Is noncoplanar plan more robust to inter-fractional variations than coplanar plan in treating bilateral HN tumors with pencil-beam scanning proton beams?

PURPOSE: Noncoplanar plans (NCPs) are commonly used for proton treatment of bilateral head and neck (HN) malignancies. NCP requires additional verification setup imaging between beams to correct residual errors of robotic couch motion, which increases imaging dose and total treatment time. This study compared the quality and robustness of NCPs with those of coplanar plans (CPs). METHODS AND MATERIALS: Under an IRB-approved study, CPs were created retrospectively for 10 bilateral HN patients previously treated with NCPs maintaining identical beam geometry of the original plan but excluding couch rotations. Plan robustness to the inter-fractional variation (IV) of both plans was evaluated through the Dose Volume Histograms (DVH) of weekly quality assurance CT (QACT) sets (39 total). In addition, delivery efficiency for both plans was compared using total treatment time (TTT) and beam-on time (BOT). RESULTS: No significant differences in plan quality were observed in terms of clinical target volume (CTV) coverage (D95) or organ-at-risk (OAR) doses (p > 0.4 for all CTVs and OARs). No significant advantage of NCPs in the robustness to IV was found over CP, either. Changes in D95 of QA plans showed a linear correlation (slope = 1.006, R2  > 0.99) between NCP and CP for three CTV data points (CTV1, CTV2, and CTV3) in each QA plan (117 data points for 39 QA plans). NCPs showed significantly higher beam delivery time than CPs for TTT (539 ± 50 vs. 897 ± 142 s; p < 0.001); however, no significant differences were observed for BOT. CONCLUSION: NCPs are not more robust to IV than CPs when treating bilateral HN tumors with pencil-beam scanning proton beams. CPs showed plan quality and robustness similar to NCPs while reduced treatment time (∼6 min). This suggests that CPs may be a more efficient planning technique for bilateral HN cancer proton therapy.

Docetaxel radiosensitizes castration-resistant prostate cancer by downregulating CAV-1.

PURPOSE: Docetaxel (DXL), a noted radiosensitizer, is one of the few chemotherapy drugs approved for castration-resistant prostate cancer (CRPC), though only a fraction of CRPCs respond to it. CAV-1, a critical regulator of radioresistance, has been known to modulate DXL and radiation effects. Combining DXL with radiotherapy may create a synergistic anticancer effect through CAV-1 and improve CRPC patients' response to therapy. Here, we investigate the effectiveness and molecular characteristics of DXL and radiation combination therapy in vitro. MATERIALS AND METHODS: We used live/dead assays to determine the IC50 of DXL for PC3, DU-145, and TRAMP-C1 cells. Colony formation assay was used to determine the radioresponse of the same cells treated with radiation with/without IC50 DXL (4, 8, and 12 Gy). We performed gene expression analysis on public transcriptomic data collected from human-derived prostate cancer cell lines (C4-2, PC3, DU-145, and LNCaP) treated with DXL for 8, 16, and 72 hours. Cell cycle arrest and protein expression were assessed using flow cytometry and western blot, respectively. RESULTS: Compared to radiation alone, combination therapy with DXL significantly increased CRPC death in PC3 (1.48-fold, p < .0001), DU-145 (1.64-fold, p < .05), and TRAMP-C1 (1.13-fold, p < .05) at 4 Gy of radiation. Gene expression of CRPC treated with DXL revealed downregulated genes related to cell cycle regulation and upregulated genes related to immune activation and oxidative stress. Confirming the results, G2/M cell cycle arrest was significantly increased after treatment with DXL and radiation. CAV-1 protein expression was decreased after DXL treatment in a dose-dependent manner; furthermore, CAV-1 copy number was strongly associated with poor response to therapy in CRPC patients. CONCLUSIONS: Our results suggest that DXL sensitizes CRPC cells to radiation by downregulating CAV-1. DXL + radiation combination therapy may be effective at treating CRPC, especially subtypes associated with high CAV-1 expression, and should be studied further.

