CN112449602A

CN112449602A - Methods of using ultra-high dose rate radiation and therapeutic agents

Info

Publication number: CN112449602A
Application number: CN201980048106.XA
Authority: CN
Inventors: R·帕里; E·阿贝尔; S·格德哈尼; S·曼斯菲尔德; P·库佩利安; D·昆蒂亚
Original assignee: Varian Medical Systems Inc
Current assignee: Varian Medical Systems Inc
Priority date: 2018-07-19
Filing date: 2019-07-19
Publication date: 2021-03-05
Also published as: WO2020018904A9; WO2020018904A1; EP3823618A1

Abstract

Methods for treating tumors by administering FLASH radiation and a therapeutic agent to a patient having cancer are disclosed. This approach provides the dual benefit of anti-tumor efficacy plus normal tissue protection when the therapeutic agent is combined with FLASH radiation to treat cancer patients. The methods described herein also allow for classification of patients into groups for receiving optimized radiation therapy in combination with therapeutic agents based on patient-specific biomarker signatures. Radiation therapy planning methods and systems incorporating FLASH radiation and therapeutic agents are also provided.

Description

Methods of using ultra-high dose rate radiation and therapeutic agents

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No.62/700,783 entitled "Methods of Use of Ultra-High Dose Rate Radiation and Therapeutic Agents", filed 2019, 7/19, the entire contents of which are hereby incorporated by reference for all purposes.

Background

Radiation therapy is a key treatment modality for cancer patients. Radiation can be delivered to the tumor with sub-millimeter accuracy while leaving the normal tissue largely unaffected, eventually resulting in tumor cells being killed. However, the ability of tumor cells to evade the cell killing effects of radiation and/or to develop resistance mechanisms can counteract the tumor cell killing effects of radiation therapy, potentially limiting the therapeutic efficacy of radiation therapy to treat cancer. Furthermore, the potential for toxicity to normal tissues may impact the therapeutic window of radiation therapy as a therapeutic modality.

Radiation-induced tumor cell death results in the release of tumor antigens from lysed cells, increases MHC-1 expression on antigen presenting cells, and increases the diversity of T cell populations within tumors. These and other factors are critical to initiating the activation of the human autoimmune system to eliminate cancer cells. Immunomodulators are being explored to activate the human body's own immune system, but are known to have limitations of monotherapy (e.g., patient response rate). The response rate of an immunomodulator when used as a monotherapy is in the range of 20% to 30% of the target patient population. Due to systemic normal tissue toxicity, combinatorial approaches such as the use of two immunomodulators or an immunomodulator with a targeted anticancer drug have limitations.

Disclosure of Invention

Treatment of tumors with ultra-high dose rate radiation (e.g., FLASH RT) can improve the therapeutic window by reducing normal tissue side effects while maintaining tumor toxicity compared to conventional RT. Side effects of normal tissue can be reduced by increasing the dose rate or by limiting the time that normal tissue is exposed to radiation. An increased therapeutic window may be enforced by the continuous rotational delivery of multiple discrete fields or ionizing radiation. Reduction of side effects on normal tissue may also allow for increased dose in the tumor, resulting in enhanced tumor killing and control.

Conventional radiation induces stromal, immune, and vascular changes that can counteract the tumor cell killing effects of the radiation. It was unexpectedly found that FLASH irradiation had a different effect on the microenvironment, ultimately resulting in a tumor environment with more or improved immunocompetence. As a result, FLASH irradiation in combination with therapeutic agents can increase tumor cell killing while minimizing normal tissue toxicity. In some embodiments, the therapeutic agent is an immunomodulator, an anti-aging agent, a radiosensitizer, and/or a nanoparticle.

A variety of mechanisms are employed to promote immunosuppression in the tumor microenvironment, including but not limited to the recruitment of regulatory T cells (Tregs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs). In addition, anti-inflammatory components such as transforming (or tumor) growth factor beta (TGF-beta) and IL-10 inhibit the cytolytic activity of cytotoxic T Cells (CTLs). TAMs and MDSCs modify the metabolic environment of the tumor microenvironment by producing arginase and nitric oxide which deplete L-arginine, a nutrient essential for T cell function. In addition, aberrant tumor angiogenesis leads to hypoxia, which initiates the recruitment of immunosuppressive bone marrow cells. Inhibitory myeloid cells produce reactive oxygen and nitrogen species that modify the receptors for cytotoxic lymphocytes (CTLs) in lymphoid organs and the tumor itself, thereby affecting the ability of CTLs to home to the tumor and kill tumor cells.

When patients are treated with FLASH RT, the immunosuppressive microenvironment has no opportunity to develop adequately. In cases where the immunosuppressive tumor microenvironment is severely restricted, the immune response to dying tumor cells will be enhanced, which will eventually improve tumor cell killing.

Other factors that play a role in the immunosuppressive microenvironment include tumor-infiltrating macrophages that can form a phenotypic transformation of M1-M2 that can affect the killing of tumor cells. These effects are not seen in the case of FLASH RT. Another benefit of FLASH RT is that it protects circulating immune cells from radiation-induced toxicity, thereby enhancing immune-related tumor cell killing.

The methods described herein provide the dual benefits of anti-tumor efficacy and normal tissue protection when the therapeutic agent is combined with FLASH radiation to treat cancer patients. The methods described herein can be used to treat local and metastatic cancers by FLASH radiation therapy to deliver highly conformal doses, and immunomodulators, to tumors. This combination therapy has the effect of improving the efficacy of local and systemic radiation therapy as well as the efficacy of immunomodulators, while minimizing toxicity to normal tissues.

Protons deposit energy most densely at the ends of their pathways, a mechanism that has been exploited to have advantages in cancer radiation therapy (British Journal of Radiotherapy in 2006 by Jones et al, Jakel radiation Protection therapy in 2009). The profile of absorbed dose as a function of penetration depth has a maximum at the position before the particles stop, supporting therapeutic targeting of the tumor at a specific depth. In contrast, the dose deposition for electron/photon beam radiotherapy is greatest near the entrance surface of the tissue (e.g., skin), and then decreases exponentially with tissue depth. Due to these physical properties, proton therapy allows high dose deposition in deep tumors.

The inventors have surprisingly determined that the unexpected advantages of proton energy deposition depend not only on the absolute dose delivered to the target, but also on the dose rate (speed) used to deliver the dose. Unexpectedly, it was observed that depending on the biology of the proton dose rate, for example when protons are delivered at different dose rates, different biological pathways are activated. The dose rate dependent nature of proton deposition is of great importance for the development of cancer therapies and novel treatment options, including dose rate dependent combination therapies.

Accordingly, in one aspect, provided herein is a method for treating a tumor in a subject having cancer, the method comprising administering to the tumor an effective amount of ultra-high dose rate (FLASH) radiation and a therapeutic agent. In some embodiments, the method reduces damage to normal tissue as compared to administration of conventional radiation (e.g., a dose of 0.5 Gy/second) to the tumor. In some embodiments, the dermatitis, pulmonary fibrosis, or lymphocyte apoptosis is reduced as compared to administration of conventional radiation.

In some embodiments, the FLASH radiation is administered at a dose rate equal to or greater than 40Gy/sec, or the dose is administered in 1 second or less. In some embodiments, FLASH radiation is applied in a single pulse or multiple pulses. In some embodiments, the FLASH radiation comprises or consists of protons. In some embodiments, the FLASH radiation does not include electrons.

In some embodiments, the therapeutic agent is an immunomodulatory agent, a radiosensitizer, or a nanoparticle.

In some embodiments, the therapeutic agent is a mitotic spindle inhibitor, a DNA damage repair and response inhibitor, a MAPK pathway inhibitor, an epithelial to mesenchymal (EMT) inhibitor, an activator of T helper type1 (TH1) lymphocytes, an activator of the PTEN pathway and an inhibitor of the TGF- β pathway, an activator of the type1 interferon signaling pathway, an activator of dendritic cell maturation, an inhibitor of CD47/SIRP- α, or an inhibitor of arene receptor (ahR).

In some embodiments, the mitotic spindle inhibitor is selected from CDK4/6 inhibitors, AURKA inhibitors, TPX2-AURKA complex inhibitors, or taxanes.

In some embodiments, the DNA damage repair and response inhibitor is selected from the group consisting of: a PARP inhibitor, a RAD51 inhibitor, or an inhibitor of a DNA damage response kinase selected from CHCK1, ATM or ATR.

In some embodiments, the MAPK pathway inhibitor is an inhibitor of EGFR, MEK, BRAF, or ERK.

In some embodiments, the EMT inhibitor is a TGF β pathway inhibitor selected from a compound, small molecule, antibody, or fragment thereof that binds TGF- β.

In some embodiments, the activator of T helper type1 (TH1) lymphocytes is a cytokine, a toll-like receptor agonist, a STAT3 modulator, a derivative derived from inactivated bacteria/or parasites (including but not limited to listeria monocytogenes, leishmania major and toxoplasma, mycobacterium tuberculosis) or compounds thereof that trigger interferon gamma or IL-12 production, enterococcal enterotoxin B, and unmethylated CpG nucleotides that activate TH1 responses in vivo, or a gene therapy system including bacterial or viral based gene expression systems that result in the production of IL2, IL-12, and IFN-gamma when injected at the tumor site.

In some embodiments, the activator of the PTEN pathway is selected from an mTOR inhibitor selected from rapamycin, temsirolimus, everolimus, sirolimus or AP-2357, ubuliximab, rituximab, sunitinib (induced PTEN), trastuzumab and pertuzumab (increase PTEN by Src inhibition), resistin (p38MAPK modulator, increase PTEN), simvastatin (NF0-kB inhibitor), lovastatin and rosiglitazone (PPAR-gamma modulator), NVP-AEW541 (IGF-1R modulator that increases PTEN), and PP1 herbicidin (Src inhibitor) (see, e.g., Expert Opm Ther Pat.2013, 5 months, 23(5),569-580 to bosani et al).

In some embodiments, the inhibitor of the TGF- β pathway is selected from a compound, small molecule, antibody, or fragment thereof that binds TGF- β.

In some embodiments, the activator of a type1 interferon signaling pathway is a STING agonist, a Toll-like receptor (TLR) agonist, or a MAVS agonist.

In some embodiments, the activator of dendritic cell maturation is a synthetic peptide vaccine. In some embodiments, the inhibitor of CD 47/SIRP-a is selected from an antibody or fragment thereof, or a small molecule compound that inhibits the CD 47/DSIRP-a interaction.

In some embodiments, the nanoparticles have a high effective atomic number or comprise gold or gadolinium.

In some embodiments, the ahR inhibitor is SR1, CH-223191, UM729, or galangin.

In some aspects, provided herein is a method for treating a tumor in a subject having cancer, the method comprising administering to the tumor ionizing FLASH radiation and an immunomodulator, wherein the immunomodulator is selected from the group consisting of: inhibitors of inhibitory checkpoint molecules, activators of stimulatory checkpoint molecules, chemokine inhibitors, inhibitors of macrophage Migration Inhibitory Factor (MIF), growth factors, cytokines, interleukins, interferons, antibodies that bind to cells of the immune system, cellular immunomodulators, vaccines, oncolytic viruses, and any combination thereof. It was surprisingly found that administration of an immunomodulator when combined with FLASH irradiation increased the anti-tumor response.

In some embodiments, the inhibitor of an inhibitory checkpoint molecule is a small molecule drug or an antibody or fragment thereof that specifically binds to and inhibits the activity of an inhibitory checkpoint molecule, wherein the inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, BTLA, A2aR, B7-H2, B7-H3, B7-H4, B7-H6, CD47, CD48, CD160, CD244(2B4), CHK1, CHK2, CGEN-15049, ILT-2, ILT-4, LAG-3, VISTA, gp49B, PIR-B, TIGIT, TIM1, TIM2, TIM3, TIM4 and KIR and their ligands. In some embodiments, the activator of a stimulatory checkpoint molecule is a small molecule drug, a polypeptide-based activator, or a polynucleotide-based activator that specifically binds to and increases the activity of the stimulatory checkpoint molecule, wherein the stimulatory checkpoint molecule is selected from the group consisting of: b7-1(CD80), B7-2(CD86), 4-1BB (CD137), OX-40(CD134), HVEM, inducible costimulatory molecule (ICOS), glucocorticoid-induced tumor necrosis factor receptor (GITR), CD27, CD28, CD40 and their ligands. In certain instances, the chemokine inhibitor is a small molecule drug or an antibody or fragment thereof that specifically binds to a chemokine (or a receptor thereof) and inhibits chemokine activity. In some embodiments, the chemokine is selected from the group consisting of: CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CXCL 24, and CXCL 24. In some embodiments, the chemokine inhibitor binds to a chemokine receptor selected from the group consisting of: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR 7. In some cases, the inhibitor of MIF is a small molecule drug or antibody or fragment thereof that specifically binds to MIF and inhibits MIF activity.

In some aspects, provided herein is a method for treating a tumor in a subject having cancer, the method comprising administering FLASH radiation and an immunomodulator to the tumor. In some embodiments, the immunomodulatory agent may be an antibody or antibody fragment that targets CD44, MMP9, ALDH1a1, vimentin, hyaluronic acid, β catenin, MFG-E8, CD68, TGF β pathway-associated biomarkers, or any combination thereof.

Also provided herein are improved methods for treating a tumor, the methods comprising administering to a subject having cancer an immunomodulator and FLASH radiation. Such combination therapy may elicit an increased anti-cancer response compared to immunomodulator monotherapy or conventional radiation monotherapy.

Immune modulators that bind to FLASH include, but are not limited to, checkpoint inhibitors, co-stimulators, broad immune modulators that modulate the adenosine pathway or STING (stimulating interferon gene) pathway, bispecific antibodies that target both immune cell antigens and cancer antigens, and cell therapy methods (e.g., Chimeric Antigen Receptor (CAR) therapy).

In some embodiments, the positive effects of FLASH RT are enhanced by combining it with the delivery of immunotherapeutics as a concomitant adjuvant or a novel adjuvant process. Thus, when used in combination with an immunomodulatory agent, the immunomodulatory properties of FLASH RT described herein can be enhanced and provide further benefits to a patient or subject in need of treatment. A combination of FLASH RT and immunotherapy can be incorporated into the radiation treatment plan as well as the treatment itself.

The combination of FLASH RT and immunomodulators described herein can also be combined with a personalized drug treatment regimen. Thus, in some embodiments, the method comprises detecting expression of one or more biomarkers in the subject. In some embodiments, the one or more biomarkers are selected from CD44, MMP9, ALDH1a1, vimentin, hyaluronic acid, beta catenin, MFG-E8, CD68, t GF β, TGF β pathway-associated biomarkers, or any combination thereof. In some embodiments, the method includes radiologic information, such as tumor phenotype. In some embodiments, the method includes functional imaging information including, but not limited to, PET, SPECT, and fMRI.

