Verification of a Novel Minimally Invasive Device for the Isolation of Rare Circulating Tumor Cells (CTC) in Cancer Patients' Blood.

Circulating tumor cells (CTCs) exist in low quantities in the bloodstream in the early stages of cancers. It, therefore, remains a technical challenge to isolate them in large enough quantities for a precise diagnosis and downstream analysis. We introduce the BMProbe™, a minimally invasive device that isolates CTCs during a 30-minute incubation in the median cubital vein. The optimized geometry of the device creates flow conditions for improved cell deposition. The CTCs are isolated using antibodies that are bound to the surface of the BMProbe™. In this study, flow experiments using cell culture cells were conducted. They indicate a 31 times greater cell binding efficiency of the BMProbe™ compared to a flat geometry. Further, the functionality of isolating CTCs from patient blood was verified in a small ex vivo study that compared the cell count from seven non-small-cell lung carcinoma (NSCLC) patients compared to nine healthy controls with 10 mL blood samples. The median cell count was 1 in NSCLC patients and 0 in healthy controls. In conclusion, the BMProbe™ is a promising method to isolate CTCs in large quantities directly from the venous bloodstream without removing blood from a patient. The future step is to verify the functionality in vivo.

International harmonization of procedures for measuring and analyzing of vessel underwater radiated noise.

The habitat of the endangered southern resident killer whale (SRKW) overlaps major international shipping lanes near the Port of Vancouver, British Columbia. Shipping is a dominant source of underwater noise, which can hinder SRKW key life functions. To reduce environmental pressure on the SRKWs, Vancouver Fraser Port Authority offers incentives for quieter ships. However, the absence of a widely accepted underwater radiated noise (URN) measurement procedure hinders the determination of relative quietness. We review URN measurement procedures, summarizing results to date from two Canadian-led projects aimed at improving harmonization of shallow-water URN measurement procedures: One supports the International Organization for Standardization (ISO) in the development of a URN measurement standard; the other supports the alignment of URN measurement procedures developed by ship classification societies. Weaknesses in conventional shallow-water URN metrics are identified, and two alternative metrics proposed. Optimal shallow-water measurement geometry is identified.

Evaluating the predictive strength of underwater noise exposure criteria for marine mammals.

The aim of underwater noise exposure criteria in a regulatory context is to identify at what received levels noise-induced effects are predicted to occur, so that those effects may be appropriately considered in an evaluation or mitigation context under the respective regulatory regime. Special emphasis has been given to hearing related impairment of marine mammals due to their high sensitivity to and reliance on underwater sound. Existing regulations of underwater noise show substantial qualitative and quantitative discrepancies. A dataset acquired during an experiment that induced temporary threshold shift (TTS) in a harbor porpoise (Phocoena phocoena) from Lucke, Siebert, Lepper, and Blanchet [(2009). J. Acoust. Soc. Am. 125, 4060-4070] was reanalyzed to see if various exposure criteria predicted TTS differently for high-frequency cetaceans. This provided an unambiguous quantitative comparison of predicted TTS levels for the existing noise exposure criteria used by regulatory bodies in several countries. The comparative evaluation of the existing noise exposure criteria shows substantial disagreement in the predicted levels for onset for auditory effects. While frequency-weighting functions evolved to provide a better representation of sensitivity to noise exposure when compared to measured results at the criteria's onset, thresholds remain the most important parameter determining a match between criteria and measured results.

Techniques for distinguishing between impulsive and non-impulsive sound in the context of regulating sound exposure for marine mammals.

Regulations designed to mitigate the effects of man-made sounds on marine mammal hearing specify maximum daily sound exposure levels. The limits are lower for impulsive than non-impulsive sounds. The regulations do not indicate how to quantify impulsiveness; instead sounds are grouped by properties at the source. To address this gap, three metrics of impulsiveness (kurtosis, crest factor, and the Harris impulse factor) were compared using values from random noise and real-world ocean sounds. Kurtosis is recommended for quantifying impulsiveness. Kurtosis greater than 40 indicates a sound is fully impulsive. Only sounds above the effective quiet threshold (EQT) are considered intense enough to accumulate over time and cause hearing injury. A functional definition for EQT is proposed: the auditory frequency-weighted sound pressure level (SPL) that could accumulate to cause temporary threshold shift from non-impulsive sound as described in Southall, Finneran, Reichmuth, Nachtigall, Ketten, Bowles, Ellison, Nowacek, and Tyack [(2019). Aquat. Mamm. 45, 125-232]. It is known that impulsive sounds change to non-impulsive as these sounds propagate. This paper shows that this is not relevant for assessing hearing injury because sounds retain impulsive character when SPLs are above EQT. Sounds from vessels are normally considered non-impulsive; however, 66% of vessels analyzed were impulsive when weighted for very-high frequency mammal hearing.