Multimodal Imaging of Pancreatic Cancer Microenvironment in Response to an Antiglycolytic Drug.

Lipid metabolism and glycolysis play crucial roles in the progression and metastasis of cancer, and the use of 3-bromopyruvate (3-BP) as an antiglycolytic agent has shown promise in killing pancreatic cancer cells. However, developing an effective strategy to avoid chemoresistance requires the ability to probe the interaction of cancer drugs with complex tumor-associated microenvironments (TAMs). Unfortunately, no robust and multiplexed molecular imaging technology is currently available to analyze TAMs. In this study, the simultaneous profiling of three protein biomarkers using SERS nanotags and antibody-functionalized nanoparticles in a syngeneic mouse model of pancreatic cancer (PC) is demonstrated. This allows for comprehensive information about biomarkers and TAM alterations before and after treatment. These multimodal imaging techniques include surface-enhanced Raman spectroscopy (SERS), immunohistochemistry (IHC), polarized light microscopy, second harmonic generation (SHG) microscopy, fluorescence lifetime imaging microscopy (FLIM), and untargeted liquid chromatography and mass spectrometry (LC-MS) analysis. The study reveals the efficacy of 3-BP in treating pancreatic cancer and identifies drug treatment-induced lipid species remodeling and associated pathways through bioinformatics analysis.

Evaluation of intrafraction couch shifts for proton treatment delivery in head-and-neck cancer patients: Toward optimal imaging frequency.

PURPOSE: Treatment planning for head-and-neck (H&N) cancer, in particular oropharynx, nasopharynx, and paranasal sinus cases, at our center requires noncoplanar proton beams due to the complexity of the anatomy and target location. Targeting accuracy for all beams is carefully evaluated by using image guidance before delivering proton beam therapy (PBT). In this study, we analyzed couch shifts to evaluate whether imaging is required before delivering each field with different couch angles. METHODS: After the Institutional Review Board approval, a retrospective analysis was performed on data from 28 H&N patients treated with PBT. Each plan was made with two-to-three noncoplanar and two-to-three coplanar fields. Cone-beam computed tomography and orthogonal kilovoltage (kV) images were acquired for setup and before delivering each field, respectively. The Cartesian (longitudinal, vertical, and lateral) and angular (pitch and roll) shifts for each field were recorded from the treatment summary on the first two fractions and every subsequent fifth fraction. A net magnitude of the three-dimensional (3D) shift in Cartesian coordinates was calculated, and a 3D vector was created from the 6 degrees of freedom coordinates for transforming couch shifts in the system coordinate to the beam's-eye view. RESULTS: A total of 3219 Cartesian and 2146 angular shift values were recorded for 28 patients. Of the Cartesian shifts, 2069 were zero (64.3%), and 1150 (35.7%) were nonzero (range, -7 to 11 mm). Of the angular shifts, 1034 (48.2%) were zero, and 1112 (51.8%) were nonzero (range, -3.0° to 3.2°). For 17 patients, the couch shifts increased toward the end of the treatment course. We also found that patients with higher body mass index (BMI) presented increased net couch shifts (p < 0.001). With BMI < 27, all overall net shift averages were <2 mm, and overall maximum net shifts were <6 mm. CONCLUSIONS: These results confirm the need for orthogonal kV imaging before delivering each field of H&N PBT at our center, where a couch rotation is involved.

Comparison of the dosimetric accuracy of proton breast treatment plans delivered with SGRT and CBCT setups.