In some aspects, provided herein is a method for treating a tumor in a subject having cancer, the method comprising administering to the tumor ionizing FLASH radiation and an immunomodulator. The method comprises the following steps: (a) determining the expression level of one or more biomarkers in a tumor sample from the subject, wherein the one or more biomarkers are selected from the group consisting of: immune cell marker(s), tumor cell marker(s), circulating marker(s), and any combination thereof; (b) comparing the expression level of the one or more biomarkers to the expression level of the one or more biomarkers in a normal tissue sample; and (c) administering a treatment comprising ionizing FLASH radiation and an immunomodulator to the tumor in the subject if the expression level of the one or more biomarkers in the tumor sample is modified compared to the expression level in the normal tissue sample. The biomarker may be CD44, MMP9, ALDH1a1, vimentin, hyaluronic acid, beta catenin, MFG-E8, CD68, TGF β pathway-related biomarkers, or any combination thereof.

In certain aspects, provided herein is a method, e.g., an in vitro method, of identifying a subject having cancer as a candidate for a treatment comprising ionizing FLASH radiation and an immunomodulator. The method comprises the following steps: (a) determining the expression level of one or more biomarkers in a tumor sample from the subject, wherein the one or more biomarkers are selected from the group consisting of: immune cell marker(s), tumor cell marker(s), circulating marker(s), imaging marker(s), and any combination thereof; (b) comparing the expression level of the one or more biomarkers to the expression level of the one or more biomarkers in a normal tissue sample; and (c) classifying the subject as a candidate for treatment comprising ionizing FLASH radiation and an immunomodulator if the expression level of the one or more biomarkers in the tumor sample is modified compared to the expression level in a normal tissue sample. The biomarker may be CD44, MMP9, ALDH1a1, vimentin, hyaluronic acid, beta catenin, MFG-E8, CD68, t GF β, TGF β pathway-associated biomarker, or any combination thereof.

In other aspects, provided herein is a method of selecting a treatment, e.g., an in vitro method, for a subject having cancer. The method comprises the following steps: (a) determining the expression level of one or more biomarkers in a tumor sample from the subject, wherein the one or more biomarkers are selected from the group consisting of: immune cell marker(s), tumor cell marker(s), circulating marker(s), and any combination thereof; (b) comparing the expression level of the one or more biomarkers to the expression level of the one or more biomarkers in a normal tissue sample; and (c) selecting a treatment comprising ionizing FLASH radiation and an immunomodulator if the expression level of the one or more biomarkers in the tumor sample is modified compared to the expression level in the normal tissue sample. The biomarker may be CD44, MMP9, ALDH1a1, vimentin, hyaluronic acid, beta catenin, MFG-E8, CD68, TGF β pathway-related biomarkers, or any combination thereof.

In some embodiments, the subject is administered ionizing FLASH radiation and/or a combination therapy comprising ionizing FLASH radiation and an immunomodulator if the expression level of CD44 is increased and/or the expression level of MFG-E8 is decreased relative to the expression level in a normal or control sample. In some embodiments, the amount of ionizing FLASH radiation and/or the amount of immunomodulator administered to the subject is increased if the expression level of CD44 is increased and/or the expression level of MFG-E8 is decreased relative to the expression level in a normal or control sample. On the other hand, if the expression level of CD44 is decreased and/or the expression level of MFG-E8 is increased relative to the expression level in a normal or control tissue sample, the amount of ionizing FLASH radiation and/or the amount of immunomodulator administered to the subject may be decreased.

In some embodiments, (a) a treatment comprising ionizing FLASH radiation and an immunomodulator is selected if the expression level of CD44 is increased and/or the expression level of MFG-E8 is decreased relative to the expression level in a normal or control sample; (b) increasing the amount of selected ionizing FLASH radiation and/or the amount of immunomodulator if the expression level of CD44 is increased and/or the expression level of MFG-E8 is decreased relative to the expression level in a normal or control sample; and (c) reducing the amount of selected ionizing FLASH radiation and/or the amount of immunomodulator if the expression level of CD44 is reduced and/or the expression level of MFG-E8 is increased relative to the expression level in a normal or control tissue sample.

In some embodiments, the subject is administered ionizing FLASH radiation and/or a combination therapy comprising ionizing FLASH radiation and an immunomodulator if the expression level of CD68 is increased relative to the expression level in a normal or control tissue sample. In some embodiments, the amount of ionizing FLASH radiation and/or the amount of immunomodulator administered to the subject is increased if the expression level of CD68 is increased relative to the expression level in a normal or control tissue sample. On the other hand, if the expression level of CD68 is decreased relative to the expression level in a normal or control tissue sample, the amount of ionizing FLASH radiation and/or the amount of immunomodulator administered to the subject may be decreased.

In some embodiments, (a) if the expression level of CD68 is elevated relative to the expression level in a normal or control tissue sample, a treatment comprising ionizing FLASH radiation and an immunomodulator is selected; (b) increasing the amount of selected ionizing FLASH radiation and/or the amount of an immunomodulator if the expression level of CD68 is increased relative to the expression level in a normal or control tissue sample; (c) the amount of selected ionizing FLASH radiation and/or the amount of immunomodulator can be reduced if the expression level of CD68 is increased relative to the expression level in a normal or control tissue sample.

In some aspects, provided herein is the use of FLASH radiation and a therapeutic agent for treating a tumor in a subject. In some embodiments, the use comprises a combination of ionizing FLASH radiation and a therapeutic agent selected from an immunomodulatory agent described herein, an anti-aging agent, and/or a radiosensitizer described herein.

In another aspect, the present disclosure provides a therapeutic agent for use in a method of treating a tumor in a subject having cancer, characterized in that the method comprises administering FLASH radiation and the therapeutic agent to the tumor. In some embodiments, a therapeutic agent in combination with ultra-high dose rate (FLASH) radiation for use in treating cancer or a tumor is provided.

In another aspect, the present disclosure provides a therapeutic agent for use in a method of treating a tumor in a subject having cancer, characterized in that the therapeutic agent is administered in combination with FLASH radiation.

In another aspect, the present disclosure provides a therapeutic agent for use in a method of treating a tumor in a subject having cancer, wherein the therapeutic agent is administered to the tumor environment of increased immune competence caused by FLASH radiation as described above.

In another aspect, the present disclosure provides a hedgehog antagonist for use in a method of treating a tumor in a subject having cancer, characterized in that the hedgehog antagonist is administered in combination with FLASH radiation.

In another aspect, provided herein is a therapeutic agent for use in a method of treating a tumor in a subject having cancer, characterized in that the method comprises:

(a) determining the expression level of one or more biomarkers in a tumor sample from the subject, wherein the one or more biomarkers are selected from the group consisting of: immune cell marker(s), tumor cell marker(s), circulating marker(s), and any combination thereof;

(b) comparing the expression level of the one or more biomarkers to the expression level of the one or more biomarkers in a normal tissue sample; and

(c) administering a treatment comprising ionizing FLASH radiation and a therapeutic agent to the tumor within the subject if the expression level of the one or more biomarkers in the tumor sample is modified compared to the expression level in the normal tissue sample.

In another aspect, a method for treating a tumor in a subject is described, the method comprising:

i) detecting expression of a biomarker described herein in a tumor microenvironment or a tumor sample from a subject, and

ii) administering to the subject an effective amount of FLASH radiation and a therapeutic agent, thereby treating the tumor.

In some embodiments, the expression level of the biomarker(s) described herein is compared to the expression level detected in normal or control (e.g., non-tumor) tissue within the subject.

i) contacting a tumor sample from a subject with an antibody that binds to a biomarker described herein;

ii) detecting the modified expression of the biomarker in the tumor sample, and

iii) administering to the subject an effective amount of FLASH radiation and a therapeutic agent, thereby treating the tumor.

In some embodiments, the modified biomarker expression comprises an increased and/or decreased expression level of the biomarker. In some embodiments, the biomarker is selected from the group consisting of: CD44, MMP9, ALDH1a1, vimentin, hyaluronic acid, beta catenin, MFG-E8, and CD 68. In some embodiments, the expression level of the biomarker CD68 is increased in a tumor environment or tumor sample. In some embodiments, the expression level of the biomarker CD44 is increased in a tumor environment or tumor sample. In some embodiments, the expression level of the biomarker CD44 is increased and the expression level of MFG-E8 is decreased in a tumor environment or tumor sample.

In some embodiments, the method further comprises detecting the expression level of a biomarker described herein in a normal tissue sample. In some embodiments, the expression level of the biomarker is detected using an antibody that binds to the biomarker(s).

In another aspect, a radiation therapy system is provided. In some embodiments, the radiation therapy system provides radiation therapy to a subject or patient in need thereof. In some embodiments, the radiation therapy system further comprises an immunotherapy system that treats the patient with a therapeutic agent in conjunction with the radiation therapy. In some embodiments, the radiation therapy is FLASH RT.

In another aspect, a radiation therapy planning system is described that is operable to generate a radiation therapy plan that further includes immunotherapy that is performed in conjunction with the radiation therapy. In some embodiments, the radiation therapy is FLASH RT.

In another aspect, a non-transitory computer-readable storage medium having computer-executable instructions for causing a computing system to perform a method for ultra-high dose rate (FLASH) radiation therapy planning for treating a tumor in combination with a described therapeutic agent is described. In some embodiments, the method includes using information related to treatment of the tumor with the therapeutic agent to adjust the beam parameters.

In some embodiments, a non-transitory computer-readable storage medium having computer-executable instructions for causing a computing system to perform a method for ultra-high dose rate (FLASH) radiation therapy planning for treating a tumor in combination with a therapeutic agent is provided. The method comprises the following steps:

accessing values of parameters from a memory of a computing system, wherein the parameters include a direction of a beam to be directed into a sub-volume in a target and a beam energy of the beam;

accessing information specifying limits of a radiation therapy plan, wherein the limits are based on a dose threshold and include limits on exposure time for each sub-volume outside of the target:

wherein the information specifying the limit comprises information about treating the tumor with a therapeutic agent; and

the values of the parameters affecting the calculated amount of dose to be delivered by the beam are adjusted until the difference between the respective total values of the sub-volumes meets a threshold.

In some embodiments, each portion of the beam that is in the target is represented as a respective set of longitudinal beam regions, and wherein the method further comprises:

for each of the beam regions, calculating an amount of dose to be delivered by the beam region, and assigning a value corresponding to the amount to the beam region; and

for each of the sub-volumes, calculating a total value for the sub-volume by adding the values of each beam region of each beam that reaches the sub-volume; wherein the adjusting further comprises adjusting a parameter affecting the calculated amount of dose to be delivered by the beam region until a difference between the respective total values of the sub-volumes satisfies a threshold.

In some embodiments, the adjusting further comprises:

determining whether the beam overlaps any other beam outside the target; and

the beam intensities of the beam segments of the beam are weighted according to how many other beams overlap the beam outside the target.

In some embodiments, the method further comprises performing dose calculations for outer subvolumes of the target, wherein said performing dose calculations comprises:

accessing values of a dose calculation factor for the outer sub-volume of the target, wherein the values of the dose calculation factor are determined according to how many beams reach the outer sub-volume of the target;

calculating a dose for the outer sub-volume of the target; and

the value of the dose calculation factor is applied to the dose calculated for the outer sub-volume of the target.

In some embodiments, the dose calculation factor reduces the dose calculated for the outer target sub-volume if only one beam reaches the outer target sub-volume.

In some embodiments, the restriction is selected from the group consisting of: a limit on the illumination time of each sub-volume in the target; a limit on dose rate for each sub-volume in the target; and a limit on the dose rate for each sub-volume outside the target.

In some embodiments, the dose threshold is also dependent on the tissue type.

In some embodiments, the beam comprises a beam type selected from the group consisting of: a proton; electrons; a photon; nuclei and ions.

In another aspect, a computer-implemented method of radiation therapy planning for treating a tumor in combination with a therapeutic agent is described. In some embodiments, the method includes determining beam parameters for ultra-high dose rate (FLASH) radiation using a prescribed dose based on a tumor's response to a therapeutic agent.

In some embodiments, a computer-implemented method of radiation therapy planning comprises:

determining a prescribed dose of ultra-high dose rate (FLASH) radiation to be delivered into and across the tumor target, wherein the prescribed dose is determined based on the tumor's response to the therapeutic agent;

accessing values of parameters including a number of beams of a plurality of beams to be directed into a sub-volume in a target, a direction of the plurality of beams, and beam energies of the plurality of beams, wherein each beam comprises a plurality of beam segments;

identifying any overlapping beams of the plurality of beams having respective beam paths that overlap outside the target;

for each beam of a plurality of beams, determining a maximum beam energy of the beam and determining a beam energy of a beam segment of the beam as a percentage of the maximum beam energy of the beam; and

for each of the overlapping beams that overlap outside the target, reducing the beam intensity of the beam segment of the overlapping beam by a dose calculation factor, wherein the beam intensities of the beam segments of the plurality of beams are determined such that the cumulative dose delivered to the target satisfies a specified dose.

In some embodiments, the method comprises:

representing each of the beams that is in the target as a respective set of longitudinal beam regions, wherein each beam region in the set has a value corresponding to a calculated amount of dose to be delivered by the beam region;

for each sub-volume in the target, summing the values of each beam region of each beam that reaches the sub-volume to determine a total value for the sub-volume to produce a corresponding total value for the sub-volume in the target; and

the values of the parameters that affect the calculated amount of dose to be delivered by the beam region are adjusted until the difference between the total values of the sub-volumes meets a threshold.

In some embodiments, the method comprises:

accessing a value of a dose calculation factor for an outer sub-volume of the target, wherein the value of the dose calculation factor is determined based on how many beams reach the outer sub-volume of the target;

calculating a dose for the outer sub-volume of the target; and

applying a value of a dose calculation factor to the dose calculated for the outer sub-volume of the target, wherein the dose calculation factor reduces the dose calculated for the outer sub-volume of the target if only one beam reaches the outer sub-volume of the target.

In some embodiments, the method includes using the dose threshold to specify a limit for the radiation therapy plan, wherein the limit is selected from the group consisting of: a limit on the illumination time of each sub-volume in the target; a limit on the illumination time of each sub-volume outside the target; a limit on dose rate for each sub-volume in the target; and a limit on the dose rate for each sub-volume outside the target.

In some embodiments, the dose threshold depends on a variety of biological factors including, but not limited to, tissue type and/or immunological profile.

In another aspect, there is provided a use of ultra-high dose rate (FLASH) radiation in combination with a therapeutic agent to treat a cancer or tumor in a subject in need thereof.

In another aspect, an ultra-high dose rate (FLASH) radiation for use in combination with a therapeutic agent in the treatment of cancer or a tumor is provided.

In any of the aspects and embodiments described herein, the therapeutic agent may be an immunomodulator, an anti-aging agent, a radiosensitizer, and/or a nanoparticle.

In another aspect, a method for reducing activation of the hedgehog signaling pathway in a subject is provided. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of ultra-high dose rate (FLASH) radiation, wherein activation of the hedgehog signaling pathway is reduced as compared to administration of conventional proton radiation therapy to the subject. In some embodiments, the subject has a cancer or tumor. In some embodiments, the method comprises administering a therapeutically effective amount of ultra-high dose rate (FLASH) radiation to a tumor in the subject. In some embodiments, FLASH radiation is administered as proton therapy, or FLASH radiation includes or consists of protons.

