Methods and systems for optimization of ultrasound-facilitated liquid biopsy

Abstract

The present disclosure is directed to devices, methods, systems. An example device comprising a memory that stores a set of instructions, and a processor coupled to the memory and configured to execute the instructions to: receive a liquid biopsy measurement for biomarker from a liquid biopsy system which is performed responsive to opening a blood-brain barrier of a patient by a focused ultrasound system; and determine a confidence value associated with the liquid biopsy measurement.

Description

METHODS AND SYSTEMS FOR OPTIMIZATION OF ULTRASOUND-FACILITATED LIQUID BIOPSYCross-Reference to Related Applications

[0001] This PCT Application claims benefit to U.S. Provisional Application No. 63 / 466,348, filed May 15, 2023, titled “Methods and Systems for Optimization of Ultrasound-Facilitated Liquid Biopsy”, and U.S. Provisional Application No. 63 / 536,770, filed September 6, 2023, titled “Methods and Systems for Optimization of Ultrasound- Facilitated Liquid Biopsy, the entirety of which are each incorporated herein by reference.Background

[0002] Liquid biopsy is a minimally invasive technique increasingly used to assist in the detection, characterization, and monitoring of tumors. Liquid biopsy methods analyze fluids such as blood and cerebral spinal fluid (CSF) for biomarkers shed for example by tumors. Circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) are examples of liquid biopsy markers gaining traction in cancer management. Liquid biopsy is challenging for brain tumors because the blood-brain barrier physically limits markers from passing into brain vasculature from brain tissue.

[0003] Targeted ultrasound can selectively and transiently open the blood-brain barrier (BBB), as taught for example in WO2018 / 026738. Ultrasound mediated opening of the BBB may elevate brain tissue-derived markers circulating in the bloodstream for subsequent detection by liquid biopsy.

[0004] Methods that optimize and / or improve the performance of such ultrasound- enhanced procedures for brain tissue, such as tumors from brain cancer, are desirable.Brief Description of the Drawings

[0005] Fig. 1 A illustrates an example ultrasound system capable of opening the blood-brain barrier, according to example embodiments.

[0006] Fig. IB illustrates an example device for providing a confidence value associated with a liquid biopsy measurement, according to example embodiments.

[0007] Figs. 2A-2B illustrates example methods for performing a liquid biopsy test in combination with the focused ultrasound procedure to open the blood-brain barrier (BBB), according to example embodiments.

[0008] Fig. 3 illustrates an example method for opening the BBB opening with, for example, the system described in Fig. 1, according to example embodiments.

[0009] Fig. 4 illustrates an example of displaying a confirmed location of the focus of the ultrasound beam that is used to open the BBB, according to example embodiments.

[0010] Fig. 5A illustrates an example decision system configured to provide confidence in the results of the liquid biopsy results when used in combination with focused ultrasound procedure to open the BBB, according to example embodiments.

[0011] Fig. 5B illustrates example decision criteria that may be used within the Fig. 5A decision system, according to example embodiments.

[0012] Fig. 6A illustrates an example focused ultrasound system capable of opening the BBB, according to example embodiments.

[0013] Fig. 6B illustrates an example method for using a normalization table that may normalize the results of the liquid biopsy, according to example embodiments.

[0014] Fig. 6C illustrates a combination of a decision system and a normalization system, according to example embodiments.

[0015] Figs. 7A-7G illustrate an example technique for identifying tissue necrosis, according to example embodiments.

[0016] Fig. 8 illustrates an example device capable of identifying tissue necrosis, according to example embodiments.

[0017] Fig. 9 illustrates an example method for distinguishing between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis, according to example embodiments.Detailed Description

[0018] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific examples in which the disclosure can be practiced. It is to be understood that other examples can be utilized, and various changes may be made without departing from the scope of the disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the disclosure is defined by the appended claims. It is to be understood that features of the various examples described herein may be combined, in part or whole, with each other, unless specifically noted otherwise.

[0019] It can be beneficial to open the blood-brain barrier (BBB) for many different applications. For example, the BBB may be opened to deliver drugs to the brain for treatment of cancers and other diseases, to obtain liquid biopsy measurements of brain tissue-derived biomarkers, and / or for research, among other purposes. In some instances, the BBB can be opened by delivering ultrasonic energy to specific region(s) in the brain which are intended to be opened. Successfully opening the BBB can increase the possibility of molecules passing from the bloodstream into tissues of the brain and from tissues of the brain into the bloodstream. However, it can be difficult to know whether or not the BBB has successfully been opened. Various embodiments of the present disclosure are directed to the use of a liquid biopsy measurement to determine a confidence value associated with the liquid biopsy measurement. The liquid biopsy measurement is obtained after delivering ultrasonic energy by a focused ultrasound system, sometimes herein referred to as “sonication”, to a location of the brain of a patient. In some embodiments, the confidence value may be indicative of confirmation of opening the BBB. In some embodiments, the confidence value may be indicative of a level of confidence of a reading of a disease biomarker (e.g., tumor biomarker) from the liquid biopsy measurement based on a reading of a brain biomarker (e.g., non-tumor or other non-disease biomarker) from the liquid biopsy measurement. As further described herein, the confidence value may be based on information from the focused ultrasound system which is indicative of the location of the opening of the BBB and the liquid biopsy measurement from a liquid biopsy system. In various embodiments, the BBB opening may be confirmed by comparing a concentration of a brain biomarker to a threshold.

[0020] Some embodiments are directed to differentiating between necrosis tissue and tumor (or other diseased) tissue. Distinguishing between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis using a magnetic resonance (MR) image can be difficult as the blood-brain barrier can be leaky which results in necrosis tissue and tumor tissue appearing similar on the MR image. As an example, a tumor can reoccur at or near the site where a surgery may have been performed to remove the tumor at the time of the initial finding. As the BBB can be somewhat leaky, when imaged with MRI in the presence of an MRI contrast agent, the brightness of the images corresponding to the location of the tumors increases. Thus, in MRI images, brain tumors can appear bright. In some implementations, the treatment following the initial diagnosis may have included radiation and the tissue in such areas may have necrosed or undergone necrosis.In some implementations, where tumor recurrence is occurring or tissue is necrosed or undergoing necrosis, each of these regions may appear bright in MRI imaging. Thus, radiation necrosis and a true tumor recurrence may present similarly in MR images and may potentially lead to an incorrect diagnosis and / or a delayed diagnosis of tumor recurrence. Embodiments of the present disclosure are directed to distinguishing between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis by delivering ultrasonic energy to a location of the brain after injecting a patient with microbubbles and analyzing ultrasound echo signals in response thereto. Tumor tissue may have some vascularity and necrosis tissues may not, which can result in the microbubbles not traversing through the necrosis tissue and traversing through, and being contained by, the tumor tissue. As the tumor tissue may contain the microbubbles (e.g., as injected into the patient through a peripheral intravenous injection) and the necrosis tissue may not contain microbubbles, or may contain minimal microbubbles, harmonic components of the ultrasound echo signals may be used to differentiate between necrosis tissue and tumor (or other diseased) tissue, as further described herein.

[0021] Fig. 1 A illustrates an example focused ultrasound system that opens the BBB non-invasively, according to example embodiments. The focused ultrasound system (FUS) 100 as shown by Fig. 1 A may include at least some of substantially the same features and attributes, and / or otherwise be implemented, as described in US 11,534,630, issued on December 27, 2022, and entitled “Ultrasound Guided Opening of Blood-Brain Barrier”, which is hereby incorporated herein by reference in its entirety for its teaching. For example, the FUS 100 may include an ultrasound cap 106 for placement on the head of a patient 101, components for providing a contrast agent to the patient 101 (e.g., stand 103, bag containing the contrast agent 104, electronically controlled valve 105, a cable 108 coupling the valve 105 to the device 102), the device 102 having a processor for controlling the FUS 100 (sometimes referred to as the “control device”), and components for connecting the cap 106 to the device 102 (e.g., cable 107, cable interface 109). The ultrasound cap 106 may include at some of substantially the same features and attributes, and / or otherwise be implemented, as described in more detail in US Publication No. 2023 / 0082109, published on March 16, 2023, and entitled “Ultrasound Transducer Assembly”, which is hereby incorporated herein by reference in its entirety for its teaching. In brief, the ultrasound cap 106 may include several types of transducer components: (1) a low-frequency transducer configured to provide the energy to open theblood-brain barrier, (2) a high-frequency transducer set or imaging array configured to image through the thin areas of the skull to image structures of the brain; the ultrasound images obtained from the high-frequency set may be aligned with the images obtained from another modality such as magnetic resonance (MR) where the energy for imaging can penetrate the skull, and / or (3) monitoring transducers which may be used in a feedback loop to open the BBB. In various embodiments, the monitoring transducers may be used to provide safe opening of the BBB, by capturing or receiving signals coming from structures within the brain and presenting these signals for further analysis by other components of the FUS 100.

[0022] For the purposes of this disclosure, in some embodiments, the low-frequency range may be between 0.200 megahertz (MHz) to 10 MHz and, in some embodiments, may be between 0.25 MHz to 5 MHz. The high-frequency range may be between 2MHz to 5MHz, for example. The monitoring transducers may operate with a bandwidth 100 kHz to 10 MHz, for example.

[0023] Fig. IB illustrates an example device for providing a confidence value associated with a liquid biopsy measurement, according to example embodiments. The device 110 may form part of the FUS 100 of Fig. 1 A, part of the liquid biopsy system (such as 552 of Fig. 5 A), a separate device from the FUS and the liquid biopsy system (such as 554 of Fig. 5 A), or may be distributed among any combination of the FUS, the liquid biopsy system, and a separate device from the FUS and liquid biopsy system. For example, as further described herein, the processor 112 and memory 114 may include processing and memory resources which are distributed on the FUS, the liquid biopsy system, and / or a separate device, such as via a Cloud-computing system. In some embodiments, the processor 112 and memory 114 may form part of computing device(s) which are local to or remotely located to the FUS and liquid biopsy system.

[0024] As shown by Fig. IB, the device comprises a processor 112 and memory 114. The memory 114 may include a computer-readable storage medium storing a set of instructions 116, 118. The memory 114 (as well as memory 814 of Fig. 8) may include Read-Only Memory (ROM), Random-Access Memory (RAM), Electrically Erasable Programmable Read-Only Memory (EEPROM), flash memory, a solid state drive, Electrically Programmable Read Only Memory aka write once memory (EPROM), physical fuses and e-fuses, and / or discrete data register sets. In some embodiments, memory 114 may be a non-transitory storage medium, where the term “non-transitory”does not encompass transitory propagating signals. The processor 112 may be coupled to the memory 114 to execute the instructions 116, 118 to perform the actions, as further described below.

[0025] At 116, the processor 112 may receive a liquid biopsy measurement for biomarker from a liquid biopsy system which is performed responsive to opening a BBB of a patient by a focused ultrasound system. As further described below, the liquid biopsy measurement may identify a concentration of a biomarker, such as a brain biomarker. In some examples, the liquid biopsy measurement may identify a concentration of plurality of biomarkers, including the brain biomarker and a tumor biomarker, among other molecules such as circulating tumor DNA (ctDNA). A brain biomarker, as used herein, refers to or includes a brain tissue biomarker that is associated with the opening of the BBB, and which is not associated with a disease state, such as a tumor and may be referred to as a “non-disease brain biomarker”. Example brain biomarkers include glial fibrillary acidic protein (GFAP) and myelin basic protein (MPB), among others. In some embodiments, the processor 112 may further receive information from the FUS indicative of the location of the opening of the BBB. For example, the information from the FUS may identify the location of the opening of the BBB.

[0026] At 118, the processor 112 may determine a confidence level associated with the liquid biopsy measurement. In some embodiments the processor 112 may determine the confidence value based on a location of the opening of the BB after sonication by the FUS and confirmation of opening of the BBB after sonication by the FUS based on the liquid biopsy measurement. For example, the processor 112 may determine the confidence value based on a focus location of a transmitted ultrasound beam relayed by the FUS, such as received from the FUS. Said differently, the confidence value may be determined based on information from the FUS identifying the location of the opening of the BBB and the liquid biopsy measurement from the liquid biopsy system.

[0027] In some embodiments, the confidence value is determined based on a comparison of a concentration of the biomarker, as measured by the liquid biopsy system, to a threshold. The threshold may be stored on the memory 114. For example, the threshold may be stored as a table, such as a LUT stored on the memory 114. The LUT may include expected concentrations of a plurality of brain biomarkers, such as different types of brain biomarkers including the brain biomarker and other biomarkers, when the BBB is opened.

[0028] In some embodiments, the processor 112 may receive the liquid biopsy measurement including a measured concentration of the biomarker from the liquid biopsy system and may output the confidence value based on the comparison of the biomarker to the threshold. In some embodiments, the processor 112 receives the liquid biopsy measurement including a measured concentration of the biomarker comprising a brain biomarker and a measured concentration of a disease biomarker from the liquid biopsy system and outputs the confidence value of the measured concentration of the disease biomarker based on the comparison of the measured concentration of the brain biomarker to the threshold. In some embodiments, the processor 112 determines the confidence value further based on a focus location of a transmitted ultrasound beam relayed by the FUS and a confirmation of the BBB opening based on the concentration of the biomarker being equal to or greater than the threshold.

