Advancing Biomarker Development Through Convergent Engagement: Summary Report of the 2nd International Danube Symposium on Biomarker Development, Molecular Imaging and Applied Diagnostics; March 14-16, 2018; Vienna, Austria.

Here, we report on the outcome of the 2nd International Danube Symposium on advanced biomarker development that was held in Vienna, Austria, in early 2018. During the meeting, cross-speciality participants assessed critical aspects of non-invasive, quantitative biomarker development in view of the need to expand our understanding of disease mechanisms and the definition of appropriate strategies both for molecular diagnostics and personalised therapies. More specifically, panelists addressed the main topics, including the current status of disease characterisation by means of non-invasive imaging, histopathology and liquid biopsies as well as strategies of gaining new understanding of disease formation, modulation and plasticity to large-scale molecular imaging as well as integrative multi-platform approaches. Highlights of the 2018 meeting included dedicated sessions on non-invasive disease characterisation, development of disease and therapeutic tailored biomarkers, standardisation and quality measures in biospecimens, new therapeutic approaches and socio-economic challenges of biomarker developments. The scientific programme was accompanied by a roundtable discussion on identification and implementation of sustainable strategies to address the educational needs in the rapidly evolving field of molecular diagnostics. The central theme that emanated from the 2nd Donau Symposium was the importance of the conceptualisation and implementation of a convergent approach towards a disease characterisation beyond lesion-counting "lumpology" for a cost-effective and patient-centric diagnosis, therapy planning, guidance and monitoring. This involves a judicious choice of diagnostic means, the adoption of clinical decision support systems and, above all, a new way of communication involving all stakeholders across modalities and specialities. Moreover, complex diseases require a comprehensive diagnosis by converging parameters from different disciplines, which will finally yield to a precise therapeutic guidance and outcome prediction. While it is attractive to focus on technical advances alone, it is important to develop a patient-centric approach, thus asking "What can we do with our expertise to help patients?"

Southern Ocean Biogeochemical Float Deployment Strategy, With Example From the Greenwich Meridian Line (GO-SHIP A12).

Biogeochemical Argo floats, profiling to 2,000-m depth, are being deployed throughout the Southern Ocean by the Southern Ocean Carbon and Climate Observations and Modeling program (SOCCOM). The goal is 200 floats by 2020, to provide the first full set of annual cycles of carbon, oxygen, nitrate, and optical properties across multiple oceanographic regimes. Building from no prior coverage to a sparse array, deployments are based on prior knowledge of water mass properties, mean frontal locations, mean circulation and eddy variability, winds, air-sea heat/freshwater/carbon exchange, prior Argo trajectories, and float simulations in the Southern Ocean State Estimate and Hybrid Coordinate Ocean Model (HYCOM). Twelve floats deployed from the 2014-2015 Polarstern cruise from South Africa to Antarctica are used as a test case to evaluate the deployment strategy adopted for SOCCOM's 20 deployment cruises and 126 floats to date. After several years, these floats continue to represent the deployment zones targeted in advance: (1) Weddell Gyre sea ice zone, observing the Antarctic Slope Front, and a decadally-rare polynya over Maud Rise; (2) Antarctic Circumpolar Current (ACC) including the topographically steered Southern Zone chimney where upwelling carbon/nutrient-rich deep waters produce surprisingly large carbon dioxide outgassing; (3) Subantarctic and Subtropical zones between the ACC and Africa; and (4) Cape Basin. Argo floats and eddy-resolving HYCOM simulations were the best predictors of individual SOCCOM float pathways, with uncertainty after 2 years of order 1,000 km in the sea ice zone and more than double that in and north of the ACC.

Comprehensive Evaluation of the MBT STAR-BL Module for Simultaneous Bacterial Identification and ß-Lactamase-Mediated Resistance Detection in Gram-Negative Rods from Cultured Isolates and Positive Blood Cultures.

