Asuc_0142 of Actinobacillus succinogenes 130Z is the l-aspartate/C4-dicarboxylate exchanger DcuA.

Anaerobic bacteria often use antiporters DcuB (malate/succinate antiport) or DcuA (l-aspartate/succinate antiport) for the excretion of succinate during fumarate respiration. The rumen bacterium Actinobacillus succinogenes is able to produce large amounts of succinate by fumarate respiration, using the DcuB-type transporter DcuE for l-malate/succinate antiport. Asuc_0142 was annotated as a second DcuB-type transporter. Deletion of Asuc_0142 decreased the uptake rate for l-[14C]aspartate into A. succinogenes cells. Properties of transport by heterologously expressed Asuc_0142 were investigated in an Escherichia coli mutant deficient of anaerobic C4DC transporters. Expression of Asuc_0142 resulted in high uptake activity for l-[14C]fumarate or l-[14C]aspartate, but the former showed a strong competitive inhibition by l-aspartate. In E. coli loaded with l-[14C]aspartate, [14C]succinate or [14C]fumarate, extracellular C4DCs initiated excretion of the intracellular substrates, with a preference for l-aspartateex/succinatein or l-aspartateex/fumaratein antiport. These findings indicate that Asuc_0142 represents a DcuA-type transporter for l-aspartate uptake and l-aspartateex/C4DCin antiport, differentiating it from the DcuB-type transporter DcuE for l-malateex/succinatein antiport. Sequence analysis and predicted structural characteristics confirm structural similarity of Asuc_0142 to DcuA, and Asuc_0142 was thus re-named as DcuAAs. The bovine rumen fluid contains l-aspartate (99.6 µM), whereas fumarate and l-malate are absent. Therefore, bovine rumen colonisers depend on l-aspartate as an exogenous substrate for fumarate respiration. A. succinogenes encodes HemG (protoporphyrinogen oxidase) and PyrD (dihydroorotate dehydrogenase) for haem and pyrimidine biosynthesis. The enzymes require fumarate as an electron acceptor, suggesting an essential role for l-aspartate, DcuAAs, and fumarate respiration for A. succinogenes growing in the bovine rumen.

The dosimetric impact of titanium implants in spinal SBRT using four commercial treatment planning algorithms.

To evaluate the dosimetric impact of titanium implants in spine SBRT using four dose calculation algorithms. Twenty patients with titanium implants in the spine treated with SBRT without density override (DO) were selected. The clinical plan for each patient was created in Pinnacle and subsequently imported into Eclipse (AAA and AcurosXB) and Raystation (CC) for dose evaluation with and without DO to the titanium implant. We renormalized all plans such that 90% of the tumor volume received the prescription dose and subsequently evaluated the following dose metrics: (1) the maximum dose to 0.03 cc (Dmax), dose to 99% (D99%) and 90% (D90%) of the tumor volume; (2) Dmax and volumetric metrics of the spinal cord. For the same algorithm, plans with and without DO had similar dose distributions. Differences in Dmax, D99% and D90% of the tumor were on average <2% with slightly larger variations up to 5.58% in Dmax using AcurosXB. Dmax of the spinal cord for plans calculated with DO increased but the differences were clinically insignificant for all algorithms (mean: 0.36% ± 0.7%). Comparing to the clinical plans, the relative differences for all algorithms had an average of 1.73% (-10.36%-13.21%) for the tumor metrics and -0.93% (-9.87%-10.95%) for Dmax of the spinal cord. A few cases with small tumor and spinal cord volumes, dose differences of >10% in both D99% and Dmax of the tumor, and Dmax of the spinal cord were observed. For all algorithms, the presence of titanium implants in the spine for most patients had minimal impact on dose distributions with and without DO. For the same plan calculated with different algorithms, larger differences in volumetric metrics of >10% could be observed, impacted by dose gradient at the plan normalization volume, tumor volumes, plan complexity, and partial voxel volume interpolation.

Synergism of AZD6738, an ATR Inhibitor, in Combination with Belotecan, a Camptothecin Analogue, in Chemotherapy-Resistant Ovarian Cancer.

