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Featured Articles
Point-of-care molecular test for Zika infection
The recent outbreak of Zika virus (ZIKV) infection has led to a serious threat to public health. Rapid and sensitive diagnostics for ZIKV infection are crucial because Zika infection is usually mild and often asymptomatic, but may have serious consequences to infants born to infected mothers. We report on a simple, sensitive, inexpensive, point-of-care diagnostic technology for rapid detection of ZIKV in saliva. We use a chemically heated cup for isothermal amplification without the need for electrical power. The detection results can be directly read out by eye.
by Dr Michael G. Mauk, Dr Jinzhao Song, Prof. Haim H. Bau, Dr Changchun Liu*
Introduction
The recent emergence of the Zika virus (ZIKV) pandemic has underscored the need for a point-of-care (POC) test that can detect viral infections, fostering widespread, accurate and timely diagnostics [1, 2]. Zika infection is often asymptomatic or has comparatively mild symptoms, such as fever and chills that are common to many other infections. However, as is widely known, Zika infection during pregnancy entails substantial risks for the newborn, including severe birth defects [3]. Hence, it is highly desirable to screen women of child-bearing age and their partners for exposure to the ZIKV. Zika episodes have created bottlenecks in conventional laboratory-based diagnostic.
Zika is spread primarily by mosquitos, but sexual and perinatal transmission, as well as transmission via blood transfusions have also been reported. Specific applications for Zika diagnostics thus include determining whether infection has occurred over the course of a pregnancy, whether sexual partners harbour infection, safeguarding the blood supply and tracking the geographic range of Zika infections [4]. In order to inform investment decisions on prevention, control and response, Lee et al. [5] modelled the economic burden for various scenarios of Zika emergence across six US states, estimating total costs (direct medical, Medicaid, productivity losses) ranging from 0.5 to 2 billion US dollars.
Ideally, a POC technology will enable rapid diagnostics tests that can be performed outside of centralized laboratories such as, for example, in doctors or dentists offices, pharmacies, rural clinics, school infirmaries, border crossings or, ultimately, as an over-the-counter test for home use. Thus, the availability of a mass-produced, inexpensive POC diagnostics device with easy-to-interpret test results, and that could be used in any almost any locale by non-specialists with minimal training, would greatly expand capabilities and options for screening, surveillance, diagnostics, monitoring and therapy.
The recent emergence of the Zika virus (ZIKV) pandemic has underscored the need for a point-of-care (POC) test that can detect viral infections, fostering widespread, accurate and timely diagnostics [1, 2]. Zika infection is often asymptomatic or has comparatively mild symptoms, such as fever and chills that are common to many other infections. However, as is widely known, Zika infection during pregnancy entails substantial risks for the newborn, including severe birth defects [3]. Hence, it is highly desirable to screen women of child-bearing age and their partners for exposure to the ZIKV. Zika episodes have created bottlenecks in conventional laboratory-based diagnostic.
Zika is spread primarily by mosquitos, but sexual and perinatal transmission, as well as transmission via blood transfusions have also been reported. Specific applications for Zika diagnostics thus include determining whether infection has occurred over the course of a pregnancy, whether sexual partners harbour infection, safeguarding the blood supply and tracking the geographic range of Zika infections [4]. In order to inform investment decisions on prevention, control and response, Lee et al. [5] modelled the economic burden for various scenarios of Zika emergence across six US states, estimating total costs (direct medical, Medicaid, productivity losses) ranging from 0.5 to 2 billion US dollars.
Ideally, a POC technology will enable rapid diagnostics tests that can be performed outside of centralized laboratories such as, for example, in doctors or dentists offices, pharmacies, rural clinics, school infirmaries, border crossings or, ultimately, as an over-the-counter test for home use. Thus, the availability of a mass-produced, inexpensive POC diagnostics device with easy-to-interpret test results, and that could be used in any almost any locale by non-specialists with minimal training, would greatly expand capabilities and options for screening, surveillance, diagnostics, monitoring and therapy.
Current diagnostics technology
According to the World Health Organization (WHO), the pipeline diagnostic ZIKV kits can be categorized into: (i) antibody/antigen-based immunoassay and (ii) nucleic acid-based molecular diagnostics [6]. Immunoassay (antibody detection) for ZIKV infection utilizes envelope proteins and NS1 as targets. The major challenge is that these antibodies cross-react with other highly homologous flaviviruses such as dengue, resulting in non-specific test results [7]. IgM and IgG antibodies, typically emerge, respectively, ~4 and ~10 days after infection, but are usually undetectable until >7–14 days post-infection. Moreover, antibody responses during pregnancy may differ from those in non-pregnant individuals [8], which may adversely impact the effectiveness of immunoassay tests. Moreover, antibody tests may not discriminate between recent and historic exposure. The Food and Drug Administration (FDA) recently authorized for emergency use of the IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (Zika MAC-ELISA) to detect ZIKV [8]. However, this assay requires a lab-format with delays in generating results given current demand. More importantly, these assays are a readout for exposure to ZIKV, whereas active virus infection is not determined. Currently, several companies are developing lateral flow-based rapid diagnostic test for ZIKV antibody detection [9, 10].