[0029] As further described below, the confidence value may be indicative of a confidence of the BBB opening. For example, confidence value may be indicative of a level of confidence that the BBB opened at the intended location. In some embodiments, the biomarker may be a brain biomarker, as previously described. The threshold may be an expected concentration of the brain biomarker if the BBB is open. For example, the FUS may identify the location of the BBB opening, and the brain biomarker concentration may be used to verify the BBB has opened. Verifying the BBB is opened may be used for a variety of purposes, such as, but not limited to, research. Embodiments are not limited to a single brain biomarker and may include a plurality of brain biomarkers. Further, the liquid biopsy measurement may include other biomarkers and information, such as ctDNA analysis.

[0030] In some embodiments, the confidence value may be indicative of a confidence of a disease biomarker. In such embodiments, the biomarker may include a plurality of biomarkers including a brain biomarker and a disease biomarker, such as a tumor biomarker. For example, the confidence value may be indicative of a level of confidence of a reading of the disease biomarker based on a reading of the brain biomarker from the liquid biopsy measurement, and optionally, the BBB opening at the intended location. In some such embodiments, the confidence value may be indicative of a qualitative confidence of the liquid biopsy results. Embodiments are not limited to a single brain biomarker and / or a single disease biomarker and may include a plurality of brainbiomarkers and / or a plurality of disease biomarkers. Further, the liquid biopsy measurement may include other biomarkers and information.

[0031] In some embodiments, the processor 112 is to determine the confidence value using a set of criteria to evaluate the results of the liquid biopsy, as performed by the liquid biopsy system, being applicable to a region of interest. The set of criteria may be associated with identification of an opening location of the BBB as relayed by the FUS and a concentration of the biomarker based on the liquid biopsy measurement. In some embodiments, the processor 112 may assess the opening location for overlapping with (or overlapping less than a threshold amount with) the region of interest.

[0032] In some embodiments, the processor 112 may assess the concentration of the biomarker at least prior to and after sonication by the FUS. For example, the processor 112 may compare the concentration of the biomarker prior to and to assess at least one of: (i) an increase in the concentration after sonication compared to prior to sonication indicative of a higher confidence value as compared to the biomarker (e.g., the brain biomarker) concentration not increasing, and (ii) the concentration after sonication being above a threshold indicative of a higher confidence value as compared to the biomarker (e.g., the brain biomarker) concentration being less than the threshold.

[0033] In some embodiments, the processor 112 may assess whether the opening location is overlapping with a region of interest, which indicates a higher confidence value of a reading of the liquid biopsy measurement pertaining to a disease biomarker as compared to the opening location not overlapping or overlapping less than a threshold amount with the region of interest. However, embodiments are not so limited.

[0034] In some embodiments, the processor 112 may output the confidence value as a message to a user, such as on a user display of a computer device 102 of Fig. 1 A.

[0035] In some embodiments, the processor 112 may adjust a parameter of the liquid biopsy based on information derived about the opening of the BBB. For example, the processor may output data indicative of the adjusted parameter to the liquid biopsy system. The adjusted parameter may comprise a read depth, and the processor 112 may adjust the read depth based on a volume of the opening of the BBB. In some embodiments, the adjusted parameter comprises a variant calling parameter.

[0036] In some embodiments, the processor 112 may adjust the read depth based on at least one of the volume of the opening of the BBB, an expected concentration of the biomarker, and a concentration of the biomarker determined by the liquid biopsy. In someembodiment, the biomarker may include the brain biomarker in addition to other biomarkers, such as the tumor biomarker, cell-free DNA (cfDNA), ctDNA, or a combination thereof. The processor 112 may determine the adjustment to the read depth using at least one of a LUT, an estimated opening of the BBB, and the expected concentration of the biomarker.

[0037] In some embodiments, the processor 112 may normalize results of the liquid biopsy using a normalization table, as further described herein.

[0038] As further described herein, embodiments are directed to systems which include the device 110 of Fig. IB or portions thereof. For example, the system may include a FUS configured to open the BBB of a patient, a liquid biopsy system configured to obtain the liquid biopsy measurement for the biomarker responsive to the opening of the BBB, and the processor which is configured to receive the liquid biopsy measurement from the liquid biopsy system, and determine the confidence value associated with the liquid biopsy measurement, as described above. The processor of the system may perform any of the above-described variations, as described in connection with Fig. IB. As previously described, the processor may form part of the FUS, the liquid biopsy system, and / or a decisions system.

[0039] Figs. 2A-2B illustrates example methods for performing a liquid biopsy test in combination with the focused ultrasound procedure to open the BBB, according to example embodiments. The focused ultrasound methods illustrated by Figs. 2A-2B may be conducted, for example, with the system and / or the device as described in connection with Figs. 1A-1B.

[0040] As shown by the example method 222 of FIG. 2A, at 222, an initial or first blood sample is obtained. The blood is processed and is analyzed to determine the concentration of (at least one) biomarker. Several laboratory or commercial systems are available that can be used to determine the concentration of biomarkers. One example of such a system is called the Guardant360™ test from Guardant Health. The first blood sample results in a baseline biomarker concentration. At 224, the blood-brain barrier is opened with focused ultrasound. When the blood-brain barrier is opened, biomarkers may enter into the blood circulation from the brain tissue. At 226, a second blood draw is obtained and analyzed to determine biomarker concentration(s). At 226, the biomarker concentrations may be compared manually or through an automated analysis, and a summary or conclusion is provided. This summary or conclusion may provide guidanceabout the status of the health of the patient. At 228, the summary or conclusion may also provide information to the physician or medical technician in terms of choosing therapeutic options. Thus, it can be beneficial (e.g., desirable) to have confidence in the summary or conclusion at 228 of the method illustrated by 228.

[0041] Examples of 224 in Fig. 2A is explained in more detail in Fig. 3 which outlines the focused ultrasound-based BBB opening according to example embodiments with the configurations, for example, described in Fig. 1 A. Fig. 3 illustrates an example method for opening the BBB opening with the system described in Fig. 1 A, according to example embodiments. Other configurations or systems for focused ultrasound-based BBB opening may also be used. At 330, the patient may obtain a diagnostic scan of the head anatomy such as an MRI scan. At 332, based on the scans obtained at 330, the physician determines a treatment plan. For some diseases related to the brain, such as brain cancer, the physician may determine that the patient may benefit from focused ultrasound-based BBB opening. For the ultrasound-based BBB opening procedure, the physician typically specifies or draws a region-of-interest (ROI) in the images obtained at 330. The BBB is typically to be opened within the ROI. At 334, the FUS takes the ROI and the diagnostic scans (from 330) as input and performs several calculations. A number of such calculations are described, for example, in the by US Patent US 11,534,630, granted on December 27, 2022, and entitled “Ultrasound Guided Opening of Blood-Brain Barrier; as well as PCT Publication No. WO2024 / 025927, published on February 1, 2024, and entitled “Rapid Calculation of Parameters for Delivering Ultrasound Energy to Selected Locations in the Brain”, and PCT Publication No. WO2024 / 025928, published on February 1, 2024, and entitled “Methods and Systems for Confirming Focus of Ultrasound Beams”, which are hereby each incorporated herein by reference in their entireties for their teachings. Briefly, the ultrasound system calculates the position of the ultrasound cap in relation to the patient’s internal cranial anatomy non-invasively. Subsequently, the location and timing of a set of elements (e.g., the transmit parameters) that emit low-frequency waves are determined, and the using the ROI as one of the inputs. The patient may then be injected with an ultrasound contrast agent (e.g., microbubbles). Additionally, the location of the transmit focus of the low-frequency ultrasound beam may be confirmed before the ultrasound system applies the full power sonication to open the BBB. Following this focus confirmation, low-frequency ultrasound is transmitted from these elements. The BBB is then opened in the region around and including the focus of the low-frequency ultrasoundbeam emitted by these selected low-frequency elements. The BBB may open in and around the focus due to the acoustic pressure being typically high in these regions. If the pressure is above a lower threshold (called the “opening threshold”) and is lower than an upper threshold (called the “damage threshold”), then the BBB opens safely at 336.

[0042] Typically, when a focused ultrasound system (such as that generated by the system of Fig. 1A, for example) is deployed to open the BBB, the definitive confirmation that the BBB is opened may be obtained by performing an MRI. However, performing an MRI is not always practical; the MRI machine may not be available for several hours after BBB opening procedure and if too much time has passed, the BBB, which may have been opened, may start to close again. In other circumstances, the MRI machine may not be available on site near where the focused ultrasound procedure is performed. For such cases, it is advantageous to at least know if the transmit focus was in the appropriate location, as described, for example in PCT Publication No. WO2024 / 025928. When the focused ultrasound procedure is followed by a blood draw for the purposes of a liquid biopsy, confirmation of where the BBB opening may have been opened gains even more prominence, at 338, as the results of the liquid biopsy may determine the treatment path for the patient.

[0043] Thus, in some embodiments, subsequent to confirming where the location of the transmit focus is, the location of the focus may be displayed on the screen along with the physician-specified ROI. In some embodiments, the locations of the focus location may be displayed on a screen also along with the physician specified ROI. In some embodiments, the focus location may include multiple disparate focus locations. In some embodiments, the percent / fraction or some other indicia of the physician-specified ROI that overlaps with the focal area or region of the ultrasound beam used to open the BBB may be determined and suitably output to a user. In some embodiments, messages may be generated and output from the ultrasound system. These messages may include various status messages, for example, the messages may state that the actual focus location within the brain is within the specified ROI or may state the results of the computation, such as the percent / fraction or other indicia of physician-specified ROI that is estimated to be overlapping with the focal area or region of the ultrasound beam.

[0044] Fig. 2B illustrates another example method for performing a liquid biopsy test in combination with the focused ultrasound procedure to open the BBB, according to example embodiments. The method 221 may include an implementation of the methodsdescribed by Figs. 2-3 and / or implemented using the FUS 100 and / or device 110 described by Figs. 1A-1B.

[0045] As shown by Fig. 2B, the example method 221 comprises opening a bloodbrain barrier of a patient by a FUS, at 223, and responsive to the opening of the bloodbrain barrier, obtaining a liquid biopsy measurement for biomarker using a liquid biopsy system, at 225. The method 221 further comprises determining a confidence value associated with the liquid biopsy measurement, at 227. The method 221 may comprise the variations as described above in connection with Fig. IB, the common features and attributes are not repeated for ease of reference.

[0046] Fig. 4 schematically illustrates some of these concepts, including an example of displaying a confirmed location of the focus of the ultrasound beam that is used to open the BBB, according to example embodiments. In Fig. 4, an image or a representation 442 of the patient’s head anatomy is shown. This image or representation 442 may be a magnetic resonance (MR) image, a fused MR and computed tomography (CT) image, a model of the patient’s head based on the anatomy of the head, among others. The physician-specified ROI 441 is also shown. The image 442 and the ROI 441 may be the same as used at 330 and 332 in Fig. 3, for example. Within the ROI 441, several examples of the display of the confirmed location of focus of the low-frequency ultrasound beam are shown as 444, 446, 448, and 449. Each of these focus locations 444, 446, 448, and 446 is shown in Fig. 3 illustration by an elliptical region. Fig. 3 illustrates an example method to show the focus locations 444, 446, 448, and 449. In this representation, the region where the pressure is higher than a lower “opening” threshold that opens the BBB is shown. For example, a -3dB region (-3dB is approximately 0.5 of the peak value) from the peak of the focus may be shown. The transmit parameters may be arranged such that the -3dB region is at the lower threshold of BBB opening. The -3dB region may be calculated by the ultrasound system by modeling the sound propagation from the ultrasound cap into a model of the head of the patient. Thus, in example region A (444), the region shown with cross-hatching may experience BBB opening. Example region A (444) is shown entirely inside the physician specified ROI 441. In example region B (446), the location of focus is shown just slightly outside the physician specified ROI 441. In this case, the FUS may have determined prior to the start of the sonication procedure that the BBB opening region lies outside the physician specified ROI 441. The FUS may provide this information to the physician and the physician may accept and provide the permission to proceed. Exampleregions C (448) and C’ (449) show examples of positions that are altered by the displacement of the cap in relation to the patient’s head anatomy. Example region C (448) may be a focus location based on an initial position of the cap. During the sonication procedure, the relative position of the cap may change in relation to the patient anatomy. In various embodiments, the cap may include at least some of the features and attributes, as described in PCT Publication No. WO2024 / 025927). For example, new transmit parameters may be calculated rapidly without interruption of the sonication sequence. In some examples, the ultrasound system may determine that the relative displacement between the cap and the head anatomy is within a threshold and may permit the sonication procedure to continue without altering the transmit parameters. In such example embodiments, the focus position may be slightly different compared to when the sonication started. An example of such embodiments is schematically illustrated in Fig. 4 in example region C’ (449).

[0047] Where focused ultrasound is used to open the BBB, for example with the FUS 100 in Fig. 1 A and / or device 110 in Fig. IB, the area near or at the focus of the ultrasound beam (where the acoustic pressure is typically higher than elsewhere) is where the BBB opens, and the shape of the beam around the focus may be used to characterize the beam. Thus, the phrase “shape of the beam”, as used herein, may be used synonymously with the phrase “the shape of the beam around the focus” unless specified otherwise.

