HER2-HER3 heterodimer quantification by FRET-FLIM and patient subclass analysis of the COIN colorectal trial

BACKGROUND: The phase 3 MRC COIN trial showed no statistically significant benefit from adding the EGFR-target cetuximab to oxaliplatin-based chemotherapy in first-line treatment of advanced colorectal cancer. This study exploits additional information on HER2-HER3 dimerization to achieve patient stratification and reveal previously hidden subgroups of patients who had differing disease progression and treatment response. METHODS: HER2-HER3 dimerization was quantified by "FLIM Histology" in primary tumor samples from 550 COIN trial patients receiving oxaliplatin and fluoropyrimidine chemotherapy +/-cetuximab. Bayesian latent class analysis (LCA) and covariate reduction was performed to analyze the effects of HER2-HER3 dimer, RAS mutation and cetuximab on progression-free survival (PFS) and overall survival (OS). All statistical tests were two-sided. RESULTS: LCA on a cohort of 398 patients revealed two patient subclasses with differing prognoses (median OS: 1624 days [95%CI=1466-1816] vs 461 [95%CI=431-504]): Class 1 (15.6%) showed a benefit from cetuximab in OS (HR = 0.43 [95%CI=0.25-0.76]; p = 0.004). Class 2 showed an association of increased HER2-HER3 with better OS (HR = 0.64 [95%CI=0.44-0.94]; p = 0.02). A class prediction signature was formed and tested on an independent validation cohort (N = 152) validating the prognostic utility of the dimer assay. Similar subclasses were also discovered in full trial dataset (N = 1,630) based on 10 baseline clinicopathological and genetic covariates. CONCLUSIONS: Our work suggests that the combined use of HER dimer imaging and conventional mutation analyses will be able to identify a small subclass of patients (>10%) who will have better prognosis following chemotherapy. A larger prospective cohort will be required to confirm its utility in predicting the outcome of anti-EGFR treatment

Mapping femtosecond pulse front distortion and group velocity dispersion in multiphoton microscopy

Group velocity dispersion (GVD) and pulse front distortion of ultrashort pulses are of critical importance in efficient multiphoton excitation microscopy. Since measurement of the pulse front distortion due to a lens is not trivial we have developed an imaging interferometric cross-correlator which allows us to measure temporal delays and pulse-widths across the spatial profile of the beam. The instrument consists of a modified Michelson interferometer with a reference arm containing a voice-coil delay stage and an arm which contains the optics under test. The pulse replicas are recombined and incident on a 22×22 lenslet array. The beamlets are focused in a 0.5 mm thick BBO crystal (cut for Type I second harmonic generation), filtered to remove the IR component of the beam and imaged using a 500 fps camera. The GVD and pulse front distortion are extracted from the temporal stack of beamlet images to produce a low resolution spatio-temporal map

Electrically tunable fluidic lens imaging system for laparoscopic fluorescence-guided surgery

The addition of fluorescence guidance in laparoscopic procedures has gained significant interest in recent years, particularly through the use of near infrared (NIR) markers. In this work we present a novel laparoscope camera coupler based on an electrically tunable fluidic lens that permits programmable focus control and has desirable achromatic performance from the visible to the NIR. Its use extends the lower working distance limit and improves detection sensitivity, important for work with molecularly targeted fluorescence markers. We demonstrate its superior optical performance in laparoscopic fluorescence-guided surgery. In vivo results using a tumor specific molecular probe and a nonspecific NIR dye are presented

Electrically tunable fluidic lens imaging system for laparoscopic fluorescence-guided surgery

The addition of fluorescence guidance in laparoscopic procedures has gained significant interest in recent years, particularly through the use of near infrared (NIR) markers. In this work we present a novel laparoscope camera coupler based on an electrically tunable fluidic lens that permits programmable focus control and has desirable achromatic performance from the visible to the NIR. Its use extends the lower working distance limit and improves detection sensitivity, important for work with molecularly targeted fluorescence markers. We demonstrate its superior optical performance in laparoscopic fluorescence-guided surgery. In vivo results using a tumor specific molecular probe and a nonspecific NIR dye are presented

A focused scanning vertical beam for charged particle irradiation of living cells with single counted particles

The Surrey vertical beam is a new facility for targeted irradiation of cells in medium with singly counted ions. A duo-plasmatron ion source and a 2 MV Tandem accelerator supply a range of ions from protons to calcium for this beamline, with energy ranges from 0.5 to 12 MeV. A magnetic quadrupole triplet lens is used to focus the beam of ions. We present the design of this beamline, and early results showing the capability to count single ions with 98% certainty on CR-39 track etch. We also show that the beam targeting accuracy is within 5 microns and selectively target human broblasts with a <5 m carbon beam, using H2AX immuno uorescence to demonstrate which cell nuclei were irradiated. We discuss future commissioning steps necessary to achieve sub-micron targeting accuracy with this beamline

