Analysis of single-nucleotide polymorphisms in genes associated with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a rare variant of breast cancer (BC) known to be aggressive and refractory. TNBC lacks effective early diagnostic and therapeutic options leading to poorer outcomes. The genomic landscape and alterations leading to BC and TNBC are vast and unclear. Single nucleotide polymorphisms (SNPs) are a widespread form of genetic alterations with a multi-faceted impact on multiple diseases, including BC and TNBC. In this study, we attempted to construct a framework that could identify genes associated with TNBC and screen the SNPs reported in these genes using a set of computational predictors. This framework helped identify BRCA1, BRCA2, EGFR, PIK3CA, PTEN, and TP53 as recurrent genes associated with TNBC. We found 2%-29% of reported SNPs across genes to be typed pathogenic by all the predictors in the framework. We demonstrate that our framework prediction on BC samples identifies 99% of alterations as pathogenic by at least one predictor and 32% as pathogenic by all the predictors. Our framework could be an initial step in developing an early diagnosis of TNBC and potentially help improve the understanding of therapeutic resistance and sensitivity.

Characterizing placental stiffness using ultrasound shear-wave elastography in healthy and preeclamptic pregnancies.

PURPOSE: To measure the stiffness of the placenta in healthy and preeclamptic patients in the second and third trimesters of pregnancy using ultrasound shear-wave elastography (SWE). We also aimed to evaluate the effect of age, gestational age, gravidity, parity and body mass index (BMI) on placental stiffness and a possible correlation of stiffness with perinatal outcomes. METHODS: In a case-control study, we recruited a total of 47 singleton pregnancies in the second and third trimesters of which 24 were healthy and 23 were diagnosed with preeclampsia. In vivo placental stiffness was measured once at the time of recruitment for each patient. Pregnancies with posterior placentas, multiple gestation, gestational hypertension, chronic hypertension, diabetes, autoimmune disease, fetal growth restriction and congenital anomalies were excluded. RESULTS: The mean placental stiffness was significantly higher in preeclamptic pregnancies compared to controls in the third trimester (difference of means = 16.8; 95% CI (9.0, 24.5); P < 0.001). There were no significant differences in placental stiffness between the two groups in the second trimester or between the severe preeclampsia and preeclampsia without severe features groups (difference of means = 9.86; 95% CI (-5.95, 25.7); P ≥ 0.05). Peripheral regions of the placenta were significantly stiffer than central regions in the preeclamptic group (difference of means = 10.67; 95% CI (0.07, 21.27); P < 0.05), which was not observed in the control group (difference of means = 0.55; 95% CI (- 5.25, 6.35); P > 0.05). We did not identify a correlation of placental stiffness with gestational age, maternal age, gravidity or parity. However, there was a statistically significant correlation with BMI (P < 0.05). In addition, pregnancies with higher placental stiffness during the 2nd and 3rd trimesters had significantly reduced birth weight (2890 ± 176 vs. 2420 ± 219 g) and earlier GA (37.8 ± 0.84 vs. 34.3 ± 0.98 weeks) at delivery (P < 0.05). CONCLUSION: Compared to healthy pregnancies, placentas of preeclamptic pregnancies are stiffer and more heterogeneous. Placental stiffness is not affected by gestational age or the severity of preeclampsia but there is a correlation with higher BMI and poor perinatal outcomes.

Are Current Technical Exclusion Criteria for Clinical Trials of Magnetic Resonance-Guided High-Intensity Focused Ultrasound Too Restrictive?: Early Experiences at a Pediatric Hospital.

Certain technical criteria must be met to ensure the treatment safety of magnetic resonance-guided high-intensity focused ultrasound. We retrospectively reviewed how our enrollment criteria were applied from 2014 to 2017 in a clinical trial of magnetic resonance-guided high-intensity focused ultrasound ablation of recurrent malignant and locally aggressive benign solid tumors. Among the 36 screened patients between 2014 and 2017, more than one-third were excluded for technical exclusion criteria such as the anatomic location and proximity to prosthetics. Overall, patients were difficult to accrue for this trial, given the incidence of these tumors. To increase potential accrual, screening exclusion criteria could be more generalized and centered on the ability to achieve an acceptable treatment safety margin, rather than specifically excluding on the basis of general anatomic areas.