[0048] The dimensions and the shape of the ultrasound beam is dependent on the transmit parameters used, including the shape and dimensions of the low-frequency transducers that are part of the ultrasound cap shown in Fig. 1 A. In some instances, the physician may specify the ROI 441 with points rather than regions. When the shape of the beam is calculated by the FUS and presented as shown for example in Fig. 4, it may be assumed that the peak value within the -3dB region corresponds to the transmit focus location.

[0049] Since and advantageously for the patient in terms of convenience, there is no immediate MRI after the sonication process, the process of confirming the location of the focus provides assurance that the brain was targeted at the intended location. If, however, errors in calculations occur, then the BBB will not be opened at the appropriate location. The consequence of opening the BBB in an incorrect location can include the following situation: if the BBB is opened in a non-tumor area and as a consequence, the tumor biomarkers are not detected in the peripheral blood, this may provide false negativeoutput. Similarly, if the focus location is in the appropriate location coinciding with a tumor location but if the acoustic pressure is not sufficient, the BBB may not open. Again, this may lead to a false negative output by the liquid biopsy system or the combined ultrasound system and liquid biopsy system. A false negative output, as used herein, may refer to and / or include an output indicating no tumor biomarkers are detected, when tumor biomarkers should be present (such as being present at a concentration above a detection threshold value).

[0050] LIQUID BIOPSY SYSTEM

[0051] A liquid biopsy system is briefly described next. A liquid biopsy system is a diagnostic tool that detects and analyzes biomarkers including but not limited to cancer biomarkers in body fluids such as blood, urine, or cerebrospinal fluid (CSF). Cells including disease-bearing cells such as cancer cells shed various biomolecules such as DNA, RNA, proteins, and exosomes into the circulation. Blood from patients with such diseases may be obtained from a peripheral site such as the arm. Subsequently, the collected blood (or other fluid) specimen, may be analyzed by a liquid biopsy assay(s) or test(s) to detect these biomarkers. The detection may provide information about the disease status. A typical liquid biopsy system (referring to the process of blood (or other fluid) collection through test report generation) may be used for diagnosis, prognosis, and treatment monitoring by detecting a circulating biomolecule including cell-free DNA (cfDNA), cell-free RNA (cfRNA), circulating tumor cells (CTCs), proteins, exosomes and other elements.

[0052] A typical liquid biopsy system may generally go through the following: At LB1 the sample collection, at LB2: Biomarker preconcentration and detection, and at LB3: Data analysis.

[0053] LB1 : Sample Collection. At LBl, a sample is collected. Various methods may be used such as venipuncture, urine collection, or lumbar puncture. Biomarkers of interest such as ctDNA, or CTCs may be isolated.

[0054] LB2: Biomarker Detection. At LB2, the biomarkers are detected. Various techniques, such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), next-generation sequencing (NGS), digital droplet PCR (ddPCR), or mass spectrometry (MS) may be used. Detection may include qualitative detection (e.g., for the presence of a biomarker) and / or quantitative detection (e.g., for an amount of a biomarker). In some embodiments, at LB2 may further include sample preparation such asbiomarker preconcentration. Biomarker preconcentration may be particularly useful for non-nucleic acid biomarkers that cannot be amplified by PCR or other techniques.

[0055] LB3: Data Analysis. At LB3, the biomarker data is processed and results are produced. As used herein, the biomarker data may include a list of detected biomarkers (e.g., molecules). For DNA analysis, results may include a list of mutations, a list of genes in the sample, quantification of number of mutations or amount of cfDNA, etc. The data analysis step may use various bioinformatic tools, such as variant callers, fusion callers, gene expression analysis pipelines, or machine learning functions (e.g., algorithms). LB3 may further include combining biomarker data with other patient data such as, clinical / pathologic grade / stage obtained from tissue specimen(s) or imaging data to provide a comprehensive diagnosis, prognosis, therapy selection or various other patient management decisions.

[0056] As described above, at LB2 for the biomarker detection method, various techniques such as ELISA and / or NGS, among others, may be used. Example details of NGS are provided below.

[0057] A typical workflow for NGS may include the following: At NG1 : Sample preparation, at NG2: Sequencing, and at NG3: Data analysis.

[0058] NG1 : Sample Preparation: At NG1, the samples are prepared. DNA or RNA molecules are extracted from the samples and fragmented into smaller pieces, as appropriate for the selected NGS approach. Molecules called adapters are attached to the end of the fragments which allows binding of the fragment to a sequencing system.

[0059] NG2: Sequencing. At NG2, the sequence of DNA or RNA may be read by using fluorescence, electrical based techniques, etc. The fluorescence or electrical signals are created as a result of the sequencing process from the nucleotides that are used to grow the DNA or RNA strand that is used to identify the bases in the original fragment. Commercial versions of the NGS sequencing technology are available from companies such as Illumina.

[0060] NG3: Data Analysis. At NG3, the biomarker data is processed and results are produced. Results may include lists of mutations and the list of genes in the sample etc. Results may additionally include concentration of the molecules being analyzed. The data analysis step may use various bioinformatic tools, such as variant callers, fusion callers, gene expression analysis pipelines, and / or machine-learning functions, among others.

[0061] An aspect that affects the accuracy of NGS-based liquid biopsy results (e.g., accuracy in decoding the sample sequence) is the coverage or “read depth”. A parameter that may be set by the operator of the liquid biopsy system, a higher read depth increases the accuracy and confidence of the data and a lower read depth consequently decreasing the accuracy and resulting in a higher rate of sequencing errors. While greater read depth may increase the accuracy, it also may require more NGS capacity to be devoted to each sample, so less multiplexing is possible, decreasing the number of samples that may be analyzed per run. Thus, the aggregate process may be slower, even if a given instrument run i the same length.

[0062] The read depth may be adjusted depending on the type of analysis. For example, read depth may be set based on the tumor microenvironment, the complexity of the genome or transcriptome, the presence of repetitive or low-complexity regions, and / or the sensitivity and specificity requirements of the analysis, among others.

[0063] Typically, read depth is reported as a numerical value or a coverage plot showing the number of reads that cover each position along the DNA or RNA sample sequence. Higher read depths may be used, for example, when the presence of a rare variant is to be confirmed.

[0064] Read depth within an NGS may be modified in multiple ways including adjusting the sequencing parameters and making modifications to the library preparation, among other ways. The read depth may be modified by adjusting the sequencing parameters, such as the number of cycles, the read length, and the number of reads per sample, among other modifications. The read depth may also be modified by adjusting the library preparation protocol, such as the PCR cycle number, and the amount of input DNA or RNA, among others.

[0065] Another NGS parameter that may be adjusted by the user is the variant calling function, such as adjusting parameters within the variant calling function. The variant calling parameter, in some embodiments, may also or alternatively be modified based on the particular details of the study being conducted. There are several parameters and thresholds in variant calling functions (e.g., algorithms) that may be tuned to optimize the trade-off between sensitivity (the ability to detect true positive variants) and specificity (the ability to avoid false positive variants).

[0066] Some non-limiting examples of the parameters that may be adjusted in variant calling function include:1. Minimum allele frequency: This threshold parameter determines the minimum proportion of reads supporting a variant allele that is required to call a variant. Increasing this threshold increases specificity and decreases sensitivity.2. Minimum read depth: This threshold parameter determines the minimum number of reads covering a variant site that is required to call a variant. Increasing this threshold increases specificity and decreases sensitivity.3. Quality score cutoff: This threshold parameter determines the minimum quality score required for a variant call to be considered high-confidence. Increasing this threshold increases specificity and decreases sensitivity.4. Strand bias filter: This filter removes variant calls that have a biased distribution of reads on the forward or reverse strand. This filter may reduce false positives caused by sequencing errors or PCR biases.5. Base quality score recalibration: The recalibration involves re-calibrating the base quality scores based on the observed error rates in the data. This process may improve the accuracy and precision of variant calling by reducing systematic errors.

[0067] As noted, the liquid biopsy system is a diagnostic tool that may detect and analyzes biomarkers. The output of the liquid biopsy system may include data and information such as presence / absence of specific genes, mutation profiles, gene expression profiles etc. The output of the system may also be associated with a confidence value that provides information about the level of certainty associated with a particular result. The confidence value of the liquid biopsy system may be influenced by the concentration of the analytes in the sample. The relationship between confidence value and the concentration of an analyte (e.g., one or multiple) is known aprioi as a part of the characterization and the manufacture of the liquid biopsy system.

[0068] Fig. 5A illustrates an example decision system configured to provide or attach a confidence value to the results provided by a liquid biopsy system when used in combination with focused ultrasound procedure implemented by a FUS to open the BBB, according to example embodiments (for example by reducing the possibility of false positives due to the focused ultrasound procedure). The confidence value provides an indication of or an assessment of the applicability of the liquid biopsy results to the chosen or determined ROI. The system 500 may include a FUS 550, a liquid biopsy system 552, and a decision system 554 (such as the device of Fig. IB), In this system 500, the inputs551, 553, 555 may include the results of the liquid biopsy analysis (555) from a blood sample 551 and the messages or other indicia (553) from the FUS 550. As described above, the messages 553 or other indicia from the FUS 550 may include the information relating to whether the BBB opened in the appropriate location. The decision system 554 may apply a set of criteria to evaluate or calculate and to inform the user of a confidence level as to the applicability of the results of the liquid biopsy system (or the BBB opening as previously described) to the specific ROI being analyzed. An example set of criteria is now described. If the focus of the low-frequency transmit beam is in the appropriate location as determined by the FUS 550 and communicated via the messages 553 or other indicia, then the decision system 554 illustrated by Fig. 5A evaluates and displays a message or otherwise indicates that there is high confidence (at 557) in the liquid biopsy results 555 (i.e., the results are highly applicable to the ROI being examined). If the focus is not sufficiently overlapping the ROI as determined and communicated by the FUS 550, then an appropriate message may be communicated by the FUS 550 and the decision system 554 displays a message or otherwise indicates that there is low confidence (at 557) in the liquid biopsy results 555 and / or the BBB may not have opened (i.e., the results are not as applicable to the ROI being examined). The liquid biopsy results 555 may be input to the decision system 554 for at least two instances - a pre- sonication liquid biopsy result and a post-sonication liquid biopsy result from a specific ROI. The decision system 554 may include the capability to determine the changes in concentration of biomarkers for at least two instances of time for that ROI, such as a concentration of the biomarker(s) presonication and post-sonication. The results of these determinations may be used to generate the confidence level in the liquid biopsy result 555 (i.e., how applicable are the results to the specific ROI) along with the information from the FUS 550.

[0069] In some embodiments, additional or alternative information may be input into the decision system 554 to increase the quality of the confidence level determination. When the BBB is opened, the level of biomarkers that are associated with the brain tissue (and not necessarily associated with the disease status) may also be increased in the peripheral blood. Examples of such biomarkers include glial fibrillary acidic protein (GFAP) and myelin basic protein (MPB). Thus, in some embodiments, the liquid biopsy system 552 may include tests for such biomarkers. Although Fig. 5A shows one liquid biopsy system 552, more than one liquid biopsy system may be used to input data into the decision system 554. As an example, a first liquid biopsy system may be used foridentifying and obtaining the sequence of bases in the sample and a second liquid biopsy system may be used to identifying and obtaining the proteins in the same sample or a sample collected from the same patient. In some embodiments, the single liquid biopsy system 552 may provide multiple types of analysis, such as NGS and a protein or other biomarker analysis and / or which are analyzed by sub-systems of the system. As noted above, the concentration of biomarkers may be tested both prior to and after the sonication procedure implemented by FUS 550 and changes in the concentration of biomarkers may be determined. As additionally noted above, the decision system 554 may be provided the results of the liquid biopsy analysis (555) for both the time instances (i.e., prior to sonication and post-sonication) as inputs. If an increase in concentration in biomarkers is more than a predetermined threshold amount, this further evidences that the BBB opened.

[0070] Thus, the decision system 554 may include a more refined decision criteria. Examples of decision criteria are illustrated in Fig. 5B and described below.

[0071] Fig. 5B illustrates example decision criteria that may be used within the decision system of Fig. 5 A, according to example embodiments. As shown by 560, if the focus locations are confirmed to be in the appropriate locations and the concentration of the brain (non-tumor) biomarkers (for example GFAP and / or MBP) is increased between the pre- and post-sonication time points, then the confidence in BBB opening and the liquid biopsy results as applied to the specific ROI is high. This circumstance corresponds to the box in the upper left corner of Fig. 5B. As shown by 564, if the focus location is not in the appropriate location and the level of brain (non-tumor) biomarkers (e.g., GFAP and / or MBP) has increased between pre- and post-sonication, then the assumptions in the beam-formation calculations or the location of the aperture used for sonication may be incorrect. This corresponds to the box in the lower left comer of Fig. 5B. As shown by 562, if the focus location is the appropriate location and the concentration of brain (non- tumor) biomarkers did not change by a threshold amount between pre- and postsonication, this may indicate that peak negative pressure was not sufficiently high enough to open the BBB or that the functions used in the focused ultrasound system (for example to determine the voltage that is to be applied to the transducers in the ultrasound cap), are not optimized or are not working. This corresponds to the box in the upper right corner of Fig. 5B. The decision system may display a message that the results of the liquid biopsy may be inconclusive. Finally, as shown by 566, if the focus location is not in the right location and the concentration of brain (non-tumor) biomarkers did not increase betweenpre- and post-sonication, the sonication procedure may not have been successful. This corresponds to the box in the lower right corner of Fig. 5B.