In some embodiments, the expression of the gene activated by the hedgehog signaling pathway is reduced when FLASH radiation is administered to the subject as compared to administration of conventional proton radiation therapy. In some embodiments, the expression of one or more genes selected from CELSR1, TLE3, OPHN1, GPR56, PTCH1, TLE1, MYH9, RASA1, HEY1, ETS2, HEY2, LDB1, UNC5C, NF1, CDK6, VLDLR, NRP2, DPYSL2, and NRP1 is reduced when FLASH radiation is administered to a subject as compared to administration of conventional proton radiation therapy. In some embodiments, the expression of one or more proteins encoded by genes selected from the following is reduced when FLASH radiation is administered to a subject as compared to administration of conventional proton radiation therapy: CELSR1, TLE3, OPHN1, GPR56, PTCH1, TLE1, MYH9, RASA1, HEY1, ETS2, HEY2, LDB1, UNC5C, NF1, CDK6, VLDLR, NRP2, DPYSL2, and NRP 1. In some embodiments, the therapeutic agent is administered to a subject having a biological activity selected from the group consisting of SEQ ID Nos: 15-70 is reduced in expression of one or more genes encoded by the protein of the amino acid sequence selected. In some embodiments, when FLASH radiation is administered to a subject, the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID Nos: 15-70 is reduced. In some embodiments, the expression of one or more genes encoding proteins in table 7 is reduced when FLASH radiation is administered to a subject as compared to administration of conventional proton radiation therapy. In some embodiments, the expression of one or more proteins in table 7 is reduced when FLASH radiation is administered to a subject as compared to administration of conventional proton radiation therapy.

In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a hedgehog antagonist. In some embodiments, the hedgehog antagonist is selected from a Smo antagonist, a PTCH1 inhibitor, cyclopamine, Vismodegib (Vismodegib), LDE 225, saridegib, BMS 833923, LEQ 506, PF-04449913, PF-5274857, GANT61, SANT-1, Glasedigmethodology (PF-04449913), Taladegib (LY2940680), or TAK-441. In some embodiments, the subject has a cancer or tumor. In some embodiments, the method comprises administering to a tumor in the subject a therapeutically effective amount of ultra-high dose rate (FLASH) radiation and a therapeutically effective amount of a hedgehog antagonist.

Drawings

Fig. 1 shows a cross-sectional view of a sample beam geometry in one embodiment described herein.

Fig. 2 shows a representative dose surface plot as an example of the variables used to determine the dose threshold in one embodiment described herein.

FIG. 3 is a block diagram of an example of a computing system on which embodiments described herein may be implemented.

Fig. 4 is a block diagram illustrating an example of an automated radiation therapy planning system in one embodiment described herein.

Figure 5 shows the experimental design for normal tissue toxicity studies in control mice (sham) and mice treated with conventional, FLASH and split-FLASH radiation.

Figure 6 shows that FLASH has improved lung function compared to conventional radiation therapy.

Figure 7 shows that median survival of FLASH was improved compared to the conventional radiation treated group: 20.0 Gy: increase by 14%; 17.5 Gy: the increase is 18%.

Figure 8 shows the probability of improved median survival of FLASH compared to the conventional radiation treated group, and a dose rate dependent treatment window: 17.5Gy FLASH >17.5Gy CON; 17.5Gy FLASH 20,0Gy CON.

Figure 9 shows that the average dermatitis of FLASH was reduced compared to conventional irradiation: FLASH: the reduction is 34%; Split-FLASH: the reduction was 52%.

Figure 10 shows a 23% reduction in mean pulmonary fibrosis of FLASH compared to the conventional group at 17.5 Gy.

Figure 11 shows the average weight gain of conventional lungs-0.46 g versus 0.40g (m) -compared to FLASH treatment; 0.37g and 0.32g (F).

Fig. 12A-12D show venn plots of differentially expressed genes at each time point. A) DE gene at 24 hours, B) at 8 weeks, C) at 16 weeks, D) at 24 weeks, wherein the adjusted p-value is less than or greater than 0.05.

Fig. 13A-13D show the overlap of GSEA landmark pathways between the three treatment groups. A)24 hours, B)8 weeks, C)16 weeks, D) GSEA at 24 weeks with FDR-q value <0.25 and adjusted p value < 0.05. The underlined text indicates down-regulation, while the italicized text indicates up-regulation pathways, relative to the Sham-processed samples.

FIGS. 14A-14D show marker mitotic spindle enrichment maps and core-enriched genes for the Split Flash-Sham and Flash-Sham GSEA assays. Enrichment profiles for the marker mitotic spindles of the core enrichment genes for Split-Flash and FLASH A) Split-Flash and B) FLASH treatment panel gene expression heatmap. C) Overlap between Split FLASH and FLASH processing sets. D) From the gene list of figure 14C, the BOLD gene is a key participant in mitotic spindle assembly.

Fig. 15A and 15B show Flash-specific DNA repair signatures and core-enriched genes: A) (upper left): enrichment plot of DNA repair signature (bottom left): enrichment statistics of DNA repair signatures. B) Gene expression heatmap of core-enriched genes in signature DNA repair signature.

Fig. 16A-16C show the Flash specific KRAS signal rises for 8 to 24 weeks: (upper part): enrichment plots of KRAS signal rise signatures and enrichment statistics for Flash treated mice a)8 weeks, B)16 weeks, and C)24 weeks.

Fig. 17A-17C show EMT signature and core-enriched gene analysis: EMT enrichment plots and statistics for 24 weeks: A) conventional B) Flash treated cells. C) The overlap of the core enrichment gene between the conventional and Flash highlights the TGF Beta gene. See table 5 for a complete list of core-enriched genes and overlaps.

Fig. 18 shows a pie chart representing various classical pathways modulated by different irradiation schemes at 24 hours. The filtered list of genes for samples with p-value <0.05 was analyzed. The vias presented here and listed in table 6 had p values <0.05, FDR <0.1 and Z scores > 1.5. The underlined text indicates down-regulation, while the italicized text indicates up-regulation pathways, relative to the Sham-processed samples.

Fig. 19 shows a heat map of a comparative analysis of the main typical pathway adjusted by both Flash and conventional radiation processing by IPA analysis. P value 0.05, Z score > 1.5. The pathways that were found to be differentially regulated between these treatment regimens are primarily related to inflammation and immune modulation.

Figure 20 shows a pie chart representing various typical paths found at 16 weeks, adjusted by different radiation schemes. For p value<A filtered list of genes from 0.05 samples was analyzed. P-values for the pathways presented and presented herein and listed in Table 4<0.05，FDR<0.1 and Z score>1.5. With respect to the samples processed by Sham,lower marked lineThe text of (a) indicates a down-regulation, and the italic text indicates an up-regulation pathway.

Fig. 21 shows a heat map of a comparative analysis of the main classical pathway regulated by both Flash and conventional radiation therapy at 16 weeks as analyzed by IPA. P value 0.05 and Z score > 1.5. The pathways that were found to be differentially regulated between these treatment regimens are primarily related to inflammation and immune modulation.

Fig. 22 shows the results of the quapath cell assay applied to DAPI images.

Fig. 23A and 23B show (a) raw FITC images, and (B) segmented TUNEL positive cells. The fijic distribution of ImageJ was used to quantify the number of TUNEL positive cells in each field of view. Gaussian blur is first applied to the image and then the threshold is used to segment the object.

Fig. 24 is a graph showing TUNEL results. The figure shows the average counts per animal for approximately 20 animals per group (combined male and female).

Figure 25 shows differential gene expression and pathway analysis for FLAS samples versus conventional samples 24 hours after irradiation. Figure 25A shows a volcanic plot of the highest differentially expressed genes. The red dots indicate genes with FDR values less than 5%, and for clarity, the labeled genes indicate genes with expression greater than 1.3 log fold difference. Fig. 25B shows a table of the highest GSEA flags enriched between FLASH radiation and conventional radiation. Figure 25C shows enrichment maps and the top 50 leading edge (trailing edge) genes for the hallmark inflammatory response to GSEA. Figure 25D shows IPA results for the highest enrichment channel in the microarray dataset between FLASH irradiated and conventional irradiated samples.

Definition of

The term "treating" refers to administering a treatment to a tumor or a subject diagnosed with a tumor. The treatment can be administered in an amount or therapeutic dose sufficient or effective to kill tumor cells (i.e., in a therapeutically effective amount), slow the growth of the tumor, reduce the size of the tumor, or eliminate the tumor from the subject altogether. Examples of treatments include: ionizing radiation, such as FLASH radiotherapy, a therapeutic agent, an immunomodulator, an anti-aging agent, a radiosensitizer, a nanoparticle or a combination thereof. The term also includes selecting a treatment or treatment plan, and providing a treatment selection to a medical care provider or subject.

The term "therapeutic agent" refers to an agent that can be used to treat a tumor, such as a small molecule drug or a biologic drug, and can include an immunomodulator, an anti-aging agent, a radiosensitizer, or a nanoparticle. The therapeutic agent may be an agent approved by a regulatory agency for the treatment of a tumor or cancer, undergoing a clinical trial prior to regulatory approval, or undergoing a study to treat a tumor or cancer. The therapeutic agent may be combined with radiation therapy, such as conventional or FLASH radiation therapy, to treat the tumor. In some embodiments, the therapeutic agent is combined with FLASH radiation therapy to treat the tumor.

The term "ionizing radiation" refers to radiation that includes particles having sufficient kinetic energy to release electrons from atoms or molecules to produce ions. The term includes both direct ionizing radiation, such as radiation caused by atomic particles, such as alpha particles (helium nuclei), beta particles (electrons), and protons; indirect ionizing radiation, such as radiation caused by photons (including gamma rays and x-rays), is also included. Examples of ionizing radiation used in radiation therapy include high-energy x-rays, electron beams, ion beams, and proton beams.

The terms "FLASH", "FLASH radiation" or "FLASH radiation therapy (FLASH RT)" refer to ultra-high dose rate radiation that is administered to a subject or patient as therapy. In some embodiments, the dose may be administered to a subject or patient in need of treatment at an ultra-high dose rate in one to many short pulses. In some embodiments, the entire radiation dose is delivered within a total "beam on" time of no more than one second.

FLASH refers to a mode of application of ionizing radiation at a dose rate that ensures that all normal tissue radiation occurs in 1 second or less to deliver the entire dose prescription. For example, a dose prescription of 20 gray (Gy) would require a dose rate of at least 20Gy per second for a single direction of irradiation; for both irradiation directions, at least 10Gy per second will be required; for four irradiation directions, 5Gy per second would be required, and so on. The number of fields can be reduced in dose rate to meet FLASH illumination conditions as long as the fields do not overlap or deliver overlapping fields in 1 second or less.

Pulsed FLASH is a mode of applying ionizing radiation at a dose rate that results in a total active delivery time to normal tissue for a given dose of ionizing radiation in 1 second or less and allows the same normal tissue volume to be repeatedly irradiated within a single treatment session or segment. For example, 20Gy of the prescription will be delivered within an effective exposure time of 1 second or less, but may be divided into 54Gy pulses, 1 second apart per pulse, 0.2 seconds per pulse, or 20Gy per second per pulse. Another example is 10 pulses of 2Gy, with a duration of 0.1 seconds, spaced apart by 2 seconds, or 20Gy per second per pulse. Pulsed FLASH allows a free choice of duty cycle for delivery as long as a total effective delivery time of 1 second or less is implemented for a single treatment session. The pulse interval may vary from less than a second to several minutes.

Segmented FLASH is delivering FLASH or pulsed FLASH ionizing radiation over a longer time interval. After a defined split radiation therapy clinical protocol, the time interval may be hours to days, while the actual therapy delivery is FLASH or pulsed FLASH.

The term "conventional radiotherapy" or "conventional irradiation" of tissue refers to a dose of 0.5Gy per second, e.g. 20Gy within 40 seconds, 17.5Gy within 35 seconds or 15Gy within 30 seconds (0.5 Gy/sec).

The term "tumor environment" or "tumor microenvironment" refers to a direct small scale environment of an organism or a portion of an organism, particularly as a different portion of a larger environment, e.g., a direct small scale environment of a tumor. The term includes not only the tumor cells themselves, but also the associated blood vessels associated with or surrounding the tumor (including endothelial cells and smooth muscle cells), immune system cells and secreted cytokines, epithelial cells, fibroblasts, connective tissue, and/or extracellular matrix. The term also refers to the cell and extracellular environment in which the tumor resides.

The term "standard of care" or "standard radiation therapy regimen" in radiation therapy generally refers to the dose and dosing interval of ionizing radiation that is commonly accepted in the medical field as an appropriate treatment for a given tumor, based on the tumor type, size, tissue location, and other various biological parameters. The standard of care or standard of treatment varies and depends on several factors. For example, for radiation therapy of lung cancer, the standard of care includes multiple fractions (e.g., about 30 cases of low dose radiation therapy, or about 60GY over 6 weeks) or a fewer number of fractions (e.g., 1-5 fractions) of the bioactive dose administered to the tumor (e.g., 54GY in 3 fractions for peripheral tumors, 48-60GY in 4-8 fractions for central tumors).

The term "similar dose of ionizing radiation" refers to a dose of ionizing radiation that is the same, nearly the same, or substantially the same as the effective dose administered to a tumor in another subject or to a tumor in the same subject who is receiving existing treatment. The term encompasses normal and expected variations in ionizing radiation dose delivered by a medical technician in the field of administering ionizing radiation to a tumor within a subject. For example, the term encompasses effective doses administered to tumors that vary by less than 10%, less than 5%, or less than 1%. The subject may be a human or non-human animal, such as a companion animal (e.g., cat, dog) or a farm animal (e.g., cow, horse, etc.).

The term "expression level" refers to the amount or level and/or presence or absence of a biomarker described herein.

The term "small molecule drug" refers to an organic compound having a molecular weight of less than about 50kDa, less than about 10kDa, less than about 1kDa, less than about 900 daltons, or less than about 500 daltons. The term includes drugs having the desired pharmacological properties and includes compounds that can be administered orally or by injection.

The term "radiosensitizer" refers to any substance that makes tumor cells more susceptible to killing by radiation therapy. Exemplary radiosensitizers include: hypoxic radiation sensitizers such as misonidazole, metronidazole, and sodium crotinite butyrate; and DNA damage response inhibitors, such as poly (ADP) ribose polymerase (PARP) inhibitors.

The term "reduced tissue damage" or "reducing damage to normal tissue" refers to a reduction in tissue damage upon administration of FLASH radiation as compared to administration of conventional radiation to a subject. Reduced tissue damage can be determined by measuring or quantifying the response of cells or tissues to radiation, such as, but not limited to, dermatitis, fibrosis, cell death, or respiratory disorders. In some embodiments, reduced tissue damage refers to at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more reduction in tissue damage in the response of cells or tissue to radiation when FLASH is administered as compared to administration of conventional radiation to a subject.