High-Intensity Focused Ultrasound (HIFU) Triggers Immune Sensitization of Refractory Murine Neuroblastoma to Checkpoint Inhibitor Therapy.

PURPOSE: Immunotherapy promises unprecedented benefits to patients with cancer. However, the majority of cancer types, including high-risk neuroblastoma, remain immunologically unresponsive. High-intensity focused ultrasound (HIFU) is a noninvasive technique that can mechanically fractionate tumors, transforming immunologically "cold" tumors into responsive "hot" tumors. EXPERIMENTAL DESIGN: We treated <2% of tumor volume in previously unresponsive, large, refractory murine neuroblastoma tumors with mechanical HIFU and assessed systemic immune response using flow cytometry, ELISA, and gene sequencing. In addition, we combined this treatment with αCTLA-4 and αPD-L1 to study its effect on the immune response and long-term survival. RESULTS: Combining HIFU with αCTLA-4 and αPD-L1 significantly enhances antitumor response, improving survival from 0% to 62.5%. HIFU alone causes upregulation of splenic and lymph node NK cells and circulating IL2, IFNγ, and DAMPs, whereas immune regulators like CD4+Foxp3+, IL10, and VEGF-A are significantly reduced. HIFU combined with checkpoint inhibitors induced significant increases in intratumoral CD4+, CD8α+, and CD8α+CD11c+ cells, CD11c+ in regional lymph nodes, and decrease in circulating IL10 compared with untreated group. We also report significant abscopal effect following unilateral treatment of mice with large, established bilateral tumors using HIFU and checkpoint inhibitors compared with tumors treated with HIFU or checkpoint inhibitors alone (61.1% survival, P < 0.0001). This combination treatment significantly also induces CD4+CD44+hiCD62L+low and CD8α+CD44+hiCD62L+low population and is adoptively transferable, imparting immunity, slowing subsequent de novo tumor engraftment. CONCLUSIONS: Mechanical fractionation of tumors using HIFU can effectively induce immune sensitization in a previously unresponsive murine neuroblastoma model and promises a novel yet efficacious immunoadjuvant modality to overcome therapeutic resistance.

Tissue-mimicking thermochromic phantom for characterization of HIFU devices and applications.

PURPOSE: Tissue-mimicking phantoms (TMPs) are synthetic materials designed to replicate properties of biological tissues. There is a need to quantify temperature changes following ultrasound or magnetic resonance imaging-guided high intensity focused ultrasound (MR-HIFU). This work describes development, characterization and evaluation of tissue-mimicking thermochromic phantom (TMTCP) for direct visualization and quantification of HIFU heating. The objectives were to (1) develop an MR-imageable, HIFU-compatible TMTCP that reports absolute temperatures, (2) characterize TMTCP physical properties and (3) examine TMTCP color change after HIFU. METHODS AND MATERIALS: A TMTCP was prepared to contain thermochromic ink, silicon dioxide and bovine serum albumin (BSA) and its properties were quantified. A clinical MRI-guided and a preclinical US-guided HIFU system were used to perform sonications in TMTCP. MRI thermometry was performed during HIFU, followed by T2-weighted MRI post-HIFU. Locations of color and signal intensity change were compared to the sonication plan and to MRI temperature maps. RESULTS: TMTCP properties were comparable to those in human soft tissues. Upon heating, the TMTCP exhibited an incremental but permanent color change for temperatures between 45 and 70 °C. For HIFU sonications the TMTCP revealed spatially sharp regions of color change at the target locations, correlating with MRI thermometry and hypointense regions on T2-weighted MRI. TMTCP-based assessment of various HIFU applications was also demonstrated. CONCLUSIONS: We developed a novel MR-imageable and HIFU-compatible TMTCP to characterize HIFU heating without MRI or thermocouples. The HIFU-optimized TMTCP reports absolute temperatures and ablation zone geometry with high spatial resolution. Consequently, the TMTCP can be used to evaluate HIFU heating and may provide an in vitro tool for peak temperature assessment, and reduce preclinical in vivo requirements for clinical translation.

