Autofluorescence lifetime augmented reality as a means for real-time robotic surgery guidance in human patients.

Due to loss of tactile feedback the assessment of tumor margins during robotic surgery is based only on visual inspection, which is neither significantly sensitive nor specific. Here we demonstrate time-resolved fluorescence spectroscopy (TRFS) as a novel technique to complement the visual inspection of oral cancers during transoral robotic surgery (TORS) in real-time and without the need for exogenous contrast agents. TRFS enables identification of cancerous tissue by its distinct autofluorescence signature that is associated with the alteration of tissue structure and biochemical profile. A prototype TRFS instrument was integrated synergistically with the da Vinci Surgical robot and the combined system was validated in swine and human patients. Label-free and real-time assessment and visualization of tissue biochemical features during robotic surgery procedure, as demonstrated here, not only has the potential to improve the intraoperative decision making during TORS but also other robotic procedures without modification of conventional clinical protocols.

Nondestructive fluorescence lifetime imaging and time-resolved fluorescence spectroscopy detect cartilage matrix depletion and correlate with mechanical properties.

Tissue engineers utilize a battery of expensive, time-consuming and destructive techniques to assess the composition and function of engineered tissues. A nondestructive solution to monitor tissue maturation would reduce costs and accelerate product development. As a first step toward this goal, two nondestructive, label-free optical techniques, namely multispectral fluorescent lifetime imaging (FLIm) and time-resolved fluorescence spectroscopy (TRFS), were investigated for their potential in evaluating the biochemical and mechanical properties of articular cartilage. Enzymatic treatments were utilized to selectively deplete cartilage of either collagen or proteoglycan, to produce a range of matrix compositions. Samples were assessed for their optical properties using a fiber-coupled optical system combining FLIm and TRFS, their biochemical and mechanical properties and by histological staining. Single and multivariable correlations were performed to evaluate relationships among these properties. FLIm- and TRFS-derived measurements are sensitive to changes in cartilage matrix and correlate with mechanical and biochemical assays. Mean fluorescence lifetime values extracted from FLIm images (375-410 nm spectral band) showed strong, specific correlations with collagen content (R2 = 0.79, p < 0.001) and tensile properties (R2 = 0.45, p = 0.02). TRFS lifetime measurements centered at 520 nm (with a 5 nm bandwidth) possessed strong, specific correlations with proteoglycan content (R2 = 0.59, p = 0.001) and compressive properties (R2 = 0.71, p < 0.001). Nondestructive optical assessment of articular cartilage, using a combination of FLIm- and TRFS-derived parameters, provided a quantitative method for determining tissue biochemical composition and mechanical function. These tools hold great potential for research, industrial and clinical settings.

Time-resolved fluorescence spectroscopy for the diagnosis of oral lichen planus.

BACKGROUND: Lichen planus (LP) is a T-cell mediated autoimmune disorder of unknown aetiology that affects the skin, nails, oral and genital mucous membranes. Conventionally, oral LP (OLP) is diagnosed through clinical assessment and histopathological confirmation by oral biopsy. AIM: To explore the use of time-resolved fluorescence spectroscopy (TRFS) to detect fluorescence lifetime changes between lesional OLP and perilesional normal mucosa. METHODS: In this pilot study, measurements of lesional and perilesional buccal and mouth floor mucosa were conducted in vivo with a TRFS system. Histopathological findings were consistent with OLP in 8 out of 10 patients biopsied. Two patients with histopathological diagnoses of frictional hyperkeratosis and oral candidiasis, respectively, were excluded from the study. RESULTS: Our preliminary data show that lifetime values in the 360-560 nm spectral range indicate a significant differentiation between normal and diseased tissue. In contrast to the standard oral biopsy procedure, this technique is noninvasive, painless, time-efficient and safe. CONCLUSIONS: Future studies are needed to better elucidate the diagnostic capability of TRFS and to further explore the sources of fluorescence contrast. This pilot study suggests that, based on fluorescence lifetime parameters, TRFS is a very promising technology for the development of a novel OLP diagnostic technique.

In silico study of the impact of cancer stem cell dynamics and radiobiological hypoxia on tumour response to hyperfractionated radiotherapy.

