Plasma total cell-free DNA (cfDNA) is a surrogate biomarker for tumour burden and a prognostic biomarker for survival in metastatic melanoma patients

Introduction Tumour burden is a prognostic biomarker in metastatic melanoma. However, tumour burden is difficult to measure and there are currently no reliable surrogate biomarkers to easily and reliably determine it. The aim of this study was to assess the potential of plasma total cell free DNA as biomarker of tumour burden and prognosis in metastatic melanoma patients. Materials and methods A prospective biomarker cohort study for total plasma circulating cell-free DNA (cfDNA) concentration was performed in 43 metastatic melanoma patients. For 38 patients, paired blood collections and scan assessments were available before treatment and at first response evaluation. Tumour burden was calculated as the sum of volumes from three-dimensional radiological measurements of all metastatic lesions in individual patients. Results Baseline cfDNA concentration correlated with pre-treatment tumour burden (ρ = 0.52, P < 0.001). Baseline cfDNA levels correlated significantly with hazard of death and overall survival, and a cut off value of 89 pg/μl identified two distinct prognostic groups (HR = 2.22 for high cfDNA, P = 0.004). Patients with cfDNA ≥89 pg/μl had shorter OS (10.0 versus 22.7 months, P = 0.009; HR = 2.22 for high cfDNA, P = 0.004) and the significance was maintained when compared with lactic dehydrogenase (LDH) in a multivariate analysis. We also found a correlation between the changes of cfDNA and treatment-related changes in tumour burden (ρ = 0.49, P = 0.002). In addition, the ratio between baseline cfDNA and tumour burden was prognostic (HR = 2.7 for cfDNA/tumour volume ≥8 pg/(μl*cm3), P = 0.024). Conclusions We have demonstrated that cfDNA is a surrogate marker of tumour burden in metastatic melanoma patients, and that it is prognostic for overall survival.Fil: Valpione, S.. University of Manchester; Reino Unido. Christie NHS Foundation Trust; Reino UnidoFil: Gremel, G.. University of Manchester; Reino UnidoFil: Mundra, P.. University of Manchester; Reino UnidoFil: Middlehurst, P.. University of Manchester; Reino UnidoFil: Galvani, E.. Christie NHS Foundation Trust; Reino Unido. University of Manchester; Reino UnidoFil: Girotti, Maria Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. University of Manchester; Reino UnidoFil: Lee, R.J.. University of Manchester; Reino UnidoFil: Garner, G.. University of Manchester; Reino UnidoFil: Dhomen, N.. University of Manchester; Reino UnidoFil: Lorigan, P.C.. Christie NHS Foundation Trust; Reino UnidoFil: Marais, R.. University of Manchester; Reino Unid

Circulating tumor DNA predicts survival in patients with resected high risk stage II/III melanoma

Background: Patients with high-risk stage II/III resected melanoma commonly develop distant metastases. At present, we cannot differentiate between patients who will recur or those who are cured by surgery. We investigated if circulating tumor DNA (ctDNA) can predict relapse and survival in patients with resected melanoma. Patients and methods: We carried out droplet digital polymerase chain reaction to detect BRAF and NRAS mutations in plasma taken after surgery from 161 stage II/III high-risk melanoma patients enrolled in the AVAST-M adjuvant trial. Results: Mutant BRAF or NRAS ctDNA was detected (≥1 copy of mutant ctDNA) in 15/132 (11%) BRAF mutant patient samples and 4/29 (14%) NRAS mutant patient samples. Patients with detectable ctDNA had a decreased disease-free interval [DFI; hazard ratio (HR) 3.12; 95% confidence interval (CI) 1.79–5.47; P < 0.0001] and distant metastasis-free interval (DMFI; HR 3.22; 95% CI 1.80–5.79; P < 0.0001) versus those with undetectable ctDNA. Detectable ctDNA remained a significant predictor after adjustment for performance status and disease stage (DFI: HR 3.26, 95% CI 1.83–5.83, P < 0.0001; DMFI: HR 3.45, 95% CI 1.88–6.34, P < 0.0001). Five-year overall survival rate for patients with detectable ctDNA was 33% (95% CI 14%–55%) versus 65% (95% CI 56%–72%) for those with undetectable ctDNA. Overall survival was significantly worse for patients with detectable ctDNA (HR 2.63; 95% CI 1.40–4.96); P = 0.003) and remained significant after adjustment for performance status (HR 2.50, 95% CI 1.32–4.74, P = 0.005). Conclusion: ctDNA predicts for relapse and survival in high-risk resected melanoma and could aid selection of patients for adjuvant therapy

Brain microenvironment-driven resistance to immune and targeted therapies in acral melanoma.