Molecular diagnostics-based on reverse-transcription (RT)-PCR is highly specific and sensitive, and considered the gold standard for ZIKV detection. RT-PCR is effective in serum, semen, and saliva within 14 days post-infection, and possibly much longer in urine and semen [11, 12]. Importantly, a recent study has demonstrated that ZIKV is detectable in pregnant women throughout their pregnancy [3]. Indeed, the FDA has authorized the use of the Trioplex rRT-PCR laboratory test to detect ZIKV, dengue virus, and chikungunya virus RNA, under an Emergency Use Authorization (EUA) [13]. Several research groups and companies are developing multiplexed molecular assays to concurrently detect various members of the genus Flavivirus. Most of these RT-PCR kits require, however, instrumentation and are for central laboratory use only.
Instrument-free point-of-care molecular detection of ZIKV
To develop inexpensive molecular detection of ZIKV without complex instrumentation, we utilized reverse-transcription loop-mediated amplification (RT-LAMP) technology [14]. We identified highly conserved regions of the ZIKV genome and designed RT-LAMP primers for the Zika lineage that is prevalent in the Americas. To enable POC molecular diagnostics, we developed a disposable microfluidic cassette (Fig. 1a) that combines viral nucleic acid capture, concentration, isothermal amplification; and detection. Our disposable, microfluidic cassette contains multiple independent amplification reactors, each equipped with a silica-based nucleic acid isolation membrane at its inlet. The advantage of such a design is to decouple the sample volume from the reaction volume, allowing one to use relatively high sample volumes to achieve high sensitivity. Nucleic acids captured by the isolation membrane directly serve as templates in an RT-LAMP amplification process without a need for an elution step, significantly simplifying flow control.
Our microfluidic cassette can be incubated with battery power or operate electricity-free. To eliminate the need for electricity, we used a simple, thermally insulated portable cup (Fig. 1b) heated by an exothermic reaction for chip-based isothermal amplification [14]. One Mg−Fe alloy pouch, which is usually used as a heater of MRE (meal, ready-to-eat), served as the heat source. Tap water was introduced into the drawer, housing the Mg-Fe pouch, through a port in the cup lid to interact with the Mg−Fe alloy to produce heat. To isolate the amplification reactor’s temperature from variable ambient conditions, we used a phase change material (PCM) to regulate the temperature, removing the need for a thermal control circuit. An aluminium heat sink was used to enhance heat transfer from the PCM to the cassette.
We tested the utility of our POC diagnostic system with raw saliva samples spiked with various concentrations of the ZIKV. Our experiments showed that our electricity-free POC diagnostic system could detect ZIKV in saliva with the sensitivity of 5 plaque forming units (p.f.u.) of ZIKV per sample within 40 min (Fig. 1c). Our POC diagnostic system is comparable to that of the benchtop assay without a need for laboratory facilities, expensive equipment and well-trained personnel [14].
Conclusion
Zika molecular diagnostics can be performed at the point of care with a low-cost, portable system based on a microfluidic cassette that integrates nucleic acid isolation and concentration, isothermal amplification, and detection. To achieve electricity-free isothermal amplification, the cassette is combined with a chemically heated cup that generates heat with an exothermic reaction. The platform can be adapted to various sample types and sizes, and multiplex detection. This flexibility is useful in view of the evolving understanding of Zika pathology and Zika biomarker levels and their persistence in different body fluids and tissues. In the future, we plan to expand system capabilities to enable concurrent detection of multiple vector-borne diseases [15]. Our system is very suitable for resource-poor settings, where funds, centralized laboratory facilities and trained personnel are in short supply, as well as for use in remote clinics and at home.
Acknowledgment
The research reported here was supported, in part by the NIH NIDCR R21DE026700, K25AI099160, R01 CA214072, to the University of Pennsylvania.
References
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2. Tang H, Hammack C, Ogden SC, Wen Z, Qian X, et al. Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell 2016; 18(5): 587–590.
3. Driggers RW, Ho CY, Korhonen EM, Kuivanen S, Jääskeläinen AJ, et al. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. N Eng J Med 2016; 374: 2142–2151.
4. Kindhauser MK, Allen T, Frank V, Santhana RS, Dye C. Zika: the origin and spread of a mosquito-borne virus. Bull World Health Organ 2016; 94: 675–686C.
5. Lee BY, Alfaro-Murillo JA, Parpia AS, Asti L, Wedlock PT, et al. The potential economic burden of Zika in the continental United States. PLOS Neg Trop Dis 2017; 11(4): e0005531.
6. Current Zika product pipeline. World Health Organization (WHO) 2016. http://www.who.int/csr/research-and-development/zika-rd-pipeline.pdf.
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14. Song J, Mauk MG, Hackett BA, Cherry S, Bau HH, Liu C. Instrument-free point-of-care molecular detection of Zika virus. Anal Chem 2016; 88: 7289–7294.