OBJECTIVES: Advanced head and neck carcinomas (HNCs) are aggressive tumours, mainly due to hypoxia and a cancer stem cell (CSC) subpopulation. The aim of this study was to simulate tumour growth and behaviour during radiotherapy of three HNC groups (governed by different growth kinetics, hypoxia levels and CSC division pattern) to determine correlation between resistance factors and responses to hyperfractionated radiotherapy. METHODS: An in silico HNC model was developed based on biologically realistic input parameters. During radiotherapy simulation, three parameters were studied: growth kinetics, hypoxia and probability of CSC symmetrical division. Both independent and combined effects on tumour response to hyperfractionated radiotherapy were assessed. RESULTS: Oxic and very mildly hypoxic HNCs were revealed to be controlled by hyperfractionated radiotherapy, irrespective of growth kinetics and CSC division pattern. Moderately hypoxic tumours had different responses to radiotherapy: while slowly proliferating HNCs were still controllable, tumours with higher cell turnover were more resistant. In rapidly proliferating tumours, the number of fractions needed for tumour control increased exponentially with the probability of CSC symmetrical division, whereas in moderately growing HNC, this behaviour was linear. Severely hypoxic tumours could not be controlled by radiotherapy alone. Tumours with CSCs in a severely hypoxic niche required adjuvant therapies to be eradicated. CONCLUSIONS: Growth kinetics strongly influence tumour responses to treatment. Slowly growing tumours showed linear dependence between dose and hypoxia/CSC, whereas rapidly growing tumours followed exponential behaviour.

Tumour repopulation and the role of abortive division in squamous cell carcinomas during chemotherapy.

OBJECTIVES: In head and neck cancers, tumour cell repopulation during chemotherapy is one reason for treatment failure. Some of the mechanisms responsible for this repopulation are cell recruitment and abortive division. Due to lack of quantitative data in the literature regarding these mechanisms, the aim of this study was to investigate the interplay between recruitment and abortive division during cisplatin chemotherapy and to quantify the impact of these mechanisms on tumour control. MATERIALS AND METHODS: An in silico Monte Carlo tumour model was developed to simulate tumour behaviour during chemotherapy. The virtual tumour had the composition and kinetic properties of a biological tumour. Effect of cisplatin on cell cycle and repopulation mechanisms were simulated and interpreted. RESULTS: Abortive division contributed to cell production within the tumour during chemotherapy. There was a strong relationship between recruitment and tumour growth due to abortive division. This observation was supported by the value of proliferative/stem ratio, which increased from 1.3 to 36, even when using small recruitment parameters. CONCLUSIONS: While abortive division contributed towards tumour repopulation during chemotherapy, this mechanism could be controlled by daily doses of cisplatin. On the other hand, stem cells require an additional cytotoxic agent to overcome repopulation due to cell recruitment. Consequently, repopulation via abortive division during chemotherapy did not entail alterations in treatment schedule, nor dose escalation, to control the tumour.

Risk of second primary cancer after breast cancer treatment.

Technological advances in both diagnosis and treatment of breast cancer lead to early detection and better treatment management. Consequently, the population of long-term survivors is on the rise. The risk of developing second cancers among breast cancer survivors was shown to be higher than that for the general population. The aim of this work was to review the literature on the risk of second primary cancer (SPC) after breast irradiation. Pubmed search of population-based studies on SPC after breast irradiation was conducted and the findings (in terms of Standardised Incidence Ratio) were collated and discussed. Several studies confirmed the link between breast tumour irradiation and risk of SPC, showing a small, but valid risk. There are, however, confounding factors that can either underestimate or overestimate risks: misclassification of tumour status, genetic inheritance, smoking, environmental factors, and the lack of accurate data in cancer registries. While isolating these potential triggers might be difficult, this approach would allow better discernability between radiotherapy-related risks and those generated by other factors. It is also important to evaluate the current status of treatment-related late effects and to lower such risks by minimising the dose delivered to normal tissues.

Technical and dosimetric aspects of iodine-125 seed reimplantation in suboptimal prostate implants.