PURPOSE: To compare the dosimetric accuracy of surface-guided radiation therapy (SGRT) and cone-beam computed tomography (CBCT) setups in proton breast treatment plans. METHODS: Data from 30 patients were retrospectively analyzed in this IRB-approved study. Patients were prescribed 4256-5040 cGy in 16-28 fractions. CBCT and AlignRT (SGRT; Vision RT Ltd.) were used for treatment setup during the first three fractions, then daily AlignRT and weekly CBCT thereafter. Each patient underwent a quality assurance CT (QA-CT) scan midway through the treatment course to assess anatomical and dosimetric changes. To emulate the SGRT and CBCT setups during treatment, the planning CT and QA-CT images were registered in two ways: (1) by registering the volume within the CTs covered by the CBCT field of view; and (2) by contouring and registering the surface surveyed by the AlignRT system. The original plan was copied onto these two datasets and the dose was recalculated. The clinical treatment volume (CTV): V95% ; heart: V25Gy , V15Gy , and mean dose; and ipsilateral lung: V20Gy , V10Gy , and V5Gy , were recorded. Multi and univariate analyses of variance were performed to assess the differences in dose metric values between the planning CT and the SGRT and CBCT setups. RESULTS: The CTV V95% and lung V20Gy , V10Gy , and V5Gy dose metrics were all significantly (p < 0.01) lower on the QA-CT in both the CBCT and SGRT setup. The differences were not clinically significant and were, on average, 1.4-1.6% lower for CTV V95% and 1.8%-6.0% lower for the lung dose metrics. When comparing the lung and CTV V95% dose metrics between the CBCT and SGRT setups, no significant difference was observed. This indicates that the SGRT setup provides similar dosimetric accuracy as CBCT. CONCLUSION: This study supports the daily use of SGRT systems for the accurate dose delivery of proton breast treatment plans.

Proton beam therapy can achieve lower vertebral bone marrow dose than photon beam therapy during chemoradiation therapy of esophageal cancer.

Chemoradiation therapy plays an important role in both the neoadjuvant and definitive management of esophageal cancer (EC). Prior studies have suggested that advanced planning techniques can better spare organs at risk including the heart. Although multiple toxicities can result from esophageal radiotherapy, one less studied acute toxicity is that of myelosuppression, which can result, in part, from the combination of chemotherapy and incidental radiotherapy administration to the vertebral bodies (VBs), which abut the posterior aspect of the esophagus, especially in the lower thoracic esophagus. Traditionally, VB bone marrow doses are not accounted during EC radiation therapy planning. We sought to compare the doses to VBs between proton and photon radiation therapy as part of chemoradiation therapy for EC treatment. By reducing doses to the vertebrae, radiation therapy can decrease treatment-related myelosuppression, which can avoid delays or chemotherapy dose reductions in therapy, which likely affect long-term patient survival. Dose constraints are not routinely employed for bone marrow in radiation treatment planning. In our previous work, we identified thresholds to avoid grade ≥3 leukopenia, including VB V10Gy, VB V20Gy, and a mean VB dose (MVD) of 18.8 Gy. Herein we perform a retrospective dosimetric planning study comparing passive- or double-scattering proton beam therapy (PS-PBT), volumetric-modulated arc therapy (VMAT) (photon-based), and intensity-modulated radiation therapy (IMRT) (photon-based) in 25 patients with locally advanced EC who were treated originally with photon RT at our institution between 2011 and 2016. The aforementioned dose constraints were included in the retrospective planning process for PS-PBT, VMAT, and IMRT to determine the feasibility of achieving these VB constraints while maintaining reasonable target coverage and planned, consistent constraints to other organs at risk including lungs, spinal cord, and stomach. PS-PBT plans were found to achieve lower doses for VB V10Gy, V20Gy, and MVD than VMAT and static IMRT plans while achieving the same target coverage. PS-PBT resulted in lower organs at risk dosimetric parameters than the photon plans, with p < 0.0001. Student's paired t-test p-values in favor of proton therapy's ability to spare organs were as follows: for PS-PBT vs VMAT and PS-PBT vs IMRT in mean doses for lung, liver, and VB and VB V10Gy and VB V20Gy were all <0.001 (Bonferroni corrected α=0.017). One-way ANOVA found that VB doses (VB V10Gy, VB V20Gy, and MVD) were significantly lower for proton therapy (p < 0.006) among the 3 planning techniques.