BACKGROUND: Combination treatments targeting the MEK-ERK pathway and checkpoint inhibitors have improved overall survival in melanoma. Resistance to treatment especially in the brain remains challenging, and rare disease subtypes such as acral melanoma are not typically included in trials. Here we present analyses from longitudinal sampling of a patient with metastatic acral melanoma that became resistant to successive immune and targeted therapies. METHODS: We performed whole-exome sequencing and RNA sequencing on an acral melanoma that progressed on successive immune (nivolumab) and targeted (dabrafenib) therapy in the brain to identify resistance mechanisms. In addition, we performed growth inhibition assays, reverse phase protein arrays and immunoblotting on patient-derived cell lines using dabrafenib in the presence or absence of cerebrospinal fluid (CSF) in vitro. Patient-derived xenografts were also developed to analyse response to dabrafenib. RESULTS: Immune escape following checkpoint blockade was not due to loss of tumour cell recognition by the immune system or low neoantigen burden, but was associated with distinct changes in the microenvironment. Similarly, resistance to targeted therapy was not associated with acquired mutations but upregulation of the AKT/phospho-inositide 3-kinase pathway in the presence of CSF. CONCLUSION: Heterogeneous tumour interactions within the brain microenvironment enable progression on immune and targeted therapies and should be targeted in salvage treatments

Variation in inbreeding rates across the range of Northern Spotted Owls (\u3ci\u3eStrix occidentalis caurina\u3c/i\u3e): Insights from over 30 years of monitoring data

Inbreeding has been difficult to quantify in wild populations because of incomplete parentage information. We applied and extended a recently developed framework for addressing this problem to infer inbreeding rates in Northern Spotted Owls (Strix occidentalis caurina) across the Pacific Northwest, USA. Using pedigrees from 14,187 Northern Spotted Owls, we inferred inbreeding rates for 14 types of matings among relatives that produce pedigree inbreeding coefficients of F=0.25 or F=0.125. Inbreeding was most common in the Washington Cascades, where an estimated 15% of individuals are inbred. Inbreeding was lowest in western Oregon (3.5%) and northern California (2.7%), and intermediate for the Olympic Peninsula of Washington (6.1%). Estimates from the Olympic Peninsula were likely underestimates because of small sample sizes and the presence of few pedigrees capable of resolving inbreeding events. Most inbreeding resulted from matings between full siblings or half siblings, although a high rate of inbreeding from mother–son pairs was identified in the Olympic Peninsula. Geographic variation in inbreeding rates may reflect population declines and bottlenecks that have been detected in prior investigations. We show that there is strong selection against inbred birds. Only 3 of 44 inbred birds were later identified as parents (6.8%), whereas 2,823 of 10,380 birds that represented a comparable cross section of the data were later seen as reproducing parents (27.2%). Habitat loss and competition with Barred Owls (S. varia) remain primary threats to Northern Spotted Owls. However, given the negative consequences of inbreeding, Spotted Owl populations in Washington with suitable habitat and manageable numbers of Barred Owls may benefit from translocations of individuals from Oregon and California to introduce new genetic variation and reduce future inbreeding events. La endogamia ha sido dif´ıcil de cuantificar en las poblaciones silvestres debido a la falta de informaci ´on sobre los parentescos. Aplicamos y extendimos un marco conceptual recientemente desarrollado para encarar el problema de inferir las tasas de endogamia en Strix occidentalis caurina a trav´es del noroeste del Pac´ıfico, EEUU. Usando los pedigr´ıes provenientes de 14187 individuos, inferimos las tasas de endogamia para 14 tipos de apareamiento entre parientes que producen coeficientes de endogamia de pedigr´ı de F=0.25 o F=0.125. La endogamia fue ma´s com´un en las Cascadas de Washington, donde se estima que 15% de los individuos son endoga´micos. La endogamia fue menor en el oeste de Oreg´on (3.5%) y el norte de California (2.7%), e intermedia en la Pen´ınsula Ol´ımpica de Washington (6.1%). Las estimaciones de la Pen´ınsula Ol´ımpica fueron probablemente subestimadas debido a los peque ˜nos tama ˜nos de muestreo y a la presencia de pocos pedigr´ıes capaces de resolver los eventos de endogamia. La mayor´ıa de la endogamia result ´o de los apareamientos entre hermanos completos o medios hermanos, aunque se identific ´o una alta tasa de endogamia en parejas madre/hijo en la Pen´ınsula Ol´ımpica. La variaci ´on geogra´ fica en las tasas de endogamia puede reflejar disminuciones poblacionales y cuellos de botella que han sido detectados en investigaciones previas. Mostramos que hay una fuerte selecci ´on contra las aves endoga´micas. Solo tres de 44 aves endoga´micas fueron ma´s tarde identificadas como progenitores (6.8%), mientras que 2823 de 10380 aves que representaron una secci ´on transversal comparable de datos fueron vistas ma´s tarde como progenitores reproductivos (27.2%). La p´erdida de ha´bitat y la competencia con Strix varia sigue siendo la principal amenaza para S. o. caurina. Sin embargo, dadas las consecuencias negativas de la endogamia, las poblaciones de S. occidentalis en Washington con ha´bitat adecuado y n´umeros manejables de Strix varia pueden beneficiarse de traslocaciones de individuos de Oreg´on y California para introducir nueva variaci ´on gen´etica y reducir futuros eventos de endogamia