OBJECTIVE: Brachytherapy employing iodine-125 seeds is an established treatment for low-risk prostate cancers. Post-implant dosimetry (PID) is an important tool for identifying suboptimal implants. The aim of this work was to improve suboptimal implants by a subsequent iodine-125 seed top-up (reimplantation), based on the PID results. METHODS: Of 255 patients treated between 2009 and 2012, 6 were identified as having received suboptimal implants and were scheduled for seed top-up. Needle configurations and the number of top-up seeds were determined based on post-implant CT images as well as a reimplantation treatment plan. An average of 14 seeds per patient were implanted during each top-up. Dosimetric outcome was assessed via target parameters and doses received by organs at risk. RESULTS: All six patients had a successful top-up, with a 67% increase in the mean dose delivered to 90% of the prostate volume and a 40% increase in the volume that receives 100% of the prescribed dose. However, the final dosimetric assessment was based on the same seed activity, as the planning system does not account for the decay of the initially implanted seeds. Although physical dosimetry is not influenced by different seed activities (doses are calculated to infinity), the radiobiological implications might be slightly different from the situation when optimal implantation is achieved with one treatment only. CONCLUSION: Seed reimplantation in suboptimal prostate implants is feasible and leads to successful clinical outcomes. ADVANCES IN KNOWLEDGE: Suboptimal prostate implants can occur for various reasons. This work shows that seed reimplantation as salvage therapy can lead to an optimal dosimetric outcome with manageable normal tissue effects.

Evaluation of physician eye lens doses during permanent seed implant brachytherapy for prostate cancer.

Treatment of low grade prostate cancer with permanent implant of radioactive seeds has become one of the most common brachytherapy procedures in use today. The implant procedure is usually performed with fluoroscopy image guidance to ensure that the seeds are deployed in the planned locations. In this situation the physician performing the transperineal implant is required to be close to the fluoroscopy unit and dose to the eye lens may be of concern. In 1991 the International Commission on Radiological Protection (ICRP) provided a recommended dose limit of 150 mSv yr(-1) for occupational exposures to the lens of the eye. With more long term follow-up data, this limit was revised in 2011 to 20 mSv yr(-1). With this revised limit in mind, we have investigated the dose to the lens of the eye received by physicians during prostate brachytherapy seed implantation. By making an approximation of annual workload, we have related the dose received to the annual background dose. Through clinical and phantom measurements with thermoluminescent dosimeters, it was found that the excess dose to the physician's eye lens received for a conservative estimate of annual workload was never greater than 100% of the annual background dose.

Influence of stem-cell cycle time on accelerated re-population during radiotherapy in head and neck cancer.

OBJECTIVES: Tumour re-population during radiotherapy was identified as an important reason for treatment failure in head and neck cancers. The process of re-population is suggested to be caused by various mechanisms, one of the most plausible one being accelerated division of stem-cells (i.e. drastic shortening of cell cycle duration). However, the literature lacks quantitative data regarding the length of tumour stem-cell cycle time during irradiation. MATERIALS AND METHODS: The presented work suggests that if accelerated stem-cell division is indeed a key mechanism behind tumour re-population, the stem-cell cycle time can drop below 10 h during radiotherapy. To illustrate the possible implications, the mechanism of accelerated division was implemented into a Monte Carlo model of tumour growth and response to radiotherapy. Tumour response to radiotherapy was simulated with different stem-cell cycle times (between 2 and 10 h) after the initiation of radiotherapy. RESULTS: It was found that very short stem-cell cycle times lead to tumour re-population during treatment, which cannot be overcome by radiation-induced cell kill. Increasing the number of radiation dose fractions per week might be effective, but only for longer cell cycle times. CONCLUSION: It is of crucial importance to quantitatively assess the mechanisms responsible for tumour re-population, given that conventional treatment regimens are not efficient in delivering lethal doses to advanced head and neck tumours.

The use of enriched 6Li and 7Li Lif:Mg,Cu,P glass-rod thermoluminescent dosemeters for linear accelerator out-of-field radiation dose measurements.