Objective: This study evaluated the capability of a MALDI Biotyper system equipped with the newly introduced MBT STAR-BL module to simultaneously perform species identification and ß-lactamase-mediated resistance detection in bacteremia -causing bacteria isolated from cultured isolates and patient-derived blood cultures (BCs). Methods: Two hundred retrospective cultured isolates and 153 prospective BCs containing Gram-negative rods (GNR) were collected and subjected to direct bacterial identification, followed by the measurement of ß-lactamase activities against ampicillin, piperacillin, cefotaxime, ceftazidime, and meropenem using the MBT STAR-BL module. The results and turnaround times were compared with those of routine microbiological processing. All strains were also characterized by beta-lactamase PCR and sequencing. Results: Using the saponin-based extraction method, MALDI-TOF MS correctly identified bacteria in 116/134 (86.6%) monomicrobial BCs. The detection sensitivities for ß-lactamase activities against ampicillin, piperacillin, third-generation cephalosporin and meropenem were 91.3, 100, 97.9, and 100% for cultured isolates, and 80.4, 100, 68.8, and 40% for monomicrobial BCs (n = 134) respectively. The overall specificities ranged from 91.5 to 100%. Furthermore, the MBT STAR-BL and conventional drug susceptibility test results were concordant in 14/19 (73.7%) polymicrobial cultures. Reducing the logRQ cut-off value from 0.4 to 0.2 increased the direct detection sensitivities for ß-lactamase activities against ampicillin, cefotaxime and meropenem in BCs to 85.7, 87.5, and 100% respectively. The MBT STAR-BL test enabled the reporting of ß-lactamase-producing GNR at 14.16 and 47.64 h before the interim and final reports of routine BCs processing, respectively, were available. Conclusion: The MALDI Biotyper system equipped with the MBT STAR-BL module enables the simultaneous rapid identification of bacterial species and ß-lactamase-mediated resistance from BCs and cultured isolates. Adjustment of the logRQ cut-off value to 0.2 significantly increased the detection sensitivities for clinically important drug-resistant pathogens.

Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL.

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell malignancy. Here we integrated large-scale profiling data of alterations in gene expression, allelic copy number (CN), and nucleotide sequences in 111 well-characterized patients. Besides prominent signatures of T-cell activation and prevalent clonal variants, we also identify novel hot-spots for CN variability, fusion molecules, alternative transcripts, and progression-associated dynamics. The overall lesional spectrum of T-PLL is mainly annotated to axes of DNA damage responses, T-cell receptor/cytokine signaling, and histone modulation. We formulate a multi-dimensional model of T-PLL pathogenesis centered around a unique combination of TCL1 overexpression with damaging ATM aberrations as initiating core lesions. The effects imposed by TCL1 cooperate with compromised ATM toward a leukemogenic phenotype of impaired DNA damage processing. Dysfunctional ATM appears inefficient in alleviating elevated redox burdens and telomere attrition and in evoking a p53-dependent apoptotic response to genotoxic insults. As non-genotoxic strategies, synergistic combinations of p53 reactivators and deacetylase inhibitors reinstate such cell death execution.

Immunocompromised patients with metastatic cutaneous nodal squamous cell carcinoma of the head and neck: Poor outcome unrelated to the index lesion.

BACKGROUND: Immunocompromised patients with metastatic cutaneous nodal head and neck squamous cell carcinoma (HNSCC) have worse outcomes compared to the immunocompetent. The purpose of this study was to investigate the characteristics of the primary cutaneous squamous cell carcinoma (SCC), nodal pathology, and outcome between these 2 groups. METHODS: Analysis of a prospective database was performed. A 2:1 pooled analysis selected 46 immunocompetent patients matched with 23 immunocompromised patients. Overall survival (OS) and relapse-free survival (RFS) were calculated using the Kaplan-Meier method. RESULTS: No significant difference was found in the primary tumor characteristics between the 2 groups. In the immunocompromised group, RFS (hazard ratio [HR] 2.70; P = .01) and OS (HR 2.32; P = .04) were significantly worse. Extracapsular spread was present in 100% of the immunocompromised patients. CONCLUSION: No significant difference was identified in the primary cutaneous SCC between the immunocompetent and immunocompromised patients. Immunosuppression predicted worse outcome.

OSIRIS-REx Contamination Control Strategy and Implementation.