The terms "sample," "biological sample," and "tumor sample" refer to a bodily fluid, such as, but not limited to, blood, serum, plasma, or urine, and/or cells or tissue obtained from a subject or patient. In some embodiments, the sample is a formalin-fixed and paraffin-embedded tissue or tumor sample. In some embodiments, the sample is a frozen tissue or tumor sample. In some embodiments, the tumor sample may be a biopsy comprising tumor cells from a tumor. In some embodiments, the subject or patient is an animal or mammal. In some embodiments, the subject or patient is a human.

The term "about" refers to any value described herein or a change in a measured value of an administered dose described herein that is commonly encountered by one of ordinary skill in the art. Thus, the term "about" includes plus or minus 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 percent changes in any value described herein. Any value described herein is to be considered as modified by the term "about", whether or not the term "about" is used. Any numerical range recited herein includes the endpoints and all values between the endpoints. For example, a range of about 1 to 10 includes about 1.0, about 1.1, about 1.2, … …, about 9.8, about 9.9, or about 10.0.

Detailed Description

The methods described herein provide the advantages of anti-tumor efficacy and normal tissue protection when a therapeutic agent (such as an immunomodulator, anti-aging agent, radiosensitizer, or nanoparticle) is combined with FLASH radiation to treat cancer patients. The methods described herein provide unexpected results in that FLASH radiation in combination with a therapeutic agent (e.g., an immunomodulator therapy) can increase the anti-tumor response compared to FLASH radiation therapy or therapeutic agent therapy alone (monotherapy). An increase in the anti-tumor response may enhance or increase the inhibition of tumor growth provided by either therapy alone. The methods described herein can be used to treat localized and metastatic cancers by administering FLASH radiation therapy to deliver high conformal doses as well as therapeutic agents to the tumor. The combination therapy described herein can improve both the efficacy of (local and systemic) FLASH radiation therapy and the efficacy of therapeutic agent therapy. In some embodiments, the therapeutic agent or immunomodulator also enhances the anti-cancer response when administered in combination with FLASH radiation, as compared to the administration of the therapeutic agent alone or FLASH radiation monotherapy. The methods described herein may also increase the anti-tumor response compared to treatment with conventional radiation therapy or therapeutic agent therapy alone (monotherapy).

In one aspect, a method for treating a tumor in a subject having cancer is provided, comprising administering to the tumor FLASH RT and an immunomodulator. The immune modulator may be selected from the group consisting of: inhibitors of inhibitory checkpoint molecules, activators of stimulatory checkpoint molecules, chemokine inhibitors, inhibitors of macrophage Migration Inhibitory Factor (MIF), growth factors, cytokines, interleukins, interferons, antibodies that bind to cells of the immune system (such as bispecific antibodies that bind to T cells and tumor antigens), cellular immunomodulators (such as CAR-T cells), vaccines, oncolytic viruses, and any combination thereof. In some embodiments, the inhibitor of an inhibitory checkpoint molecule is a small molecule drug or an antibody or fragment thereof that specifically binds to and inhibits the activity of an inhibitory checkpoint molecule, wherein the inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, BTLA, A2aR, B7-H2, B7-H3, B7-H4, B7-H6, CD47, CD48, CD160, CD244(2B4), CHK1, CHK2, CGEN-15049, ILT-2, ILT-4, LAG-3, VISTA, gp49B, PIR-B, TIGIT, TIM1, TIM2, TIM3, TIM4 and KIR and their ligands. In other embodiments, the activator of a stimulatory checkpoint molecule is a small molecule drug, a polypeptide-based activator, or a polynucleotide-based activator that specifically binds to and increases the activity of a stimulatory checkpoint molecule, wherein the stimulatory checkpoint molecule is selected from the group consisting of: b7-1(CD80), B7-2(CD86), 4-1BB (CD137), OX-40(CD134), HVEM, inducible costimulatory molecule (ICOS), glucocorticoid-induced tumor necrosis factor receptor (GITR), CD27, CD28, CD40 and their ligands. In certain embodiments, the chemokine inhibitor is a small molecule drug or an antibody or fragment thereof that specifically binds to a chemokine (or a receptor thereof) and inhibits chemokine activity. In some embodiments, the chemokine is selected from the group consisting of: CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CXCL 24, and CXCL 24. In some embodiments, the chemokine inhibitor binds to a chemokine receptor selected from the group consisting of: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR 7. The inhibitor of MIF may be a small molecule drug or an antibody or fragment thereof that specifically binds to MIF and inhibits MIF activity. Other inhibitors of macrophage migration may also be used. In some embodiments, the immunomodulator is an inhibitor of indoleamine 2, 3-dioxygenase (IDO).

The method may further include; (a) detecting the expression level of one or more biomarkers in a tumor sample from the subject, wherein the one or more biomarkers (e.g., 1,2, 3, 4, 5 or more biomarkers) are selected from the group consisting of: immune cell marker(s), tumor cell marker(s), circulating marker(s), and any combination thereof; (b) comparing the expression level of the one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) to the expression level of the one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in a normal tissue sample; and (c) treating the tumor with FLASH RT and an immunomodulator if the expression level of the one or more biomarkers (e.g., 1,2, 3, 4, 5 or more biomarkers) is modified compared to the expression level in a normal tissue sample. In some cases, the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) is modified if the expression level of the at least one biomarker is decreased, or the expression level of the at least one biomarker is increased and the expression level of the at least one biomarker is decreased, as compared to the expression level in a normal tissue sample. The expression levels of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) can be ranked or weighted.

In some embodiments, the immune cell biomarker(s) or tumor cell biomarker(s) or circulating biomarker(s) is a polynucleotide or protein. The detecting step may be performed by using an assay selected from the group consisting of: immunohistochemistry, ELISA, Western analysis, HPLC, proteomics, PCR, RT-PCR, Northern analysis, and microarray.

The tumor sample may be a biopsy comprising tumor cells. The normal tissue sample may include non-tumor cells from the same tissue type as the tumor.

In another aspect, provided herein is a method of treating a tumor in a subject having cancer, the method comprising: (a) determining the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in a tumor sample from a subject, wherein the one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) are selected from the group consisting of: immune cell marker(s), tumor cell marker(s), circulating marker(s), and any combination thereof; (b) comparing the expression level of the one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) to the expression level of the one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in a normal tissue sample; and (c) administering a treatment comprising FLASH RT and a therapeutic agent to the tumor in the subject if the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in the tumor sample is modified compared to the expression level in the normal tissue sample.

In some cases, the step of administering FLASH RT comprises contacting the tumor with a radiosensitizer. FLASH RT can be administered at a higher dose compared to a standard treatment regimen if the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in the tumor sample is modified compared to the expression level in the normal tissue sample. FLASH RT can be administered as a macro-fractionated radiation therapy if the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in the tumor sample is modified compared to the expression level in the normal tissue sample. In other cases, FLASH RT is administered as a super-segmentation radiation therapy if the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in the tumor sample is modified compared to the expression level in the normal tissue sample.

In yet another aspect, provided herein is a method of identifying a subject with cancer as a candidate for treatment comprising FLASH RT and a therapeutic agent. The method comprises the following steps: (a) determining the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in a tumor sample from a subject, wherein the one or more biomarkers are selected from the group consisting of: immune cell marker(s), tumor cell marker(s), circulating marker(s), imaging marker(s), and any combination thereof: (b) comparing the expression level of the one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) to the expression level of the one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in a normal tissue sample; and (c) classifying the subject as a candidate for treatment comprising FLASH RT and a therapeutic agent if the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5 or more biomarkers) in the tumor sample is modified compared to the expression level in the normal tissue sample. In some cases, the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) is modified if the expression level of the at least one biomarker is increased, or the expression level of the at least one biomarker is decreased, or the expression level of the at least one biomarker is increased and the expression level of the at least one biomarker is decreased, as compared to the expression level in a normal tissue sample. In some cases, the expression levels of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) are ranked or weighted. In some cases, the method further comprises performing functional imaging of the tumor.

In another aspect, provided herein is a method of selecting a treatment for a subject having cancer, the method comprising: (a) determining the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in a tumor sample from a subject, wherein the one or more biomarkers are selected from the group consisting of: immune cell marker(s), tumor cell marker(s), circulating marker(s), and any combination thereof; (b) comparing the expression level of the one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) to the expression level of the one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in a normal tissue sample; and (c) selecting a treatment method comprising FLASH RT and a therapeutic agent if the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5 or more biomarkers) in the tumor sample is modified compared to the expression level in the normal tissue sample. In some embodiments, the method comprises performing functional imaging of the tumor; and selecting a treatment comprising FLASH RT and a therapeutic agent based on functional imaging of the tumor. In some cases, FLASH RT comprises contacting the tumor with a radiosensitizer.

In any of the above aspects and embodiments, the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) is modified if the expression level of the at least one biomarker is increased, or the expression level of the at least one biomarker is decreased, or the expression level of the at least one biomarker is increased and the expression level of the at least one biomarker is decreased, as compared to the expression level in a normal tissue sample. The expression levels of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) can be ranked or weighted.

In any of the above aspects and embodiments, if the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in the tumor sample is modified compared to the expression level in the normal tissue sample, FLASH RT is administered at a higher dose compared to the standard treatment regimen. In certain instances, FLASH RT is administered as a large fraction radiation therapy if the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in the tumor sample is modified compared to the expression level in the normal tissue sample. In other cases, FLASH RT is administered as a super-segmentation radiation therapy if the expression level of one or more biomarkers (e.g., 1,2, 3, 4, 5, or more biomarkers) in the tumor sample is modified compared to the expression level in the normal tissue sample.

In any of the above aspects and embodiments, the therapeutic agent is an immunomodulatory agent selected from the group consisting of: inhibitors of inhibitory checkpoint molecules, activators of stimulatory checkpoint molecules, chemokine inhibitors, inhibitors of macrophage Migration Inhibitory Factor (MIF), growth factors, cytokines, interleukins, interferons, antibodies that bind to cells of the immune system, cellular immunomodulators, vaccines, oncolytic viruses, and any combination thereof.

In any of the above aspects and embodiments, the methods described herein can further comprise performing functional imaging of the tumor prior to administering FLASH RT and/or the therapeutic agent.

Ionizing radiation (e.g., FLASH RT) and the therapeutic agent can be administered simultaneously. Alternatively, the FLASH RT and the therapeutic agent may be administered sequentially.

In another aspect, a kit (kit) is provided. The kit comprises reagents capable of detecting the expression of the biomarkers described herein. In some embodiments, the kit includes reagents capable of detecting expression of a nucleic acid (e.g., RNA) of a biomarker. For example, a kit can include oligonucleotide primers capable of amplifying nucleic acids expressed by a biomarker gene described herein. In some embodiments, the kit further comprises an oligonucleotide probe that hybridizes to the biomarker nucleic acid or amplified biomarker nucleic acid, or complement thereof. Methods of amplifying and detecting nucleic acids are well known in the art and may include PGR, RT-PCR real-time PCR and quantitative real-time PCR, Northern analysis, sequencing of expressed nucleic acids, and hybridization of expressed and/or amplified nucleic acids to microarrays. In some embodiments, the kit comprises reagents capable of detecting protein expression by the biomarkers described herein. In some embodiments, the agent is an antibody that specifically binds to the biomarker protein. Methods for detecting protein expression are well known in the art and include immunoassay, ELISA, Western analysis, and proteomic techniques.

In some embodiments of any of the above aspects and embodiments, the difference in the expression level of each biomarker in the tumor sample as compared to the expression level in normal tissue is increased or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the expression level of each biomarker in a tumor sample is increased or decreased by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more relative to the expression level in normal tissue.

In some embodiments, the average and/or ranked expression level of all biomarkers in a tumor sample is increased or decreased relative to the expression level in normal tissue. Thus, in some embodiments, the average and/or ranked expression level of all biomarkers in a tumor sample is increased or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the expression level in normal tissue. In some embodiments, the expression level in normal tissue is normalized to a control or baseline level. It will be appreciated that the expression level may also be compared to the expression level in a tumor sample before, after or during treatment, course of treatment or treatment plan. Thus, in some embodiments, the expression level of each biomarker in a tumor sample is increased or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the expression level in the tumor sample before, during, or after treatment.

Further, with respect to any of the above aspects and embodiments, the one or more biomarkers can include or consist of any combination of biomarkers, e.g., any biomarker described herein, any combination of two or more biomarkers, any combination of three or more biomarkers, any combination of four or more biomarkers, any combination of five or more biomarkers, any combination of six or more biomarkers, and any combination of seven or more biomarkers.

In another aspect, the expression level of at least one, two, three, four or more of the biomarkers described herein is determined. A combination of the expression levels of two or more biomarkers (e.g., 2,3, 4, 5, 6, or more biomarkers) can indicate that a subject having cancer is more sensitive to radiation than a control subject. A reduced or reduced dose of radiation may be administered to the subject compared to a standard dose. In other cases, if a combination of expression levels of two or more biomarkers (e.g., 2,3, 4, 5, 6, or more biomarkers) can indicate that a subject with cancer is less sensitive to radiation than a control subject. A subject that is less sensitive to radiation may be administered an increased dose, a large fractionated dose, or a super-fractionated dose of radiation. Alternatively, radiation therapy may be administered in combination with an immunomodulatory agent such as, but not limited to, an anti-TIM 4 antibody, an anti-MFG-E8 antibody, an anti-M199 antibody, and any combination thereof.

In some embodiments, the biomarker is CD44, MFG-E8, CD68, t GF β, or any combination thereof. In certain embodiments, if the first biomarker has a high expression level and the second biomarker has a low expression level in a sample obtained from a subject with cancer relative to a control sample, it can be predicted that radiation treatment monotherapy can result in failure of local tumor control. As such, the biomarker profile (profile) may indicate that radiation therapy should be administered to the subject in conjunction with an immunomodulatory agent. Alternatively, the biomarker profile may indicate that the radiation dose should be increased (i.e., over the standard regimen dose). For example, if the level of CD44 is higher and the level of MFG-E8 is lower in a tumor sample of a subject compared to a control sample, it can be predicted that radiation therapy alone will not result in a clinical response. In other words, tumor samples with high levels of CD44 and low levels of MFG-E8 may be insensitive or less sensitive to ionizing radiation or FLASH radiation therapy. In certain instances, the biomarker profiles described herein indicate that the subject should receive an increased dose of radiation and/or a combination therapy comprising FLASH RT and an immunomodulatory agent, such as an anti-TIM 4 antibody, an anti-MFG-E8 antibody, an anti-M199 antibody, and any combination thereof.

In other embodiments, the subject may have a clinical response to ionizing radiation or FLASH monotherapy if the level of CD44 is lower compared to the normal sample and/or the level of MFG-E8 is higher compared to the normal sample. In certain instances, it is predicted that subjects with low levels of CD44 and/or high levels of MFG-E8 may be sensitive to ionizing radiation or FLASH radiation therapy.