(6)LiF:Mg,Cu,P and (7)LiF:Mg,Cu,P glass-rod thermoluminescent dosemeters (TLDs) were used for measurements of out-of-field photon and neutron doses produced by Varian iX linear accelerator. Both TLDs were calibrated using 18-MV X-ray beam to investigate their dose-response sensitivity and linearity. CR-39 etch-track detectors (Luxel+, Landauer) were employed to provide neutron dose data to calibrate (6)LiF:Mg,Cu,P TLDs at various distances from the isocentre. With cadmium filters employed, slow neutrons (<0.5 eV) were distinguished from fast neutrons. The average in-air photon dose equivalents per monitor unit (MU) ranged from 1.5±0.4 to 215.5±94.6 µSv at 100 and 15 cm from the isocentre, respectively. Based on the cross-calibration factors obtained with CR-39 etch-track detectors, the average in-air fast neutron dose equivalents per MU range from 10.6±3.8 to 59.1±49.9 µSv at 100 and 15 cm from the isocentre, respectively. Contribution of thermal neutrons to total neutron dose equivalent was small: 3.1±7.2 µSv per MU at 15 cm from the isocentre.

Out-of-field neutron and leakage photon exposures and the associated risk of second cancers in high-energy photon radiotherapy: current status.

Determination and understanding of out-of-field neutron and photon doses in accelerator-based radiotherapy is an important issue since linear accelerators operating at high energies (>10 MV) produce secondary radiations that irradiate parts of the patient's anatomy distal to the target region, potentially resulting in detrimental health effects. This paper provides a compilation of data (technical and clinical) reported in the literature on the measurement and Monte Carlo simulations of peripheral neutron and photon doses produced from high-energy medical linear accelerators and the reported risk and/or incidence of second primary cancer of tissues distal to the target volume. Information in the tables facilitates easier identification of (1) the various methods and measurement techniques used to determine the out-of-field neutron and photon radiations, (2) reported linac-dependent out-of-field doses, and (3) the risk/incidence of second cancers after radiotherapy due to classic and modern treatment methods. Regardless of the measurement technique and type of accelerator, the neutron dose equivalent per unit photon dose ranges from as low as 0.1 mSv/Gy to as high as 20.4 mSv/Gy. This radiation dose potentially contributes to the induction of second primary cancer in normal tissues outside the treated area.

The role of amifostine in the treatment of head and neck cancer with cisplatin-radiotherapy.

Although head and neck cancer is not one of the most common cancers, it is a debilitating disease with poor prognosis and considerable post-treatment normal tissue toxicity. The unremitting search to increase the therapeutic ratio between tumour control and late normal tissue injury led to the adoption of altered fractionation schedules. While the increase in acute toxicity can be managed with appropriate medical support, damage produced to late responding tissues is usually irreversible, therefore clinically unacceptable. By altering the conventionally fractionated radiotherapy both loco-regional control and overall survival are increased. Moreover, phase III randomized trials indicated that the combined administration of cisplatin and radiotherapy further improves treatment outcome. Although the uptake of cisplatin in normal cells is not amplified by the combined modality treatment, cisplatin, by itself is a highly cytotoxic agent. Therefore, the need for normal tissue protection has arisen. Amifostine is a selective, radio-protective drug used in both radiotherapy and chemotherapy to reduce normal tissue toxicity. This paper provides an overview of clinical trials employing cisplatin-radiotherapy treatment for advanced head and neck cancer with specific focus on normal tissue toxicity. The emerging role of radioprotectors and furthermore, the effectiveness of amifostine in combined cisplatin-radiotherapy trials are presented.

Efficient Monte Carlo modelling of individual tumour cell propagation for hypoxic head and neck cancer.