Epithelial ovarian cancer remains the leading cause of mortality among all gynecologic malignancies owing to recurrence and ultimate development of chemotherapy resistance in the majority of patients. In the chemotherapy-resistant ovarian cancer preclinical model, we investigated whether AZD6738 (an ataxia telangiectasia and Rad3-related (ATR) inhibitor) could synergize with belotecan (a camptothecin analog and topoisomerase I inhibitor). In vitro, both chemotherapy-resistant and chemotherapy-sensitive ovarian cancer cell lines showed synergistic anti-proliferative activity with a combination treatment of belotecan and AZD6738. The combination also demonstrated synergistic tumor inhibition in mice with a chemotherapy-resistant cell line xenograft. Mechanistically, belotecan, a DNA-damaging agent, increased phospho-ATR (pATR) and phospho-Chk1 (pChk1) in consecutive order, indicating the activation of the DNA repair system. This consequently induced G2/M arrest in the cell cycle analysis. However, when AZD6738 was added to belotecan, pATR and pChk1 induced by belotecan alone were suppressed again. A cell cycle analysis in betotecan showed a sub-G1 increase as well as a G2/M decrease, representing the release of G2/M arrest and the induction of apoptosis. In ascites-derived primary cancer cells from both chemotherapy-sensitive and -resistant ovarian cancer patients, this combination was also synergistic, providing further support for our hypothesis. The combined administration of ATR inhibitor and belotecan proved to be synergistic in our preclinical model. This combination warrants further investigation in a clinical trial, with a particular aim of overcoming chemotherapy resistance in ovarian cancer.

Adaptation of Methanosarcina barkeri 227 as acetate scavenger for succinate fermentation by Actinobacillus succinogenes.

Acetate is the main by-product from microbial succinate production. In this study, we performed acetate removal by Methanosarcina barkeri 227 for succinate fermentation by Actinobacillus succinogenes 130Z. The acetoclastic methanogen M. barkeri requires similar environmental factors to A. succinogenes, and the conditions required for co-cultivation were optimized in this study: gas used for anaerobicization, strain adaptation, medium composition, pH adjustment, and inoculation time points. M. barkeri 227 was adapted to acetate for 150 days, which accelerated the acetate consumption to 9-fold (from 190 to 1726 mmol gDW-1 day-1). In the acetate-adapted strain, there was a noticeable increase in transcription of genes required for acetoclastic pathway-satP (acetate transporter), ackA (acetate kinase), cdhA (carbon monoxide dehydrogenase/acetyl-CoA synthase complex), and mtrH (methyl-H4STP:CoM methyltransferase), which was not induced before the adaptation process. The activities of two energy-consuming steps in the pathway-acetate uptake and acetate kinase-increased about 3-fold. This acetate-adapted M. barkeri could be successfully applied to succinate fermentation culture of A. succinogenes, but only after pH adjustment following completion of fermentation. This study suggests the utility of M. barkeri as an acetate scavenger during fermentation for further steps towards genetic and process engineering.

Cyclo(Phe-Pro) produced by Vibrio species passes through biological membranes by simple diffusion.

Cyclo(Phe-Pro) (cFP), produced by the Vibrio species, plays the dual roles of being a signaling molecule and a virulence factor. Acting modes of this compound have recently been characterized at the molecular level. Nevertheless, the method by which this compound passes across biological membranes remains obscure. Using radiolabeled cFP, we examined the kinetics of transport for this compound across membranes using V. vulnificus, Escherichia coli, and sheep red blood cells. We observed that cFP was taken up by these cells in a concentration-dependent manner and was not affected by the addition of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), suggesting that cFP is taken up by passive transport. The kinetics of uptake of cFP by the above three types of cells revealed no significant differences, indicating that no specific protein is involved in this process. When the intracellular accumulation of cFP in the tested cells was measured, the concentrations did not exhibit significant differences between the 1-min and 10-min time points after cFP was added to the culture. In contrast, the intracellular concentration of fumarate, which is well known to be taken up by cells via active transport, was significantly higher at the 10-min than at the 1-min time point after addition. Taken together, this study shows that cFP is a diffusible molecule that does not require energy for transportation across biological membranes, and that cFP does not need membrane machinery in order to cross membranes and consequently act as a virulence factor or signal. KEY POINTS: • Kinetics of cFP uptake into cells of V. vulnificus, E. coli, or RBS was studied. • The uptake was not saturated and required no energy, indicating passive transport. • The lack of cell specificity in cFP uptake means no specific protein is needed. • Therefore, the cFP moves across the biological membrane by simple diffusion.

Radio-immune response modelling for spatially fractionated radiotherapy.