In some embodiments, a subject is predicted to have reduced survivability after radiation monotherapy if the subject's tumor has a high level of CD68 compared to a control sample. As such, a combination therapy comprising FLASH RT and an immunomodulator can be administered to the subject. In other cases, a subject may have a clinical response to radiation monotherapy if the subject's tumor has a lower level of CD68 compared to a control sample. It is predicted that the subject is sensitive to radiation. In some cases, it may be indicated that a low dose or reduced dose of radiation should be administered to the subject compared to the standard regimen dose.

FLASH radiation

FLASH radiation can be applied to tissue (e.g., tumor cells) in a single pulse or any sequence of pulses, where the interval between pulses is less than one second to several minutes. The dose of FLASH radiation can range from about 40Gy/sec to greater than 500 Gy/sec. In an embodiment, FLASH radiation allows dose deposition up to 200 centimeters in living tissue at FLASH rate. In some embodiments, the dose of FLASH radiation is sufficient to kill tumor cells located up to 200 cm deep in human or animal tissue, for example from 1Gy to more than 500Gy, with a total beam on delivery time of no more than 1 second. Each pulse within the FLASH sequence may be continuous or a sequence of shorter micropulses. In some embodiments, the dose per pulse is at least 0.1Gy, and the dose rate per pulse is at least 40Gy/sec (i.e., 0.1Gy within 2.5ms, 4Gy within 10ms, 20Gy within 0.5 seconds, etc.) and is up to 1e7Gy/sec as specified herein. The pulse duty cycle (i.e., the time interval) may have any value from 1 to 1 e-9.

In some embodiments, FLASH radiation is delivered using high energy particles or waves such as x-rays, gamma rays, electron beams, or protons. In some embodiments, FLASH radiation is delivered as proton therapy. In some embodiments, electrons are not used to deliver FLASH radiation. In some embodiments, the proton therapy is delivered according to the following schedule: FLASH:

1. 20Gy within 0.5 sec (40 Gy/sec)

2. 17.5Gy (40 Gy/sec) in 0.4375 sec

3. 15Gy within 0.375 second (40 Gy/second)

Pulse type FLASH

1. 10 1.5Gy pulses in 10 seconds with a duty cycle of 0.1 or 10%

2. 10 1.75Gy pulses in 10 seconds with a duty cycle of 0.1 or 10%

3. 10 pulses of 2Gy in 10 seconds with a duty cycle of 0.1 or 10%

In contrast, conventional proton radiation therapy can be delivered on the following schedule:

1. 20Gy within 40 seconds (0.5Gy/sec)

2. 17.5Gy within 35 seconds (0.5Gy/sec)

3. 15Gy within 30 seconds (0.5Gy/sec)

FLASH irradiation using electrons delivered by a linear electron accelerator is described, for example, in "ultra high dose-rate lasers and tumor tissue" of sci.trans.med.6, 24hra 93 (2014). A system for generating FLASH radiation is described in U.S. patent application No.15/089,330 (filed 4/1/2016), which is incorporated by reference herein in its entirety for all purposes. Thus, in some embodiments, the radiation therapy or therapy system is a proton pen beam scanning system that may be used for FLASH RT. More specifically, the system may include an accelerator and a beam delivery system and a nozzle that may be aimed toward the object. The nozzle includes a beam energy adjuster configured to adjust the beam by: for example, different thicknesses of material may be placed in the path of the beam to affect the energy of particles in the beam. The nozzle is capable of rapidly adjusting the particles in the beam to create a scanned beam (as opposed to a scattered beam) that delivers an entire relatively high therapeutic radiation dose in the target volume. For example, a dose of four gray or more may be delivered along a specified beam direction (e.g., a given ray) in less than one second.

Each ray is part of a scan pattern and irradiates the tissue along a different line segment ("target line segment") through the target volume. A high dose that can be delivered along a target line segment in a short time may be referred to as a "shot". In one embodiment, the shots may be adjusted in energy (intensity) or range and delivered to the target volume in a spread bragg peak (SOBP) that provides a uniform and appropriately modified dose for the entire target line segment.

Other types of radiation therapy systems that can be used for FLASH RT are described in co-pending provisional application Serial No. 62/434,053 entitled "Dynamic Three-Dimensional Compensator" filed on 12/14/2016, the entire contents of which are incorporated herein by reference.

In some embodiments, a radiation therapy or treatment system includes an accelerator, a beam delivery system, and a beam shaping system. In one embodiment, the accelerator may be a Radio Frequency (RF) based (linear accelerator, cyclotron, or synchrotron) or laser based accelerator. The accelerator may deliver the dose in a pulsed, continuous or quasi-continuous manner. The beam transmission may include magnetic elements (dipole, quadrupole, multipole), electrostatic elements, and slits or collimators. Beam shaping can be performed by a combination of magnetic, electrostatic and mechanical elements, with the aim of maximizing the conformality of the dose to the tumor. Embodiments include systems capable of delivering 3D conformal dose to a tumor in a single field or as a combination of multiple beamlets.

The FLASH RT dose may be delivered as a single dose (single fraction), or the total dose may be divided into multiple fractions that are delivered over time. In one embodiment, the medical technician may continuously monitor the tumor using, for example, x-ray or Magnetic Resonance Imaging (MRI) to help determine when to turn the beam on when the target is in a defined target field to further ensure optimal dose conformality.

FLASH RT may use Very High Energy Charged Particles (VHECPs) including but not limited to electrons, protons and heavy ions, high energy photons (x-rays, gamma rays) or any high energy neutral particles such as neutrons. High energy is defined as any energy that allows deposition of doses up to 200 cm in living tissue at FLASH rate. Intensity Modulated (IM) FLASH RT and Modulated Arc (MA) FLASH RT provide methods for achieving optimal dose conformality.

Radiation therapy planning system

In Intensity Modulated Radiation Therapy (IMRT), such as Intensity Modulated Particle Therapy (IMPT), the beam intensity varies across each treatment region (target) within the patient. Instead of treating with a relatively large and uniform field, the patient is treated with a plurality of smaller beams (e.g., beam segments or beamlets), each of which may have its own energy and/or intensity value, and each of which may be delivered from a different direction or angle (which may be referred to as a beam geometry). Because there are many possible beam geometries, number of beams, and ranges of beam energies or intensities across and within each beam, there are virtually an unlimited number (or at least a very large number) of potential treatment plans, and thus, consistently and effectively generating and evaluating high quality treatment plans is beyond the capabilities of humans and relies on the use of computer systems, particularly in view of the time constraints associated with using radiation therapy to treat diseases such as cancer, and particularly in view of the large number of patients who are receiving or need to receive radiation therapy over any given period of time.

Fig. 1 shows a cross-sectional view of possible beam geometries for a tumor (target 710) and surrounding tumor microenvironment 720. In fig. 1, the prescribed dose to be delivered into and across the target is determined. Each portion of the target may be represented by at least one 3D element (called voxel); a portion may include more than one voxel. A portion of a voxel or target may also be referred to herein as a sub-volume; the sub-volume may include one or more portions or one or more voxels. As will be described in detail below, each portion or voxel may receive radiation from one or more beams delivered from different directions. The specified dose defines, for example, a dose value or a minimum dose value and a maximum dose value for each portion or voxel of the target. In one embodiment, the prescribed dose is the same for all portions (sub-volumes or voxels) of the target, such that a uniform dose is prescribed for the entire target.

In fig. 1, the direction for delivering the beam 705-707 into the target 710 (e.g., gantry angle relative to the patient or target, or nozzle direction relative to the patient or target) is determined. The beam may be a proton beam, electron beam, photon beam, ion beam, or nuclear beam. Determining the beam direction may include determining the number of beams (the number of directions from which the beams are to be delivered). The paths of the beams may or may not overlap within the target and may or may not overlap outside the target. Generally, when generating a radiation therapy plan, one goal is to determine a beam path that minimizes the irradiation dose for each sub-volume or voxel of tissue outside the target. In some cases, organs considered important by radiation oncologists may need to receive less dose than other organs in the vicinity. Thus, conventional treatment plans are driven by a specified threshold of calculated integrated dose to the tumor and surrounding organs.

The present disclosure provides additional criteria for determining organ dose thresholds outside of the target 710. Figure 2 contains a 3-dimensional surface plot of dose versus time and another variable. The dose delivered to the tumor will depend on more variables, but two variables are chosen for the purpose of describing the embodiments without increasing complexity. In general, the dose surface may depend on any number of variables, and will generally be n-dimensional, where n is greater than or equal to 1. In the case of tumor microenvironment 720, the other parameter may be immune sensitivity, tumor reactivity to an immunomodulator, or some other biological parameter. While in general the shape of the surface may vary, fig. 2 shows an increasing dose threshold with decreasing exposure time, and a slightly increasing dose threshold with increasing "immunobiological indicators".

In one embodiment, the temporal constraints may be satisfied by the geometry of the beam configuration. Ideally, each sub-volume or voxel outside the target intersects at most only a single beam. If some overlap between the beam paths is allowed, then ideally each sub-volume or voxel outside the target intersects no more than two beams, and most intersect only a single beam. In one embodiment, as a means to achieve the above goal, the beam direction is determined such that the total amount of overlap between beam paths outside the target is minimized. In one such embodiment, the directions are determined such that the beam paths overlap within the target and such that a total amount of overlap of the beam paths outside the target is less than a total amount of overlap of the beam paths within the target. In another such embodiment, the directions are determined such that the beam paths do not overlap at all outside the target. The beam paths may be in the same plane or in different planes.

The beam (705-707) is shown passing through the patient, but the disclosure is not so limited. The beam may also terminate within the target, like an ion beam and a proton beam. In this case, the beam energy determines the endpoint within the target, which the calculation engine and optimizer need to take into account. Otherwise, all other optimization parameters are the same.

Although the operations in fig. 1 are shown as occurring in a certain order and in series, the disclosure is not so limited. These operations may be performed in a different order and/or in parallel, and they may also be performed in an iterative manner, as the number of beams (and corresponding number of directions), beam directions, and beam energies or intensities (and/or beam segment energies or intensities) used to deliver a specified dose are interrelated. As described above, due to the different parameters that need to be considered, the range of values of those parameters, the interrelationships of those parameters, the need for a treatment plan that minimizes risk to the patient but is effective, and the need for rapid generation of a high quality treatment plan, it is important that the optimizer model 150 executed consistently on the computing system 106 (FIG. 3) be used for radiation treatment planning as disclosed herein.

Although multiple beams are shown in fig. 1, this does not mean that all beams must be delivered simultaneously or at overlapping time periods, although they may be. The number of beams delivered at any one time depends on the number of gantries or nozzles in the radiation therapy system and the treatment plan.

In operation, in one embodiment, the beam segments are delivered sequentially. For example, beam segment 705 is delivered to the target (on) and then off, then beam segment 706 is turned on and then off, then beam segment 707 is turned on and then off, and so on. Each beam segment may be on for only one second of time (in milliseconds). In one embodiment, all beams 705-707 may be turned on simultaneously.

As shown in fig. 1, a subvolume can be traversed by more than two beams, in which case the cumulative dose for that subvolume is represented by adding the appropriate values for each beam that reaches it. Such sub-volumes are referred to as overlap regions 730. That is, a total value is determined for each sub-volume in the target 710 by adding together the values of each beam region of each beam that reaches the sub-volume. The dose in the overlap region can also be optimized by the constraints defined by the surfaces in fig. 2.

The optimizer model may adjust parameters that affect the calculated dose delivered to the target 710 to achieve a satisfactorily uniform cumulative dose across the target 710. A satisfactory uniform cumulative dose is indicated when all of the total values for each sub-volume in the target 710 are the same or when the difference between the total values for each sub-volume meets a threshold.

The threshold may be, for example, a value specifying a range of allowed differences or specifying a maximum allowed difference.

Dose limitations may include, but are not limited to: a limit on the irradiation time of each sub-volume (voxel) in the target (e.g., treatment time <0.5 seconds for each voxel of the target tissue); a limitation on the exposure time of each sub-volume (voxel) outside the target (e.g., treatment time <0.5 seconds for each voxel of normal tissue); a limit on the dose rate per sub-volume (voxel) in the target (e.g., dose rate >40Gy/sec per voxel of target tissue); and a limit on the dose rate per sub-volume (voxel) outside the target (e.g., dose rate >40Gy/sec per voxel of normal tissue); dose and dose rate limitations in the tumor microenvironment. Generally, these limitations aim to minimize the amount of time to irradiate normal tissue and maximize the immune response.

In summary, the embodiments described herein improve radiation therapy planning and treatment itself by: extending FLASH RT to a broader therapeutic platform and target site and optimizing delivery of FLASH RT to enhance immune response. The generated treatment plans described herein protect normal tissue more effectively from radiation by designing the amplitude (and in some cases the integral) of the dose to the normal tissue (outside the target) that is reduced (if not minimized) compared to conventional techniques (even for non-FLASH dose rates). When used with FLASH dose rates, patient motion management is simplified. While still a complex task to find a balance between competing and related parameters, treatment planning is simplified over conventional planning. The techniques described herein may be used for stereotactic radiosurgery as well as stereotactic body radiotherapy with single or multiple metastases.

In some embodiments, the methods described herein may be used with other types of external beam radiotherapy, such as, but not limited to, IMPT, Intensity Modulated Radiation Therapy (IMRT), Image Guided Radiation Therapy (IGRT), RapidArc^TMRadiotherapy, stereotactic radiotherapy (SBRT)And stereotactic ablative radiation therapy (SABR).

Fig. 3 shows a block diagram of an example of a computing system 100 that may be used for radiation treatment planning. In its most basic configuration, system 100 includes at least one processing unit 102 and memory 104. This most basic configuration is illustrated in fig. 3 by dashed line 106. The system 100 may also have additional features and/or functionality. For example, system 100 may also include additional storage (removable and/or non-removable) including, but not limited to, magnetic or optical disks or tape. Such additional storage is illustrated in FIG. 3 by removable storage 108 and non-removable storage 120. System 100 may also contain communication connection(s) 122 that allow the device to communicate with other devices, such as in a networked environment using logical connections to one or more remote computers.

The system 100 also includes input device(s) 124 such as keyboard, mouse, pen, voice input device, touch input device, etc. Output device(s) 126 such as a display device, speakers, printer, etc. may also be included.

In the example of fig. 3, memory 104 includes computer-readable instructions, data structures, program modules, etc. associated with treatment planning system 150. However, treatment planning system 150 may alternatively reside in any one of the computer storage media used by system 100, or may be distributed across some combination of computer storage media, or may be distributed across some combination of networked computers.

The treatment planning system 150 may be used to formulate a radiation treatment plan for a particular patient by receiving patient-specific information (e.g., geometric information) that is input to and processed by the treatment planning system. The input patient-specific information may include virtually any combination of parameters that may affect the radiation therapy plan. For example, patient-specific information may be organized as a vector or data structure, including characteristic elements of: the size and shape of the target volume; a location of the target volume; size and shape of the organ at risk; the type of organ at risk; a portion of the target volume overlapping the organ; and a portion of the organ that overlaps the target volume.