Mechanical fractionation of tissues using microsecond-long HIFU pulses on a clinical MR-HIFU system.

PURPOSE: High intensity focussed ultrasound (HIFU) can non-invasively treat tumours with minimal or no damage to intervening tissues. While continuous-wave HIFU thermally ablates target tissue, the effect of hundreds of microsecond-long pulsed sonications is examined in this work. The objective of this study was to characterise sonication parameter-dependent thermomechanical bioeffects to provide the foundation for future preclinical studies and facilitate clinical translation. METHODS AND MATERIALS: Acoustic power, number of cycles/pulse, sonication time and pulse repetition frequency (PRF) were varied on a clinical magnetic resonance imaging (MRI)-guided HIFU (MR-HIFU) system. Ex vivo porcine liver, kidney and cardiac muscle tissue samples were sonicated (3 × 3 grid pattern, 1 mm spacing). Temperature, thermal dose and T2 relaxation times were quantified using MRI. Lesions were histologically analysed using H&E and vimentin stains for lesion structure and viability. RESULTS: Thermomechanical HIFU bioeffects produced distinct types of fractionated tissue lesions: solid/thermal, paste-like and vacuolated. Sonications at 20 or 60 Hz PRF generated substantial tissue damage beyond the focal region, with reduced viability on vimentin staining, whereas H&E staining indicated intact tissue. Same sonication parameters produced dissimilar lesions in different tissue types, while significant differences in temperature, thermal dose and T2 were observed between the parameter sets. CONCLUSION: Clinical MR-HIFU system was utilised to generate distinct types of lesions and to produce targeted thermomechanical bioeffects in ex vivo tissues. The results guide HIFU research on thermomechanical tissue bioeffects, inform future studies and advice sonication parameter selection for direct tumour ablation or immunomodulation using a clinical MR-HIFU system.

Magnetic Resonance Imaging-guided High-intensity Focused Ultrasound Applications in Pediatrics: Early Experience at Children's National Medical Center.

Magnetic resonance imaging-guided high-intensity focused ultrasound (MR-HIFU) is a novel technology that integrates magnetic resonance imaging with therapeutic ultrasound. This unique approach provides a completely noninvasive method for precise thermal ablation of targeted tissues with real-time imaging feedback. Over the past 2 decades, MR-HIFU has shown clinical success in several adult applications ranging from treatment of painful bone metastases to uterine fibroids to prostate cancer and essential tremor. Although clinical experience in pediatrics is relatively small, the advantages of a completely noninvasive and radiation-free therapy are especially attractive to growing children. Unlike elderly patients, young children must deal with an entire lifetime of negative effects related to collateral tissue damage associated with invasive surgery, side effects of chemotherapy, and risk of secondary malignancy due to radiation exposure. These reasons provide a clear rationale and strong motivation to further advance clinical utility of MR-HIFU in pediatrics. We begin with an introduction to MR-HIFU technology and the clinical experience in adults. We then describe our early institutional experience in using MR-HIFU ablation to treat symptomatic benign, locally aggressive, and metastatic tumors in children and young adults. We also review some limitations and challenges encountered in treating pediatric patients and highlight additional pediatric applications which may be feasible in the near future.

Technical aspects of osteoid osteoma ablation in children using MR-guided high intensity focussed ultrasound.