[0072] Referring back to Fig. 5 A, in variations of the above, in some implementations, such as tumors of a certain volume (e.g., larger than 1cm3), the predetermined threshold for the increase in biomarker concentrations before and after the sonication procedure may be modified according to the ROI size. In this context, the ROI size may be specified, for example, as an area parameter or a volume parameter. The ROI size may be determined by the FUS. The decision system 554 may have access to a LUT, where biomarker pre-to-post sonication increase thresholds may be stored according to ROI size or the decision system 554 may be otherwise configured to determine an appropriate biomarker pre-to-post sonication increase threshold based on ROI size. The LUT can store a range of ROI sizes. The actual ROI size may not correspond to an ROI size within the LUT. In such embodiments, the threshold for next lower ROI size may be utilized or a threshold may be interpolated between LUT values.

[0073] As previously described, the decision system 554 may comprise a standalone computer that may include a computing infrastructure, memory and a display and ports for communicating information. Alternatively, the decision system may comprise part of the FUS 550 for example as part of the system in Fig. 1 A. The decision system 554 may also be included as part of the liquid biopsy system 552 (for example, the computations may be carried out at LB3).

[0074] In some embodiments, the physician may read the display messages or other indicia from the decision system 554 and use such information appropriately (along with other possible information) to choose the best treatment course for the patient.

[0075] In some embodiments, the liquid biopsy analysis is informed by the FUS 550 used for opening the BBB. Information obtained by and / or from the FUS 550 may be used to select the parameters used for the liquid biopsy analysis. Example information that may be conveyed from the FUS 550 is the volume of the BBB opening. Other information may also be conveyed from the FUS 550 to the liquid biopsy system 552, such as that described herein.

[0076] When the BBB is opened in the brain, biomarkers are expected to cross into the blood stream from the surrounding brain tissue. For example, an increase in concentration of biomarkers is expected in the peripheral blood supply. If the region where the BBB is opened has a tumor, then the biomarkers may be both tumor and non-tumorrelated. In the example of the biomarker of cfDNA, the tumor related cfDNA is called the ctDNA. Non-tumor related cfDNA may originate from one or multiple sources, such as necrotic cells, endothelial cells, or apoptotic cells that may be in the brain tissue in the vicinity of the BBB opening. In some embodiments, the FUS 550 provides an estimate of an amount (e.g., how much) or an increase in ctDNA or non-tumor cfDNA that may be expected in the peripheral blood for the specific volume of BBB planned to be opened. The liquid biopsy system 552 may use this estimate to adjust the read depth to ensure the proper biomarker data analysis.

[0077] NGS panel assays (non-tumor informed panels) typically need to observe a minimum number of mutations to determine if a mutation is present. This number is called the allelic fraction, e.g., the number of times a mutated base is observed, divided by the total number of times any base is observed at the locus. Different genes have different thresholds of an expected tumor fraction depending upon mutation location, type of mutation, and tumor type. In most cancers, the non-tumor cfDNA is contributed by white blood cells that are shedding their germline DNA into the blood, which adds considerable cfDNA concentrations compared to the ctDNA. In most cancer patients, allelic fraction or (ctDNA:cfDNA ratios) range from 0. l%-10%. In the focused ultrasound-based BBB opening, as described above, opening the BBB (regardless of whether a tumor exists at the BBB opening region or not), may increase the non-tumor cfDNA concentration. Thus, the denominator in the allelic fraction may be affected by the non-tumor cfDNA. Based upon the BBB opening volume, the amount of non-tumor cfDNA may be large enough to disrupt the diagnostic accuracy of the NGS panel. The diagnostic accuracy may be increased by increasing the read depth. Thus, the information provided by the FUS 550 with regard to the BBB opening volume may be used to adjust the read depth of the liquid biopsy system.

[0078] Fig. 6A illustrates an example system including a FUS capable of opening the BBB. The system 600 may include a FUS 650 and liquid biopsy system 652. The FUS 650 of Fig. 6A may include an implementation of the system illustrated by Fig. 1A and / or may be combined with a liquid biopsy system 652, in some embodiments. The figure also illustrates an output 661 from the FUS 650 coupled or sent into the input of the liquid biopsy system 652. In this example, the output 661 of the FUS 650 is the BBB opening volume. Variations of example FUS output(s) 661 provided to the liquid biopsy system 652 are further described below. The liquid biopsy system 652 may also be provided theblood sample 651 as input. In addition, an optional “parameter adjust” input 667 to the liquid biopsy system 652 is also shown. The optional parameter adjust input 667 may be used to modify the parameters such as read depth of the liquid biopsy system 652. The output 662 of the liquid biopsy system 652 is the results of the blood sample analysis.

[0079] The BBB opening volume may be used to estimate the cfDNA due to nontumor sources. This may be performed in various methods. In a non-limiting example method, a predetermined table may be stored with the FUS 650 in which the opening volume (actual or estimated) is associated with the amount of the specific biomarker (e.g., cfDNA) increase that is expected in the peripheral blood when the BBB is open with focused ultrasound. In some embodiments, the data in the table may be entered a priori based on independent research or other methods. In some embodiments, the table may also be made more detailed in that it may contain an estimated increase in the specific biomarker that is dependent on at least one additional or alternative factor. As non-limiting examples, such additional or alternative factors include type of brain disease, age, sex, location in the brain, stage of disease, etc. This table may reside within the computation environment of the FUS 650, such as illustrated by Fig. 6B.

[0080] Fig. 6B illustrates an example method for using a normalization table that may normalize the results of the liquid biopsy, according to example embodiments. The method 663 may be implemented using the system 600. For example, in Fig. 6B, the table 664 called the “Normalization table” is illustrated within the FUS 650 . This normalization table 664 may contain the relationship between estimated or actual opening volume 661 and the expected increase in the brain biomarker 665 in the peripheral blood after a safe opening of BBB. As described above, the table 664 may be multi-dimensional and may include a number of additional or alternative factors (other than opened BBB volume). In various embodiments, the output of this normalization table 664 is the expected increase in the brain biomarker in the peripheral blood.

[0081] In some embodiments, the normalization table 664 may reside within the liquid biopsy system 652. For example, the normalization table 664 may reside within the liquid biopsy system 652 described previously and may be used with at LB3 of the system at described previously. The normalization table 664 may additionally or alternatively be implemented within another computer system (a third computer system) that takes the BBB opening volume as input and outputs the expected increase of the brain biomarker(s). In some embodiments, the decision system of Figs. 5 A and 5B may be combined with thenormalization system of Figs. 6A and 6B. In such embodiments, the normalization system may be implemented within the decision system, such as shown by Fig. 6C.

[0082] Fig. 6C illustrates a combination of a decision system and a normalization system, according to example embodiments. In the system 670 and / or method of Fig. 6C, the decision system 654 receives input 653, 655 from the FUS 650 and the liquid biopsy system 652. Based on the opening volume messages (e.g., 653), the decision system 654 may automatically adjust the read depth or other parameters of the liquid biopsy system 652. Once the liquid biopsy test is carried out (e.g., with pre- and post-sonication samples), the results (655) are sent to the decision system 654. The results may be qualified, such as according to table shown in Fig. 5B. The final display messages may be displayed on the display mechanism of the decision system 654 as shown by the output 672.

[0083] While the concept of modifying the read depth on the basis of the characteristics of the planned or achieved sonication is described above, other liquid biopsy parameters may also be modified similarly based on characteristics of the planned or achieved sonication. Variant calling is one such liquid biopsy parameter. As described above, in some embodiments, the accuracy of the sequencing may be modified by the cfDNA from non-tumor cells. As an example, the minimum allele frequency may be decreased if a large volume of non-tumor cfDNA is to be expected. A large volume of non-tumor may occur if, for example, the sonicated volume is larger than the tumor volume. This implies that more variant alleles have to be found to support the variant call. Said differently, if the non-tumor cfDNA by chance has the same variant that physicians are interested in, then it is more appropriate to increase the minimum allele frequency to support a higher confidence result. As another example, if the sonicated volume and the tumor volume are more similar, the ratio of ctDNA to cfDNA may be closer to 1. In such a case, it may be appropriate to increase the expected allelic frequency.

[0084] In the context of Fig. 6C, the FUS 650 may send data and communication to the decision system 654 which may in turn calculate or determine the parameter(s) 667 used in the liquid biopsy system 652. The value or values of such parameter(s) 667 may be passed to the liquid biopsy system 652 and used in the appropriate locations with the processing steps. The liquid biopsy results 655 modified by the parameters may be output to the liquid biopsy system 655.

[0085] In some embodiments, the BBB opening volume may be optimized depending on a threshold confidence value (e.g., a desired and / or needed confidence level). Earlier it was noted that the relationship between the concentration and the confidence value may be known apriori. This relationship may be stored electronically within the FUS 650 in the form of a look up table. During the planning stage prior to the ultrasound procedure to open the BBB, the physician may refer to the table of relationship and specify an ROI with a size that corresponds to the threshold confidence value. If the actual tumor is smaller than the desired ROI for a specific confidence value, then the physician may make the ROI fit the tumor; however in this case, the FUS 650 may send a message to the decision system that conveys the information about the expected confidence value per the size of the ROI selected. In some embodiments, if the tumor is large, the ROI may be smaller than the tumor for the threshold confidence value. Once the ROI size is established, the FUS 650 may decide how the transmit foci are placed to achieve the BBB opening region that matches the ROI as closely as possible.

[0086] DISTINGUISHING TUMOR RECURRENCE AND TISSUE NECROSIS

[0087] In some embodiments, the methods, devices, and systems described herein may be used to distinguish between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis. As an example, in some patients, such as patients with glioblastoma, a tumor may reoccur at or near the site where a surgery may have been performed to remove the tumor at the time of the initial finding. Typically, in such recurrent tumors and in all brain tumors in general, the BBB is somewhat leaky; when imaged with MRI in the presence of an MRI contrast agent (e.g., gadolinium), the brightness of the images corresponding to the location of the tumors increases. The BBB being leaky may include the disease damaging the BBB tissue enough to disrupt the (normal) anatomy of the tight junctions making up the BBB but not in a way that lets large molecule drugs or biomarkers through the BBB. To explain this more, a healthy BBB would not let the MRI contrast agent pass through; however, brain cancers degrade the integrity of the BBB allowing the MRI contrast through from the blood into the tissue. Thus in MR images, brain tumors may appear bright. This is at least one basis of tumor diagnosis in the brain. However, in some implementations, the treatment following the initial diagnosis may have included radiation and the tissue in such areas may have necrosed or undergone necrosis. In some implementations, where tumor recurrence is occurring or tissue is necrosed or undergoing necrosis, each of these regions may appearbright in MRI imaging. Thus, radiation necrosis and a true tumor recurrence may present similarly in MRI imaging (i.e., regions in an MRI corresponding to either of these circumstances may look bright) and may potentially lead to an incorrect diagnosis, or, more commonly, a delayed diagnosis of tumor recurrence.

[0088] Another factor that makes the confirmation of necrosed tissue challenging is the biology of necrosed tissue. If a region is necrotic, for example from radiation necrosis, the tissue may be devoid of vasculature which implies that this region may have diminished perfusion. This further means that the focused ultrasound method that uses peripherally infused microbubbles to open the BBB, may not work appropriately as this region may be devoid of vasculature or contain damaged vasculature. As an example, in a non-necrosed region where the vasculature is present and allows microbubbles to flow through, the harmonic components of the signals from the microbubbles may be used in a feedback loop to adjust the input excitation to the transmitting transducers. However, if tissue is necrosed, the harmonic components may not be present or may be highly diminished. The feedback loop may not work adequately under these circumstances, potentially leading to the transmission of more ultrasound energy into the tissue than necessary or desirable.

[0089] Figs. 7A-7G illustrate an example technique for identifying tissue necrosis, according to example embodiments. In some embodiments, it may be possible to distinguish between tumor recurrence and radiation necrosis. This concept is described in connection with Fig. 7A. This concept may be applied independently or may be applied before and / or after the BBB is attempted to be opened in a region which is potentially necrosed. In Fig. 7A, a patient’s head 781 is shown along with the ultrasound cap 706 (and coupling material 780), such as that previously described in connection with Fig. 1 A. As described above, this cap 706 and related instrumentation may direct ultrasound energy to the patient specified region-of-interest (ROI) 783 of the brain 782 in order to open the BBB temporarily. When the appropriate electrical energy is applied to the transducers in the cap 706, the ultrasound energy generated by the transducers opens the BBB when microbubbles are present within the circulatory system of the patient. To determine if a region of tissue is necrotic, in some embodiments, the transmit ultrasound beam may be focused at least one location; different techniques are described whether one or multiple focal locations are used. If multiple locations are used, the locations may be within and / or outside the ROI 783. The ROI 783 may be specified in different ways which are describedfurther herein. The ultrasound echo signals from the tissue, which may or may not have sufficient blood flow, may be analyzed from each location of the transmit focus. By analyzing the harmonic content of these signals, it can be determined if a region is necrotic, as further described below.