OSIRIS-REx will return pristine samples of carbonaceous asteroid Bennu. This article describes how pristine was defined based on expectations of Bennu and on a realistic understanding of what is achievable with a constrained schedule and budget, and how that definition flowed to requirements and implementation. To return a pristine sample, the OSIRIS-REx spacecraft sampling hardware was maintained at level 100 A/2 and <180 ng/cm2 of amino acids and hydrazine on the sampler head through precision cleaning, control of materials, and vigilance. Contamination is further characterized via witness material exposed to the spacecraft assembly and testing environment as well as in space. This characterization provided knowledge of the expected background and will be used in conjunction with archived spacecraft components for comparison with the samples when they are delivered to Earth for analysis. Most of all, the cleanliness of the OSIRIS-REx spacecraft was achieved through communication among scientists, engineers, managers, and technicians.

Human papillomavirus (HPV) and somatic EGFR mutations are essential, mutually exclusive oncogenic mechanisms for inverted sinonasal papillomas and associated sinonasal squamous cell carcinomas.

Background: Inverted sinonasal (Schneiderian) papilloma (ISP) is a locally aggressive neoplasm often associated with sinonasal squamous cell carcinoma (SNSCC). While the etiology of ISP is not well understood, human papillomavirus (HPV) has been detected in a subset of cases. Our group recently identified activating somatic EGFR mutations in the majority of ISP and ISP-associated SNSCC. However, the relationship between EGFR mutations and HPV infection has not been explored. Patients and methods: We evaluated 58 ISP and 22 ISP-associated SNSCC (including 13 patients with matched ISP/SNSCC samples), as well as 14 SNSCC without clinical or pathologic evidence of an associated ISP. Formalin-fixed, paraffin-embedded samples were evaluated for EGFR mutations using Sanger sequencing and for HPV infection using GP5+/GP6+ PCR. HPV subtyping based on the L1 sequence was done for HPV positive cases including temporally distinct tumors for four patients. Clinicopathologic data including progression free survival was also analyzed. Results: All ISP and ISP-associated SNSCC demonstrated either an EGFR mutation or HPV infection. HPV and EGFR mutation were mutually exclusive in all cases of ISP-associated SNSCC and all but one ISP; this case was only weakly HPV positive, and analysis of a prior temporally distinct ISP specimen from this patient failed to show HPV infection, suggesting transient infection/incidental colonization. HPV subtypes in ISP and ISP-associated SNSCC were predominantly low-risk, in contrast with SNSCC without ISP association, which showed frequent high-risk HPV. All paired ISP and associated SNSCC samples demonstrated concordant HPV status and EGFR genotypes. ISP progression to SNSCC was significantly associated with the presence of HPV infection and the absence of an EGFR mutation (log-rank = 9.620, P = 0.002). Conclusions: Collectively our data show that EGFR mutations and HPV infection represent essential, alternative oncogenic mechanisms in ISP and ISP-associated SNSCC.

Next-generation IgVH sequencing CLL-like monoclonal B-cell lymphocytosis reveals frequent oligoclonality and ongoing hypermutation.

Chronic lymphocytic leukemia (CLL) develops from CLL-like monoclonal B-cell lymphocytosis (MBL) which represents a low-level asymptomatic expansion of cells that phenotypically resemble CLL. Although antigen selection plays a key role during CLL development, it is not known whether this occurs in early MBL or only during progression to CLL. Recent studies suggested that MBL sometimes displays oligoclonality, but these used techniques with limited sensitivity and specificity and were not conclusive. In this study, we combine cell sorting and next-generation sequencing of rearranged immunoglobulin heavy chain variable (IgVH) genes to thoroughly assess the VH repertoire and oligoclonality of purified MBL cells. Clonal functional rearrangements or clonotypes were identified in 29 of 30 sequenced cases, with 7 or 24% having two clonotypes with unrelated CDR3 sequences. In four of the seven cases with unrelated clonotypes, VH segments from the same family were used. In addition, 6 of 29 cases showed clear evidence of ongoing VH gene hypermutation with three of these being among the seven with unrelated clonotypes. This study conclusively shows that MBL cases often contain multiple B-cell clones, the first to report ongoing VH gene mutation in MBL, and that antigen selection appears to occur in early MBL.

Oncogenic Y641 mutations in EZH2 prevent Jak2/ß-TrCP-mediated degradation.