BACKGROUND: Osteoid osteoma (OO) is a painful bone tumour occurring in children and young adults. Magnetic resonance imaging-guided high intensity focussed ultrasound (MR-HIFU) allows non-invasive treatment without ionising radiation exposure, in contrast to the current standard of care treatment with radiofrequency ablation (RFA). This report describes technical aspects of MR-HIFU ablation in the first 8 paediatric OO patients treated in a safety and feasibility clinical trial (total enrolment of up to 12 patients). MATERIALS AND METHODS: OO lesions and adjacent periosteum were treated with MR-HIFU ablation in 5-20 sonications (sonication duration = 16-48 s, frequency = 1.2 MHz, acoustic power = 20-160 W). Detailed treatment workflow, patient positioning and coupling strategies, as well as temperature and tissue perfusion changes were summarised and correlated. RESULTS: MR-HIFU ablation was feasible in all eight cases. Ultrasound standoff pads were shaped to conform to extremity contours providing acoustic coupling and aided patient positioning. The energy delivered was 10 ± 7 kJ per treatment, raising maximum temperature to 83 ± 3 °C. Post ablation contrast-enhanced MRI showed ablated volumes ranging 0.46-19.4 cm3 extending further into bone (7 ± 4 mm) than into soft tissue (4 ± 6 mm, p = 0.01, Mann-Whitney). Treatment time ranged 30-86 min for sonication and 160 ± 40 min for anaesthesia. No serious treatment-related adverse events were observed. Complete pain relief with no medication occurred in 7/8 patients within 28 days following treatment. CONCLUSIONS: MR-HIFU ablation of painful OO appears technically feasible in children and it may become a non-invasive and radiation-free alternative for painful OO. Therapy success, efficiency, and applicability may be improved through specialised equipment designed more specifically for extremity bone ablation.

Comparison of Noninvasive High-Intensity Focused Ultrasound with Radiofrequency Ablation of Osteoid Osteoma.

OBJECTIVE: To evaluate clinical feasibility and safety of magnetic resonance imaging-guided high-intensity focused ultrasound (MR-HIFU) treatment of symptomatic osteoid osteoma and to compare clinical response with standard of care treatment. STUDY DESIGN: Nine subjects with radiologically confirmed, symptomatic osteoid osteoma were treated with MR-HIFU in an institutional review board-approved clinical trial. Treatment feasibility and safety were assessed. Clinical response was evaluated in terms of analgesic requirement, visual analog scale pain score, and sleep quality. Anesthesia, procedure, and recovery times were recorded. This MR-HIFU group was compared with a historical control group of 9 consecutive patients treated with radiofrequency ablation. RESULTS: Nine subjects (7 male, 2 female; 16 ± 6 years) were treated with MR-HIFU without technical difficulties or any serious adverse events. There was significant decrease in their median pain scores 4 weeks within treatment (6 vs 0, P < .01). Total pain resolution and cessation of analgesics were achieved in 8 of 9 patients after 4 weeks. In the radiofrequency ablation group, 9 patients (8 male, 1 female; 10 ± 6 years) were treated in routine clinical practice. All 9 demonstrated complete pain resolution and cessation of medications by 4 weeks with a significant decrease in median pain scores (9 vs 0, P < .001). One developed a second-degree skin burn, but there were no other adverse events. Procedure times and treatment charges were comparable between the 2 groups. CONCLUSION: This pilot study shows that MR-HIFU treatment of osteoid osteoma refractory to medical therapy is feasible and can be performed safely in pediatric patients. Clinical response is comparable with standard of care treatment but without any incisions or exposure to ionizing radiation. TRIAL REGISTRATION: ClinicalTrials.govNCT02349971.

Boiling histotripsy lesion characterization on a clinical magnetic resonance imaging-guided high intensity focused ultrasound system.