Objective.Radiation-induced cell death is a complex process influenced by physical, chemical and biological phenomena. Although consensus on the nature and the mechanism of the bystander effect were not yet made, the immune process presumably plays an important role in many aspects of the radiotherapy including the bystander effect. A mathematical model of immune response during and after radiation therapy is presented.Approach.Immune response of host body and immune suppression of tumor cells are modelled with four compartments in this study; viable tumor cells, T cell lymphocytes, immune triggering cells, and doomed cells. The growth of tumor was analyzed in two distinctive modes of tumor status (immune limited and immune escape) and its bifurcation condition.Main results.Tumors in the immune limited mode can grow only up to a finite size, named as terminal tumor volume analytically calculated from the model. The dynamics of the tumor growth in the immune escape mode is much more complex than the tumors in the immune limited mode especially when the status of tumor is close to the bifurcation condition. Radiation can kill tumor cells not only by radiation damage but also by boosting immune reaction.Significance.The model demonstrated that the highly heterogeneous dose distribution in spatially fractionated radiotherapy (SFRT) can make a drastic difference in tumor cell killing compared to the homogeneous dose distribution. SFRT cannot only enhance but also moderate the cell killing depending on the immune response triggered by many factors such as dose prescription parameters, tumor volume at the time of treatment and tumor characteristics. The model was applied to the lifted data of 67NR tumors on mice and a sarcoma patient treated multiple times over 1200 days for the treatment of tumor recurrence as a demonstration.

Radio-Immune Response Modelling for Spatially Fractionated Radiotherapy.

Radiation-induced cell death is a complex process influenced by physical, chemical and biological phenomena. Strong dose gradient may intensify the complexity and reportedly creates significantly more cell death known as bystander effect. Although consensus on the nature and the mechanism of the bystander effect were not yet made, the immune process presumably plays an important role in many aspects of the radiotherapy including the bystander effect. Immune response of host body and immune suppression of tumor cells are modelled with four compartments in this study; viable tumor cells, T cell lymphocytes, immune triggering cells, and doomed cells. The growth of tumor was analyzed in two distinctive modes of tumor status (immune limited and immune escape) and its bifurcation condition. Tumors in the immune limited mode can grow only up to a finite size, named as terminal tumor volume analytically calculated from the model. The dynamics of the tumor growth in the immune escape mode is much more complex than the tumors in the immune limited mode especially when the status of tumor is close to the bifurcation condition. Radiation can kill tumor cells not only by radiation damage but also by boosting immune reaction. The model demonstrated that the highly heterogeneous dose distribution in spatially fractionated radiotherapy (SFRT) can make a drastic difference in tumor cell killing compared to the homogeneous dose distribution. SFRT can not only enhance but also moderate the cell killing depending on the immune response triggered by many factors such as dose prescription parameters, tumor volume at the time of treatment and tumor characteristics. The model was applied to the lifted data of 67NR tumors on mice and a sarcoma patient treated multiple times over 1200 days for the treatment of tumor recurrence as a demonstration.

Dosimetric evaluation of adult and paediatric brain tumours planned using mask-based cobalt-60 fractionated stereotactic radiotherapy compared to linear accelerator-based volumetric modulated arc therapy.

INTRODUCTION: We conducted a study to evaluate the dosimetric feasibility of mask-based cobalt-60 fractionated stereotactic radiotherapy (mcfSRT) with the Leksell Gamma Knife® Icon™ device. METHODS: Eleven patients with intracranial tumours were selected for this dosimetry study. These patients, previously treated with volumetric arc therapy (VMAT), were re-planned using mcfSRT. Target volume coverage, conformity/gradient indices, doses to organs at risk and treatment times were compared between the mcfSRT and VMAT plans. Two-sided paired Wilcoxon signed-rank test was used to compare differences between the two plans. RESULTS: The V95 for PTV was similar between fractionated mcfSRT and VMAT (P = 0.47). The conformity index and gradient indices were 0.9 and 3.3, respectively, for mcfSRT compared to 0.7 and 4.2, respectively, for VMAT (P < 0.001 and 0.004, respectively). The radiation exposure to normal brain was lower for mcfSRT across V10, V25 and V50 compared with VMAT (P = 0.007, <0.001 and <0.001, respectively). The median D0.1cc for optic nerve and chiasm as well as the median D50 to the hippocampi were lower for mcfSRT compared to VMAT. Median beam-on time for mcfSRT was 9.7 min per fraction, compared to 0.9 min for VMAT (P = 0.002). CONCLUSION: mcfSRT plans achieve equivalent target volume coverage, improved conformity and gradient indices, and reduced radiation doses to organs at risk as compared with VMAT plans. These results suggest superior dosimetric parameters for mcfSRT plans and can form the basis for future prospective studies.

Assessment of intra-fraction motion during frameless image guided Gamma Knife stereotactic radiosurgery.