15. Song J, Liu C, Mauk MG, Rankin SC, Lok JB, et al. Two-stage isothermal enzymatic amplification for concurrent multiplex molecular detection. Clin Chem 2017; 63(3): 714–722.
The authors
Michael G Mauk PhD, Jinzhao Song PhD, Haim H. Bau PhD, Changchun Liu* PhD
Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
*Corresponding author
E-mail: lchangc@seas.upenn.edu
RAS pathway biomarkers for breast cancer prognosis
Metastatic breast cancer is a highly heterogeneous, rapidly evolving and devastating disease that challenges our ability to find curative therapies. RAS pathway activation is an understudied research area in breast cancer. EGFR/RAS pathway activation is prevalent in breast cancer with poor prognosis. The prognostic RAS pathway biomarkers can be used to identify resistant tumour clones, stratify patients and guide therapies.
by L. L. Siewertsz van Reesema, M. P. Lee, Dr V. Zheleva, Dr J. S. Winston,
Dr C. F. O’Connor, Prof. R. R. Perry, Prof. R. A. Hoefer and Prof. A. H. Tang
Introduction
Breast cancer currently represents the second leading cause of cancer-related deaths among women in the United States [1]. In 2016 alone, an estimated 246 660 women will be diagnosed and an additional 40 450 women are expected to succumb to the disease process [1, 2]. By 2030, there are projected to be 294 000 new cases, making breast cancer a growing public health concern [3–5]. The increased breast cancer screenings, major technical advancements in breast cancer imaging and early detection, and targeted anti-cancer therapies have contributed to significant decreases in morbidity and mortality since the 1970s, and now more than 90% of patients with early-stage breast cancer have expected survival of much more than 5 years [2, 5]. Despite these amazing advancements, the prognosis for patients with high-grade, locally advanced and metastatic breast cancers remains poor with average survival less than 2 years [2, 6–8]. State-of-the-art treatments, such as anti-HER2 therapy, anti-ER therapy, anti-PI3K and anti-mTOR therapy, tumour genome-guided combination therapies, stem cell therapy, and anti-CTLA-4/PD1 immunotherapy, alone or in combination, are not curative in eradicating metastatic breast cancer [9–14]. The clinical reality is that despite similar clinical diagnoses and presentation, patients often display diverse tumour response to standard therapies. The intrinsic diversity and evolving heterogeneity of their mammary tumours can become more pronounced in relapsed or metastatic tumours after therapeutic intervention and clonal selection by ineffective treatment. This de novo and acquired tumour heterogeneity leads to diverse tumour responses in neoadjuvant and adjuvant setting following standard-of-care therapies, which in turn leads to varied clinical outcome and disparity in patient survival [6, 15, 16].
Currently, there are no reliable clinical biomarkers that can be used to consistently predict survival of patients with late-stage or metastatic breast cancers [6]. Classically utilized breast cancer biomarkers, such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) molecular subtypes, do not correlate with survival outcomes nor do they predict tumour responses to guided therapies in invasive disease [6]. Patients with malignant tumours are often subjected to full regimens of toxic therapies followed by a period of uncertainty awaiting to learn the response of their tumours and efficacy of their treatments. Unfortunately, some patients may be over-treated, which compromises their long-term quality of life, or, conversely, some patients may be under- or incorrectly treated and thus miss a critical window of opportunity to change prescribed anticancer regimens if therapy-resistant tumours persist after aggressive therapy. There is a pressing need to identify reliable, sensitive, and robust prognostic biomarkers to closely monitor tumour response in real-time, and, importantly, to determine whether first-line standard therapies prescribed are effective against high-risk and invasive mammary tumours, given diverse tumour response and intrinsic tumour heterogeneity [7, 16–21].
Current prognostic biomarkers used in breast cancer
Clinicopathological parameters such as patient age, TMN (tumour size, lymph node status, metastasis) staging, tumour grade and histology, and molecular subtype of breast tumours have become commonplace in justifying medical decision making and prescribing treatment modalities. Therapies for invasive and high-risk breast cancer (luminal, basal-like, HER2-positive and triple-negative breast cancer) are routinely selected based on tumour ER, PR and HER2 status, in addition to aforementioned clinicopathological parameters [22–25]. The American Society of Clinical Oncology (ASCO) updated their clinical practice guidelines this year and according to the panel, only the current gold-standard biomarkers ER, PR, and HER2 should be used to guide treatment decisions, despite the fact they do not correlate with survival outcomes, nor predict tumour response to standard-of-care therapies [26].
ER-positive breast tumours account for the majority of cases worldwide [27]. Estrogen is the main stimulation for growth in the mammary tumours allowing this clinical biomarker to direct treatment planning and assess the benefit of mainstay anti-hormone therapies such as tamoxifen and aromatase inhibitors. The expression of PR is frequently associated with ER-positive tumours where less than 1% of mammary tumours are PR-positive and ER negative [27]. Furthermore, PR expression has shown strong prognostic value with high level PR expression in invasive breast tumours displaying better outcomes to anti-hormone treatment when compared to low level PR expression [28]. Current guidelines put forth by the ASCO recommend ER and PR testing of all new cases of primary and distant recurrent breast tumours [29].