[0090] Figs. 7A-7D illustrate this concept. In Fig. 7A, a “Tissue region-of-interesf ’ is schematically shown. The physician may want to determine if this ROI 783 is necrotic. In Fig. 7A, three positions of the transmit beam focus are shown, i.e., in this example, three transmit beams are used and are successively focused at Position 1, Position 2, and Position 3, respectively. The monitoring transducers that are part of the ultrasound cap 706 may also be focused at the locations of the transmit focus. The monitoring transducers may continually receive the signals (echo signals) coming back from the tissue and from the microbubbles from within the tissue. These signals are analyzed; in particular, the harmonic content of these signals may be determined. In some embodiments, a low-power transmit mode may be utilized for the transmit beams to distinguish between tumor recurrence and radiation necrosis. The low-power transmit mode is such that the in-situ pressure at the focal site is less than the “opening” threshold pressure for opening the BBB. For example, at 250KHz transmit frequency, the transmit transducers may be excited such that the in-situ pressure at the focal location is less than 0.2 MPa which may not open the BBB. Alternatively, a low concentration of microbubbles may also be used such as 1 / 5 or 1 / 10 of the typical clinical concentration used to open the BBB. The low microbubble concentration may be used with normal ultrasound power (such that the in- situ pressure is limited to around 0.3 - 0.5 MPa at 250 kHz). In addition, low power ultrasound transmission mode may be used with a low concentration of microbubbles as well. These conditions may be selected such that no BBB opening is caused while performing the necrotic confirmation procedure.

[0091] Several signal analysis techniques may be used to determine if a region is necrotic. For example, the ratio of the amplitude at second harmonic frequency to amplitude at the fundamental may be analyzed. Referring to Figs. 7A-7D, in Fig. 7A, the transmit ultrasound beam is focused at Position 1. Fig. 7B demonstrates an example of the result after the received return signal from the Position 1 focus is analyzed. Fig. 2B shows the received signal amplitude to have a frequency content at the second harmonic (2fo) frequency and the (fundamental) first harmonic (fo) frequency. A ratio of the amplitude at 2fo to the amplitude at fo is shown. The signal may have other frequency components aswell. These other frequency are not shown in the Figs. 7B-7D for clarity. The second harmonic to first (fundamental) harmonic ratio (■ ■) from Position 1 may be stored in |^(fo)| memory residing within the system (see Fig. 1 A). Here the notation A(2fo) and the notation A(fo) stands for the amplitude of the signals at 2fo and at fo respectively. The transmit beam and the monitoring transducers are subsequently focused on Position 2 which is within the ROI 783. If this tissue were necrotic, then the second harmonic to firstW o)l(fundamental) harmonic ratio ( ) for Position 2 may be different compared to that|^(fo)| from Position 1 as the harmonic signal may be significantly smaller or non-existent. This is shown Fig. 7C. The ratio from Position 2 may also be stored in memory. The transmit beam may be focused on additional locations either within the ROI 783 or outside the ROI783. In some embodiments, the average of the amplitude ratios (i.e., ratios of the amplitude at second harmonic to first harmonic or fundamental signals) for the focal regions outside the ROI 783 may be found and compared to the average of the corresponding ratios for the focal regions inside the ROI 783. If the comparison is such that the ratio is below a suitable threshold, then it may be concluded that the ROI 783 is necrotic. Thus, in some embodiments, the average of the second harmonic to firstIW(fundamental) harmonic amplitude ratio (■ ) for the regions outside the ROI 783 may Wo) I be 1 : 1 and the average of the second harmonic to first (fundamental) harmonic to amplitude ratio ('f^°2') for the regions within the ROI may be 0.25 : 1. This may be the case if the ROI 783 were necrotic. The threshold may be set at different levels. For example, if the second harmonic to first (fundamental) harmonic amplitude ratio ( W / b)lWo) I of the signals arising from within the ROI 783 is less that 50% of the level for the signals from outside the ROI 783, a decision may be made that the ROI 783 is necrotic. Other thresholds may be used. An advantage of the configuration where multiple transmit and receive focal zones are used with low power, is that by placing these focal zones in the vicinity of the ROI 783 and then sweeping the zones through the ROI 783, the approximate size of the necrotic region may be determined. This is illustrated in Fig. 7E.The size may be estimated by storing the second harmonic to first (fundamental) harmonicIWO amplitude ratio ( ) for each of the multiple locations, and by storing the locations C / b)l themselves and then overlaying a representation of the second harmonic to first(fundamental) harmonic ratio on the diagnostic image 785 (for example, the diagnosticMRI). Thus, in Fig. 7E, the inner region 789 is shown by a hatch pattern slanting from top left to bottom right. In this region 789, the second harmonic to first (fundamental) harmonic amplitude ratio ) may be < 0.5: 1 indicating the approximate size of theWo) I necrotic region. In Fig. 7E, an outer region 787 is also shown but the display of the outer region 787 is optional. In this example, regions (789) where the second harmonic to first(fundamental) harmonic amplitude ratio is larger than 05: 1 is shown by a different crosshatch pattern.

[0092] In the embodiments described above, multiple transmit and receive focal points are used. In some embodiments, one focal region is used that is placed within theROI to be examined. If this region is necrotic, as described above, the blood volume and therefore the second harmonic signals may be diminished or non-existent. In such embodiments, instead of comparing the second harmonic to first (fundamental amplitudes) harmonics from adjacent locations, the comparison of the second harmonic amplitude at the one focus location may be made to a threshold value which may be predetermined and which may be stored in memory. For such embodiments, although a low power transmit mode may be used, it may be beneficial to use normal power transmit mode. However, using normal power transmit mode may result in opening the BBB if the tissue is non- necrotic and non-cancerous. To overcome opening the BBB of a healthy section of the tissue (i.e., if the tissue is non-necrotic and if the tissue is non-cancerous), the single focal location technique may optionally be used with another technique. Here, the in-situ pressure may be ramped up from a low value with the transmit focus placed within theROI. Noting that the in-situ pressure is a function of the input excitation voltage, the second harmonic to fundamental amplitude ratio may be stored for every value of the input excitation voltage. As the excitation voltage is ramped up, if the ROI is necrotic, the second harmonic amplitude level may not increase in value commensurate with the increased excitation. Thus, several values of the second harmonic amplitude may be analyzed and stored internally. Fig. 7F demonstrates an example of such a graph constructed from these values. The graph plots the second harmonic to first (fundamental)W harmonic amplitude ratio ) as a function of increasing excitation voltage. The Wo) I example measurement points are shown by “x” . A line called the “measured line” is fitted with these measurement points. In this example, the slope of the measured line is close to0, indicating that this region is (possibly) necrotic. A “reference line” is also included in Fig. 7F to demonstrate that the slope may be >0 and for signals that emanate from non- necrotic regions. Thus, in the system and / or device described in Figs. 1 A-1B, a value of the slope may be predetermined and stored in the system and / or device. The predetermined slope may be set with values such as ’A or 14 . If the slope of the measured line is less than the predetermined slope, the system may output a message that the tissue in question may be necrotic. While Fig. 7F shows a linear line, this line may be non-linear in various embodiments.

[0093] In some embodiments, a physician may initially specify the ROI which is to be examined. The physician may specify the ROI using, for example, the MRI images of the patient. In some embodiments, the ROI for the current ultrasound procedure (such as the procedure to determine if a tissue region is necrotic) may be specified from a prior or a previously conducted procedure, and / or not by a physician. As an example, a previously conducted procedure may involve a physician specifying a ROI in an MRI image of a patient scheduled to be treated with radiation for a brain metastatic tumor. The previous MRI and the ROI associated with that previous treatment may be imported into the system for the current session. The current session may include obtaining a new MRI. The previous MRI may be aligned and mapped to the current MRI; subsequently, the previous ROI may also be aligned and mapped to the current MRI. The newly mapped ROI maybe be input to the concept described above. Once the coordinates of the ROI are specified, the guidance of the ultrasound energy may be conducted as described herein.

[0094] Fig. 7G illustrates an example method for identifying tissue necrosis in a flow chart, according to example embodiments. In the example method, the physician may determine if a region in the brain is necrotic or if the region is experiencing a recurrent tumor growth. In some embodiments, the order of the steps may differ from that shown in Fig. 7G.

[0095] The “preplanning stage” at 790, is typically accomplished prior to the time the patient is present in the location where the ultrasound procedure is planned to occur. Some or many of the preplanning tasks may be carried out in a preplanning computer. It is to be noted that the preplanning computer may be a standalone computer or may be integrated within the FUS. For the purposes of clarity, the preplanning stage tasks, at 790, may be grouped into five example tasks. These are:1. Import previously obtained image of patient (e.g., diagnostic MRI). Other non-image data (e.g., the second-harmonic to fundamental amplitude ratio) may also be imported from a previously conducted procedure or study. Prior image data may be stored in a preplanning computer and / or in the FUS. The non-image data may be stored within the main FUS as some of this data may be used while the sonication procedure to open the BBB is occurring. Import or specify ROI. As specified above, the ROI may be specified by a physician, for example on the preplanning computer. Additionally or alternatively, the ROI may be imported from a previously conducted procedure. Regardless of the method for specifying the ROI, the system may ask for the physician’s approval of the ROI before proceeding. The physician may use a tool provided by the system for modifying / specifying the ROI. For exploring whether a region of the tissue is necrotic or has recurrent disease, the ROI may be assumed to be the region that the physician is interested in, regardless of how the ROI is input. A user (typically a physician) selects which method is used to confirm recurrence or necrotic region. Multiple methods and variations are described above. An example method uses a low power technique and a transmission sequence that involves focusing the transmit beam at multiple locations that are placed both inside and outside the ROI. Another example method uses one focal region inside the ROI and either ramping in-situ pressure or the pressure that may open the BBB (in a healthy tissue). The low power setting may change with the frequency of transmissions but for a frequency of around 250 kHz, the transducers may be excited such that the in-situ pressure at the focal regions are less than about 0.2-0.3 MPa. The normal power setting which opens the BBB for the same frequency is approximately >0.3-0.4 MPa and <0.5 MPa. Other ranges may be used. As used herein, a transmission sequence is defined as a set of low-frequency transmit beams. The set of transmit beams may be divided into at least one sub-set, where each sub-set may be focused at a specific region within the brain. Each sub-set may include at least one transmission events, where each transmission event may be 10ms long, repeated at a frequency of 1 Hz for a duration of 2 min. Other parameters may be used. Depending on inputs including the input of the method of recurrent / necrotic confirmation and the ROI, the ultrasound system executes preplanning steps including calculating the beamforming parameters for the at least one locationwhere low-frequency beam is to be guided.5. Ultrasound system may display results of preplanning to obtain approval from the physician before proceeding.

[0096] Patient Preparation at 791. The tasks may be divided into at least three groups:1. Patient is seated in a chair, such as an infusion chair, patient positioning on a bed is not precluded and may be addressed by the system, and preparation may include placing a catheter for injection of microbubbles.2. Appropriate components of the ultrasound system, such as cap, gel, conforming jacket may be placed on the patient.3. Initial calibration steps may be performed, such as checking for air bubbles. This check may be done visually; alternatively, an electronic method such as a transmit and receive sequence from one or multiple elements on the cap may also be used.

[0097] Registration and adjustment calculations, at 792. The tasks may be divided into at least two groups:1. A registration step may be performed to register coordinate frame of the cap to the patient’s anatomy using previously obtained MR and / or CT images.2. Additional beamforming calculations may optionally be performed to accommodate for the actual position of the cap. A method to perform the additional beamforming calculations may include at least some of substantially the same features and attributes as described in PCT Publication No. WO2024 / 025927).

[0098] Low-frequency (LF) ultrasound transmission and collection of data at 793. The tasks may be divided into at least two groups:1. Depending on the transmission sequence determined in the preplanning stage, the ultrasound system commences the procedure for identifying a recurrent or necrotic region, and2. In parallel, as the transmission sequence is commenced, the echo data from the monitoring transducers in the cap are received, digitized, and stored in the ultrasound system memory for analysis.

[0099] Data Analysis at 794. Depending on the transmission sequence, data analysis is performed to determine if a region is necrotic or the tumor is recurrent. The data analysis may be different depending on the type of transmission sequence used as described above.

[0100] Results display and further analysis at 795.1. The results of the analysis may be displayed. This results display may be in the form of a message such as “Based on the analysis, minimal or no enhancement was seen in the ultrasound signals from the ROI. Thus this region is suspected to be necrotic”. The results may be graphical in nature. For example, the system may display the previously acquired MR image and display the second harmonic to fundamental ratio for the regions that were sonicated. A color map may be used to encode the values of the ratios for different focal locations. If the overlayed color is same or nearly the same for the ROI and surrounding areas, that is an indication that the tissues are similar (i.e., non-necrotic and possibly containing a recurrent disease).2. The results may also be provided as messages that may dictate further action. These messages may be displayed on the system screen or may be input to a different system. As an example, if the low-power method is used and the analysis is suggestive of a recurrent tumor, the physician may choose to open the BBB at the ROI and perform a genomic analysis to determine the genetic makeup of the tumor.

[0101] Fig. 8 illustrates an example device capable of identifying tissue necrosis, according to example embodiments. The device 810 may include an example implementation or at least some of the features and attributes of the device 810 as previously described in connection with FIG. 8. For example, the device 810 may form part of the FUS and / or a decision system. The device 810 includes a memory 814 storing a set of instructions 896, 898 and a processor 812 coupled to the memory 814 to execute the instructions 896, 898 to perform the following.