As frameless stereotactic radiosurgery increase in use, the aim of this study was to evaluate intra-fraction motion through cone-beam CT (CBCT) and high-definition motion management (HDMM) systems. Intra-fraction motion measured between localization, repeat localization and post-treatment CBCTs were correlated to intra-faction motion indicated by the HDMM files using the Pearson coefficient (r). A total of 302 plans were reviewed from 263 patients (114 male, 149 female); 216 pairs of localization-repeat localization, and 260 localization-post-treatment CBCTs were analyzed against HDMM logs. We found the magnitude of intra-fraction motion detected by the HDMM system were larger than the corresponding CBCT results.

Preclinical Platform Using a Triple-negative Breast Cancer Syngeneic Murine Model to Evaluate Immune Checkpoint Inhibitors.

BACKGROUND/AIM: To evaluate the feasibility of syngeneic mouse models of breast cancer by analyzing the efficacy of immune checkpoint inhibitors (ICIs) and potential predictive biomarkers. MATERIALS AND METHODS: To establish the murine triple-negative breast cancer (TNBC) models, JC, 4T1, EMT6, and E0771 cells were subcutaneously implanted into female syngeneic mice. When the tumor reached 50-100 mm3, each mouse model was divided into a treatment (using a murine PD-1 antibody) and a no-treatment control group. The treatment group was further divided into the responder and non-responder groups. Potential predictive biomarkers were evaluated by analyzing serum cytokines, peripheral blood T cells and tumor infiltrating immune cells. RESULTS: The EMT6 model showed the highest tumor response rate (54%, 6/11) of the syngeneic models: 4T1 (45%, 5/11), JC (40%, 4/10), or E0771 (23%, 3/13). Early changes in tumor size at 7 days post-PD-1 inhibitor treatment predicted the final efficacy of the PD-1 inhibitor. Peripheral blood CD8+ and CD4+ T cells with or without Ki67 expression at 7 days post-PD-1 inhibitor treatment were higher in the finally designated responder group than in the non-responder group. At the time of sacrifice, analyses of tumor infiltrating lymphocytes consistently supported these results. We also demonstrated that retro-orbital blood sampling procedures (baseline, 7 days post-treatment, time of sacrifice) were safe for serum cytokine analyses, suggesting that our preclinical platform may be used for biomarker research using serum cytokines. CONCLUSION: Our syngeneic mouse model of TNBC is a feasible preclinical platform to evaluate ICI efficacy combined with other drugs and predictive biomarkers in the screening process of immune-oncology drug development.

Implementing failure mode and effect analysis to improve the safety of volumetric modulated arc therapy for total body irradiation.

PURPOSE: Volumetric-modulated arc therapy for total body irradiation (VMAT-TBI) is a novel radiotherapy technique that has been implemented at our institution. The purpose of this work is to investigate possible failure modes (FMs) in the treatment process and to develop a quality control (QC) program for VMAT-TBI following TG-100 guidelines. METHODS: We formed a multidisciplinary team to map out the complete treatment process of VMAT-TBI following the AAPM TG-100 guidelines. This process map gives a visual representation of the VMAT-TBI workflow from the CT simulation, image processing, contouring, treatment planning, to treatment delivery. From the process map, potential FMs were identified. The occurrence (O), detectability (D), and severity of impact (S) of each FM were assigned according to scoring criteria (1-10) by the multidisciplinary team. A risk priority number (RPN) was calculated from average O, S, and D of each FM (RPN = O x S x D). High risk FMs were identified as 20% of the FMs having the highest RPN scores. After the FMEA analysis, fault-tree analysis (FTA) was performed for each major step of the treatment process to determine the effects of potential failures to the treatment outcome. Effective QC methods were identified to prevent the high risk failures and to improve the safety of the VMAT-TBI program. RESULTS: We identified a total of 55 sub-processes and 128 FMs from the VMAT-TBI workflow. The top five high-risk FMs were: (1) Prescription and/or OAR constraints changed during planning and not communicated to the planner, (2) Patient moves or breathes too heavily during the upper body CT scan (3) Patient moves during the lower body CT scan, (4) Treatment planning system not calculating total body DVH metrics correctly for TBI, (5) Improper optimization criteria used or not sufficient optimization, resulting in suboptimal dose coverage, OAR sparing or excessive hotspots during treatment planning. Two FMs have average severity scores ≥8: Incorrect PTV subdivision/isocenter placement and Prescription and/or OAR constraints changed during planning and not communicated to the planner. Quality assurance and QC interventions including staff training, standard operating procedures, and quality checklists were implemented based on the FMEA and FTA. CONCLUSION: FM and effect analysis was performed to identify high-risk FMs of our VMAT-TBI program. FMEA and FTA were effective in identifying potential FMs and determining the best quality management (QM) measures to implement in the VMAT-TBI program.