HER2 status has been identified as a strong indicator of patient overall survival. Overexpression of the HER2 oncogene leads to higher chances of relapse with shorter survival times. The development of anti-HER2 therapies, such as trastuzumab, in treating HER2 have been shown to have major benefit and therefore HER2 status is tested in the majority of breast cancer diagnoses [30]. Other emerging biomarkers such as Ki67 have a possible prognostic role in invasive breast cancers [31]. While the exact function is unknown, Ki67 is expressed in cycling tumour cells and it has strong correlation with tumour grade [31, 32]. Approaches to combining Ki67 with established biomarkers ER, PR, and HER2 are now being used to further distinguish breast cancers into clinical subtypes. However, the ASCO did not recommend using Ki67 to guide adjuvant therapies [29].
The ASCO has recognized the clinical utility of six multigene expression assays for specific subgroups of patients, including OncotypeDX (Genomic Health), EndoPredict (Sividon Diagnostics), PAM50 (Prosigna Breast Cancer Prognostic Gene Signature Assay), Breast Cancer Index, urokinase plasminogen activator, and plasminogen activator inhibitor type 1 [33–37]. Most of these assays are recommended for patients with early-stage breast cancers only; none of these multi-gene assays were encouraged for patients with HER2-positive or triple-negative cancers, in addition to late-stage or metastatic cancers [34, 35]. The development of these promising gene signature-based molecular assessment tools for breast cancer is encouraging for the personalization of breast cancer therapies; however, there still remains an unmet medical need for patients with locally advanced and metastatic breast cancers.
The potential role of RAS pathway proteins in the prognosis and treatment of breast cancer
The oncogenic K-RAS carries the most common gain-of-function mutations in human cancer (30% of all human cancers) and its oncogenic role has been well-established in many types of human cancers [38, 39]. Thus, focusing on this tumour-promoting RAS signalling pathway is a logical choice of investigation for prognostic biomarker discovery and novel anticancer therapy development against oncogenic K-RAS-driven human cancer [40, 41]. The RAS pathway has been an intensely studied area of cancer research due to its activation of downstream effectors resulting in tumour proliferation, survival and motility (Fig. 1A). The loss of its most essential downstream signalling gatekeeper, SIAH (seven in absentia homologue), impeded oncogenic K-RAS-driven human pancreatic cancer and lung cancer [40, 42, 43]. Although oncogenic K-RAS mutations are rarely detected in mammary tumours (observed in about 5% of patients), genomic studies have indicated that the EGFR/HER2/K-RAS ‘pathway’ is activated in a large proportion of aggressive and malignant breast cancers [44, 45]. EGFR/HER2/K-RAS activation has been correlated with shortened survival, resistance to therapy, and tumour relapse despite aggressive treatments in breast cancer [44–47]. The mechanism and function of persistent RAS pathway activation remains elusive in breast cancer. This lack of mechanistic understanding, along with the low mutation rate of oncogenic RAS/RAF/MARK detected in mammary tumours, may contribute to the tumour-driving RAS pathway activation being understudied in the area of breast cancer research. It should also be noted that activation of RAS pathway in mammary tissues of animal models is adequate to induce oncogenic transformation and malignancy [48, 49]. Therefore, although there may not be any detectable RAS mutations in high-grade, locally advanced and metastatic breast tumours, alternative RAS pathway activation mechanisms of the RAS pathway downstream effectors may be present, promoting mammary tumorigenesis and metastasis.
Since RAS pathway activation is a major and essential signalling hub to promote human malignancies, we investigated whether EGFR/HER2/RAS pathway biomarker expression can be added to evaluate therapy efficacy and predict patient survival in breast cancer (Fig. 1A). It has been established that expression of SIAH, the most downstream signalling module and the most evolutionarily conserved component of the RAS signalling pathway, reflects RAS pathway activation, cell proliferation, and tumour growth [40, 42, 43]. In a retrospective study, 364 matched pairs of breast tumour specimens from 182 patients who received neoadjuvant systemic therapy (NST) were analysed to determine whether SIAH and/or EGFR outperform ER, PR, HER2, and Ki67 as a prognostic biomarker in locally advanced and metastatic breast cancer. The prognostic power of SIAH and EGFR, alone or in combination, is comparable to the clinical gold standards of clinical predictors (lymph node positivity, mammary tumour size, grade, stage and molecular subtypes in combination), and imaging-guided technology [50]. The activation/inactivation of the tumour-promoting RAS pathway biomarkers, SIAH and EGFR, is associated with tumour progression/regression in mammary tumours post-neoadjuvant systemic therapies (NST). We found that a marked reduction in SIAH/EGFR expression post-NST would indicate effective therapy and increased survival, while persistent high SIAH/EGFR expression post-NST would indicate ineffective therapy and decreased survival [50]. These results suggest that NST-induced reduction of SIAH and EGFR expression may be used as two useful surrogate prognostic biomarkers to quantify therapeutic efficacy, determine tumour responses, detect emerging resistant clones, and predict patient survival in high-grade and aggressive molecular subtype of breast cancer in the neoadjuvant setting in the future (Fig. 1B).