[0102] At 896, the processor 812 may analyze an ultrasound echo signal from tissue responsive to an ultrasound beam focused to a location of brain of a patient. At 898, the processor 812 may distinguish between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis based on the analysis. As described above, the tissue experiencing tumor recurrence or experiencing radiation necrosis may include brain tissue and the tumor may be a brain tumor.

[0103] In some embodiments, the ultrasound beam is focused to the location and the patient contains injected microbubbles. The processor 812 may analyze the ultrasound echo signal from tissue responsive to the ultrasound beam. As described above, the tumortissue may contain microbubbles and necrosis tissue may not contain microbubbles or may contain less microbubbles than the tumor tissue (e.g., minimal).

[0104] In some embodiments, the ultrasound beam may be output at a power (e.g., voltage) that is insufficient to open the BBB, which may be referred to as “low power”. In some embodiments, the ultrasound beam is output at a power insufficient to open the BBB and / or microbubbles are provided at a concentration that is insufficient to open the BBB. However, embodiments are not so limited and relatively higher power and / or high concentrations of microbubbles may be used.

[0105] In some embodiments, the ultrasound beam comprises a plurality of ultrasound beams focused to a plurality of locations and the ultrasound echo signal comprises a plurality of ultrasound echo signals, and the processor 812 may analyze the plurality of ultrasound echo signals from the tissue responsive to the plurality of ultrasound beams.

[0106] In some embodiments, the processor 812 may assess harmonic components of the ultrasound echo signal to distinguish tissue experiencing tumor recurrence from tissue experiencing radiation necrosis. The harmonic components may include first and second harmonics. For example, the processor 812 may assess the harmonic components by: (i) assessing the harmonic components of the plurality of ultrasound echo signals derived from the plurality of locations, wherein the plurality of locations are adjacent one another and are respectively associated with a region of interest and regions outside the region of interest; or (ii) assessing the harmonic components of the ultrasound echo signal derived from the location, wherein assessment comprising comparing the harmonic components to a threshold.

[0107] In some embodiments, the processor 812 may assess the harmonic components by determining a ratio of the second harmonic to the first harmonic, and comparing the ratio of the second harmonic to the first harmonic to the threshold, as described above.

[0108] In some embodiments, the processor 812 may determine the ratio of the second harmonic to the first harmonic for each of the plurality of ultrasound echo signals derived from the plurality of locations, and compare the ratio for each of the plurality of ultrasound echo signals derived from the plurality of locations to the threshold. The processor 812 may further distinguish between the tissue experiencing tumor recurrence and tissue experiencing radiation necrosis by: (i) identifying tissue in locations of the plurality of locations with ratios of the second harmonic to the first harmonic being above the threshold as tissue experiencing tumor recurrence; and (ii) identifying tissue inlocations of the plurality of locations with ratios of the second harmonic to the first harmonic being below the threshold as tissue experiencing radiation necrosis.

[0109] In some embodiments, the processor 812 may store the ratios and the locations of the plurality of locations associated with the tissue experiencing radiation necrosis. In some embodiments, the processor 812 may determine a size of a necrosed region (in the brain) based on the stored ratios and locations.

[0110] In some embodiments, the ultrasound beam focused to the location comprises a plurality of ultrasound beams of different power strengths focused to the location and the ultrasound echo signal comprises a plurality of ultrasound echo signals which are responsive to the plurality of ultrasound beams. In some such embodiments, the processor 812 may: (i) assess harmonic components of the plurality of ultrasound echo signals derived from the location, the harmonic components including first and second harmonics; (ii) determine a ratio of the second harmonic to the first harmonic for each of the plurality ultrasound of echo signals derived from the location; and (iii) identify the tissue within the location as being tissue experiencing tumor recurrence or tissue experiencing radiation necrosis based on a change in the ratio across the plurality of ultrasound echo signals.

[0111] In some embodiments, the processor 812 may identify the tissue as being necrosis tissue in response to the ratio not increasing or increasing below a threshold across the plurality of ultrasound beams of different power strengths.

[0112] Various embodiments are directed to systems which include the device 810 or portions thereof. For example, the system may include a FUS configured to open the BBB of a patient, BBB, and the processor which is arranged with the FUS and configured to analyze an ultrasound echo signal from tissue responsive to the ultrasound beam focused to the location of a brain of a patient, and distinguish between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis based on the analysis, as described above. The processor of the system may perform any of the above-described variations, as described in connection with Fig. 8. As previously described, the processor may form part of the FUS and / or a decisions system.

[0113] Fig. 9 illustrates an example method for distinguishing between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis, according to example embodiments. The method 997 may include an implementations of any of the methods illustrated by FIG. 7A-7G and / or may be implemented using the device 810 of Fig. 8.

[0114] As shown by Fig. 9, the example method 997 comprises opening a blood-brain barrier of a patient by a FUS, at 998, and responsive to the opening of the blood-brain barrier, obtaining a liquid biopsy measurement for biomarker using a liquid biopsy system, at 999. The method may further comprise the variations as described above in connection with Fig. 8, the common features and attributed not being repeated for ease of reference.

[0115] As used, herein, “patient” refers to or includes human and other veterinary subjects, including not limited to mammals. A patient may be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Other example patients include domesticated animals (e.g., cats, dogs), livestock (e.g., cattle, horses, pigs, sheep, goats), and laboratory animals (e.g., monkeys, mouse, rabbit, rat, guinea pig, pigs, fruit fly). In some embodiments, the patient may be afflicted with a disease or disorder.

[0116] Unless the context clearly requires otherwise, throughout the description and the claims:• “comprise”, “comprising”, and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”;• “connected”, “coupled”, or any variant thereof, means any connection or coupling, either direct or indirect, between two or more elements; the coupling or connection between the elements may be physical, logical, or a combination thereof• “herein”, “above”, “below”, and words of similar import, when used to describe this specification, shall refer to this specification as a whole, and not to any particular portions of this specification;• “or”, in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list;• singular forms “a”, “an”, and “the” also include the meaning of any appropriate plural forms.

[0117] Words indicating directions (e.g., “left”, “right”, “front”, “back”, “top”, and “bottom”) used herein may depend on the orientation of the apparatus illustrated. The subject matter described herein may assume alternative orientations.

[0118] Embodiments may be implemented using specifically designed hardware, configurable hardware, programmable data processors configured by the provision ofsoftware (which may optionally comprise firmware) capable of executing on the processors, special purpose computers or data processors that are specifically programmed, configured, or constructed to perform at least one step in a method as explained in detail herein and / or combinations of two or more of these. Examples of specifically designed hardware are: logic circuits, application-specific integrated circuit (ASICS), large scale integrated circuits (LSIs), very large scale integrated circuits (VLSIs), among others. Examples of configurable hardware are: at least one programmable logic device such as programmable array logic (PALSs), programmable logic arrays (PLAs), and field programmable gate arrays (FPGAs). Examples of programmable data processors are: microprocessors, digital signal processors (DSPs), embedded processors, graphics processors, math co-processors, general purpose computers, server computers, cloud computers, mainframe computers, and / or computer workstations, among others. A processor in a control circuit for a device may implement example by executing instructions in a program memory accessible to the processors.

[0119] Processing may be centralized or distributed. Where processing is distributed, information including software and / or data may be kept centrally or distributed. Such information may be exchanged between different functional units by way of a communications network, such as a Local Area Network (LAN), Wide Area Network (WAN), or the Internet, wired or wireless data links, electromagnetic signals, or other data communication channel.

[0120] For example, while processes or blocks are presented in a given order, alternative examples may perform routines having steps, or employ systems having blocks, in a different order, and some processes or blocks may be deleted, moved, added, subdivided, combined, and / or modified to provide alternative or subcombinations. Each of these processes or blocks may be implemented in a variety of different ways. While processes or blocks are at times shown as being performed in series, these processes or blocks may instead be performed in parallel, or may be performed at different times.

[0121] In addition, while elements are at times shown as being performed sequentially, they may instead be performed simultaneously or in different sequences. It is therefore intended that the following claims are interpreted to include all such variations as are within their intended scope.

[0122] Software and other modules may reside on servers, workstations, personal computers, tablet computers, image data encoders, image data decoders, PDAs, color-grading tools, video projectors, audio-visual receivers, displays (such as televisions), digital cinema projectors, media players, and other devices suitable for the purposes described herein. Those skilled in the art will appreciate that aspects of the system may be practiced with other communications, data processing, or computer system configurations, including: Internet appliances, hand-held devices (including personal digital assistants (PDAs)), wearable computers, all manner of cellular or mobile phones, multi-processor systems, microprocessor-based or programmable consumer electronics (e.g., video projectors, audio-visual receivers, displays, such as televisions), set-top boxes, colorgrading tools, network PCs, mini-computers, and mainframe computers, among others.

[0123] The invention may also be provided in the form of a program product. The program product may comprise any non-transitory medium which carries a set of computer-readable instructions which, when executed by a data processor, cause the data processor to execute a method of the invention. Program products according to the invention may be in any of a wide variety of forms. The program product may comprise, for example, non-transitory media such as magnetic data storage media including floppy diskettes, hard disk drives, optical data storage media including CD ROMs, DVDs, electronic data storage media including ROMs, flash RAM, EPROMs, hardwired or preprogrammed chips (e.g., EEPROM semiconductor chips), or nanotechnology memory, among others. The computer-readable signals on the program product may optionally be compressed or encrypted.

[0124] In some embodiments, the invention may be implemented in software. “Software” includes any instructions executed on a processor, and may include (but is not limited to) firmware, resident software, and / or microcode, among others. Both processing hardware and software may be centralized, distributed, or a combination thereof, in whole or in part. For example, software and other modules may be accessible via local memory, via a network, via a browser or other application in a distributed computing context, or via other means suitable for the purposes described above.

[0125] Where a component (e.g., a software module, processor, assembly, device, circuit, etc.) is referred to above, unless otherwise indicated, reference to that component (or a “means”) should be interpreted as including as equivalents of the component which performs the function of the described component (i.e., that is functionally equivalent), including components which are not structurally equivalent to the disclosed structure which performs the function in the illustrated embodiments.

[0126] Specific examples of systems, methods and apparatus have been described herein for purposes of illustration. These are only examples. The technology provided herein may be applied to systems other than the example systems described above. Many alterations, modifications, additions, omissions, and permutations are possible within the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and / or acts with equivalent features, elements and / or acts; mixing and matching of features, elements and / or acts from different embodiments; combining features, elements and / or acts from embodiments as described herein with features, elements and / or acts of other technology; and / or omitting combining features, elements and / or acts from described embodiments.

[0127] Various features are described herein as being present in “some embodiments”. Such features are not mandatory and may not be present in all embodiments. Embodiments of the invention may include zero, any one or any combination of two or more of such features. This is limited only to the extent that certain ones of such features are incompatible with other ones of such features in the sense that it would be impossible for a person of ordinary skill in the art to construct a practical embodiment that combines such incompatible features. Consequently, the description that “some embodiments” possess feature A and “some embodiments” possess feature B should be interpreted as an express indication that the inventors also contemplate embodiments which combine features A and B, unless the description states otherwise or features A and B are fundamentally incompatible.

Claims

CLAIMS1. A device comprising: a memory that stores a set of instructions; and a processor coupled to the memory and configured to execute the instructions to: receive a liquid biopsy measurement for biomarker from a liquid biopsy system which is performed responsive to opening a blood-brain barrier of a patient by a focused ultrasound system; and determine a confidence value associated with the liquid biopsy measurement.

2. The device of claim 1, wherein the processor is configured to execute the instructions to determine the confidence value associated with the liquid biopsy measurement based on at least one of: a location of the opening of the blood-brain barrier after sonication by the focused ultrasound system; and confirmation of the opening of the blood-brain barrier after sonication by the focused ultrasound system based on the liquid biopsy measurement.

3. The device of claim 2, wherein the processor is configured to execute the instructions to receive information from the focused ultrasound system indicative of the location of the opening of the blood-brain barrier.

4. The device of claim 1, wherein the processor is configured to execute the instructions to determine the confidence value based on a focus location of a transmitted ultrasound beam relayed by the focused ultrasound system.

5. The device of claim 1, wherein the processor is configured to execute the instructions to determine the confidence value based on a comparison of a concentration of the biomarker as measured by the liquid biopsy system to a threshold.

6. The device of claim 5, wherein the biomarker comprises a brain biomarker.

7. The device of claim 5, wherein the biomarker comprises a plurality of biomarkers including a brain biomarker and a disease biomarker.

8. The device of claim 5, wherein the threshold is an expected concentration of the biomarker comprising a brain biomarker.

9. The device of claim 5, wherein the processor is configured to execute the instructions to identify the threshold using a look up table (LUT) stored on the memory, wherein the LUT comprises expected concentrations of a plurality of biomarkers.

10. The device of claim 5, wherein the processor is configured to execute the instructions to receive the liquid biopsy measurement including a measured concentration of the biomarker from the liquid biopsy system and to output the confidence value based on the comparison of the concentration of the biomarker to the threshold.

11. The device of claim 5, wherein the processor is configured to execute the instructions to receive the liquid biopsy measurement including a measured concentration of the biomarker comprising a brain biomarker and a measured concentration of a disease biomarker from the liquid biopsy system and to output the confidence value of the measured concentration of the disease biomarker based on the comparison of the measured concentration of the brain biomarker to the threshold.