The treatment planning system 150 may be used to predict dose parameters for a treatment plan corresponding to a particular patient. The dose treatment planning system 150 may include a Dose Volume Histogram (DVH) estimation model, where the predicted quantity is a dose volume histogram. In other embodiments, the treatment planning system 150 also generates predictions based on a histogram of distance to target (DTH), which represents the distance from a region of interest (ROI) to the radiation target. In general, the treatment planning system 150 is implemented as any model suitable for predicting the dose (as a dose histogram or spatial 3D dose distribution) for radiation treatment planning.

As described above, the radiation therapy planning system 150 may consider both the spatial and temporal domains. For example, a time-dependent component may be included in a treatment plan with IM or MA FLASH RT for VHECP.

In some embodiments, the treatment planning system 150 considers a combination of FLASH RT and immunotherapy. Treatment planning system 150 incorporates immunotherapy into the treatment plan, including how immunotherapy affects dosage regimens and the effect of immunotherapy on radiation delivery and dosage rates. In general, the treatment planning system 150 determines a final radiation treatment plan that integrates FLASH RT and immunotherapy. For example, the treatment planning system 150 can iteratively evaluate the FLASH aspect of radiation therapy and the immunotherapy/biological aspect of radiation therapy to generate a final radiation treatment plan that can optimize the combination of the two aspects.

For example, the planner defines a set of quality metrics. For planning purposes, metrics are defined such that smaller values are preferred over larger values. The planner also defines a relative priority or weight (wi) for each quality metric. Then, the task of making the best plan is expressed as a quadratic cost function C: sum (wi (Qi-Qi)2), where Qi is the value of the quality metric that can be achieved for a particular treatment plan. The best plan is determined by minimizing the cost function C.

Another approach to assist the planner is to use a multi-criteria optimization (MCO) approach for treatment planning. Pareto surface navigation is an MCO technique that facilitates the exploration of tradeoffs between clinical goals. For a given set of clinical goals, a treatment plan is considered pareto-optimal if it meets the goals and none of the metrics improves without worsening at least one other metric. A set of pareto optimal plans defines pareto surfaces associated with a set of clinical objectives. Motion along the pareto surface results in a trade-off between clinical goals; some metrics will improve at the expense of deteriorating one or more other metrics. The planner may navigate along the pareto surface and select the final (optimized) radiation treatment plan that appears best according to criteria applied by the planner, or may automatically select a treatment plan based on its proximity to the pareto surface.

Metrics associated with the combination of FLASH RT and immunotherapy include, for example, metrics associated with target homogeneity, critical organ retention, prescribed dose, dose delivery rate, normal and tumor tissue toxicity, and the like.

Fig. 4 is a block diagram illustrating an embodiment of an automated radiation therapy treatment planning system 150. The system 150 comprises an input interface 210 for receiving patient specific information (data) 201 and an output interface 230. System 150 may be implemented in whole or in part on/using computing system 100 (fig. 3) as a software program, hardware logic, or a combination thereof.

The radiation therapy treatment planning system 150 also receives or accesses FLASH RT parameters and metrics, as well as immunotherapy parameters and metrics. Examples of metrics are described herein. FLASH RT parameters may include, for example, beam type, beam energy, angle of the beam relative to the patient/target volume, beam shape, dose delivery schedule, total dose and exposure time for any given normal tissue volume outside the tumor, and dose and exposure time within the tumor microenvironment that may optimize the immune response. Immunotherapy parameters may include, for example, the type of drug or immunomodulator administered to the subject, the tumor antigen, neoantigen or antibody administered, the dosage, and the delivery schedule (e.g., before, after, and/or during FLASH RT).

The treatment planning system 150 generates prediction results, such as achievable dose distribution predictions. A treatment plan may then be generated based on the prediction. In one embodiment, the prediction outcome is accompanied by parameters indicative of the quality of the prediction, such as the reliability of the outcome, the projected complexity of the prediction, and the probability of the outcome.

In some embodiments, the radiation therapy planning system described herein will generate predictions as follows: a lower effective dose of radiation, such as FLASH radiation, can be administered to a subject based on the subject's response to immunotherapy. For example, in some embodiments, the radiation therapy planning system(s) will predict that if a tumor of the subject produces a favorable response to immunotherapy, e.g., the tumor shrinks after immunotherapy, a lower effective dose of radiation, e.g., FLASH radiation, may be administered to the subject.

In some embodiments, the radiation therapy planning system described herein will generate predictions as follows: the effective dose of radiation (e.g., FLASH radiation) is varied based on parameters that enhance the immune response (e.g., increase delivery or activity of an immunomodulator) to more effectively treat a tumor in a subject.

Biomarkers for radiation therapy selection

The biomarkers described herein can be used to stratify patients to receive personalized, customized radiation therapy (e.g., FLASH RT) in combination with immunomodulators. Biomarkers can also be used to monitor the efficacy of immune modulator therapy on cancer patients. Biomarkers include, but are not limited to, one or more immune cell biomarkers, one or more tumor cell biomarkers, one or more circulating biomarkers, one or more imaging biomarkers, and any combination thereof. For example, immune cell biomarkers can provide information about the location and/or activity of a particular cell population (such as a T cell population). The immune cell biomarker or tumor cell biomarker may be a genetic biomarker, a polynucleotide biomarker, or a protein biomarker. In some embodiments, an immune cell biomarker is a specific polynucleotide (e.g., RNA and microrna) or protein that is expressed at a higher level by a specific immune cell than a non-immune cell or a different type of immune cell. Similarly, a tumor cell biomarker can be a specific polynucleotide (e.g., RNA and microrna) or protein that is expressed at a higher level by a tumor cell than a non-tumor cell. For example, a tumor cell biomarker can be a protein or polynucleotide encoding a protein associated with proliferation and/or metastasis of a tumor cell. In some cases, the protein may be involved in angiogenesis or other processes activated by tumor cells. The tumor biomarker may be an oncogene or a tumor suppressor. In some cases, a tumor cell biomarker is a genetic variation, genetic mutation, Copy Number Variation (CNV), Single Nucleotide Polymorphism (SNP), or the like, that is present in a tumor cell but not in a non-tumor cell. In some embodiments, the circulating biomarker is an exosome (i.e., a cell-derived vesicle that can be found in a bodily fluid). Examples of useful biomarkers include those described in U.S. patent application publication No.20160024594, the disclosure of which is incorporated herein by reference for all purposes.

The set of biomarkers may include, but is not limited to, CD44, lactococcus EGF factor 8(MFG-E8), CD68, and TGF β. CD44 is a cell surface glycoprotein that plays a role in cell proliferation, cell-cell interactions, cell adhesion, and cell migration of various cell types, including lymphocytes and cancer cells. The human CD44 polypeptide sequence is described in, for example, GenBank accession No. np _ 000601. The human CD44 mRNA (encoding) sequence is described in, for example, GenBank accession No. nm — 000610. lactococcus-EGF factor 8 protein (MFG-E8) is a macrophage-produced protein that promotes tumor cell apoptosis and phagocytosis. The human MFG-E8 polypeptide sequence is described, for example, in GenBank accession No. np _ 005919. The human MFG-E8 mRNA (coding) sequence is described in, for example, GenBank accession No. nm — 005928. CD68 is a 110-kD transmembrane glycoprotein that is highly expressed by human monocytes and tissue macrophages. The protein is predominantly localized to lysosomes and endosomes, with a smaller fraction circulating to the cell surface. It is a type I integral membrane protein with a highly glycosylated extracellular domain and binds to tissue and organ specific lectins or selectins. CD68 is also a member of the scavenger receptor family. The human CD68 polypeptide sequence is described in, for example, GenBank accession No. np _ 001242. Human CD68mRNA (coding) sequence is described in, for example, GenBank accession No. nm _ 001251. TGF is a cytokine involved in cell growth, cell proliferation, cell differentiation, apoptosis, homeostasis, and many other cellular processes. Human TGF β polypeptide sequences are described, for example, in GenBank accession No. np _ 000651. The human TGF β mRNA (coding) sequence is described in, for example, GenBank accession No. nm — 000660.

It will be appreciated that the expression level of each of the biomarkers described herein in a patient sample may be increased or decreased relative to the expression level of the tumor biomarker in a normal or control tissue sample. For example, the expression level of one tumor biomarker in a tumor sample can be increased as compared to the expression level in normal tissue, while the expression level of a second biomarker in a tumor sample can be decreased as compared to the expression level in normal tissue. The expression level may also be based on the average, combination or sum of the expression levels of all tumor biomarkers in the patient sample. For example, the expression level of each biomarker in a patient sample may be ranked or weighted to produce a ranking value (which may be a normalized value, e.g., set to 1) that is higher or lower than the normal tissue value.

In some embodiments, biomarker expression is determined in a biological sample from a subject having a tumor. In some embodiments, the biological sample is a tumor sample. The tumor sample may be a biopsy comprising tumor cells from a tumor. In some embodiments, the biological sample comprises a bodily fluid from the subject, such as, but not limited to, blood, serum, plasma, or urine, and/or cells or tissues. In some embodiments, the biological sample is a formalin-fixed and paraffin-embedded tissue or tumor sample. In some embodiments, the biological sample is a frozen tissue or tumor sample. Thus, in some embodiments, one or more steps of the methods described herein are performed in vitro. For example, in some embodiments, biomarker expression is determined in vitro.

In some embodiments, the normal tissue sample comprises non-tumor cells from the same tissue type as the tumor. In some embodiments, the normal tissue sample is obtained from the same subject diagnosed with a tumor. The normal tissue sample may also be a control sample of the same tissue type from a different subject. The expression level of a normal tissue sample may also be an average or mean value obtained from a population of normal tissue samples.

The expression level of the biomarkers described herein can be determined using any method known in the art. For example, the expression level can be determined by detecting the expression of a nucleic acid (e.g., RNA, mRNA, or microrna) or a protein encoded by the nucleic acid.

Exemplary methods for detecting the expression level of a nucleic acid include, but are not limited to, Northern analysis, Polymerase Chain Reaction (PCR), reverse transcription PGR (RT-PCR), real-time PCR, quantitative real-time PCR, and DNA microarrays.

Exemplary methods for detecting the expression level of a protein (e.g., polypeptide) include, but are not limited to, immunohistochemistry, ELISA, Western analysis, HPLC, and proteomic assays. In some embodiments, protein expression levels are determined by immunohistochemistry using the Allred method to assign a score (see, e.g., "Allred, d.c., Connection9:4-5,2005," incorporated herein by reference). For example, formalin fixed, paraffin embedded tissue is contacted with an antibody that specifically binds to the biomarkers described herein. The bound antibody is detected using a detectable label or a secondary antibody, such as a colorimetric label (e.g., an enzymatic substrate produced by HRP or AP), coupled to a detectable label. Antibody positive signals were scored by estimating the proportion of positive tumor cells and their mean staining intensity. Both the scale and intensity scores are combined into an overall score that is used to weight both factors.

In some embodiments, the protein expression level is determined by digital pathology. Digital pathology methods include scanning images of tissue on a solid support, such as a slide. The slide is scanned into a complete slide image using a scanning device. The scanned images are typically stored in an information management system for archiving and retrieval. Image analysis tools can be used to obtain objective quantitative measurements from digital slides. For example, the area and intensity of immunohistochemical staining may be analyzed using appropriate image analysis tools. The digital pathology system may include scanners, analytics (visualization software, information management system, and image analysis platform), storage, and communications (sharing services, software). Digital pathology Systems are available from a number of commercial suppliers, such as Aperio Technologies, Inc (a subsidiary of leica microscopy Systems, Inc.) and Ventana Medical Systems, Inc (now part of Roche). Expression levels can be quantified by commercial service providers, including Flagship Biosciences (CO), Pathology, Inc. (CA), Quest Diagnostics (NJ), and Premier Laboratory LLC (CO).

In some embodiments, imaging of tumors (such as functional imaging) is also used to identify or select cancer patients that should receive the combination therapies described herein. Non-limiting examples of functional imaging include single photon emission computed tomography, optical imaging, ultrasound, Positron Emission Tomography (PET), Computed Tomography (CT), perfusion computed tomography, Magnetic Resonance Imaging (MRI), functional magnetic resonance imaging, magnetic resonance spectroscopy imaging, dynamic contrast enhanced imaging, diffusion weighted imaging, blood oxygen level dependent imaging, magnetic resonance spectroscopy, magnetic resonance lymphography, and any combination thereof. Any type of functional imaging, such as multi-modal imaging, may be performed to characterize the tumor, to determine the spectrum of the tumor, the extent of the tumor, the volume of the tumor, and/or to assess the microenvironment of the tumor (e.g., the environment surrounding the tumor). Functional imaging may aid in the selection of an optimal treatment regimen and/or monitoring response to treatment.

Method for selecting a treatment course

The expression level of the biomarker can be used to determine or select a course of treatment for a subject diagnosed with a tumor. For example, in some embodiments, the treatment comprises administering ionizing radiation, such as FLASH RT, to a tumor within the subject. Ionizing radiation may also be administered to the entire subject or a portion thereof, particularly where the tumor is dispersed or mobile. In some embodiments, the treatment further comprises contacting the tumor with a radiosensitizer. In some embodiments, the treatment further comprises administering to the subject a compound or biologic, such as an antibody, that inhibits the immune checkpoint pathway. In some embodiments, the treatment comprises administering a FLASH radiation therapy regimen in combination with an immunomodulator.

The course of treatment may be selected based on the expression level of the biomarker. For example, the expression level may be used to determine whether radiation therapy is appropriate for the subject (i.e., for making a pass/fail decision on radiation therapy). Furthermore, if the expression level of the biomarker is increased relative to a normal or control value, the effective radiation dose to the tumor may be increased and/or the segmentation schedule modified accordingly. In some embodiments, the effective dose of FLASH RT to the tumor is increased. The radiation dose to the blood vessels that feed the tumor can also be increased. In some cases, large fraction radiation therapy is administered. Alternatively, hyperfractionation radiation therapy is administered. In some embodiments, FLASH radiation therapy is provided in combination with immunomodulatory therapy.

In some embodiments, if the expression level of the biomarker is increased relative to a normal or control value, the treatment may comprise administering ionizing radiation, such as FLASH RT, to the tumor. In some embodiments, if the expression level of the biomarker is reduced relative to a normal or control value, the treatment may comprise reducing the amount of ionizing radiation or FLASH RT administered to the tumor.

Treatment may also include modifying existing treatment procedures. For example, in some embodiments, existing treatment procedures are modified to increase the effective dose of ionizing radiation (such as FLASH radiation) administered to the tumor. In some embodiments, the effective dose of ionizing radiation, such as FLASH radiation, is increased by increasing the amount of ionizing radiation administered to the tumor and/or contacting the tumor with a radiosensitizer. In some embodiments, existing treatment procedures are modified to reduce the effective dose of ionizing radiation administered to the tumor. In some embodiments, the treatment comprises modifying a standard radiation therapy regimen in combination with administration of an immunomodulator.