Highly Selective Capture Surfaces on Medical Wires for Fishing Tumor Cells in Whole Blood.

The detection of circulating tumor cells (CTCs) in the blood of cancer patients is a challenging task. CTCs are, especially at the early stages of cancer development, extremely rare cells hidden in a vast background of regular blood cells. We describe a new strategy for the isolation of CTCs from whole blood. The key component is a medical wire coated with a multilayer assembly that allows highly specific capture of EpCAM (epithelial cell adhesion molecule) positive CTCs from blood. The assembly is generated in a layer-by-layer fashion through photochemically induced C,H insertion reactions and consists of a protective layer, which shields the contacting solution from the metal, a protein resistant layer, which prevents nonspecific interactions with proteins and a layer containing the EpCAM antibodies. In vitro experiments show that these surfaces can capture tumor cells from whole blood with enrichment factors (specifically vs nonspecifically bound cells) of up to about 3000 compared to the number of leucocytes in the blood. The purity of the isolated cells is greater than 90%. After "fishing" them from the blood, the cells, still bound to the wire, can be genetically analyzed. This demonstrates that this strategy might prove useful for next generation sequencing.

Aerial low-frequency hearing in captive and free-ranging harbour seals (Phoca vitulina) measured using auditory brainstem responses.

The hearing sensitivity of 18 free-ranging and 10 captive harbour seals (Phoca vitulina) to aerial sounds was measured in the presence of typical environmental noise through auditory brainstem response measurements. A focus was put on the comparative hearing sensitivity at low frequencies. Low- and mid-frequency thresholds appeared to be elevated in both captive and free-ranging seals, but this is likely due to masking effects and limitations of the methodology used. The data also showed individual variability in hearing sensitivity with probable age-related hearing loss found in two old harbour seals. These results suggest that the acoustic sensitivity of free-ranging animals was not negatively affected by the soundscape they experienced in the wild.

The Use of a New CellCollector to Isolate Circulating Tumor Cells from the Blood of Patients with Different Stages of Prostate Cancer and Clinical Outcomes - A Proof-of-Concept Study.

BACKGROUND AND METHODS: Circulating tumor cells (CTCs) constitute a useful approach for personalized medicine. Nevertheless, the isolation of these cells remains very challenging because they rarely circulate in the blood. Another current problem is the cancer-specific characterization of these cells, which requires a method that allows for the molecular and immunocytochemical profiling of all captured cells. The purpose of our proof of concept study was to investigate the use of a medical wire (CellCollector, GILUPI) to isolate CTCs in the blood of prostate cancer (PCa) patients, which allowed CTCs to be counted and molecularly characterized. Forty-three PCa patients in different stages and 11 control subjects were studied. Some randomized samples were used to detect tumor-associated transcripts, such as prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA) and epidermal growth factor receptor (EGFR), in the isolated CTCs. RESULTS: The mean CTC counts were 4.6 CTCs [range, 0-8] in patients with localized PCa, 16.8 CTCs [range, 10-25] in patients with locally advanced PCa, and 26.8 CTCs [range, 0-98] in patients with metastatic PCa. The median follow-up time was 24 months, and there was a significant difference in the cancer-specific survival rates. Patients with CTC counts under 5 CTCs lived significantly longer (p = 0.035) than patients with more than 5 CTCs. We also demonstrated that the captured CTCs could be molecularly characterized. We detected tumor-associated transcripts of EGFR and PSMA in patients with metastatic PCa in 42.8% and 14.3% of the analyzed samples, respectively. CONCLUSION: Our results indicate that the sensitive isolation and molecular characterization of CTCs can be achieved ex vivo using the wire. Patients with more than 5 CTCs had a mortality risk that was 7.0 times greater that of those with fewer than 5 CTCs (hazard ratio 7.0 95%, CI 1.1-29.39). This proof of concept was required for the approval of the use of the CellCollector in a clinical study for the in vivo isolation of CTCs from the blood stream of PCa patients by the Federal Institute for Drugs and Medical devices (Germany, BfArM).