PURPOSE: High intensity focused ultrasound (HIFU) is a non-invasive therapeutic technique that can thermally ablate tumors. Boiling histotripsy (BH) is a HIFU approach that can emulsify tissue in a few milliseconds. Lesion volume and temperature effects for different BH sonication parameters are currently not well characterized. In this work, lesion volume, temperature distribution, and area of lethal thermal dose were characterized for varying BH sonication parameters in tissue-mimicking phantoms (TMP) and demonstrated in ex vivo tissues. METHODS: The following BH sonication parameters were varied using a clinical MR-HIFU system (Sonalleve V2, Philips, Vantaa, Finland): acoustic power, number of cycles/pulse, total sonication time, and pulse repetition frequency (PRF). A 3×3×3 pattern was sonicated inside TMP's and ex vivo tissues. Post sonication, lesion volumes were quantified using 3D ultrasonography and temperature and thermal dose distributions were analyzed offline. Ex vivo tissues were sectioned and stained with H&E post sonication to assess tissue damage. RESULTS: Significant increase in lesion volume was observed while increasing the number of cycles/pulse and PRF. Other sonication parameters had no significant effect on lesion volume. Temperature full width at half maximum at the end of sonication increased significantly with all parameters except total sonication time. Positive correlation was also found between lethal thermal dose and lesion volume for all parameters except number of cycles/pulse. Gross pathology of ex vivo tissues post sonication displayed either completely or partially damaged tissue at the focal region. Surrounding tissues presented sharp boundaries, with little or no structural damage to adjacent critical structures such as bile duct and nerves. CONCLUSION: Our characterization of effects of HIFU sonication parameters on the resulting lesion demonstrates the ability to control lesion morphologic and thermal characteristics with a clinical MR-HIFU system in TMP's and ex vivo tissues. We demonstrate that this system can produce spatially precise lesions in both phantoms and ex vivo tissues. The results provide guidance on a preliminary set of BH sonication parameters for this system, with a potential to facilitate BH translation to the clinic.

Development of a 3D Printed, Bioengineered Placenta Model to Evaluate the Role of Trophoblast Migration in Preeclampsia.

Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality. Current research suggests that the impaired trophoblastic invasion of maternal spiral arteries contributes significantly to the development of PE. However, the pathobiology of PE remains poorly understood, and there is a lack of treatment options largely due to ineffective experimental models. Utilizing the capability of bioprinting and shear wave elastography, we developed a 3D, bioengineered placenta model (BPM) to study and quantify cell migration. Through BPM, we evaluated the effect of epidermal growth factor (EGF) on the migratory behavior of trophoblast and human mesenchymal stem cells. Our results demonstrate a positive correlation between cell migration rates and EGF concentration. These results indicate that a feasible ex vivo placental model can be bioprinted to examine cellular, molecular, and pharmacologic interactions. In addition, EGF clearly affects the celluar migration, a potential therapeutic agent to treat preeclampsia. We envision that our ex vivo tissue modeling approach can be readily transferred to study other normal biologic and abnormal pathologic processes such as fibroblast migration in wound healing and stem cell homing.

A novel application of musculoskeletal ultrasound imaging.

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

Real-time measurement of rectus femoris muscle kinematics during drop jump using ultrasound imaging: a preliminary study.

We have developed an office based vector tissue Doppler imaging (vTDI) that can be used to quantitatively measure muscle kinematics using ultrasound. The goal of this preliminary study was to investigate if vTDI measures are repeatable and can be used robustly to measure and understand the kinematics of the rectus femoris muscle during a drop jump task. Data were collected from 8 healthy volunteers. Vector TDI along with a high speed camera video was used to better understand the dynamics of the drop jump. Our results indicate that the peak resultant vector velocity of the rectus femoris immediately following landing was repeatable across trials (intraclass correlation coefficient=0.9).The peak velocity had a relatively narrow range in 6 out of 8 subjects (48-62 cm/s), while in the remaining two subjects it exceeded 70 cm/s. The entire drop jump lasted for 1.45 0.27 seconds. The waveform of muscle velocity could be used to identify different phases of the jump. Also, the movement of the ultrasound transducer holder was minimal with peak deflection of 0.91 0.54 degrees over all trials. Vector TDI can be implemented in a clinical setting using an ultrasound system with a research interface to better understand the muscle kinematics in patients with ACL injuries.

Knee joint angular velocities and accelerations during the patellar tendon jerk.

Tendon jerk (TJ) is one of the most commonly used clinical tests in differential diagnosis of human motor disorders. There remains some ambiguity in the physiological interpretation of the test, especially with respect to its association to the functional status of patients. The TJ test inputs a non-physiological stimuli, but it is unclear to what degree the kinematics generated during the TJ test exceed the ranges that muscles encounter in activities of daily living (ADLs). The aim of our pilot study was to determine the range of angular knee kinematics (angular velocities and accelerations) corresponding to the muscle stretch elicited by TJ. We measured the longitudinal kinematics (velocities and accelerations) of the rectus femoris muscle in vivo using vector tissue Doppler imaging, an ultrasound-based method, and measured the angular kinematics of the knee in response to tendon taps with an electrogoniometer. We concluded that muscle longitudinal elongation accelerations elicited during the standard TJ test exceed angular accelerations (104.40-4534.20 rads⁻²) encountered in typical ADLs, but the velocities (0.82-6.21 rads⁻¹) elicited do not exceed those elicited by ADLs.