In some embodiments, if the level of one or more biomarkers described herein is elevated in the tumor environment, the effective dose of ionizing radiation (such as FLASH radiation) administered to the tumor is increased. For example, the effective dose of ionizing radiation is increased compared to the standard of treatment for a subject who does not have an elevated level of biomarker(s) in the tumor environment. This applies to subjects not currently undergoing radiation therapy, as well as to methods of modifying existing treatment protocols for subjects undergoing radiation therapy. Thus, if the subject has been subjected to radiation therapy directed to a tumor, the effective dose of ionizing radiation may be increased from the current effective dose. The radiation therapy may be modified to reduce the constraint on adjacent healthy tissue. For example, if biomarker levels in the tumor environment indicate a need for more aggressive radiation therapy, the treatment plan may be modified to reduce the constraints on the boundary between healthy tissue and tumor tissue. This will lead to a trade-off between damaging some healthy tissue to kill more tumorous tissue.

In some embodiments, the treatment comprises a combination of radiation therapy and an immunomodulator (including a radiosensitizer). In some embodiments, the treatment comprises a combination of FLASH radiation therapy and an immunomodulator (including a radiosensitizer). In some embodiments, the effective dose of ionizing radiation administered to a tumor does not change (e.g., relative to standard of care or relative to an existing course of treatment) when an immunomodulatory agent is administered to a subject. For example, in some embodiments, the same or a similar effective dose of ionizing radiation is administered to the subject as that administered to a subject who does not have an elevated level of one or more biomarkers described herein in the tumor environment, and the immune modulator is further administered to the subject. In some embodiments, the effective dose of ionizing radiation administered to the tumor is based on a standard of care for the subject that does not have an elevated biomarker level(s) in the tumor environment, and the immune modulator agent is further administered to the subject. In some embodiments involving existing therapeutic procedures, an effective dose of ionizing radiation is maintained at the current effective dose, and if the level of one or more biomarkers described herein is elevated in the tumor environment, an anti-cancer agent is administered to the subject in combination with the ionizing radiation.

In some embodiments, a treatment plan is formulated and/or modified based on the expression levels of the biomarkers described herein.

The course of treatment may also be selected by using an algorithm (e.g., a computer-implemented algorithm) that analyzes or determines the expression level of a biomarker in a tumor sample relative to the level in a normal sample. The algorithm may be a linear regression algorithm that includes biomarker expression levels and coefficients (i.e., weights) for combining the expression levels. In some embodiments, the algorithm includes a least squares fit for calculating the coefficients. If the algorithm determines that the expression level of the biomarker in the tumor sample is increased or decreased relative to the normal sample, an appropriate course of treatment can be assigned. In some embodiments, the algorithm is a non-parametric regression tree. In some embodiments, a treatment comprising FLASH radiation and an immunomodulator is selected depending on the algorithm determining that the expression level of the biomarker in the tumor sample is increased or decreased relative to a normal sample. In some embodiments, the data is analyzed using standard statistical methods to determine which biomarkers are most predictive of clinical survival or failure of local tumor control.

In some embodiments, the methods described herein are computer-implemented methods. In some embodiments, the computer-implemented method comprises a linear regression model that assigns a ranking or weighting value to the expression levels of the biomarkers described herein. In some embodiments, the present disclosure provides a computer-readable medium that provides instructions for causing a computer to perform the methods described herein. For example, the medium may provide instructions for causing a computer to assign a ranking or weighting value to the expression levels of the biomarkers described herein.

Radiation therapy

The expression levels of the tumor biomarkers described herein can be used to optimize treatment of patients with radiation therapy (such as FLASH RT). For example, the therapeutic dose of radiation administered to a tumor or a subject can be adjusted based on the expression level of the biomarker. The effective dose of ionizing radiation will vary with the type of tumor and the stage of the cancer to be treated. Effective dosages may also vary based on the other treatment modalities being administered to the patient (e.g., chemotherapy and surgical treatment) and whether radiation therapy is being administered pre-or post-operatively. Typically, the conventional therapeutically effective dose for solid epithelial tumors is about 60 to 80 gray (Gy), while the therapeutically effective dose for lymphomas is about 20 to 40 Gy. Typically, the prophylactic dose may be 45-60 Gy. For FLASH irradiation, the therapeutically effective treatment dose for solid epithelial tumors may range from about 20 to 200 gray (Gy), while the therapeutically effective dose for lymphomas may range from about 10 to 200Gy, while the prophylactic dose is 5-500 Gy.

The therapeutic dose may be delivered in divided fashion. Segmentation refers to the spreading of the total radiation dose over time (e.g., days, weeks, or months). The dose delivered in each fraction may be about 1.5-2Gy per day. The treatment plan may include one or more fractions of treatments per day, every other day, weekly, etc., depending on the treatment needs of each patient. For example, a large split schedule includes dividing the total dose into several relatively large doses, and administering the doses at least one day apart. An exemplary large fractionated dose is 3Gy to 20Gy per fraction. An exemplary segmentation schedule that may be used to treat lung cancer is continuous hyper-segmentation accelerated radiation therapy (CHART), which consists of three small segments per day.

In some embodiments, FLASH RT comprises contacting a tumor within the subject with a radiosensitizer. Exemplary radiosensitizers include hypoxic radiosensitizers, such as misonidazole, metronidazole, and clocetin trans-sodium, which are compounds that help increase the diffusion of oxygen into hypoxic tumor tissue. Radiosensitizers can also be DNA damage response inhibitors that interfere with Base Excision Repair (BER), Nucleotide Excision Repair (NER), mismatch repair (MMR), repair by recombination including Homologous Recombination (HR) and non-homologous end joining (NHEJ), and direct repair mechanisms. The SSB repair mechanisms include BER, NER or MMR pathways, while the DSB repair mechanisms include HR and NHEJ pathways. Irradiation causes DNA breaks that would be fatal if not repaired in time. Single strand breaks are repaired by a combination of BER, NER and MMR mechanisms using the intact DNA strand as a template. The dominant pathway for SSB repair is BER using a series of related enzymes called poly (ADP-ribose) polymerase (PARP). Thus, radiosensitizers may include DNA damage response inhibitors, such as poly (ADP) ribose polymerase (PARP) inhibitors.

The biomarkers described herein can be used to develop and modify treatment plans for patients diagnosed with tumors or cancers. The treatment plan may include visualizing or measuring the volume of the tumor that needs to be irradiated, the optimal or effective dose of radiation to be administered to the tumor, and the maximum dose that prevents damage to nearby healthy tissue or organs at risk. Algorithms may be used for treatment planning and include dose calculation algorithms based on the particular radiation therapy technique parameters employed (e.g., gantry angle, MLC leaf position, etc.), and search algorithms that adjust system parameters between dose calculations using various techniques to optimize treatment outcome. Exemplary dose calculation algorithms include various Monte Carlo ("MC") techniques and pencil beam convolution ("PBC"). Exemplary search algorithms include various simulated annealing ("SA") techniques, algebraic inverse processing plans ("AITP"), and simultaneous iterative inverse processing plans ("SIITP"). Such techniques and others are included within the scope of the present disclosure.

The treatment planning algorithm may be implemented as part of an integrated treatment planning software package that provides additional features and capabilities. For example, a dose calculation algorithm and a search algorithm may be used to optimize a set of flux maps at each gantry angle, where a separate leaf sequencer is used to calculate the leaf motion required to deliver the leaves. Alternatively, the leaf motion and other machine parameters may be optimized directly using dose calculation algorithms and search algorithms. Eclipse provided by the assignee of the present application^TMThe treatment planning system includes such an integrated software program. Methods for optimizing treatment regimens are described in U.S. Pat. No.7,801,270, which is incorporated herein by reference.

In some embodiments, the biomarkers described herein can be used to monitor the progression of tumor control after FLASH radiation therapy. For example, the expression levels of the biomarkers before and after ionizing FLASH radiation therapy can be compared. In some embodiments, if the expression level of the biomarker increases after radiation therapy, this suggests that the size of the tumor continues to increase. Thus, radiation therapy can be modified based on monitoring tumor growth using the biomarkers described herein.

The biomarkers described herein can be used with any radiation therapy technique known in the art. Radiation therapy techniques include external beam radiation therapy ("EBRT") and intensity modulated radiation therapy ("IMRT"), which may be administered by a radiation therapy system, such as a linear accelerator, equipped with a multi-leaf collimator ("MLC"). The use of multi-leaf collimators and IMRT allows the treatment of a patient from multiple angles while changing the shape and dose of the radiation beam, thereby avoiding over-irradiation of nearby healthy tissue. Other exemplary radiation therapy techniques include stereotactic radiotherapy (SBRT), volume modulated arc therapy, three-dimensional conformal radiotherapy ("3D conformal" or "3 DCRT"), Image Guided Radiotherapy (IGRT). Radiation therapy techniques may also include Adaptive Radiation Therapy (ART), a form of IGRT that may modify the treatment method during the course of radiation therapy to optimize the dose distribution according to patient anatomical changes and the shape of organs and tumors. Another radiation therapy technique is brachytherapy. In brachytherapy, a radiation source is implanted in a subject such that the radiation source is in close proximity to a tumor. As used herein, the term radiotherapy should be broadly construed and is intended to include various techniques for irradiating a patient, including the use of photons (such as high energy x-rays and gamma rays), particles (such as electron beams and proton beams), FLASH RT, and radiosurgery techniques. Moreover, any method of providing conformal radiation to a target volume is intended to be within the scope of the present disclosure.

Therapeutic agents

The FLASH radiation therapy described herein can be administered in combination with one or more therapeutic agents. Examples of therapeutic agents include immunomodulators, anti-aging agents, radiosensitizers, and nanoparticles.

In some embodiments, the therapeutic agent is a mitotic spindle inhibitor. In some embodiments, radiation therapy is combined with mitotic spindle inhibitors such as cyclin inhibitors, e.g., CDK4/6 inhibitors (e.g., Pablociclib), AURKA inhibitors (such as the small molecules Alisertib and Tozasertib), blocking TPX2-AURKA complexes such as complex disruptors (GSK1070916) (Asteriti et al, 2015; Janecek et al, 2016) and taxanes (such as docetaxel, paclitaxel).

In some embodiments, the therapeutic agent is a DNA repair and response pathway inhibitor. In some embodiments, FLASH radiation therapy is used in combination with: PARP inhibitors (e.g., Talazoparib, Rucaparib, Olaparib) (Lord and Ashworth, 2016; Murai et al, 2012), RAD51 inhibitors (RI-1), or inhibitors of DNA damage response kinases (such as CHCK1(AZD7762), ATM (KU-55933, KU-60019, NU7026, VE-821), and ATR (NU 7026).

In some embodiments, the therapeutic agent is an inhibitor of the KRAS and/or MAPK pathway. In some embodiments, FLASH radiation therapy is combined with an inhibitor upstream and/or downstream of KRAS. The upstream inhibitor may target EGFR (erlotinib, gefitinib, lapatinib, afatinib) and/or SHP2 (RMC-4550). Downstream inhibitors include inhibitors of MEK (trametinib, Serlumitinib, PD0325901), BRAF (Dabrafenib, Werafenib, PLX-4720) or ERK (SCH772984, LY3214996, GDC-0994).

In some embodiments, the therapeutic agent is an inhibitor of epithelial to mesenchymal (EMT) conversion. TGF-. beta.is a known driver of EMT. Thus, in some embodiments, FLASH radiation therapy is combined with an inhibitor of EMT transformation, such as a small molecule inhibitor (e.g., SD-208, LY2109761, LY21157299) or an antibody or fragment thereof that binds TGF- β.

In some embodiments, the therapeutic agent induces or is an activator of the TH1 pathway. The T helper type1 (Th1) cell is CD4⁺A subset of effector T cells, and play a key role in immune responses as well as cancer immunotherapy. Th1 subtype cells are involved in activation of Antigen Presenting Cells (APC) and recruitment of macrophages or mast cells to the tumor site [1]. They are also involved in cytokines in the tumor microenvironmentDirect activation, which may lead to enhanced killing of tumor cells [2]. This pathway is inhibited by Flash radiation compared to untreated mice. Thus, in some embodiments, FLASH radiation therapy is combined with an activator of the TH1 pathway (such as an adjuvant that induces TH1 activity). Adjuvants that can be used in combination with FLASH radiation include, but are not limited to: 1) cytokines associated with Th1 activation, such as IL-12, IFN-alpha, beta or gamma, IL-2, IL-18, IL-27, CD80 (drug targets abatacept, beletacept, glaximab), ICAM1 and TNF-alpha [3](ii) a 2) Toll-like receptor agonists directed against TLR4 and TLR9, such as monophosphoryl lipid a and bacillus calmette-guerin, Agatolimod, ISS-1018, HYB2055 and MGN 1703; 3) STAT3 modulators, such as danvatissen and OPB-31121; and 4) inactivated bacteria/parasites or derivatives thereof that trigger interferon gamma or IL-12 production, including but not limited to Listeria monocytogenes, Leishmania major and Toxoplasma gondii, Mycobacterium tuberculosis, staphylococcal enterotoxin B and unmethylated CpG nucleotides that activate the Th1 response in the body [4 ]]. In addition, gene therapy systems including bacterial or viral based gene expression systems that result in the production of IL2, IL-12 and IFN- γ when injected at the tumor site can be used to activate Th1 responses.

In some embodiments, the therapeutic agent is an activator of phosphatase and tenascin-homologous Protein (PTEN) pathways, which are protective against cancer. The PTEN pathway is activated in Flash-irradiated tissue, and therefore Flash radiation is expected to have a protective effect on tissue compared to conventional radiation. The inventors also observed a reduction in pulmonary fibrosis in Flash compared to conventionally irradiated mice, and thus Flash irradiation can reduce pulmonary fibrosis through activation of the PTEN pathway. Thus, in some embodiments, FLASH radiation therapy is combined with activators of the PTEN pathway. Activators/agonists of the PTEN pathway are described in US 2011/0189169a1, and include mTOR inhibitors, such as rapamycin (r) (i.e., (r))

Sirolimus, ATC code L04AA10, commercially available from Whitberg) and chemical analogs thereof, such as CCI-779 (sirolimus, anatomical therapy)Chemical (ATC) code L01XE09, commercially available from Wyeth; RAD-001 (everolimus, ATC code L04AA18, commercially available from Nowa pharmaceutical Co.) and AP-2357 (Clin. cancer Res.12: 679,2006 to Granville et al), which are incorporated herein by reference. Additional activators of PTEN activity include Src inhibitors, p38MAPK modulators, NF-kB inhibitors, PPAR-gamma modulators, and IGF-1R modulators; ubuliximab, rituximab, sunitinib (induced PTEN), trastuzumab and pertuzumab (increase PTEN by Src inhibition), resistin (p38MAPK modulator, increase PTEN), simvastatin (NF-kB inhibitor), lovastatin and rosiglitazone (PPAR- γ modulator), NVP-AEW541 (IGF-1R modulator that increases PTEN) and PP1 herbimycin (Src inhibitor) (see Expert Opin Ther Pat,23 (5): 569-.