Assessing auditory evoked potentials of wild harbor porpoises (Phocoena phocoena).

Testing the hearing abilities of marine mammals under water is a challenging task. Sample sizes are usually low, thus limiting the ability to generalize findings of susceptibility towards noise influences. A method to measure harbor porpoise hearing thresholds in situ in outdoor conditions using auditory steady state responses of the brainstem was developed and tested. The method was used on 15 live-stranded animals from the North Sea during rehabilitation, shortly before release into the wild, and on 12 wild animals incidentally caught in pound nets in Denmark (inner Danish waters). Results indicated that although the variability between individuals is wide, the shape of the hearing curve is generally similar to previously published results from behavioral trials. Using 10-kHz frequency intervals between 10 and 160 kHz, best hearing was found between 120 and 130 kHz. Additional testing using one-third octave frequency intervals (from 16 to 160 kHz) allowed for a much faster hearing assessment, but eliminated the fine scale threshold characteristics. For further investigations, the method will be used to better understand the factors influencing sensitivity differences across individuals and to establish population-level parameters describing hearing abilities of harbor porpoises.

Auditory sensitivity in aquatic animals.

A critical concern with respect to marine animal acoustics is the issue of hearing "sensitivity," as it is widely used as a criterion for the onset of noise-induced effects. Important aspects of research on sensitivity to sound by marine animals include: uncertainties regarding how well these species detect and respond to different sounds; the masking effects of man-made sounds on the detection of biologically important sounds; the question how internal state, motivation, context, and previous experience affect their behavioral responses; and the long-term and cumulative effects of sound exposure. If we are to better understand the sensitivity of marine animals to sound we must concentrate research on these questions. In order to assess population level and ecological community impacts new approaches can possibly be adopted from other disciplines and applied to marine fauna.

Impacts of Underwater Noise on Marine Vertebrates: Project Introduction and First Results.

The project conducts application-oriented research on impacts of underwater noise on marine vertebrates in the North and Baltic Seas. In distinct subprojects, the hearing sensitivity of harbor porpoises and gray seals as well as the acoustic tolerance limit of harbor porpoises to impulsive noise from pile driving and stress reactions caused by anthropogenic noise is investigated. Animals are equipped with DTAGs capable of recording the actual surrounding noise field of free-swimming harbor porpoises and seals. Acoustic noise mapping including porpoise detectors in the Natura 2000 sites of the North and Baltic Seas will help to fully understand current noise impacts.

Development of a Model to Assess Masking Potential for Marine Mammals by the Use of Air Guns in Antarctic Waters.

We estimated the long-range effects of air gun array noise on marine mammal communication ranges in the Southern Ocean. Air gun impulses are subject to significant distortion during propagation, potentially resulting in a quasi-continuous sound. Propagation modeling to estimate the received waveform was conducted. A leaky integrator was used as a hearing model to assess communication masking in three species due to intermittent/continuous air gun sounds. Air gun noise is most probably changing from impulse to continuous noise between 1,000 and 2,000 km from the source, leading to a reduced communication range for, e.g., blue and fin whales up to 2,000 km from the source.

Enumeration and Molecular Characterization of Tumor Cells in Lung Cancer Patients Using a Novel In Vivo Device for Capturing Circulating Tumor Cells.