A total inverse planning paradigm: Prospective clinical trial evaluating the performance of a novel MR-based 3D-printed head immobilization device.

Background and purpose: Brain radiotherapy (cnsRT) requires reproducible positioning and immobilization, attained through redundant dedicated imaging studies and a bespoke moulding session to create a thermoplastic mask (T-mask). Innovative approaches may improve the value of care. We prospectively deployed and assessed the performance of a patient-specific 3D-printed mask (3Dp-mask), generated solely from MR imaging, to replicate a reproducible positioning and tolerable immobilization for patients undergoing cnsRT. Material and methods: Patients undergoing LINAC-based cnsRT (primary tumors or resected metastases) were enrolled into two arms: control (T-mask) and investigational (3Dp-mask). For the latter, an in-house designed 3Dp-mask was generated from MR images to recreate the head positioning during MR acquisition and allow coupling with the LINAC tabletop. Differences in inter-fraction motion were compared between both arms. Tolerability was assessed using patient-reported questionnaires at various time points. Results: Between January 2020 - July 2022, forty patients were enrolled (20 per arm). All participants completed the prescribed cnsRT and study evaluations. Average 3Dp-mask design and printing completion time was 36 h:50 min (range 12 h:56 min - 42 h:01 min). Inter-fraction motion analyses showed three-axis displacements comparable to the acceptable tolerance for the current standard-of-care. No differences in patient-reported tolerability were seen at baseline. During the last week of cnsRT, 3Dp-mask resulted in significantly lower facial and cervical discomfort and patients subjectively reported less pressure and confinement sensation when compared to the T-mask. No adverse events were observed. Conclusion: The proposed total inverse planning paradigm using a 3D-printed immobilization device is feasible and renders comparable inter-fraction performance while offering a better patient experience, potentially improving cnsRT workflows and its cost-effectiveness.

Fast transit portal dosimetry using density-scaled layer modeling of aSi-based electronic portal imaging device and Monte Carlo method.

PURPOSE: Fast and accurate transit portal dosimetry was investigated by developing a density-scaled layer model of electronic portal imaging device (EPID) and applying it to a clinical environment. METHODS: The model was developed for fast Monte Carlo dose calculation. The model was validated through comparison with measurements of dose on EPID using first open beams of varying field sizes under a 20-cm-thick flat phantom. After this basic validation, the model was further tested by applying it to transit dosimetry and dose reconstruction that employed our predetermined dose-response-based algorithm developed earlier. The application employed clinical intensity-modulated beams irradiated on a Rando phantom. The clinical beams were obtained through planning on pelvic regions of the Rando phantom simulating prostate and large pelvis intensity modulated radiation therapy. To enhance agreement between calculations and measurements of dose near penumbral regions, convolution conversion of acquired EPID images was alternatively used. In addition, thickness-dependent image-to-dose calibration factors were generated through measurements of image and calculations of dose in EPID through flat phantoms of various thicknesses. The factors were used to convert acquired images in EPID into dose. RESULTS: For open beam measurements, the model showed agreement with measurements in dose difference better than 2% across open fields. For tests with a Rando phantom, the transit dosimetry measurements were compared with forwardly calculated doses in EPID showing gamma pass rates between 90.8% and 98.8% given 4.5 mm distance-to-agreement (DTA) and 3% dose difference (DD) for all individual beams tried in this study. The reconstructed dose in the phantom was compared with forwardly calculated doses showing pass rates between 93.3% and 100% in isocentric perpendicular planes to the beam direction given 3 mm DTA and 3% DD for all beams. On isocentric axial planes, the pass rates varied between 95.8% and 99.9% for all individual beams and they were 98.2% and 99.9% for the composite beams of the small and large pelvis cases, respectively. Three-dimensional gamma pass rates were 99.0% and 96.4% for the small and large pelvis cases, respectively. CONCLUSIONS: The layer model of EPID built for Monte Carlo calculations offered fast (less than 1 min) and accurate calculation for transit dosimety and dose reconstruction.

A new conformity and dose gradient distance measure for stereotactic radiosurgery of brain metastasis.