A Monte Carlo tumour model has been developed to simulate tumour cell propagation for head and neck squamous cell carcinoma. The model aims to eventually provide a radiobiological tool for radiation oncology clinicians to plan patient treatment schedules based on properties of the individual tumour. The inclusion of an oxygen distribution amongst the tumour cells enables the model to incorporate hypoxia and other associated parameters, which affect tumour growth. The object oriented program FORTRAN 95 has been used to create the model algorithm, with Monte Carlo methods being employed to randomly assign many of the cell parameters from probability distributions. Hypoxia has been implemented through random assignment of partial oxygen pressure values to individual cells during tumour growth, based on in vivo Eppendorf probe experimental data. The accumulation of up to 10 million virtual tumour cells in 15 min of computer running time has been achieved. The stem cell percentage and the degree of hypoxia are the parameters which most influence the final tumour growth rate. For a tumour with a doubling time of 40 days, the final stem cell percentage is approximately 1% of the total cell population. The effect of hypoxia on the tumour growth rate is significant. Using a hypoxia induced cell quiescence limit which affects 50% of cells with and oxygen levels less than 1 mm Hg, the tumour doubling time increases to over 200 days and the time of tumour growth for a clinically detectable tumour (10(9) cells) increases from 3 to 8 years. A biologically plausible Monte Carlo model of hypoxic head and neck squamous cell carcinoma tumour growth has been developed for real time assessment of the effects of multiple biological parameters which impact upon the response of the individual patient to fractionated radiotherapy.

Hybrid Optical-Ultrasonic Technique for Biomedical Diagnostics.

We report the development of a diagnostic system combining time-resolved fluorescence spectroscopy and ultrasound backscatter microscopy and its application in diagnosis of tumors and atherosclerotic disease. This system allows for concurrent evaluation of distinct compositional, functional, and micro-anatomical features of normal and diseased tissues.

New methods for time-resolved fluorescence spectroscopy data analysis based on the Laguerre expansion technique--applications in tissue diagnosis.

OBJECTIVES: A new deconvolution method for the analysis of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data is introduced and applied for tissue diagnosis. METHOD: The intrinsic TR-LIFS decays are expanded on a Laguerre basis, and the computed Laguerre expansion coefficients (LEC) are used to characterize the sample fluorescence emission. The method was applied for the diagnosis of atherosclerotic vulnerable plaques. RESULTS: At a first stage, using a rabbit atherosclerotic model, 73 TR-LIFS in-vivo measurements from the normal and atherosclerotic aorta segments of eight rabbits were taken. The Laguerre deconvolution technique was able to accurately deconvolve the TR-LIFS measurements. More interesting, the LEC reflected the changes in the arterial biochemical composition and provided discrimination of lesions rich in macrophages/foam-cells with high sensitivity (> 85%) and specificity (> 95%). At a second stage, 348 TR-LIFS measurements were obtained from the explanted carotid arteries of 30 patients. Lesions with significant inflammatory cells (macrophages/foam-cells and lymphocytes) were detected with high sensitivity (> 80%) and specificity (> 90%), using LEC-based classifiers. CONCLUSION: This study has demonstrated the potential of using TR-LIFS information by means of LEC for in vivo tissue diagnosis, and specifically for detecting inflammation in atherosclerotic lesions, a key marker of plaque vulnerability.

Miniaturized side-viewing imaging probe for fluorescence lifetime imaging (FLIM): validation with fluorescence dyes, tissue structural proteins and tissue specimens.

We report a side viewing fibre-based endoscope that is compatible with intravascular imaging and fluorescence lifetime imaging microscopy (FLIM). The instrument has been validated through testing with fluorescent dyes and collagen and elastin powders using the Laguerre expansion deconvolution technique to calculate the fluorescence lifetimes. The instrument has also been tested on freshly excised unstained animal vascular tissues.

Scheduling cisplatin and radiotherapy in the treatment of squamous cell carcinomas of the head and neck: a modelling approach.

The aim of the present work was to implement the kinetics of cisplatin into a previously developed tumour growth model and to simulate the combined cisplatin-radiotherapy treatment with the emphasis on time sequencing and scheduling of drug and radiation. An investigation into whether the effect of cisplatin-radiation is determined by independent cell kill or by cisplatin-produced radiosensitization was also undertaken. It was shown that cisplatin administered before radiation conferred similar tumour control to the post-radiation sequencing of the drug. The killing effect of the combined modality treatment on tumour increased with the increase in cell recruitment. Furthermore, the individual cell kill produced by the two cytotoxins led to an additive only tumour response when the treatments were given concurrently, suggesting that for a synergistic effect, cisplatin must potentiate the effect of radiation, through the radiosensitizing mechanisms addressed in the literature. It was concluded that the optimal timing of cisplatin should be close to radiation. The model showed that daily administration of cisplatin led to a 35% improvement of tumour control as compared to radiation alone, while weekly cisplatin has improved radiotherapy by only 6%.