Conclusion
Early stage breast cancer is highly responsive to commonly prescribed standard of care therapies with excellent long-term survival. Locally advanced and metastatic breast cancer has a much worse prognosis despite aggressive chemo- and radiation therapies and locoregional surgical interventions. This disparity in prognosis underlines the acute need to tailor therapy and stratify patients in order to improve patient survival. The discovery of incorporating tumour heterogeneity-independent, therapy-responsive and tumour-driven RAS pathway biomarkers is prognostic in breast cancer, have a clear clinical impact to benefit breast cancer patients with locally advanced and metastatic diseases.
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The authors
Lauren L. Siewertsz van Reesema†*1 BS; Michael P. Lee†1 MS; Vasilena Zheleva2 MD; Janet S. Winston3 MD; Caroline F. O’Connor4 MD; Roger R. Perry2 MD, FACS; Richard A. Hoefer5,6,7 DO, FACS; Amy H. Tang*4,8 PhD
1Eastern Virginia Medical School, Norfolk, VA 23507, USA
2Department of Surgery, Eastern Virginia Medical School, Norfolk, VA 23507, USA
3Sentara Pathology and Pathology Sciences Medical Group, Sentara Norfolk General Hospital (SNGH), Norfolk, VA 23507, USA
4Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23507, USA
5Sentara Cancer Network, 6Dorothy G. Hoefer Comprehensive Breast Center, 7Sentara CarePlex Hospital, Newport News, Virginia 23606, USA
8Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, VA 23507, USA
†Authors contributed equally to this publication.
*Corresponding author
E-mail: Tangah@evms.edu
Literature Review: Tumour Markers
DcR3, TFF3 and Midkine are novel serum biomarkers in small intestinal neuroendocrine tumours
Edfeldt K, Daskalakis K, Bäcklin C, et al. Neuroendocrinology 2016; https://doi.org/10.1159/000452891
Small intestinal neuroendocrine tumours (SI-NETs) are amine- and peptide producing neoplasms. Most patients display metastases at the time of diagnosis, they have an unpredictable individual disease course and the tumours are often therapy resistant. Chromogranin A (CgA) and 5-hydroxyindoleacetic acid (5-HIAA) are the clinically most used biomarkers today, but there is a great need for novel diagnostic and prognostic biomarkers and new therapeutic targets. Sixty-nine biomarkers were screened in serum from 23 SI-NET patients and 23 healthy controls using multiplex PLA (proximity ligation assay). A refined method, PEA (proximity extension assay), was used to analyse 76 additional biomarkers. Statistical testing and multivariate classification were performed. Immunohistochemistry and ELISA assays were performed in an extended cohort. Using PLA, 19 biomarkers showed a significant difference in serum concentrations between patients and controls, and PEA revealed difference in concentrations in 13 proteins. Multivariate classification analysis revealed decoy receptor 3 (DcR3), trefoil factor 3 (TFF3) and Midkine to be good biomarkers for disease, which was confirmed by ELISA analysis. All three biomarkers were expressed in tumour tissue. DcR3 concentrations were elevated in patients with stage IV disease. High concentrations of DcR3 and TFF3 were correlated to poor survival. DcR3, TFF3 and Midkine exhibited elevated serum concentrations in SI-NET patients compared to healthy controls, and DcR3 and TFF3 were associated with poor survival. DcR3 seems to be a marker for liver metastases while TFF3 and Midkine may be new diagnostic biomarkers for SI-NETs.
Integration of multiple “OMIC” biomarkers: a precision medicine strategy for lung cancer
Robles AI, Harris CC. Lung Cancer 2017; 107: 50–58
More than half of all new lung cancer diagnoses are made in patients with locally advanced or metastatic disease, at which point therapeutic options are scarce. It is anticipated, however, that the widespread use of Low-Dose Computed Tomography (LDCT) screening, will lead to a greater proportion of lung cancers being diagnosed at an early, operable, stage. Still, the overall rate of recurrence for surgically treated Stage I lung cancer patients is up to 30% within 5 years of diagnosis. Thus, the identification and clinical application of biomarkers of early stage lung cancer are a pressing medical need. The integrative analysis of “omic,” clinical and epidemiological data for single patients is a core principle of precision medicine. Through rigorous bioinformatics and statistical analyses we have identified biomarkers of early-stage lung cancer based on DNA methylation, expression of mRNA and miRNA, inflammatory cytokines, and urinary metabolites. Beyond a more comprehensive understanding of the molecular taxonomy of lung cancer, these biomarkers can have very practical implications in the context of unmet clinical needs of early stage lung cancer patients: First, current guidelines for LDCT screening broadly include individuals based on age and history of heavy smoking. Tumour-derived circulating biomarkers in the blood and urine associated with lung cancer risk could narrow and prioritize individuals for LDCT screening. Second, a high number of nodules are identified by LDCT, of which fewer than 5% are finally diagnosed as lung cancer. Biomarkers may help discriminate malignant nodules from benign or indolent lesions. Third, the expected rise in the numbers of lung cancer patients diagnosed at an early stage will necessitate new treatment options. Circulating, urinary and tissue-based biomarkers that molecularly categorize Stage I patients after tumour resection can help identify high-risk patients who may benefit from adjuvant chemotherapy or innovative immunotherapy regimens.