12. The device of claim 10, wherein the processor is configured to execute the instructions to determine the confidence value based on a focus location of a transmitted ultrasound beam relayed by the focused ultrasound system and a confirmation of the blood-brain barrier opening based on the concentration of the biomarker being equal to or greater than the threshold.

13. The device of claim 1, wherein the processor is configured to execute the instructions to output the confidence value as a message to a user.

14. The device of claim 13, wherein the confidence value is indicative of qualitative confidence in the liquid biopsy results.

15. The device of claim 1, wherein the processor is configured to execute the instructions to determine the confidence value using a set of criteria to evaluate results of the liquid biopsy measurement, as performed by a liquid biopsy system, being applicable to a region of interest.

16. The device of claim 1, wherein the device forms part of: a focused ultrasound system, the liquid biopsy system, or a decision system.

17. The device of claim 1, wherein the processor is configured to execute the instructions to determine the confidence value using a set of criteria associated with identification of an opening location of the blood-brain barrier as relayed by the focused ultrasound system and a concentration of the biomarker based on the liquid biopsy measurement.

18. The device of claim 17, wherein processor is configured to execute the instructions to assess the opening location for overlapping with a region of interest.

19. The device of claim 17, wherein processor is configured to execute the instructions to assess the concentration of the biomarker at least prior to and after sonication.

20. The device of claim 19, wherein the processor is configured to execute the instructions to compare the concentration of the biomarker prior to and after sonication and to assess for at least one of: an increase in the concentration after sonication compared to prior to sonication indicative of a higher confidence value as compared to the biomarker concentration not increasing; and the concentration after sonication being above a threshold indicative of a higher confidence value as compared to the biomarker concentration being less than the threshold.

21. The device of claim 20, wherein the confidence value is indicative of a level of confidence that the blood-brain barrier opened at an intended location.

22. The device of claim 20, wherein the biomarker comprises a plurality of biomarkers including a brain biomarker and a disease biomarker, and wherein the confidence value is indicative of a level of confidence of a reading of the disease biomarker based on a reading of the brain biomarker from the liquid biopsy measurement.

23. The device of claim 20, wherein the processor is configured to execute the instructions to assess whether the opening location is overlapping with a region of interest, which indicates a higher confidence value of a reading of the liquid biopsy measurement pertaining to a disease biomarker as compared to the opening location not overlapping or overlapping less than a threshold amount with the region of interest.

24. The device of claim 1, wherein the processor is configured to execute the instructions to adjust a parameter of the liquid biopsy system based on information derived about the opening of the blood-brain barrier.

25. The device of claim 24, wherein the processor is configured to execute the instructions to output data indicative of the adjusted parameter to the liquid biopsy system.

26. The device of claim 24, wherein the adjusted parameter comprises a read depth, and the processor is configured to execute the instructions to adjust the read depth based on a volume of the opening of the blood-brain barrier.

27. The device claim 26, wherein the processor is configured to execute the instructions to adjust the read depth based on at least one of: the volume of the opening of the blood-brain barrier, an expected concentration of the biomarker, and a concentration of the biomarker determined by the liquid biopsy measurement.

28. The device of claim 27, wherein the biomarker comprises a brain biomarker, cell- free DNA (cfDNA), circulating tumor DNA (ctDNA), or a combination thereof.

29. The device of claim 27, wherein the processor is configured to execute the instructions to determine the adjustment to the read depth using at least one of a look uptable (LUT), an estimated opening of the blood-brain barrier, and the expected concentration of the biomarker.

30. The device of claim 24, wherein the adjusted parameter comprises a variant calling parameter.

31. The device of claim 1, wherein the processor is configured to execute the instructions to normalize results of the liquid biopsy measurement using a normalization table.

32. A method comprising: opening a blood-brain barrier of a patient by a focused ultrasound system; responsive to the opening of the blood-brain barrier, obtaining a liquid biopsy measurement for biomarker using a liquid biopsy system; and determining a confidence value associated with the liquid biopsy measurement.

33. The method of claim 32, wherein determining the confidence value associated with the liquid biopsy measurement is based on at least one of: a location of the opening of the blood-brain barrier after sonication by the focused ultrasound system; and confirmation of the opening of the blood-brain barrier after sonication by the focused ultrasound system based on the liquid biopsy measurement.

34. The method of claim 33, comprising receiving information from the focused ultrasound system to indicative of the location of the opening of the blood-brain barrier.

35. The method of claim 32, wherein determining the confidence value associated with the liquid biopsy measurement is based on a focus location of a transmitted ultrasound beam relayed by the focused ultrasound system.

36. The method of claim 32, wherein determining the confidence value associated with the liquid biopsy measurement comprises comparing a concentration of the biomarker as measured by the liquid biopsy system to a threshold.

37. The method of claim 36, wherein the biomarker comprises a brain biomarker.

38. The method of claim 36, wherein the biomarker comprises a plurality of biomarkers including a brain biomarker and a disease biomarker.

39. The method of claim 36, wherein the threshold is an expected concentration of the biomarker comprising a brain biomarker which is not associated with a disease.

40. The method of claim 36, comprising identifying the threshold using a look up table (LUT) stored on memory, wherein the LUT comprises expected concentrations of a plurality of biomarkers.

41. The method of claim 36, comprising receiving the liquid biopsy measurement including a measured concentration of the biomarker from the liquid biopsy system and outputting the confidence value based on the comparison of the biomarker to the threshold.

42. The method of claim 36, comprising receiving the liquid biopsy measurement including a measured concentration of the biomarker comprising a brain biomarker and a measured concentration of a disease biomarker from the liquid biopsy system and outputting the confidence value of the measured concentration of the disease biomarker based on the comparison of the measured concentration of the brain biomarker to the threshold.

43. The method of claim 41, comprising determining the confidence value based on a focus location of a transmitted ultrasound beam relayed by the focused ultrasound system and a confirmation of the blood-brain barrier opening based on the concentration of the biomarker being equal to or greater than the threshold.

44. The method of claim 32, comprising outputting the confidence value as a message to a user.

45. The method of claim 44, wherein the confidence value is indicative of qualitative confidence in the liquid biopsy results.

46. The method of claim 32, comprising determining the confidence value applying a set of criteria to evaluate a confidence of results of the liquid biopsy measurement, as performed by the liquid biopsy system, being applicable to a region of interest.

47. The method of claim 32, wherein the method is performed using at least one of: a focused ultrasound system, the liquid biopsy system, and a decision system.

48. The method of claim 32, comprising determining the confidence value using a set of criteria associated with identification of an opening location of the blood-brain barrier as relayed by the focused ultrasound system and a concentration of the biomarker based on the liquid biopsy measurement.

49. The method of claim 48, comprising assessing the opening location for overlapping with a region of interest.

50. The method of claim 48, comprising assessing the concentration of the biomarker at least prior to and after sonication.

51. The method of claim 50, comprising comparing the concentration of the biomarker prior to and after sonication and to assess for at least one of: an increase in the concentration after sonication compared to prior to sonication indicative of a higher confidence value as compared to the biomarker concentration not increasing; and the concentration after sonication being above a threshold indicative of a higher confidence value as compared to the biomarker concentration being less than the threshold.

52. The method of claim 51, wherein the confidence value is indicative of a level of confidence that the blood-brain barrier opened at an intended location.

53. The method of claim 51, wherein the biomarker comprises a plurality of biomarkers including a brain biomarker and a disease biomarker, and wherein the confidence value is indicative of a level of confidence of a reading of the disease biomarker based on a reading of the brain biomarker from the liquid biopsy measurement.

54. The method of claim 51, comprising assessing whether the opening location overlaps with a region of interest, which indicates a higher confidence value of a reading of the liquid biopsy measurement pertaining to a disease biomarker as compared to the opening location not overlapping or overlapping less than a threshold amount with the region of interest.

55. The method of claim 32, comprising adjusting a parameter of the liquid biopsy system based on information derived about the opening of the blood-brain barrier.

56. The method of claim 55, comprising outputting data indicative of the adjusted parameter to the liquid biopsy system.

57. The method of claim 55, wherein the adjusted parameter comprises a read depth, and the method comprises adjusting the read depth based on a volume of the opening of the blood-brain barrier.

58. The method claim 57, comprising adjusting the read depth based on at least one of: the volume of the opening of the blood-brain barrier, an expected concentration of the biomarker, and a concentration of the biomarker determined using the liquid biopsy measurement.

59. The method of claim 58, wherein the biomarker comprises a brain biomarker, cell- free DNA (cfDNA), circulating tumor DNA (ctDNA), or a combination thereof.

60. The method of claim 58, comprising determining the adjustment to the read depth using at least one of a look up table (LUT), an estimated opening of the blood-brain barrier, and the expected concentration of the biomarker.

61. The method of claim 55, wherein the adjusted parameter comprises a variant calling parameter.

62. The method of claim 32, further comprising normalizing results of the liquid biopsy measurement using a normalization table.

63. A system comprising: a focused ultrasound system configured to open a blood-brain barrier of a patient; a liquid biopsy system configured to obtain a liquid biopsy measurement for biomarker responsive to the opening of the blood-brain barrier; and a processor configured to: receive the liquid biopsy measurement for the biomarker from the liquid biopsy system; and determine a confidence value associated with the liquid biopsy measurement.

64. The system of claim 63, wherein the processor is configured to determine the confidence value associated with the liquid biopsy measurement based on at least one of: a location of the opening of the blood-brain barrier after sonication by the focused ultrasound system; and confirmation of the opening of the blood-brain barrier after sonication by the focused ultrasound system based on the liquid biopsy measurement.

65. The system of claim 64, wherein the processor is configured to receive information from the focused ultrasound system indicative of the location of the opening of the bloodbrain barrier.

66. The system of claim 63, wherein the processor is configured to determine the confidence value based on a focus location of a transmitted ultrasound beam relayed by the focused ultrasound system.

67. The system of claim 63, wherein the processor is configured to determine the confidence value by comparing a concentration of the biomarker as measured by the liquid biopsy system to a threshold.

68. The system of claim 67, wherein the biomarker comprises a brain biomarker.

69. The system of claim 67, wherein the biomarker comprises a plurality of biomarkers including a brain biomarker and a disease biomarker.

70. The system of claim 67, wherein the threshold is an expected concentration of the biomarker comprising a brain biomarker.

71. The system of claim 67, wherein the processor is configured to identify the threshold using a look up table (LUT) stored on memory, wherein the LUT comprises expected concentrations of a plurality of biomarkers.

72. The system of claim 67, wherein the processor to receive the liquid biopsy measurement including a measured concentration of the biomarker comprising a brain biomarker and a measured concentration of a disease biomarker from the liquid biopsy system and to output the confidence value of the measured concentration of the disease biomarker based on the comparison of the measured concentration of the brain biomarker to the threshold.

73. The system of claim 67, wherein the processor is configured to receive the liquid biopsy measurement including a measured concentration of the biomarker from the liquid biopsy system and to output the confidence value based on the comparison of the concentration of the biomarker to the threshold.

74. The system of claim 73, wherein the processor is configured to determine the confidence value based on a focus location of a transmitted ultrasound beam relayed by the focused ultrasound system and a confirmation of the blood-brain barrier opening based on the concentration of the biomarker being equal to or greater than the threshold.

75. The system of claim 63, wherein the processor is configured to output the confidence value as a message to a user.

76. The system of claim 75, wherein the confidence value is indicative of qualitative confidence in the liquid biopsy measurement.

77. The system of claim 63, wherein the processor is configured to determine the confidence value applying a set of criteria to evaluate a confidence of results of the liquid biopsy measurement, as performed by a liquid biopsy system, being applicable to a region of interest.

78. The system of claim 63, wherein the processor forms part of: the focused ultrasound system, the liquid biopsy system, or a decision system.

79. The system of claim 63, wherein processor is configured to determine the confidence value using a set of criteria associated with identification an opening location of a bloodbrain barrier as relayed by the focused ultrasound system and a concentration of the biomarker based on the liquid biopsy measurement.

80. The system of claim 79, wherein processor is configured to assess the opening location for overlapping with a region of interest.

81. The system of claim 79, wherein processor is configured to assess the concentration of the biomarker at least prior to and after sonication.

82. The system of claim 81, wherein the processor is configured to compare the concentration of the biomarker prior to and after sonication and to assess for at least one of: an increase in the concentration after sonication compared to prior to sonication indicative of a higher confidence value as compared to the biomarker concentration not increasing; and the concentration after sonication being above a threshold indicative of a higher confidence value as compared to the biomarker concentration being less than the threshold.

83. The system of claim 82, wherein the confidence value is indicative of a level of confidence that the blood-brain barrier opened at an intended location.

84. The system of claim 82, wherein the biomarker comprises a plurality of biomarkers including a brain biomarker and a disease biomarker, and wherein the confidence value is indicative of a level of confidence of a reading of the disease biomarker based on a reading of the brain biomarker from the liquid biopsy measurement.

85. The system of claim 82, wherein the processor is configured to assess whether the opening location is overlapping with a region of interest, which indicates a higher confidence value of a reading of the liquid biopsy measurement pertaining to a disease biomarker as compared to the opening location not overlapping or overlapping less than a threshold amount with the region of interest.