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase ß-TrCP (FBXW1). ß-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs ß-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of ß-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2(Y641) mutants recurrently implicated in lymphoma pathogenesis are unable to bind ß-TrCP. Further, endogenous EZH2(Y641) mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that ß-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival.

Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.

MMSET stimulates myeloma cell growth through microRNA-mediated modulation of c-MYC.

Multiple myeloma (MM) represents the malignant proliferation of terminally differentiated B cells, which, in many cases, is associated with the maintenance of high levels of the oncoprotein c-MYC. Overexpression of the histone methyltransferase MMSET (WHSC1/NSD2), due to t(4;14) chromosomal translocation, promotes the proliferation of MM cells along with global changes in chromatin; nevertheless, the precise mechanisms by which MMSET stimulates neoplasia remain incompletely understood. We found that MMSET enhances the proliferation of MM cells by stimulating the expression of c-MYC at the post-transcriptional level. A microRNA (miRNA) profiling experiment in t(4;14) MM cells identified miR-126* as an MMSET-regulated miRNA predicted to target c-MYC mRNA. We show that miR-126* specifically targets the 3'-untranslated region (3'-UTR) of c-MYC, inhibiting its translation and leading to decreased c-MYC protein levels. Moreover, the expression of this miRNA was sufficient to decrease the proliferation rate of t(4;14) MM cells. Chromatin immunoprecipitation analysis showed that MMSET binds to the miR-126* promoter along with the KAP1 corepressor and histone deacetylases, and is associated with heterochromatic modifications, characterized by increased trimethylation of H3K9 and decreased H3 acetylation, leading to miR-126* repression. Collectively, this study shows a novel mechanism that leads to increased c-MYC levels and enhanced proliferation of t(4;14) MM, and potentially other cancers with high MMSET expression.

NPM-ALK signals through glycogen synthase kinase 3ß to promote oncogenesis.

Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70-80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3ß as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3ß. Expression of NPM-ALK in 293T cells led to an increase of pS(9)-GSK3ß (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3ß levels. Phosphorylation of pS(9)-GSK3ß by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3ß substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3ß. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3ß inhibition. Furthermore, stable knockdown of GSK3ß conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS(9)-GSK3ß and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS(9)-GSK3ß and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S(9)-GSK3ß by PI3K/AKT. The subsequent inhibition of GSK3ß activity results in accumulation of CDC25A and Mcl-1, which confers the advantage of growth and protection from apoptosis. These findings provide support for the role of GSK3ß as a mediator of NPM-ALK oncogenesis.

Comparative analysis of urban atmospheric aerosol by particle-induced X-ray emission (PIXE), proton elastic scattering analysis (PESA), and aerosol mass spectrometry (AMS).

A multifaceted approach to atmospheric aerosol analysis is often desirable in field studies where an understanding of technical comparability among different measurement techniques is essential. Herein, we report quantitative intercomparisons of particle-induced X-ray emission (PIXE) and proton elastic scattering analysis (PESA), performed of fline under a vacuum, with analysis by aerosol mass spectrometry (AMS) carried out in real-time during the MCMA-2003 Field Campaign in the Mexico City Metropolitan Area. Good agreement was observed for mass concentrations of PIXE-measured sulfur (assuming it was dominated by SO4(2-)) and AMS-measured sulfate during most of the campaign. PESA-measured hydrogen mass was separated into sulfate H and organic H mass fractions, assuming the only major contributions were (NH4)2SO4 and organic compounds. Comparison of the organic H mass with AMS organic aerosol measurements indicates that about 75% of the mass of these species evaporated under a vacuum. However approximately 25% of the organics does remain under a vacuum, which is only possible with low-vapor-pressure compounds, and which supports the presence of high-molecular-weight or highly oxidized organics consistent with atmospheric aging. Approximately 10% of the chloride detected by AMS was measured by PIXE, possibly in the form of metal-chloride complexes, while the majority of Cl was likely present as more volatile species including NH4Cl. This is the first comparison of PIXE/PESA and AMS and, to our knowledge, also the first report of PESA hydrogen measurements for urban organic aerosols.

Detection of the c-kit D816V mutation in systemic mastocytosis by allele-specific PCR.