The past and future roles of competition and habitat in the range-wide occupancy dynamics of Northern Spotted Owls

Slow ecological processes challenge conservation. Short-term variability can obscure the importance of slower processes that may ultimately determine the state of a system. Furthermore, management actions with slow responses can be hard to justify. One response to slow processes is to explicitly concentrate analysis on state dynamics. Here, we focus on identifying drivers of Northern Spotted Owl (Strix occidentalis caurina) territorial occupancy dynamics across 11 study areas spanning their geographic range and forecasting response to potential management actions. Competition with Barred Owls (Strix varia) has increased Spotted Owl territory extinction probabilities across all study areas and driven recent declines in Spotted Owl populations. Without management intervention, the Northern Spotted Owl subspecies will be extirpated from parts of its current range within decades. In the short term, Barred Owl removal can be effective. Over longer time spans, however, maintaining or improving habitat conditions can help promote the persistence of northern spotted owl populations. In most study areas, habitat effects on expected Northern Spotted Owl territorial occupancy are actually greater than the effects of competition from Barred Owls. This study suggests how intensive management actions (removal of a competitor) with rapid results can complement a slower management action (i.e., promoting forest succession)

Lateral orbitofrontal cortex anticipates choices and integrates prior with current information

Adaptive behavior requires integrating prior with current information to anticipate upcoming events. Brain structures related to this computation should bring relevant signals from the recent past into the present. Here we report that rats can integrate the most recent prior information with sensory information, thereby improving behavior on a perceptual decision-making task with outcome-dependent past trial history. We find that anticipatory signals in the orbitofrontal cortex about upcoming choice increase over time and are even present before stimulus onset. These neuronal signals also represent the stimulus and relevant second-order combinations of past state variables. The encoding of choice, stimulus and second-order past state variables resides, up to movement onset, in overlapping populations. The neuronal representation of choice before stimulus onset and its build-up once the stimulus is presented suggest that orbitofrontal cortex plays a role in transforming immediate prior and stimulus information into choices using a compact state-space representation

Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA.

BACKGROUND: The application of precision medicine in oncology requires in-depth characterisation of a patient's tumours and the dynamics of their responses to treatment. PATIENTS AND METHODS: We used next-generation sequencing of circulating cell-free DNA (cfDNA) to monitor the response of a KIT p.L576P-mutant metastatic vaginal mucosal melanoma to sequential targeted, immuno- and chemotherapy. RESULTS: Despite a KIT mutation, the response to imatinib was mixed. Unfortunately, tumours were not accessible for molecular analysis. To study the mechanism underlying the mixed clinical response, we carried out whole-exome sequencing and targeted longitudinal analysis of cfDNA. This revealed two tumour subclones; one with a KIT mutation that responded to imatinib and a second KIT-wild-type subclone that did not respond to imatinib. Notably, the subclones also responded differently to immunotherapy. However, both subclones responded to carboplatin/paclitaxel, and although the KIT-wild-type subclone progressed after chemotherapy, it responded to subsequent re-administration of paclitaxel. CONCLUSION: We show that cfDNA can reveal tumour evolution and subclonal responses to therapy even when biopsies are not available