Epigenetic alterations as biomarkers in pancreatic ductal adenocarcinoma
Syren P, Andersson R, Bauden M, Ansari D. Scand J Gastroenterol 2017; 52(6–7): 668–673
Pancreatic ductal adenocarcinoma (PDAC) prognosis remains very poor and has only marginally improved during the last decades. Epigenetic alterations have been the focus of many recent studies and offer valuable options for PDAC detection, prognosis and treatment. DNA methylation, histone modifications and microRNA (miR) level changes can be used as biomarkers. These alterations occur early in carcinogenesis and may be specific for PDAC. Additionally, epigenetic alterations can be analysed from cell-free DNA, free-circulating nucleosomes or shed tumour cells in blood. High-throughput methods are available for miR and DNA methylation level detection. In particular, multiple promising miR level changes have been discovered. No single epigenetic biomarker that offers a sufficient specificity has been discovered yet, but patterns containing multiple independent biomarkers exist.
Blood-based and urinary prostate cancer biomarkers: a review and comparison of novel biomarkers for detection and treatment decisions
Hendriks RJ, van Oort IM, Schalken JA. Prostate Cancer Prostatic Dis 2017; 20(1): 12–19
BACKGROUND: The diagnosis of prostate cancer (PCa) is currently based on serum PSA testing and/or abnormal digital rectal examination and histopathologic evaluation of prostate biopsies. The main drawback of PSA testing is the lack of specificity for PCa. To improve early detection of PCa more specific biomarkers are needed. In the past few years, many new promising biomarkers have been identified; however, to date, only a few have reached clinical practice.
METHODS: In this review, we discuss new blood-based and urinary biomarker models that are promising for usage in PCa detection, follow-up and treatment decision-making. These include Prostate Health Index (PHI), prostate cancer antigen 3 (PCA3), four-kallikrein panel (4K), transmembrane protease serine 2-ERG (TMPRSS2-ERG), ExoDx Prostate Intelliscore, SelectMDx and the Mi-Prostate score. Only few head-to-head studies are available for these new fluid-based biomarkers and/or models. The blood-based PHI and urinary PCA3 are two US Food and Drug Administration-approved biomarkers for diagnosis of PCa. In the second part of this review, we give an overview of published studies comparing these two available biomarkers for prediction of (1) PCa upon prostate biopsy, (2) pathological features in radical prostatectomy specimen and (3) significant PCa in patients eligible for active surveillance.
RESULTS: Studies show opposing results in comparison of PHI with PCA3 for prediction of PCa upon initial and repeat prostate biopsy. PHI and PCA3 are able to predict pathological findings on radical prostatectomy specimen, such as tumour volume and Gleason score. Only PHI could predict seminal vesicle invasion and only PCA3 could predict multifocality. There is some evidence that PHI outperforms PCA3 in predicting significant PCa in an active surveillance population.
CONCLUSIONS: Future research should focus on independent validation of promising fluid-based biomarkers/models, and prospective comparison of biomarkers with each other.
CONCLUSIONS: Future research should focus on independent validation of promising fluid-based biomarkers/models, and prospective comparison of biomarkers with each other.
Using novel biomarkers to triage young adult women with minor cervical lesions: a cost-effectiveness analysis
Pedersen K, Sørbye SW, Kristiansen IS, Burger EA. BJOG. 2017; 124(3): 474–484
OBJECTIVE: To evaluate the short-term consequences and cost-effectiveness associated with the use of novel biomarkers to triage young adult women with minor cervical cytological lesions.
DESIGN: Model-based economic evaluation using primary epidemiological data from Norway, supplemented with data from European and American clinical trials.
SETTING: Organised cervical cancer screening in Norway.
POPULATION: Women aged 25–33 years with minor cervical cytological lesions detected at their primary screening test.
METHODS: We expanded an existing simulation model to compare 12 triage strategies involving alternative biomarkers (i.e. reflex human papillomavirus (HPV) DNA/mRNA testing, genotyping, and dual staining) with the current Norwegian triage guidelines.
MAIN OUTCOME MEASURES: The number of high-grade precancers detected and resource use (e.g. monetary costs and colposcopy referrals) for a single screening round (3 years) for each triage strategy. Cost-efficiency, defined as the additional cost per additional precancer detected of each strategy compared with the next most costly strategy.
RESULTS: Five strategies were identified as cost-efficient, and are projected to increase the precancer detection rate between 18 and 57%, compared with current guidelines; however, the strategies did not uniformly require additional resources. Strategies involving HPV mRNA testing required fewer resources, whereas HPV DNA-based strategies detected >50% more precancers, but were more costly and required twice as many colposcopy referrals compared with the current guidelines.