"Broadbeam" irradiation of mammalian cells using a vertical microbeam facility

A "broadbeam" facility is demonstrated for the vertical microbeam at Surrey's Ion Beam Centre, validating the new technique used by Barazzuol et al. (Radiat Res 177:651-662, 2012). Here, droplets with a diameter of about 4 mm of 15,000 mammalian cells in suspension were pipetted onto defined locations on a 42-mm-diameter cell dish with each droplet individually irradiated in "broadbeam" mode with 2 MeV protons and 4 MeV alpha particles and assayed for clonogenicity. This method enables multiple experimental data points to be rapidly collected from the same cell dish. Initially, the Surrey vertical beamline was designed for the targeted irradiation of single cells with single counted ions. Here, the benefits of both targeted single-cell and broadbeam irradiations being available at the same facility are discussed: in particular, high-throughput cell irradiation experiments can be conducted on the same system as time-intensive focused-beam experiments with the added benefits of fluorescent microscopy, cell recognition and time-lapse capabilities. The limitations of the system based on a 2 MV tandem accelerator are also discussed, including the uncertainties associated with particle Poisson counting statistics, spread of linear energy transfer in the nucleus and a timed dose delivery. These uncertainties are calculated with Monte Carlo methods. An analysis of how this uncertainty affects relative biological effect measurements is made and discussed. © 2013 Springer-Verlag Berlin Heidelberg

The development of technology for effective respiratory-gated irradiation using an image-guided small animal irradiator

The development of image-guided small animal irradiators represents a significant improvement over standard irradiators enabling preclinical treatments to mimic radiotherapy in humans. The ability to deliver tightly collimated targeted beams, in conjunction with gantry or animal couch rotation, has the potential to maximise tumour dose while sparing normal tissues. However the current commercial platforms do not incorporate respiratory gating as required for accurate and precise targeting in organs subject to breath-related motions, which may be up to the order of 5 mm in mice. Therefore a new treatment head assembly for the Xstrahl Small Animal Radiation Research Platform (SARRP) has been designed. This includes a fast x-ray shutter subsystem, a motorised beam hardening filter assembly, an integrated transmission ionisation chamber to monitor beam delivery, a kinematically positioned removable beam collimator and a targeting laser exiting the centre of the beam collimator. The x-ray shutter not only minimises timing errors but also allows beam gating during imaging and treatment, with irradiation only taking place during the breathing cycle when tissue movement is minimal. The breathing-related movement is monitored by measuring, using a synchronous detector/lock-in amplifier that processes diffuse reflectance light from a modulated light source. Following thresholding of the resulting signal, delays are added around the inhalation/exhalation phases, enabling the ‘no movement’ period to be isolated and to open the x-ray shutter. Irradiation can either be performed for a predetermined time of x-ray exposure, or through integration of current from the transmission monitor ionisation chamber (corrected locally for air density variations). The ability to successfully deliver breathing gated x-ray irradiations has been demonstrated by comparing movies obtained using planar x-ray imaging with and without breathing gating, in addition to comparing dose profiles observed from a collimated beam on EBT3 radiochromic film mounted on the animals’ chest. Altogether, the development of breathing gated irradiation facilitates improved dose delivery during animal movement and constitutes an important new tool for preclinical radiotherapy studies. This arrangement is particularly well suited for treatments of orthotopic tumours or other targets within the chest and abdomen where breathing related movement is significant

Irradiation at ultra-high (FLASH) dose rates reduces acute normal tissue toxicity in the mouse gastrointestinal system

Purpose Preclinical studies using ultra-high dose rate (FLASH) irradiation have demonstrated reduced normal tissue toxicity compared to conventional dose rate (CONV) irradiation, although this finding is not universal. We investigated the effect of temporal pulse structure and average dose rate of FLASH compared to CONV irradiation, on acute intestinal toxicity. Materials and Methods Whole abdomens of C3H mice were irradiated with a single fraction to various doses, using a 6 MeV electron linear accelerator (LINAC), with single pulse FLASH (dose rate =2-6 × 106 Gy/s) or conventional (CONV; 0.25 Gy/s) irradiation. At 3.75 days post-irradiation, fresh feces were collected for 16S rRNA sequencing to assess changes in the gut microbiota. A Swiss roll-based crypt assay was used to quantify acute damage to the intestinal crypts to determine how tissue toxicity was affected by the different temporal pulse structures of FLASH delivery. Results We found statistically significant improvements in crypt survival for mice irradiated with FLASH at doses between 7.5 and 12.5 Gy, with a dose modifying factor of 1.1 for FLASH (7.5 Gy, p < 0.01; 10 Gy, p < 0.05; 12.5 Gy, p < 0.01). This sparing effect was lost when the delivery time was increased, either by increasing the number of irradiation pulses or by prolonging the time between two successive pulses. Sparing was observed for average dose rates of ≥280 Gy/s. Fecal microbiome analysis showed that FLASH irradiation caused fewer changes to the microbiota than CONV irradiation. Conclusions This study demonstrates that FLASH irradiation can spare mouse small intestinal crypts and reduce changes in gut microbiome composition compared to CONV irradiation. The higher the average dose rate, the larger the FLASH effect, which is also influenced by temporal pulse structure of the delivery