Neural antecedents of self-initiated actions in secondary motor cortex

The neural origins of spontaneous or self-initiated actions are not well understood and their interpretation is controversial. To address these issues, we used a task in which rats decide when to abort waiting for a delayed tone. We recorded neurons in the secondary motor cortex (M2) and interpreted our findings in light of an integration-to-bound decision model. A first population of M2 neurons ramped to a constant threshold at rates proportional to waiting time, strongly resembling integrator output. A second population, which we propose provide input to the integrator, fired in sequences and showed trial-to-trial rate fluctuations correlated with waiting times. An integration model fit to these data also quantitatively predicted the observed inter-neuronal correlations. Together, these results reinforce the generality of the integration-to-bound model of decision-making. These models identify the initial intention to act as the moment of threshold crossing while explaining how antecedent subthreshold neural activity can influence an action without implying a decision.info:eu-repo/semantics/publishedVersio

Towards pain-free diagnosis of skin diseases through multiplexed microneedles: biomarker extraction and detection using a highly sensitive blotting method

Immunodiagnostic microneedles provide a novel way to extract protein biomarkers from the skin in a minimally invasive manner for analysis in vitro. The technology could overcome challenges in biomarker analysis specifically in solid tissue, which currently often involves invasive biopsies. This study describes the development of a multiplex immunodiagnostic device incorporating mechanisms to detect multiple antigens simultaneously, as well as internal assay controls for result validation. A novel detection method is also proposed. It enables signal detection specifically at microneedle tips and therefore may aid the construction of depth profiles of skin biomarkers. The detection method can be coupled with computerised densitometry for signal quantitation. The antigen specificity, sensitivity and functional stability of the device were assessed against a number of model biomarkers. Detection and analysis of endogenous antigens (interleukins 1α and 6) from the skin using the device was demonstrated. The results were verified using conventional enzyme-linked immunosorbent assays. The detection limit of the microneedle device, at ≤10 pg/mL, was at least comparable to conventional plate-based solid-phase enzyme immunoassays

A systematic search strategy identifies cubilin as independent prognostic marker for renal cell carcinoma

$\textbf{Background:}$ There is an unmet clinical need for better prognostic and diagnostic tools for renal cell carcinoma (RCC). $\textbf{Methods:}$ Human Protein Atlas data resources, including the transcriptomes and proteomes of normal and malignant human tissues, were searched for RCC-specific proteins and cubilin (CUBN) identified as a candidate. Patient tissue representing various cancer types was constructed into a tissue microarray ($n$ = 940) and immunohistochemistry used to investigate the specificity of CUBN expression in RCC as compared to other cancers. Two independent RCC cohorts ($n$ = 181; $n$ = 114) were analyzed to further establish the sensitivity of CUBN as RCC-specific marker and to explore if the fraction of RCCs lacking CUBN expression could predict differences in patient survival. $\textbf{Results:}$ CUBN was identified as highly RCC-specific protein with 58% of all primary RCCs staining positive for CUBN using immunohistochemistry. In venous tumor thrombi and metastatic lesions, the frequency of CUBN expression was increasingly lost. Clear cell RCC (ccRCC) patients with CUBN positive tumors had a significantly better prognosis compared to patients with CUBN negative tumors, independent of T-stage, Fuhrman grade and nodal status (HR 0.382, CI 0.203–0.719, $P$ = 0.003). $\textbf{Conclusions:}$ CUBN expression is highly specific to RCC and loss of the protein is significantly and independently associated with poor prognosis. CUBN expression in ccRCC provides a promising positive prognostic indicator for patients with ccRCC. The high specificity of CUBN expression in RCC also suggests a role as a new diagnostic marker in clinical cancer differential diagnostics to confirm or rule out RCC.This work was supported by the Swedish Cancer Society and the Knut and Alice Wallenberg Foundation. The work of DJH and GDS was funded by the Chief Scientist Office (grant number ETM37), Renal Cancer Research Fund and Kidney Cancer Scotland