplexed SRM assay. The interlaboratory coefficient of variation (CV) was less than 10% for 25 of 39 peptides quantified (12 peptides were not quantified based upon hydrophobicity) and exhibited CVs less than 20% for the remaining peptides. In this report, we demonstrate that previously developed research platforms for SRM assays can be improved and optimized for deployment in clinical research environments.

The use of kallikrein-related peptidases as adjuvant prognostic markers in colorectal cancer.

Several members of the human tissue kallikrein-related peptidase (KLK) family are emerging cancer biomarkers. The aim of this study was to analyse the expression of a panel of KLKs in colorectal cancer and to find out if the multiparametric combination of them can increase the accuracy of prediction of patients survival beyond the traditional clinical information. Nine KLKs (KLK5-8, KLK10, KLK11, KLK13-15) were measured using ELISA assays in cytosolic extracts of 122 colon cancer tissues and their nearby normal mucosa, obtained during surgery. The mean levels of almost all KLKs in tumour tissues were significantly different from their counterparts of normal tissue (P<0.0001). KLK 5, 6, 7, 13, 14 were significantly associated with overall survival in univariate analysis, but after adjusting for age, TNM and differentiation stage, only KLK5 (HR: 1.24 (95% CI: 1.05-1.47)), KLK7 (HR: 1.57 (95% CI: 1.04-2.37)) and KLK14 (HR: 1.43 (95% CI: 1.05-1.94)) remained significant. Addition of a panel of selected KLK markers to clinical parameters gave an increment in AUC of 0.86 beyond the clinical factors at year 1, showing that it can increase the accuracy of prediction of overall survival beyond the traditional clinical information, particularly the short-term (1 year) survival after surgery.