The role of post-implant dosimetry in the quality assessment of prostate implants. The RAH experience.

The quality of a prostate implant using radioactive I-125 seeds can be assessed by performing post-implant dosimetry, which therefore has to be a reliable evaluation tool. At the RAH, we have noticed large variations in post-implant dosimetric parameters compared to the pre-implant data. The purpose of this study was to investigate the cause of these differences. Post-implant dosimetry was performed 4 weeks after implant on 15 prostate cancer patients. The CT images of the pelvis were exported to the planning system, where contouring of the target (prostate) was executed by 6 clinicians, followed by the post-implant dosimetry performed by a physicist. Pre- and post-implant dosimetric parameters were analyzed and compared. The average target volume based on the ultrasound measurements was 35.8 cc. Post-implant CT volumes were determined and averaged over the 15 patients by each clinician, and their average values vary from 28.9 cc to 67.9 cc. Beside the under/overestimation of the target on the CT there was also a "shift" in the target base on the ultrasound image. By comparing pre-implant and post-implant dosimetric parameters we have encountered a significant discrepancy between target volumes based on ultrasound image and CT image. It was concluded that the accuracy of target coverage was partially connected to the poorer quality of the CT image compared to the ultrasound scan, patient's anatomy, but mostly to the poor implantation of the base.

Diagnosis of vulnerable atherosclerotic plaques by time-resolved fluorescence spectroscopy and ultrasound imaging.

In this study, time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonography were applied to detect vulnerable (high-risk) atherosclerotic plaque. A total of 813 TR-LIFS measurements were taken from carotid plaques of 65 patients, and subsequently analyzed using the Laguerre deconvolution technique. The investigated spots were classified by histopathology as thin, fibrotic, calcified, low-inflamed, inflamed and necrotic lesions. Spectral and time-resolved parameters (normalized intensity values and Laguerre expansion coefficients) were extracted from the TR-LIFS data. Feature selection for classification was performed by either analysis of variance (ANOVA) or principal component analysis (PCA). A stepwise linear discriminant analysis algorithm was developed for detecting inflamed and necrotic lesion, representing the most vulnerable plaques. These vulnerable plaques were detected with high sensitivity (>80%) and specificity (>90%). Ultrasound (US) imaging was obtained in 4 carotid plaques in addition to TR-LIFS examination. Preliminary results indicate that US provides important structural information of the plaques that could be combined with the compositional information obtained by TR-LIFS, to obtain a more accurate diagnosis of vulnerable atherosclerotic plaque.

Tumour resistance to cisplatin: a modelling approach.

Although chemotherapy has revolutionized the treatment of haematological tumours, in many common solid tumours the success has been limited. Some of the reasons for the limitations are: the timing of drug delivery, resistance to the drug, repopulation between cycles of chemotherapy and the lack of complete understanding of the pharmacokinetics and pharmacodynamics of a specific agent. Cisplatin is among the most effective cytotoxic agents used in head and neck cancer treatments. When modelling cisplatin as a single agent, the properties of cisplatin only have to be taken into account, reducing the number of assumptions that are considered in the generalized chemotherapy models. The aim of the present paper is to model the biological effect of cisplatin and to simulate the consequence of cisplatin resistance on tumour control. The 'treated' tumour is a squamous cell carcinoma of the head and neck, previously grown by computer-based Monte Carlo techniques. The model maintained the biological constitution of a tumour through the generation of stem cells, proliferating cells and non-proliferating cells. Cell kinetic parameters (mean cell cycle time, cell loss factor, thymidine labelling index) were also consistent with the literature. A sensitivity study on the contribution of various mechanisms leading to drug resistance is undertaken. To quantify the extent of drug resistance, the cisplatin resistance factor (CRF) is defined as the ratio between the number of surviving cells of the resistant population and the number of surviving cells of the sensitive population, determined after the same treatment time. It is shown that there is a supra-linear dependence of CRF on the percentage of cisplatin-DNA adducts formed, and a sigmoid-like dependence between CRF and the percentage of cells killed in resistant tumours. Drug resistance is shown to be a cumulative process which eventually can overcome tumour regression leading to treatment failure.