AIMS: The c-kit D816V activating mutation is found in >80% of cases of systemic mastocytosis (SM) and represents a potential drug target. Furthermore, because D816V is one of the diagnostic criteria for SM, it is clinically relevant to determine whether the mutation is present. Traditional techniques such as DNA sequencing are often not sensitive enough to detect mutations in low-abundance tumour cells, including SM. Here, an allele-specific assay to detect the D816V mutation in DNA from archived formalin-fixed paraffin-embedded tissues is described. METHODS: A two-tube PCR format was employed to amplify c-kit exon 17 as a control and an allele-specific reaction to selectively amplify the D816V mutant allele using standard oligonucleotides. A D816V-mutant plasmid control was generated by site-directed mutagenesis of wild-type cells. 14 cases of SM, one D816V-positive seminoma sample, and 35 cases without SM were analysed using the assay. RESULTS: The assay successfully amplified D816V in the mutant plasmid control, 13/14 cases of SM, and confirmed D816V in a seminoma sample. In addition, D816V was not amplified in 35/35 cases without SM. Serial dilution experiments demonstrated sensitivity down to <1%. CONCLUSION: A sensitive, specific and cost-effective assay to detect the D816V mutation in archived formalin-fixed paraffin-embedded tissues from cases of SM has been developed.

Assessment of bony resection margins in oral cancer using elastic scattering spectroscopy: a study on archival material.

Oral squamous cell carcinoma has been shown to infiltrate local bone necessitating either a local or segmental resection for clearance. The current decalcification process takes several weeks before histological examination of the margins is possible. The aim of this study was to determine whether elastic scattering spectroscopy (ESS) could be used to identify bone resection margins positive for tumour. We used an ESS biopsy (optical biopsy) system to assess formalin fixed bone margins resected for squamous cell carcinoma of the oral cavity and compared the results with the histopathological diagnosis. Archival specimens obtained in oral cancer resections over the last 10 years were used, the ESS spectra were obtained from residual resection margins immediately adjacent to the area from which sections were cut and the results correlated with the histopathological diagnosis. Three hundred and forty-one spectra were used in this study taken from the mandibular specimens of 21 patients, of which 231 spectra were taken from histologically positive sites and the rest were of normal tissue. Two different sets of spectra were obtained and using a linear discriminant analysis, a sensitivity of 87% and a specificity of 80% were obtained. These results suggest that ESS may identify tumour involvement of resection margins. This study, on formalin fixed tissue, was shown to be reliable and may significantly reduce pathology workload. If these findings can be applied in vivo, this would be an accurate and instant mechanism for assessment of margins in clinical practice.

Validation of cDNA microarray gene expression data obtained from linearly amplified RNA.

BACKGROUND: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail. METHODS: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template. CONCLUSIONS: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.

Solution-based scanning for single-base alterations using a double-stranded DNA binding dye and fluorescence-melting profiles.

DNA molecules differing by as little as a single-base substitution have traditionally been distinguished by gel electrophoresis-based methodologies that exploit differences in the sequence-specific properties of double-stranded DNA (dsDNA) such as melting temperature and secondary conformational configuration. By comparison, solution-based fluorescence methods using sequence-specific probes are limited to detecting mutations restricted to very short segments of DNA ( approximately 20 bp). We describe a solution-based fluorescence method that discriminates between wild-type and mutant sequences using a dsDNA binding dye, and interrogates a region of >200 nucleotides. This method is based on melting theory and entails fluorescence monitoring of the melting temperatures of GC-clamped amplicons subjected to gradual and progressive thermal denaturation in the presence of a constant concentration of urea. Heterozygous samples are easily identified by the lower melting temperatures of the less thermodynamically stable heteroduplex mismatches from the wild-type:mutant DNA hybrids as compared to the more stable wild-type Watson-Crick duplexes. All of the four possible sets of mismatches (A.G/T.C, T.G/A.C, G.G/C.C, and T.T/A.A) represented in 17 heterozygous mutations distributed throughout the length of 20 different amplicons (104 to 212 bp), were distinguished from the wild-type by their altered melting profiles. This methodology is advantageous in that it obviates gel electrophoresis or labeled oligonucleotide probes. Significantly, it expands the region of interrogation for detection of single-base changes using fluorescence-based methods in solution, and is amenable for automation and adaptation to high-throughput systems.