Variation of V105% between pre- and postmerged subfields in field-in-field hypofractionated breast radiotherapy plans.

Hypofractionated whole-breast irradiation has emerged as a viable alternative to conventional fractionation. In the field-in-field forward planning technique, a merged plan with 2 to 4 segmental fields is the final plan delivered to the machine. As per the ASTRO guidelines for the hypofractionation regimen, the volume of breast tissue receiving V105% of the prescription dose should be less than 200 cc. However, we have noticed substantial changes to this volume (change in V105% between -55 cc and + 47.1 cc) after merging the subfields. This study compares the V105% of 29 breast plans before and after merging the subfields.

In vivo longitudinal imaging of RNA interference-induced endocrine therapy resistance in breast cancer.

Endocrine therapy resistance in breast cancer is a major obstacle in the treatment of patients with estrogen receptor-positive (ER+) tumors. Herein, we demonstrate the feasibility of longitudinal, noninvasive and semiquantitative in vivo molecular imaging of resistance to three endocrine therapies by using an inducible fluorescence-labeled short hairpin RNA (shRNA) system in orthotopic mice xenograft tumors. We employed a dual fluorescent doxycycline (Dox)-regulated lentiviral inducer system to transfect ER+ MCF7L breast cancer cells, with green fluorescent protein (GFP) expression as a marker of transfection and red fluorescent protein (RFP) expression as a surrogate marker of Dox-induced tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown. Xenografted MCF7L tumor-bearing nude mice were randomized to therapies comprising estrogen deprivation, tamoxifen or an ER degrader (fulvestrant) and an estrogen-treated control group. Longitudinal imaging was performed by a home-built multispectral imaging system based on a cooled image intensified charge coupled device camera. The GFP signal, which corresponds to number of viable tumor cells, exhibited excellent correlation to caliper-measured tumor size (P << .05). RFP expression was substantially higher in mice exhibiting therapy resistance and strongly and significantly (P < 1e-7) correlated with the tumor size progression for the mice with shRNA-induced PTEN knockdown. PTEN loss was strongly correlated with resistance to estrogen deprivation, tamoxifen and fulvestrant therapies.

Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase.

INTRODUCTION: Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance. METHODS: Altered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy. RESULTS: Moderate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression. CONCLUSIONS: Moderate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.

Targeting pancreatic cancer with magneto-fluorescent theranostic gold nanoshells.

AIM: We report a magneto-fluorescent theranostic nanocomplex targeted to neutrophil gelatinase-associated lipocalin (NGAL) for imaging and therapy of pancreatic cancer. MATERIALS & METHODS: Gold nanoshells resonant at 810 nm were encapsulated in silica epilayers doped with iron oxide and the near-infrared (NIR) dye indocyanine green, resulting in theranostic gold nanoshells (TGNS), which were subsequently conjugated with antibodies targeting NGAL in AsPC-1-derived xenografts in nude mice. RESULTS: Anti-NGAL-conjugated TGNS specifically targeted pancreatic cancer cells in vitro and in vivo providing contrast for both NIR fluorescence and T2-weighted MRI with higher tumor contrast than can be obtained using long-circulating, but nontargeted, PEGylated nanoparticles. The nanocomplexes also enabled highly specific cancer cell death via NIR photothermal therapy in vitro. CONCLUSION: TGNS with embedded NIR and magnetic resonance contrasts can be specifically targeted to pancreatic cancer cells with expression of early disease marker NGAL, and enable molecularly targeted imaging and photothermal therapy.

Characteristic spectral features of the polarized fluorescence of human breast cancer in the wavelet domain.