Purpose: Competing radiosurgery plans are compared based on their conformity and gradient of dose distribution to the target volume (TV). Most widely used quality metrics such as new conformity index (NCI) and gradient index (GI) are known to have strong volume dependency on the TV of interest. A simple quality measure without the volume dependency is presented for evaluating stereotactic radiosurgery plans, expressed in distance dimension compared to the unit-less volume ratio used in NCI and GI. Methods and Materials: The conformity distance measure (CDM) is defined as the effective radius of the union volume subtracted by that of the intersection volume, where volume operations are on TV and prescription isodose volume (PIV). Gradient distance measure (GDM) is defined as the effective radius of 50% PIV (low dose volume of the plan) subtracted by that of corresponding ideal low dose volume (iLDV). Volume independency and consistent sensitivity of CDM and GDM on PIV displacement and dose spillage are analyzed using a simple two-sphere model. 2429 cases of Gamma Knife and 76 cases of Linac based radiosurgery plans for brain metastasis were retrospectively studied to demonstrate volume independency of the new measures and their implication on target coverage. Results: The sensitivity of NCI on PIV displacement and dose spillage was inversely proportional to the effective radius of the target volume, while the sensitivity of CDM on target motion and dose spillage was constant regardless the target volume. The iLDV for 50% PIV was approximately 2.4 times of PIV based on previous Linac based radiosurgery/IMRT/VMAT plans and single shot analysis from Gamma Knife (GK), ICON. Although NCI ranged from 1 to 14.7 for GK plans and from 1.2 to 20.8 for VMAT plans showing strong volume dependency, CDM showed negligible volume dependency of less than 2.1 mm for more than 90% cases and peak frequency was at 0.8 mm. CDM was correlated well with target coverage as a function of PIV displacement regardless of target volume. Target coverage, V100, was larger than 95% when PIV displacement is less than CDM. Conclusions: The new conformity and gradient measure, CDM and GDM are proposed in this paper. The new measures are volume independent which is preferred for reliable evaluation of the radiosurgery plan quality over wide range of radiosurgery targets. As represented by distance dimension similar to PTV margin, the new measures may be more adequate for image guided radiosurgery applications.

Combination of Abemaciclib following Eribulin Overcomes Palbociclib-Resistant Breast Cancer by Inhibiting the G2/M Cell Cycle Phase.

Breast cancer remains a leading cancer burden among women worldwide. Acquired resistance of cyclin-dependent kinase (CDK) 4/6 inhibitors occurs in almost all hormone receptor (HR)-positive subtype cases, comprising 70% of breast cancers, although CDK4/6 inhibitors combined with endocrine therapy are highly effective. CDK4/6 inhibitors are not expected to cooperate with cytotoxic chemotherapy based on the basic cytotoxic chemotherapy mode of action that inhibits rapidly proliferating cells. The palbociclib-resistant preclinical model developed in the current study investigated whether the combination of abemaciclib, CDK4/6 inhibitor with eribulin, an antimitotic chemotherapy could be a strategy to overcome palbociclib-resistant HR-positive breast cancer. The current study demonstrated that sequential abemaciclib treatment following eribulin synergistically suppressed CDK4/6 inhibitor-resistant cells by inhibiting the G2/M cell cycle phase more effectively. The current study showed the significant association of the pole-like kinase 1 (PLK1) level and palbociclib resistance. Moreover, the cumulative PLK1 inhibition in the G2/M phase by each eribulin or abemaciclib proved to be a mechanism of the synergistic effect. The synergistic antitumor effect was also supported by in vivo study. The sequential combination of abemaciclib following eribulin merits further clinical trials to overcome resistance to CDK4/6 inhibitors in HR-positive breast cancer.

Is there a volume threshold of brain metastases for Linac-based stereotactic radiotherapy?

PURPOSE: To investigate whether there is a volume threshold in target volume of brain metastases below which a small cone size and sharp penumbra in Gamma Knife (GK) may provide improved plan quality when compared to Volumetric Modulated Arc Therapy (VMAT)-based stereotactic radiosurgery (SRS). METHODS: For patients treated on GK SRS for brain metastases in 2018-2019 in our institution, 121 patients with two and three targets were identified. Twenty-six patients with two or three brain metastases (total of 76 lesions) were selected for this study. Two VMAT plans, SmartArc (Pinnacle) and HyperArc (Eclipse), were generated retrospectively for each patient. Plan quality was evaluated based on RTOG conformity index (CI), Paddick gradient index (GI), normal tissue (NT) V12Gy and V4.5Gy. By using the receiver operating characteristic (ROC) curve for both VMAT plans (SmartArc and HyperArc) and metrics of RTOG CI and NT V12Gy, we compared GK plans to SmartArc and HyperArc plans separately to determine the threshold volume. RESULTS: For SmartArc plans, both ROC curve analyses showed a threshold volume of 0.4 cc for both CI and NT V12Gy. For HyperArc plans, the threshold volumes were 0.2 cc for the CI and 0.5 cc for NT V12Gy. GK plans produced improved dose distribution compared to VMAT for targets ≤0.4 cc, but HyperArc was found to have competing results with GK in terms of CI and NT V12Gy. For targets > 0.4 cc, both SmartArc and HyperArc showed better plan quality when compared to the GK plans. CONCLUSIONS: Target volumes ≤0.4 cc may require a small cone size and sharp penumbra in GK while for target volumes >0.4 cc, VMAT-based SRS can provide improved overall plan quality and faster treatment delivery.