PURPOSE: The use of circulating tumor cells (CTC) as "liquid biopsy" is limited by the very low yield of CTCs available for subsequent analyses. Most in vitro approaches rely on small sample volumes (5-10 mL). EXPERIMENTAL DESIGN: Here, we used a novel approach, the GILUPI CellCollector, which enables an in vivo isolation of CTCs from peripheral blood. In total, 50 lung cancer patients were screened in two subsequent device applications before and after therapy (n = 185 applications). RESULTS: By in vivo isolation, 58% (108/185) of the patients were positive for ≥1 CTC (median, 5 CTCs; range, 1-56 cells) as compared with 27% (23/84; range, 1-300 cells) using the FDA-cleared CellSearch system. Furthermore, we could show that treatment response during therapy was associated with significant decreases in CTC counts (P = 0.001). By dPCR, mutations in the KRAS and EGFR genes relevant for treatment decisions could be detected in CTCs captured by in vivo isolation and confirmed in the primary tumors of the same patients. CONCLUSIONS: In vivo isolation of CTCs overcomes blood volume limitations of other approaches, which might help to implement CTC-based "liquid biopsies" into clinical decision making. Clin Cancer Res; 22(9); 2197-206. ©2015 AACR.

Communication masking in marine mammals: A review and research strategy.

Underwater noise, whether of natural or anthropogenic origin, has the ability to interfere with the way in which marine mammals receive acoustic signals (i.e., for communication, social interaction, foraging, navigation, etc.). This phenomenon, termed auditory masking, has been well studied in humans and terrestrial vertebrates (in particular birds), but less so in marine mammals. Anthropogenic underwater noise seems to be increasing in parts of the world's oceans and concerns about associated bioacoustic effects, including masking, are growing. In this article, we review our understanding of masking in marine mammals, summarise data on marine mammal hearing as they relate to masking (including audiograms, critical ratios, critical bandwidths, and auditory integration times), discuss masking release processes of receivers (including comodulation masking release and spatial release from masking) and anti-masking strategies of signalers (e.g. Lombard effect), and set a research framework for improved assessment of potential masking in marine mammals.

In-air evoked potential audiometry of grey seals (Halichoerus grypus) from the North and Baltic Seas.

In-air anthropogenic sound has the potential to affect grey seal (Halichoerus grypus) behaviour and interfere with acoustic communication. In this study, a new method was used to deliver acoustic signals to grey seals as part of an in-air hearing assessment. Using in-ear headphones with adapted ear inserts allowed for the measurement of auditory brainstem responses (ABR) on sedated grey seals exposed to 5-cycle (2-1-2) tone pips. Thresholds were measured at 10 frequencies between 1-20 kHz. Measurements were made using subcutaneous electrodes on wild seals from the Baltic and North Seas. Thresholds were determined by both visual and statistical approaches (single point F-test) and good agreement was obtained between the results using both methods. The mean auditory thresholds were ≤40 dB re 20 µPa peak equivalent sound pressure level (peSPL) between 4-20 kHz and showed similar patterns to in-air behavioural hearing tests of other phocid seals between 3 and 20 kHz. Below 3 kHz, a steep reduction in hearing sensitivity was observed, which differed from the rate of decline in sensitivity obtained in behavioural studies on other phocids. Differences in the rate of decline may reflect influence of the ear inserts on the ability to reliably transmit lower frequencies or interference from the structure of the distal end of the ear canal.

Seasonal migrations of North Atlantic minke whales: novel insights from large-scale passive acoustic monitoring networks.

BACKGROUND: Little is known about migration patterns and seasonal distribution away from coastal summer feeding habitats of many pelagic baleen whales. Recently, large-scale passive acoustic monitoring networks have become available to explore migration patterns and identify critical habitats of these species. North Atlantic minke whales (Balaenoptera acutorostrata) perform seasonal migrations between high latitude summer feeding and low latitude winter breeding grounds. While the distribution and abundance of the species has been studied across their summer range, data on migration and winter habitat are virtually missing. Acoustic recordings, from 16 different sites from across the North Atlantic, were analyzed to examine the seasonal and geographic variation in minke whale pulse train occurrence, infer information about migration routes and timing, and to identify possible winter habitats. RESULTS: Acoustic detections show that minke whales leave their winter grounds south of 30° N from March through early April. On their southward migration in autumn, minke whales leave waters north of 40° N from mid-October through early November. In the western North Atlantic spring migrants appear to track the warmer waters of the Gulf Stream along the continental shelf, while whales travel farther offshore in autumn. Abundant detections were found off the southeastern US and the Caribbean during winter. Minke whale pulse trains showed evidence of geographic variation, with longer pulse trains recorded south of 40° N. Very few pulse trains were recorded during summer in any of the datasets. CONCLUSION: This study highlights the feasibility of using acoustic monitoring networks to explore migration patterns of pelagic marine mammals. Results confirm the presence of minke whales off the southeastern US and the Caribbean during winter months. The absence of pulse train detections during summer suggests either that minke whales switch their vocal behaviour at this time of year, are absent from available recording sites or that variation in signal structure influenced automated detection. Alternatively, if pulse trains are produced in a reproductive context by males, these data may indicate their absence from the selected recording sites. Evidence of geographic variation in pulse train duration suggests different behavioural functions or use of these calls at different latitudes.