86. The system of claim 63, wherein the processor is configured to adjust a parameter of the liquid biopsy system based on information derived about the opening of the bloodbrain barrier.

87. The system of claim 86, wherein the processor is configured to output data indicative of the adjusted parameter to the liquid biopsy system.

88. The system of claim 86, wherein the adjusted parameter comprises a read depth, and the processor is configured to adjust the read depth based on a volume of the opening of the blood-brain barrier.

89. The system claim 88, wherein the processor is configured to adjust the read depth based on at least one of: the volume of the opening of the blood-brain barrier, an expected concentration of the biomarker, and a concentration of the biomarker determined by the liquid biopsy measurement.

90. The system of claim 89, wherein the biomarker comprises a brain biomarker, a cell- free DNA (cfDNA), circulating tumor DNA (ctDNA), or a combination thereof.

91. The system of claim 89, wherein the processor is configured to determine the adjustment to the read depth using at least one of a look up table (LUT), an estimated opening of the blood-brain barrier, and the expected concentration of the biomarker.

92. The system of claim 86, wherein the adjusted parameter comprises a variant calling parameter.

93. The system of claim 63, wherein the processor is configured to normalize results of the liquid biopsy measurement using a normalization table.

94. A device comprising: a memory that stores a set of instructions; and a processor coupled to the memory and configured to execute the instructions to: analyze an ultrasound echo signal from tissue responsive to an ultrasound beam focused to a location of brain of a patient; and distinguish between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis based on the analysis.

95. The device of claim 94, wherein the tissue experiencing tumor recurrence includes brain tissue and the tumor is a brain tumor.

96. The device of claim 94, wherein the ultrasound beam comprises a plurality of ultrasound beams focused to a plurality of locations and the ultrasound echo signal comprises a plurality of ultrasound echo signals, and the processor is configured to execute the instructions to analyze the plurality of ultrasound echo signals from the tissue responsive to the plurality of ultrasound beams.

97. The device of claim 94, wherein the ultrasound beam is focused to the location, the patient contains injected microbubbles, and the processor is configured to execute the instructions to analyze the ultrasound echo signal from tissue responsive to the ultrasound beam.

98. The device of claim 97, wherein the ultrasound beam is output at a power insufficient to open the blood-brain barrier.

99. The device of claim 94, wherein the processor is configured to execute the instructions to assess harmonic components of the ultrasound echo signal to distinguish tissue experiencing tumor recurrence from tissue experiencing radiation necrosis.

100. The device of claim 99, wherein the ultrasound beam comprises a plurality of ultrasound beams focused to a plurality of locations and the ultrasound echo signal comprises a plurality of ultrasound echo signals, and the processor is configured to execute the instructions to analyze the plurality of ultrasound echo signals from the tissue responsive to the plurality of ultrasound beams.

101. The device of claim 100, wherein the processor is configured to execute the instructions to assess the harmonic components by: assessing the harmonic components of the plurality of ultrasound echo signals derived from the plurality of locations, the harmonic components including first and second harmonics, wherein the plurality of locations are adjacent one another and are respectively associated with a region of interest and regions outside the region of interest; or assessing the harmonic components of the ultrasound echo signal derived from the location, the assessment comprising comparing the harmonic components to a threshold, wherein the harmonic components include the first and second harmonics.

102. The device of claim 101, wherein the processor is configured to execute the instructions to assess the harmonic components by: determining a ratio of the second harmonic to the first harmonic; and comparing the ratio of the second harmonic to the first harmonic to the threshold.

103. The device of claim 102, wherein the processor is configured to execute the instructions to: determine the ratio of the second harmonic to the first harmonic for each of the plurality of ultrasound echo signals derived from the plurality of locationscompare the ratio for each of the plurality of ultrasound echo signals; and distinguish between the tissue experiencing tumor recurrence and tissue experiencing radiation necrosis by: identifying tissue in locations of the plurality of locations with ratios of the second harmonic to the first harmonic being above the threshold as tissue experiencing tumor recurrence; and identifying tissue in locations of the plurality of locations with ratios of the second harmonic to the first harmonic being below the threshold as tissue experiencing radiation necrosis.

104. The device of claim 103, wherein the processor is configured to execute the instructions to: store the ratios and the locations of the plurality of locations associated with the tissue experiencing radiation necrosis.

105. The device of claim 104, wherein the processor is configured to execute the instructions to determine a size of a necrosed region based on the stored ratios and locations.

106. The device of claim 94, wherein at least one of the ultrasound beam is output at a power insufficient to open the blood-brain barrier and microbubbles are provided at a concentration that is insufficient to open the blood-brain barrier.

107. The device of claim 94, wherein the ultrasound beam focused to the location comprises a plurality of ultrasound beams of different power strengths focused to the location and the ultrasound echo signal comprises a plurality of ultrasound echo signals which are responsive to the plurality of ultrasound beams, wherein the processor is configured to execute the instructions to: assess harmonic components of the plurality of ultrasound echo signals derived from the location, the harmonic components including first and second harmonics; determine a ratio of the second harmonic to the first harmonic for each of the plurality of ultrasound echo signals derived from the location; andidentify the tissue within the location as being tissue experiencing tumor recurrence or tissue experiencing radiation necrosis based on a change in the ratio across the plurality of ultrasound echo signals.

108. The device of claim 107, wherein the processor is configured to execute the instructions to identify the tissue as being necrosis tissue in response to the ratio not increasing or increasing below a threshold across the plurality of ultrasound beams of different power strengths.

109. A method comprising: analyzing an ultrasound echo signal from tissue responsive to an ultrasound beam focused to a location of brain of a patient; and distinguishing between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis responsive based on the analysis.

110. The method of claim 109, wherein the tissue experiencing tumor recurrence includes brain tissue and the tumor is a brain tumor.

111. The method of claim 109, wherein the ultrasound beam comprising a plurality of ultrasound beams focused to a plurality of locations and the ultrasound echo signal comprises a plurality of ultrasound echo signals, the method comprising analyzing the ultrasound echo signals from the tissue responsive to the plurality of ultrasound beams.

112. The method of claim 109, comprising injecting microbubbles to the patient and, in response, focusing the ultrasound beam to the location and analyzing the ultrasound echo signal from tissue responsive to the ultrasound beam.

113. The method of claim 112, comprising outputting the ultrasound beam at a power insufficient to open the blood-brain barrier.

114. The method of claim 109, comprising assessing harmonic components of the ultrasound echo signal and distinguishing tissue experiencing tumor recurrence from tissue experiencing radiation necrosis using the assessment.

115. The method of claim 114, wherein the ultrasound beam comprising a plurality of ultrasound beams focused to a plurality of locations and the ultrasound echo signal comprises a plurality of ultrasound echo signals, and the method further comprises analyzing the plurality of ultrasound echo signals from the tissue responsive to the plurality of ultrasound beams.

116. The method of claim 115, wherein assessing the harmonic components comprises: assessing the harmonic components of the plurality of ultrasound echo signals derived from the plurality of locations, the harmonic components including first and second harmonics, wherein the plurality of locations are adjacent one another and are respectively associated with a region of interest and regions outside the region of interest; or assessing the harmonic components of the ultrasound echo signal derived from the location, the assessment comprising comparing the harmonic components to a threshold, wherein the harmonic components include the first and second harmonics.

117. The method of claim 116, wherein assessing the harmonic components comprises: determining a ratio of the second harmonic to the first harmonic; and comparing the ratio of the second harmonic to the first harmonic to the threshold.

118. The method of claim 117, comprising: determining the ratio of the second harmonic to the first harmonic for each of the plurality of ultrasound echo signals derived from the plurality of locations; and comparing the ratio for each of the plurality of ultrasound echo signals to the threshold; and distinguishing between the tissue experiencing tumor recurrence and tissue experiencing radiation necrosis by: identifying tissue in locations of the plurality of locations with ratios of the second harmonic to the first harmonic being above the threshold as tissue experiencing tumor recurrence; andidentifying tissue in locations of the plurality of locations with ratios of the second harmonic to the first harmonic being below the threshold as tissue experiencing radiation necrosis.

119. The method of claim 118, further comprising storing the ratios and the locations of the plurality of locations associated with the tissue experiencing radiation necrosis.

120. The method of claim 119, further comprising determining a size of necrosed region based on the stored ratios and locations.

121. The method of claim 109, comprising at least one of: outputting the ultrasound beam at a power insufficient to open the blood-brain barrier; and providing microbubbles at a concentration that is insufficient to open the blood-brain barrier.

122. The method of claim 109, wherein the ultrasound beam focused to the location comprises a plurality of ultrasound beams of different power strengths focused to the location and the ultrasound echo signal comprises a plurality of ultrasound echo signals which are responsive to the plurality of ultrasound beams, the method further comprising: assessing harmonic components of the ultrasound echo signals derived from the location, the harmonic components including first and second harmonics; determining a ratio of the second harmonic to the first harmonic for each of the plurality of ultrasound echo signals derived from the location; and identifying the tissue within the location as being tissue experiencing tumor recurrence or tissue experiencing radiation necrosis based on a change in the ratio across the plurality of ultrasound echo signals.

123. The method of claim 122, further comprising identifying the tissue as being necrosis tissue in response to the ratio not increasing or increasing below a threshold across the plurality of ultrasound beams of different power strengths.

124. A system comprising:a focused ultrasound system configured to focus an ultrasound beam to a location; and a processor arranged with the focused ultrasound system and configured to: analyze an ultrasound echo signal from tissue responsive to the ultrasound beam focused to the location of a brain of a patient; and distinguish between tissue experiencing tumor recurrence and tissue experiencing radiation necrosis based on the analysis.

125. The system of claim 124, wherein the tissue experiencing tumor recurrence includes brain tissue and the tumor is a brain tumor.

126. The system of claim 124, wherein the ultrasound beam comprising a plurality of ultrasound beams focused to a plurality of locations and the ultrasound echo signal comprises a plurality of ultrasound echo signals, and the processor is configured to analyze the plurality of ultrasound echo signals from the tissue responsive to the plurality of ultrasound beams.

127. The system of claim 124, wherein the ultrasound beam is focused to the location, the patient contains injected microbubbles, and the processor is configured to analyze the ultrasound echo signal from tissue responsive to the ultrasound beam.

128. The system of claim 127, wherein the ultrasound beam is output at a power insufficient to open the blood-brain barrier.

129. The system of claim 124, wherein the processor is configured to assess harmonic components of the ultrasound echo signal to distinguish tissue experiencing tumor recurrence from tissue experiencing radiation necrosis.

130. The system of claim 129, wherein the ultrasound beam comprising a plurality of ultrasound beams focused to a plurality of locations and the ultrasound echo signal comprises a plurality of ultrasound echo signals, and the processor to analyze the plurality of ultrasound echo signals from the tissue responsive to the plurality of ultrasound beams.

131. The system of claim 130, wherein the processor is configured to assess the harmonic components by: assessing the harmonic components of the plurality of ultrasound echo signals derived from the plurality of locations, the harmonic components including first and second harmonics, wherein the plurality of locations are adjacent one another and are respectively associated with a region of interest and regions outside the region of interest; or assessing the harmonic components of the ultrasound echo signal derived from the location, the assessment comprising comparing the harmonic components to a threshold, wherein the harmonic components include the first and second harmonics.

132. The system of claim 131, wherein the processor is configured to assess the harmonic components by: determining a ratio of the second harmonic to the first harmonic; and comparing the ratio of the second harmonic to the first harmonic to the threshold.

133. The system of claim 132, wherein the processor is configured to: determine the ratio of the second harmonic to the first harmonic for each of plurality of ultrasound echo signals derived from the plurality of locations; and compare the ratio for each of the plurality of ultrasound echo signals; and distinguish between the tissue experiencing tumor recurrence and tissue experiencing radiation necrosis by: identifying tissue in locations of the plurality of locations with ratios of the second harmonic to the first harmonic being above the threshold as tissue experiencing tumor recurrence; and identifying tissue in locations of the plurality of locations with ratios of the second harmonic to the first harmonic being below the threshold as tissue experiencing radiation necrosis.

134. The system of claim 133, wherein the processor is configured to store the ratios and the locations of the plurality of locations associated with the tissue experiencing radiation necrosis.

135. The system of claim 134, wherein the processor is configured to determine size of necrosed region based on the stored ratios and locations.

136. The system of claim 124, wherein at least one of the ultrasound beam is output at a power insufficient to open the blood-brain barrier and microbubbles are provided at a concentration that is insufficient to open the blood-brain barrier.

137. The system of claim 124, wherein the ultrasound beam focused to the location comprising a plurality of ultrasound beams of different power strengths focused to the location and the ultrasound echo signal comprises a plurality of ultrasound echo signals which are responsive to the plurality of ultrasound beams, wherein the processor is configured to: assess harmonic components of the plurality of ultrasound echo signals derived from the location, the harmonic components including first and second harmonics; determine a ratio of the second harmonic to the first harmonic for each of the plurality of ultrasound echo signals derived from the location; and identify the tissue within the location as being tissue experiencing tumor recurrence or tissue experiencing radiation necrosis based on a change in the ratio across the plurality of ultrasound echo signals.

138. The system of claim 137, wherein the processor is configured to identify the tissue as being necrosis tissue in response to the ratio not increasing or increasing below a threshold across the plurality of ultrasound beams of different power strengths.

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