CONCLUSION: Strategies involving biomarkers to triage younger women with minor cervical cytological lesions have the potential to detect additional precancers, yet the optimal strategy depends on the resources available as well as decision-makers’ and women’s acceptance of additional screening procedures.
Role of TSH receptor antibodies in the diagnosis of Graves’ disease
Hyperthyroidism can result from a number of different disorders including Graves’ disease. The diagnostic gold standard is based on radiological tests but measurement of thyroid stimulating hormone receptor antibodies plays an important role in the diagnosis of Graves’. It is important to understand the diagnostic strengths and limitations of these measurements.
by Dr Christopher Boot
Introduction
Hyperthyroidism is relatively common, with a prevalence of between 0.5 and 2 % [1]. A range of symptoms and signs are associated with hyperthyroidism because of the influence of thyroid hormones on multiple organ systems. Many of the most important manifestations are related to effects on the cardiovascular system, which may include tachycardia and arrhythmias. Untreated, hyperthyroidism is associated with significant morbidity and mortality. Hyperthyroidism can usually be diagnosed through the measurement of thyroid stimulating hormone (TSH) and free thyroxine (FT4), with TSH usually suppressed and FT4 raised [occasionally free triiodothyronine (FT3) is raised in the absence of elevated FT4].
The major causes of hyperthyroidism are Graves’ disease and toxic multinodular goitre. Other etiologies include solitary toxic adenoma and thyroiditis (Table 1). Graves’ disease is the most common cause of hyperthyroidism with most other cases due to either toxic multinodular goitre or solitary toxic nodules, which result from autonomous secretion of thyroid hormones (T4 and T3) by one or more nodules. Transient thyrotoxicosis can occur as the result of thyroiditis, secondary to viral infection or autoimmunity.
Graves’ disease is an autoimmune disease characterized by stimulation of the thyroid by TSH receptor stimulating antibodies (TRAbs). This leads to the clinical features typical of hyperthyroidism such as weight loss, heat intolerance, palpitations, anxiety, tremor and tiredness. These autoantibodies may also recognize antigens in other tissues, notably fibroblasts in the eye muscles. This can lead to growth and inflammation of fat cells and muscles around the eye leading to Graves’ orbitopathy, characterized by upper eyelid retraction, lid lag, swelling, conjunctivitis and exophthalmos.
It is important to differentiate between Graves’ disease and other causes of hyperthyroidism as the approach to treatment may depend on etiology. Current guidelines recommend that all cases of hyperthyroidism are referred to an endocrinologist for further investigation to determine the cause and a treatment plan [2, 3]. This article focuses on the role of TRAb measurements in the diagnosis of Graves’ although TRAbs also provide prognostic information [4] and have a role in assessing the risk of neonatal hyperthyroidism in pregnancies involving maternal Graves’ [5].
Diagnosis of Graves’ disease
Determining the underlying cause of hyperthyroidism relies on a combination of clinical history, physical examination, biochemical testing and imaging. Certain findings are highly suggestive of Graves’ disease such as a symmetrically enlarged, non-nodular thyroid and evidence of orbitopathy. The most commonly used imaging tests are radiolabel uptake scans, which allow visualization of a thyroid radiolabel uptake pattern. In Graves’ disease there is homogenous, increased uptake of label across the thyroid, whereas in multinodular goitre there is patchy uptake with increased uptake at the sites of the over-active nodules. Radioactive iodine has largely been replaced with technetium pertechnetate (99mTc), which mimics the behaviour of iodine but exposes patients to lower radiation doses. The recommended role for TRAbs in the diagnosis of Graves’ varies. One recommended approach is to measure TRAbs in new cases of primary hyperthyroidism and where TRAb results are positive to diagnose Graves’ disease (Fig. 1). Where TRAb results are negative, uptake scans can then be used to distinguish Graves’, toxic nodule(s) and thyroiditis [6]. However, some guidelines have recommended an uptake scan as the first-line test, with TRAbs only used in certain situations [7].
TRAb assays
There are two main categories of TRAb assays. The majority of assays in clinical use detect TRAbs in patient samples through their competition with an added TSH receptor ligand for binding of the TSH receptor. These competition-based assays are sometimes referred to as thyrotropin-binding inhibitory immunoglobulin (TBII) assays. Competition-based assays do not discriminate between stimulatory TRAbs (as found in Graves’) or non-stimulating (inhibiting or neutral) TRAbs. In cases of hyperthyroidism it is assumed that any detected TRAbs are stimulating. The second category of TRAb assay is bioassays, which detect only stimulating TRAbs.
Competition-based assays have evolved over the years. Early assays used porcine thyroid membrane extracts and detected the inhibition of binding of radiolabelled TSH to these extracts. Liquid-phase assays were developed when recombinant human TSH receptor became available and the inhibition of radiolabelled TSH to recombinant TSH receptor was detected. Further evolution of competition assays involved replacement of labelled TSH with monoclonal anti-TSH receptor antibodies as the competing ligand. Modern TRAb assays typically use fluorescent or chemiluminescent labels and can be automated allowing high throughput.