Deregulated Immune Pathway Associated with Palbociclib Resistance in Preclinical Breast Cancer Models: Integrative Genomics and Transcriptomics.

Recently, cyclin-dependent kinase (CDK) 4/6 inhibitors have been widely used to treat advanced hormone receptor-positive breast cancer. Despite promising clinical outcomes, almost all patients eventually acquire resistance to CDK4/6 inhibitors. Here, we screened genes associated with palbociclib resistance through genomics and transcriptomics in preclinical breast cancer models. Palbociclib-resistant cells were generated by exposing hormone receptor-positive breast cancer cell lines to palbociclib. Whole-exome sequencing (WES) and a mRNA microarray were performed to compare the genomic and transcriptomic landscape between both palbociclib-sensitive and resistant cells. Microarray analysis revealed 651 differentially expressed genes (DEGs), while WES revealed 107 clinically significant mutated genes. Furthermore, pathway analysis of both DEGs and mutated genes revealed immune pathway deregulation in palbociclib-resistant cells. Notably, DEG annotation revealed activation of type I interferon pathway, activation of immune checkpoint inhibitory pathway, and suppression of immune checkpoint stimulatory pathway in palbociclib-resistant cells. Moreover, mutations in NCOR1, MUC4, and MUC16 genes found in palbociclib-resistant cells were annotated to be related to the immune pathway. In conclusion, our genomics and transcriptomics analysis using preclinical model, revealed that deregulated immune pathway is an additional mechanism of CDK4/6 inhibitor resistance besides the activation of cyclin E-CDK2 pathway and loss of RB, etc. Further studies are warranted to evaluate whether immune pathways may be a therapeutic target to overcome CDK4/6 inhibitor resistance.

Verification of source and collimator configuration for Gamma Knife Perfexion using panoramic imaging.

PURPOSE: The new model of stereotactic radiosurgery system, Gamma Knife Perfexion, allows automatic selection of built-in collimation, eliminating the need for the time consuming manual collimator installation required with previous models. However, the configuration of sources and collimators inside the system does not permit easy access for the verification of the selected collimation. While the conventional method of exposing a film at the isocenter is useful for obtaining composite dose information, it is difficult to interpret the data in terms of the integrity of each individual source and corresponding collimation. The primary aim of this study was to develop a method of verifying the geometric configuration of the sources and collimator modules of the Gamma Knife Perfexion. In addition, the method was extended to make dose measurements and verify the accuracy of dose distributions calculated by the mathematical formalism used in the treatment planning system, Leksell Gamma Plan. METHODS: A panoramic view of all of 192 cobalt sources was simultaneously acquired by exposing a radiochromic film wrapped around the surface of a cylindrical phantom. The center of the phantom was mounted at the isocenter with its axis aligned along the longitudinal axis of the couch. The sizes and shapes of the source images projected on the phantom surface were compared to those calculated based on the manufacturer's design specifications. The measured dose at various points on the film was also compared to calculations using the algorithm of the planning system. RESULTS: The panoramic images allowed clear identification of each of the 192 sources, verifying source integrity and selected collimator sizes. Dose on the film surface is due to the primary beam as well as phantom scatter and leakage contributions. Therefore, the dose at a point away from the isocenter cannot be determined simply based on the proportionality of collimator output factors; the use of a dose computation algorithm is required. Scatter and leakage dose contributions from neighboring sources were calculated and found to be 6.3% (ranging from 4.5% to 7.4%), 16.7% (12.5%-19.3%), and 66.6% (38%-78%) for the 4, 8, and 16 mm collimators, respectively, at the centers of the source images. The measured average dose on films with 16 mm collimators agrees with the dose model of the treatment planning system to within 1.0%. The average doses on the film were 24.0, 60.8, and 186.2 cGy for 4, 8, and 16 mm diameter collimators, respectively, when the machine was set to deliver a reference dose of 100 Gy to the center of an 80 mm radius spherical dosimetry phantom. CONCLUSIONS: A method of simultaneously capturing and analyzing the panoramic images of 192 cobalt sources has been developed to verify the source and collimator configuration of GK systems. The method was extended to verify the dose calculation model of the treatment planning system by comparing the measured doses on the panoramic film images and the corresponding calculated doses. The method presented can play a significant role in comprehensive commissioning and routine quality assurance testing of the Gamma Knife systems.