Wavelet transform of polarized fluorescence spectra of human breast tissues is found to localize spectral features that can reliably differentiate normal and malignant tissue types. The intensity differences of parallel and perpendicularly polarized fluorescence spectra are subjected to investigation, since they are relatively free of diffusive background. A number of parameters, capturing spectral variations and subtle changes in the diseased tissues in the visible wavelength regime, are clearly identifiable in the wavelet domain. These manifest both in the average low-pass and high frequency high-pass wavelet coefficients.

Imaging tumor oxyhemoglobin and deoxyhemoglobin concentrations with ultrasound-guided diffuse optical tomography.

We present an ultrasound (US)-guided diffuse optical tomography for mapping tumor deoxyhemoglobin (deoxyHb) and oxyhemoglobin (oxyHb) concentrations in blood phantoms and in in-vivo patients. Because oxyHb and deoxyHb respond differently at different wavelengths, four laser diodes of wavelengths 740 nm, 780 nm, 808 nm and 830 nm were used in the study. Tumor model experiments were performed using phantoms of different hemoglobin oxygen saturations (14%-89%) representing hemoglobin oxygenation in tissue. Targets of different sizes and located at different depths were used to validate the accuracy of oxygen saturation estimation. The absolute deviations between the estimated hemoglobin oxygen saturations obtained from reconstructed absorption maps and oxygen measurements obtained using a pO2 electrode were less than 8% over the measured range of oxygen saturation. An inhomogeneous concentric blood phantom of deoxygenated center core and oxygenated outer shell was imaged and deoxyHb and oxyHb maps revealed corresponding distributions which correlated well with inhomogeneous deoxy- and oxy- distributions frequently seen in breast cancers. Clinical examples are given to demonstrate the utility of US-guided optical tomography in mapping heterogeneous deoxyHb and oxyHb distributions in breast cancers.

Optical scattering coefficient estimated by optical coherence tomography correlates with collagen content in ovarian tissue.

Optical scattering coefficient from ex vivo unfixed normal and malignant ovarian tissue was quantitatively extracted by fitting optical coherence tomography (OCT) A-line signals to a single scattering model. 1097 average A-line measurements at a wavelength of 1310 nm were performed at 108 sites obtained from 18 ovaries. The average scattering coefficient obtained from the normal tissue group consisted of 833 measurements from 88 sites was 2.41 mm(-1) (± 0.59), while the average coefficient obtained from the malignant tissue group consisted of 264 measurements from 20 sites was 1.55 mm(-1) (± 0.46). The malignant ovarian tissue showed significant lower scattering than the normal group (p < 0.001). The amount of collagen within OCT imaging depth was analyzed from the tissue histological section stained with Sirius Red. The average collagen area fraction (CAF) obtained from the normal tissue group was 48.4% (± 12.3%), while the average CAF obtained from the malignant tissue group was 11.4% (± 4.7%). A statistical significance of the collagen content was found between the two groups (p < 0.001). These results demonstrated that quantitative measurements of optical scattering coefficient from OCT images could be a potential powerful method for ovarian cancer detection.

Imaging tumor hypoxia by near-infrared fluorescence tomography.

We have developed a novel nitroimidazole indocyanine dye conjugate for tumor-targeted hypoxia fluorescence tomography. The hypoxia probe has been evaluated in vitro using tumor cell lines and in vivo with tumor targeting in mice. The in vitro cell studies were performed to assess fluorescence labeling differences between hypoxia and normoxia conditions. When treated with the hypoxia probe, a fluorescence emission ratio of 2.5-fold was found between the cells incubated under hypoxia compared to the cells in normoxia condition. Hypoxia specificity was also confirmed by comparing the cells treated with indocyanine dye alone. In vivo tumor targeting in mice showed that the fluorescence signals measured at the tumor site were twice those at the normal site after 150 min post-injection of the hypoxia probe. On the other hand, the fluorescence signals measured after injection of indocyanine dye were the same at tumor and normal sites. In vivo fluorescence tomography images of mice injected with the hypoxia probe showed that the probe remained for more than 5 to 7 h in the tumors, however, the images of mice injected with indocyanine only dye confirmed that the unbound dye washed out in less than 3 h. These findings are supported with fluorescence images of histological sections of tumor samples using a Li-COR scanner and immunohistochemistry technique for tumor hypoxia.