Bioassays for stimulating TRAbs detect the production of cAMP in cells incubated with patient serum. Current bioassays use Chinese hamster ovary (CHO) cells transfected with human TSH receptor. These cells produce cAMP in response to TSH receptor stimulation. cAMP can be measured by immunoassay or a luciferase reporter gene may be used to generate a chemiluminescent signal in response to increasing cAMP. TRAb bioassays are more complex and expensive than competition-based assays and less commonly used in clinical practice.
Diagnostic performance of TRAb assays
The current generation of competition-based TRAb assays are generally reported to offer a high degree of diagnostic specificity and sensitivity for Graves’ disease. A meta-analysis of clinical studies using current assays indicated a pooled specificity of 99 % and sensitivity of 97 % [8]. This high diagnostic performance has led some authors to recommend TRAbs as a first-line test to distinguish Graves’ disease from other causes of hyperthyroidism. This may lead to a quicker and more cost effective diagnosis in many cases compared to initial use of imaging tests [9]. In particular, the high diagnostic specificity achieved means that untreated, hyperthyroid patients with positive TRAbs are highly likely to have Graves’ disease so that uptake scans may not be necessary in this scenario, particularly when the clinical presentation suggests Graves’. However, a recent study that compared the diagnostic sensitivity of a number of competition-based TRAb assays found significant variability with sensitivity varying from 65 to 100 % depending on the TRAb assay used [10]. Therefore, a negative TRAb result may not always rule out Graves’ disease with a high degree of certainty.
Assessment of the diagnostic performance of TRAbs in a UK tertiary referral centre
In view of the variability in reported diagnostic sensitivity and the identification of a number of cases of apparent TRAb-negative Graves’ disease in our centre, a retrospective study of the performance of TRAbs in the diagnosis of Graves’ was carried out. The Kryptor (ThermoFisher) TRAb assay was used throughout the period of the study. Results from all TRAb requests for patients referred with a new presentation of thyrotoxicosis were gathered over 18 months. Routine diagnosis of the etiology of hyperthyroidism was based on the uptake pattern on 99mTc scintigraphy, clinical course and other features in addition to TRAb concentrations. Ninety-nine cases of Grave’s disease were identified and 131 cases where an alternative cause of thyrotoxicosis was diagnosed. There was some overlap in TRAb concentrations between patients with Graves’ and patients with other etiologies (Fig. 2). Using the diagnostic cut-off of >1.8 IU/L suggested by the manufacturers of the assay, diagnostic sensitivity was 81.8 % (18 of 99 cases of Grave’s were TRAb-negative), whereas diagnostic specificity was 99.2 %. Applying a lower cut-off of >1.2 IU/L resulted in an improved sensitivity of 88.9 % but slightly lower specificity of 97.7 %.
This data from our centre demonstrated a significant number of cases of TRAb-negative Graves’ disease among patients referred with a new presentation of thyrotoxicosis. The diagnostic sensitivity of the Kryptor TRAb assay, therefore, appears to be lower than that suggested by the manufacturer’s data (96.3 %). This could possibly be as a result of more stringent classification of Graves’ in other studies, whereas this data represents the range of patients investigated in practice, which includes cases of borderline/mild hyperthyroidism. Of the 99 cases of Graves’ disease in this study, 40 patients had a FT4 of less than 30 pmol/L. Twenty percent of patients in this group had a TRAb level of <1.0 IU/L (the lower limit of quantification for the assay). Of the remaining 59 cases of Graves’ disease with a FT4 of ≥30 pmol/L, only 5 % had a TRAb level of < 1.0 IU/L. This suggests that cases of Graves’ with milder biochemical thyrotoxicosis on presentation are more likely to be TRAb-negative. Applying a lower diagnostic cut-off than that recommended by the manufacturer may improve the sensitivity of the Kryptor TRAb assay in the diagnosis of Grave’s disease. Practice in our laboratory is now to report an ‘equivocal’ range of 1.0–1.8 IU/L in addition to a cut-off for positivity of >1.8 IU/L. This better reflects the overlap in TRAb concentrations between Graves’ and other causes of thyrotoxicosis observed in our study than a binary positive/negative threshold. However, no cut-off provided 100 % diagnostic sensitivity for Graves’ disease.
Summary
TRAb assays are useful in the differentiation of Graves’ disease from other causes of thyrotoxicosis. In particular, TRAbs appear to provide a high degree of diagnostic specificity so that hyperthyroid patients with positive TRAb results are highly likely to have Graves’. Radioactive uptake scans may, therefore, not be necessary in all cases of TRAb-positive hyperthyroidism. However, some studies (including our local data) suggest that the diagnostic sensitivity of a negative TRAb result alone is not sufficient to reliably rule out Graves’ disease. Diagnostic performance is likely to vary between TRAb assays, so assay-specific reference data should be used for interpretation.
References
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The author
Christopher Boot PhD, FRCPath
Department of Blood Sciences, Royal
Victoria Infirmary, Newcastle upon Tyne Hospitals NHS Foundation Trust,
Newcastle upon Tyne, UK
*Corresponding author
E-mail: christopher.boot@nuth.nhs.uk