Measurement of tendon velocities using vector tissue Doppler imaging: a feasibility study.

We have developed a vector Doppler ultrasound imaging method to directly quantify the magnitude and direction of muscle and tendon velocities during movement. The goal of this study was to evaluate the feasibility of using vector Tissue Doppler Imaging (vTDI) for estimating the tibialis anterior tendon velocities during dorsiflexion in children with cerebral palsy who have foot drop. Our preliminary results from this study show that tendon velocities estimated using vTDI have a strong linear correlation with the joint angular velocity estimated using a conventional 3D motion capture system. We observed a peak tendon velocity of 5.66±1.45 cm/s during dorsiflexion and a peak velocity of 8.83±2.13 cm/s during the passive relaxation phase of movement. We also obtained repeatable results from the same subject 3 weeks apart. Direct measurements of muscle and tendon velocities may be used as clinical outcome measures and for studying efficiency of movement control.

Experimental characterization of a vector Doppler system based on a clinical ultrasound scanner.

We have developed a vector Doppler system using a clinical ultrasound scanner with a research interface. In this system, vector Doppler estimation is performed by electronically dividing a linear array transducer into a transmit sub-aperture and two receive sub-apertures. The receive beams are electronically steered, and two velocity components are estimated from echoes received from the beam overlap region. The velocity vector is reconstructed from these two estimates. The goal of this study was to characterize this vector Doppler system in vitro using a string phantom with a pulsatile velocity waveform. We studied the effect of four parameters on the estimation error: beam steering angle, angle of the velocity vector, depth of the scatterer relative to the beam overlap region and the transmit focus depth. Our results show that changing these parameters have minimal effect on the velocity and angle estimates, and robust velocity vector estimates can be obtained under a variety of conditions. The mean velocity error was less than 0.06 x pulse repetition frequency. The velocity estimates are sensitive to the Doppler estimation method. Our results indicate that vector Doppler using a linear array transducer is feasible for a wide range of imaging parameters. Such a system would facilitate the investigation of complex blood flow and tissue motion in human subjects.

Measurement of rectus femoris muscle velocities during patellar tendon jerk using vector tissue doppler imaging.

We have developed a vector tissue Doppler imaging (TDI) system based on a clinical scanner that can be used to measure muscle velocities independent of the direction of motion. This method overcomes the limitations of conventional Doppler ultrasound, which can only measure velocity components along the ultrasound beam. In this study, we utilized this method to investigate the rectus femoris muscle velocities during a patellar tendon jerk test. Our goal was to investigate whether the muscle elongation velocities during a brisk tendon tap fall within the normal range of velocities that are expected due to rapid stretch of limb segments. In a preliminary study, we recruited six healthy volunteers (three men and three women) following informed consent. The stretch reflex response to tendon tap was evaluated by measuring: (1) the tapping force using an accelerometer instrumented to the neurological hammer (2) the angular velocities of the knee extension and flexion using a electrogoniometer (3) reflex activation using electromyography (EMG) and (4) muscle elongation, extension and flexion velocities using vector TDI. The passive joint angular velocity was linearly related to the passive muscle elongation velocity (R(2)=0.88). The maximum estimated joint angular velocity corresponding to muscle elongation due to tendon tap was less than 8.25 radians/s. This preliminary study demonstrates the feasibility of vector TDI for measuring longitudinal muscle velocities and indicates that the muscle elongation velocities during a clinical tendon tap test are within the normal range of values for rapid limb stretch encountered in daily life. With further refinement, vector TDI could become a powerful method for quantitative evaluation of muscle motion in musculoskeletal disorders.