Potential role of a hybrid intraoperative probe based on OCT and positron detection for ovarian cancer detection and characterization.

Ovarian cancer has the lowest survival rate of the gynecologic cancers because it is predominantly diagnosed in the late stages due to the lack of reliable symptoms and efficacious screening techniques. A novel hybrid intraoperative probe has been developed and evaluated for its potential role in detecting and characterizing ovarian tissue. The hybrid intraoperative dual-modality device consists of multiple scintillating fibers and an optical coherence tomography imaging probe for simultaneously mapping the local activities of (18)F-FDG uptake and imaging of local morphological changes of the ovary. Ten patients were recruited to the study and a total of 18 normal, abnormal and malignant ovaries were evaluated ex vivo using this device. Positron count rates of 7.5/8.8-fold higher were found between malignant and abnormal/normal ovaries. OCT imaging of malignant and abnormal ovaries revealed many detailed morphologic features that could be potentially valuable for evaluating local regions with high metabolic activities and detecting early malignant changes in the ovary. These initial results have demonstrated that our novel hybrid imager has great potential for ovarian cancer detection and characterization during minimally invasive endoscopic procedures.

Calibrating the photo-thermal response of magneto-fluorescent gold nanoshells.

We report the photothermal response and Near Infrared (NIR) imaging sensitivities of magneto-fluorescent silica core gold nanocomplexes designed for molecular image guided thermal therapy of cancer. Approximately 160 nm Silica core gold nanoshells were designed to provide NIR fluorescent and Magnetic Resonance (MR) contrast by incorporating FDA approved dye indocyanine green (ICG) and iron-oxide within an outer silica epilayer. The imaging and therapeutic sensitivity, and the stability of fluorescence contrast for 12 microliters of suspension (containing approximately 7.9 × 10(8) or 1.3 femtoMole nanoshells) buried at depths of 2-8 mm in tissue mimicking scattering media is reported.

Fluorescence imaging of vascular endothelial growth factor in tumors for mice embedded in a turbid medium.

We demonstrate the feasibility of fluorescence imaging of deeply seated tumors using mice injected with an angiogenesis tracer, a vascular endothelial growth factor conjugated with the infrared dye cyanine 7 (VEGF/Cy7). Our optical-only imaging reconstruction method separately estimates the target depth, and then applies this information to reconstruct functional information such as fluorophore concentration. Fluorescence targets with concentrations as low as sub-25 nM are well reconstructed at depths up to 2 cm in both homogeneous and heterogeneous media with this technique.

Wavelet-based characterization of spectral fluctuations in normal, benign, and cancerous human breast tissues.

Fluorescence intensity fluctuations in the visible wavelength regime in normal, benign, and cancerous human breast tissue samples are studied through wavelet transform. The analyses have been carried out in unpolarized, parallel and perpendicularly polarized channels, for optimal tissue characterization. It has been observed that polarized fluorescence data, particularly the perpendicular components, differentiate various tissue types quite well. Wavelet transform, because of its ability for multiresolution analysis, provides the ideal tool to separate and characterize fluctuations in the fluorescence spectra at different scales. We quantify these differences and find that the fluctuations in the perpendicular channel of the cancerous tissues are more randomized as compared to their normal counterparts. Furthermore, for cancerous tissues, the same is very well described by the normal distribution, which is not the case for normal and benign samples. It has also been observed that, up to a certain point, fluctuations at larger scales are more sensitive to tissue types. The differences in the average, low-pass wavelet coefficients of normal, cancerous, pericanalicular, and intracanalicular benign tissues are also pointed out.