An artificial neural network to model response of a radiotherapy beam monitoring system.

PURPOSE: The integral quality monitor (IQM) is a real-time radiotherapy beam monitoring system, which consists of a spatially sensitive large-area ion chamber, mounted at the collimator of the linear accelerator (linac), and a calculation algorithm to predict the detector signal for each beam segment. By comparing the measured and predicted signals the system validates the beam delivery. The current commercial version of IQM uses an analytic method to predict the signal, which requires a semi-empirical approach to determine and optimize various calculation parameters. The process of developing the calculation model is complex and time consuming, and moreover, the model cannot be easily generalized across various beam delivery platforms with different combinations of beam energy, beam flattening, beam shaping elements, and Linac models. Therefore, as an alternative solution, we investigated the feasibility of developing a machine learning (ML) method, using an artificial neural network (ANN), to predict the ion chamber signal. In developing an ANN, it is not necessary to explicitly account for each of the elements of beam interactions with various structures in the beam path to the ion chamber. METHODS: The ANN was designed with multilayer perceptron (MLP). The input layer consisted of multiple features, derived from the geometrical characteristics of beam segments. Gradient descent error backpropagation technique was used to train the ANN. The combined training dataset included 270 rectangular fields, and 801 clinical IMRT fields delivered using 6 MV beams on Varian TrueBeamTM and Elekta InfinityTM . Each of 12 different ANN configurations (3 different sets of input features × 4 different sets of number of hidden nodes) was simulated 10 times with randomly selected 80% of data for training and the remaining data for validation. RESULTS: Artificial neural networks with one hidden layer, consisting of 10 nodes, and 10 input features provided optimum results. Once the feature sets were extracted, the time required for the network training was on the order of a few minutes, and the time required to perform an output calculation per field was only fraction of a second. More than 95% of clinical intensity-modulated radiation therapy (IMRT) segments were calculated within ± 3.0% modeling error for Varian Truebeam (90% and ±3.3% for Elekta Infinity). A total of 3320 volumetric-modulated arc therapy (VMAT) segments from Truebeam were calculated using the ANN trained with IMRT fields. More than 95% of the cumulative VMAT beam segments were within 3.6% modeling error, similar to the performance for IMRT segments. In general the modeling error was found to be inversely proportional to the size and intensity of the beam segment. CONCLUSIONS: A prototype ANN has been developed for predicting the signals of the IQM system, with substantially less efforts compared to the analytic model. The performance of the ANN was found to be at least equivalent to that of the analytic method, in terms of average and maximum error, for 6 MV beams on both Varian TrueBeam and Elekta Infinity platforms.

Preclinical platform for long-term evaluation of immuno-oncology drugs using hCD34+ humanized mouse model.

BACKGROUND: Well-characterized preclinical models are essential for immune-oncology research. We investigated the feasibility of our humanized mouse model for evaluating the long-term efficacy of immunotherapy and biomarkers. METHODS: Humanized mice were generated by injecting human fetal cord blood-derived CD34+ hematopoietic stem cells to NOD-scid IL2rγnull (NSG) mice myeloablated with irradiation or busulfan. The humanization success was defined as a 25% or higher ratio of human CD45+ cells to mice peripheral blood mononuclear cells. RESULTS: Busulfan was ultimately selected as the appropriate myeloablative method because it provided a higher success rate of humanization (approximately 80%) and longer survival time (45 weeks). We proved the development of functional T cells by demonstrating the anticancer effect of the programmed cell death-1 (PD-1) inhibitor in our humanized mice but not in non-humanized NSG mice. After confirming the long-lasting humanization state (45 weeks), we further investigated the response durability of the PD-1 inhibitor and biomarkers in our humanized mice. Early increase in serum tumor necrosis factor α levels, late increase in serum interleukin 6 levels and increase in tumor-infiltrating CD8+ T lymphocytes correlated more with a durable response over 60 days than with a non-durable response. CONCLUSIONS: Our CD34+ humanized mouse model is the first in vivo platform for testing the long-term efficacy of anticancer immunotherapies and biomarkers, given that none of the preclinical models has ever been evaluated for such a long duration.