A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire.

The isolation of circulating tumor cells (CTCs) from the blood of patients afflicted with solid malignant tumors becomes increasingly important as it may serve as a 'liquid biopsy' with the potential of monitoring the course of the cancer disease and its response to cancer therapy, with subsequent molecular characterization. For this purpose, we functionalized a structured medical Seldinger guidewire (FSMW), normally used to obtain safe access to blood vessels and other organ cavities, with a chimeric monoclonal antibody directed to the cell surface expressed epithelial cell surface adhesion molecule (EpCAM). This medical device was optimized in vitro and its biocompatibility was tested according to the regulations for medical devices and found to be safe with no noteworthy side effects. Suitability, specificity and sensitivity of the FSMW to catch and enrich CTCs in vivo from circulating peripheral blood were tested in 24 breast cancer or non-small cell lung cancer (NSCLC) patients and in 29 healthy volunteers. For this, the FSMW was inserted through a standard venous cannula into the cubital veins of healthy volunteers or cancer patients for the duration of 30 min. After removal, CTCs were identified by immuno-cytochemical staining of EpCAM and/or cytokeratins and staining of their nuclei and counted. The FSMW successfully enriched EpCAM-positive CTCs from 22 of the 24 patients, with a median of 5.5 (0-50) CTCs in breast cancer (n=12) and 16 (2-515) CTCs in NSCLC (n=12). CTCs could be isolated across all tumor stages, including early stage cancer, in which distant metastases were not yet diagnosed, while no CTCs could be detected in healthy volunteers. In this observatory study, no adverse effects were noted. Evidently, the FSMW has the potential to become an important device to enrich CTCs in vivo for monitoring the course of the cancer disease and the efficacy of anticancer treatment.

The use of an air bubble curtain to reduce the received sound levels for harbor porpoises (Phocoena phocoena).

In December 2005 construction work was started to replace a harbor wall in Kerteminde harbor, Denmark. A total of 175 wooden piles were piled into the ground at the waters edge over a period of 3 months. During the same period three harbor porpoises were housed in a marine mammal facility on the opposite side of the harbor. All animals showed strong avoidance reactions after the start of the piling activities. As a measure to reduce the sound exposure for the animals an air bubble curtain was constructed and operated in a direct path between the piling site and the opening of the animals' semi-natural pool. The sound attenuation effect achieved with this system was determined by quantitative comparison of pile driving impulses simultaneously measured in front of and behind the active air bubble curtain. Mean levels of sound attenuation over a sequence of 95 consecutive pile strikes were 14 dB (standard deviation (s.d.) 3.4 dB) for peak to peak values and 13 dB (s.d. 2.5 dB) for SEL values. As soon as the air bubble curtain was installed and operated, no further avoidance reactions of the animals to the piling activities were apparent.

Trouble-shooting deployment and recovery options for various stationary passive acoustic monitoring devices in both shallow- and deep-water applications.

Deployment of any type of measuring device into the ocean, whether to shallow or deeper depths, is accompanied by the hope that this equipment and associated data will be recovered. The ocean is harsh on gear. Salt water corrodes. Currents, tides, surge, storms, and winds collaborate to increase the severity of the conditions that monitoring devices will endure. All ocean-related research has encountered the situations described in this paper. In collating the details of various deployment and recovery scenarios related to stationary passive acoustic monitoring use in the ocean, it is the intent of this paper to share trouble-shooting successes and failures to guide future work with this gear to monitor marine mammal, fish, and ambient (biologic and anthropogenic) sounds in the ocean-in both coastal and open waters.