In some embodiments, the therapeutic agent is an inhibitor of the TGF- β pathway. TGF-. beta.s are proteins known to be involved in a variety of normal tissue toxic effects, including pulmonary fibrosis. Bone Morphogenetic Proteins (BMPs) are members of the TGF- β superfamily and the BMP pathway is down-regulated following Flash therapy. The Flash treated mice also exhibited reduced pulmonary fibrosis compared to mice treated with conventional radiation. Thus, in some embodiments, Flash radiation therapy is combined with an inhibitor of the TGF- β pathway, such as a small molecule inhibitor (e.g., SD-208, LY2109761, LY21157299) or an antibody or fragment thereof that binds TGF- β.

In some embodiments, the therapeutic agent is an activator or inducer of type1 Interferon (IFN), which affects the development of innate and adaptive immune responses. The IFN signaling pathway was down-regulated in FLASH-treated animals compared to conventional radiation therapy. Type1 interferons modulate the innate immune response in a balanced manner that promotes antigen presentation and natural killer cell function, while inhibiting pro-inflammatory pathways and cytokine production. Type1 interferons also activate the adaptive immune system, thereby promoting the development of high affinity antigen-specific T and B cell responses and immunological memory. Type1 interferons in the microenvironment promote maturation and antigen presentation of DCs and enhance endogenous NK or CD8+ T-cell mediated anti-tumor immune responses. Thus, in some embodiments, FLASH radiation therapy is combined with an activator or inducer of type1 interferon, which is expected to create an environment in which immune cells, including T cells, can clear tumor cells.

In some embodiments, the activator or inducer of type1 interferon is an activator of the STING/cGAS pathway. Activators of STING/cGAS pathways include synthetic CDN STING agonists, small molecule STING pathway agonists, virally encoded STING pathway agonists, bacterially encoded STING pathway agonists, or STING agonist encapsulated nanoparticles and liposomes. In some embodiments, the interferon-activating agents include compounds that bind to and activate Toll-like receptors (TLRs), such as TLR4 and TLR9, and compounds that activate the MAVS pathway.

In some embodiments, the therapeutic agent is an activator of Dendritic Cell (DC) maturation. DC maturation was down-regulated after FLASH processing as described herein. The regulation of phagocytic function in dendritic cells, and thus antigen processing and presentation by innate signals, represents a key level of adaptation and integration of the innate immune system. Thus, in some embodiments, FLASH radiation therapy is combined with an activator of DC maturation (such as a synthetic peptide vaccine).

In addition, inhibitors of CD 47/SIRP-a may be useful for enhancing antigen cross-presentation by dendritic cells and increased T cell priming. Thus, in some embodiments, FLASH radiation therapy is combined with an inhibitor of CD 47/SIRP-a, an inhibitor of CD 47/SIRP-a such as an antibody, antibody derivative or fragment thereof, and a compound or small molecule that inhibits the CD 47/SIRP-a interaction.

In some embodiments, the therapeutic agent is an arene receptor (ahR) inhibitor. In some embodiments, FLASH therapy is combined with ahR inhibitors, ahR inhibitors such as SR1, CH-223191, UM729, or galangin.

Immunomodulator

The FLASH radiation therapy described herein can be administered in combination with one or more immunomodulators. Combination therapy may provide a higher anti-tumor response (positive clinical response) than administration of either therapy as monotherapy. In certain instances, the immunomodulator may be selected from the group consisting of: inhibitors of inhibitory checkpoint molecules, activators of stimulatory checkpoint molecules, chemokine inhibitors, inhibitors of macrophage Migration Inhibitory Factor (MIF), growth factors, cytokines, interleukins, interferons, antibodies that bind to cells of the immune system (such as bispecific antibodies that bind to T cells and tumor antigens), cellular immunomodulators (such as CAR-T cells), vaccines, oncolytic viruses, and any combination thereof.

Immune modulators may include small molecules and biological therapies (e.g., antibodies, fragments thereof, and derivatives thereof) that bind to molecules expressed on the surface of cells of the immune system, such as antigen presenting cells and T cells. Biological therapies may also include bispecific antibodies, fragments thereof, and derivatives thereof that bind to antigen presenting tumor cells and T cells. Immunomodulators can also include small molecules that inhibit or stimulate the immune system. In some cases, the immunomodulatory agent stimulates a CD27+ immune cell, or inhibits one or more inhibitory checkpoint molecules, including PD-1, PD-L1, PD-L2, CTLA-4, BTLA, A2aR, B7-H2, B7-H3, B7-H4, B7-H6, CD47, CD48, CD160, CD244(2B4), CHK1, CHK2, CGEN-15049, ILT-2, ILT-4, LAG-3, VISTA, gp49B, PIR-B, TIGIT, TIM1, TIM2, TIM3, TIM4, KIR, and ligands thereof, and the like. Immune checkpoint pathways and signaling molecules are described, for example, in Pardol's "Nature Rev Cancer,2012,12: 252-.

The inhibitor of the inhibitory checkpoint molecule may be an antibody or fragment thereof that specifically binds or recognizes: PD-1, PD-L1, PD-L2, CTLA-4, BTLA, A2aR, B7-H2, B7-H3, B7-H4, B7-H6, CD47, CD48, CD160, CD244(2B4), CHK1, CHK2, CGEN-15049, ILT-2, ILT-4, LAG-3, VISTA, gp49B, PIR-B, TIGIT, TLM1, TI Mm 2, TI 3, T1M4, KIR and their ligands. In some embodiments, the CTLA-4 inhibitor is selected from the group consisting of ipilimumab, tremelimumab, and the like. One non-limiting example of a small molecule immunomodulator is an inhibitor of the enzyme indoleamine 2, 3-dioxygenase (IDO). In some embodiments, the immunomodulatory agent is an inhibitor of PD-1, PD-L1, PD-L2, or CTLA-4.

In some embodiments, the PD-1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, ranibizumab, pidilizumab, AMP-244, MEDI-4736, MPDL328OA, MIH1, IBI-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, biomimetics thereof, bioaugents thereof, and bioequivalents thereof. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of: durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, KY-1003, biosimilar, bioaugmenter, and bioequivalents thereof.

In some embodiments, the activator of a stimulatory checkpoint molecule is a small molecule, an antibody or fragment thereof, a polypeptide-based activator, a polynucleotide-based activator (i.e., an aptamer), an agonist antibody or fragment thereof, or the like. The stimulatory checkpoint molecule may be B7-1(CD80), B7-2(CD86), 4-1BB (CD137), OX40(CD134), HVEM, inducible costimulatory molecule (ICOS), glucocorticoid-induced tumor necrosis factor receptor (GITR), CD27, CD28, CD40 or a ligand thereof,

in some embodiments, the chemokine inhibitor is administered as an immunomodulatory agent. The chemokine inhibitor can be a small molecule or antibody or fragment thereof that specifically binds to the chemokine (or its receptor) and inhibits its activity. In some embodiments, the chemokine is selected from the group consisting of: CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL5, CCL26, CXCL 26, cxci.2, CXCL 26, or other chemokines associated with cancer, such as transporting leukocytes to the tumor microenvironment (e.g., controlling leukocyte infiltration into the tumor). In some embodiments, the chemokine inhibitor binds to a chemokine receptor selected from the group consisting of: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR 7.

Additional examples of immunomodulatory agents include, but are not limited to, anti-TIM 4 antibodies, anti-MFG-E8 antibodies, anti-M199 antibodies, any combination thereof, and the like. In some embodiments, the immunomodulator comprises an agent (antibody or small molecule) involved in priming and activating the immune system, and comprises an agent targeting CTLA4, B7(B7-lor B7-2), PD-L1/PD-L2 or PD-1, or an agent targeting a binding interaction between CTLA4 and B7-1/B7-2 or between PD-1 and PD-L1/PD-L2. Agents targeting CTLA4, B7(B7 or B7-2), PD-L1/PD-L2, and PD-1 include antibodies, such as monoclonal antibodies, that specifically bind to these molecules. In some embodiments, the agent is an antibody that specifically binds to LAG 3, TIM1, TIM3, MFG-E8, IL-10, or phosphatidylserine.

The immunomodulatory agents described herein can be administered in a therapeutically effective dose. A therapeutically effective dose can be determined by one of ordinary skill in the art based on the type of immunomodulator administered. Dosages, routes of administration, and administration regimens described in the art can be used. Representative doses are available in the "Merck Manual Professional Edition" (see internet Merck managers.

In addition, the Dose of immunomodulator administered to an animal can be converted to an equivalent Dose for humans based on a Body Surface Area (BSA) normalization method (see, e.g., Reagan-Shaw, S. et al, 659-661(2007) of FASEB J.22, "Dose translation from animal to human subjects revisited", and the pharmacological and toxicological "guidelines for Industry-Estimating the Maximum Safe Starting Dose in Initial Clinical laboratories for Therapeutics in therapeutic health consumers"; incorporated herein by reference) at 7 months 2005 by the U.S. department of health and public service, the food and drug administration, the drug assessment and research Center (CDER). For example, the BSA-based Human Equivalent Dose (HED) can be calculated by the following formula I:

hed-animal dose in mg/kg x (animal weight in kg/human weight in kg) 0.33

Alternatively, HED can be determined by the following formula II:

HED (mg/kg) ═ animal dose (mg/kg) × (animal Km/human Km)

The Km coefficients were determined based on the following table (see "guide for Industry, id."):

table X: conversion of animal dose to human equivalent dose based on body surface area

Assume a 60kg person.

Thus, a dose of 5mg/kg in mice is equivalent to a dose of 0.4mg/kg in 60kg humans. The dose of 0.4mg/ml in a 60kg human is equal to the dose of 14.8mg/m 2.

In some embodiments, an immunomodulatory agent described herein is administered in a therapeutically effective amount for a period of time effective to treat a cancer or tumor. An effective amount of an immunomodulator described herein can be determined by one of ordinary skill in the art, and includes metered amounts for a mammal that are the following percentages of the body weight of the immunomodulator: about 0.5 to about 200mg/kg, about 0.5 to about 150mg/kg, about 0.5 to about 100mg/kg, about 0.5 to about 75mg/kg, about 0.5 to about 50mg/kg, about 0.01 to about 50mg/kg, about 0.05 to about 25mg/kg, about 0.1 to about 25mg/kg, about 0.5 to about 25mg/kg, about 1 to about 20mg/kg, about 1 to about 10mg/kg, about 20mg/kg, about 10mg/kg, about 5mg/kg, about 2.5mg/kg, about 1.0mg/kg or about 0.5mg/kg, or any range derivable therein. In some embodiments, the dose of immunomodulator is in an amount from about 0.01mg/kg to about 10mg/kg of body weight. In some embodiments, the dose of immunomodulator is from about 0.01mg/kg to about 5mg/kg of body weight or from about 0.01mg/kg to about 2.5 mg/kg. The compositions described herein may be administered in a single dose or in separate divided doses, such as 1 to 4 times per day, or once every 2,3, 4, 5, 6, weekly, or monthly. The compositions described herein may also be administered for various treatment cycles, such as 2,3, 4, 5, 6, 7,8, 9, 10 treatment cycles. Depending on the cancer to be treated, the treatment period may be of different lengths of time, for example a treatment period of 1,2, 3, 4, 5, 6, 7,8, 9 or 10 weeks. In addition, an effective amount of an immunomodulatory agent described herein can be determined during preclinical testing and clinical testing by methods known to physicians and clinicians.

Examples of the invention

The following examples are provided to illustrate, but not to limit, the claimed embodiments.

Example 1:

this example provides the results of a study comparing gene expression profiles and tissue damage in mice treated with conventional and FLASH irradiation.

Microarray methods:

sample preparation: c57BL/6 mice were irradiated at a conventional proton dose rate (1Gy/s), a Flash dose rate (40Gy/s), a Split Flash (the total dose was divided into 10 equal portions, each portion was separated by a period of 1 second, the instantaneous dose at delivery was 40Gy/s, and the overall dose rate was 4 Gy/s). 392 age and sex matched mice were treated in 6 cohorts and died at l hours-36 weeks after lung irradiation. Each queue has 4 groups: sham (false): no radiation; and (2) conventionally: 1 Gy/s; FLASH: 40Gy/s beam on; Split-FLASH: 4Gy/0.1sec, pulse delivery (see FIG. 5). Animals were euthanized 24 hours, 8 weeks, 16 weeks, or 24 weeks after irradiation and collection of the lungs. For gene expression analysis, a total of 96 lung samples (24 samples/time point) were collected, three males and three females from four different treatment groups, namely Sham, conventional 15Gy, Flash15Gy and Split Flash15Gy, respectively.

RNA isolation and quality control: total RNA was isolated using Qiagen RNase tissue microarray kit (Qiagen cat 74104). Briefly, frozen left lungs from mice (20-30mg) were homogenized in liquid nitrogen and RNA was isolated using the manufacturer's protocol. The integrity of the isolated RNA was assessed using a 2100 bioanalyzer (Agilent technique) and only samples with RNA integrity numbers of at least 7 or 28/18S ratios of 1.3 or higher were considered for further processing.

Sample amplification and labeling: fluorescent Cy3 labeling was performed as recommended by agilent technologies using a low input rapid amplification label based on monochromatic microarray gene expression analysis. 100ng of RNA from each sample was amplified and labeled using a low input rapid amplification labeling kit based on monochromatic microarray gene expression analysis, and 600ng of Cy 3-labeled cRNA was used on each patch for hybridization. Amplification and integration of Cy3 were measured using Nanodrop and only samples with specific activity greater than 8 were further used.

Gene expression profiles were generated using a Sureprint G3 mouse gene expression v2 microarray 8X 60K manufactured by Agilent technologies, Inc. (Palo alto, Calif.). Labeling and hybridization were performed following the manufacturer's protocol. Each sample was loaded in triplicate onto the agilent array for a total of 72 wells. Then, 600ng of Cy 3-labeled cRNA was hybridized on an 8X 60K array using the Agilent High-RPM Gene expression Hyb kit. Hybridization was performed in a rotary hyb oven at 10rpm at 65 ℃ for 20 hours. After washing, the arrays were scanned using an Innopsys 710 Innoscan. The resulting TIFF images were processed using the inpropsys Feature Extraction software Mapix.

Pre-processing and normalization of expression profiles: after image extraction using Mapix software, a correlation analysis between the technical copies is performed to identify any technical copies that are not related to the sibling copy. Any array that passes the QC is merged into one file by calculating the median and median background of the mean intensity of each technology copy. Data files were analyzed using R-package Limma (ritchai et al, 2015). After uploading the data, median fluorescence intensity arrays were quantile normalized to allow for comparisons across differences (Bolstad et al, 2003). To process the marked blobs, the weight of all marks having a value less than-50 is assigned to 0. This ensures that the linear fit is made without taking these points into account during the linear fit of the data.

Gene Set Enrichment Analysis (GSEA): after normalization, the data were used for GSEA analysis to identify the enrichment pathway in each treatment group (Subramanian et al, 2005). Classical GSEA analysis was run using HALLMARKS (signature) gene sets, using probe level data between treatment groups and sham. For downstream analysis, pathways with FDR-q values less than 25% and corrected p values less than or equal to 0.05 are considered for reporting and downstream purposes.

Differential expression gene analysis: to identify differentially expressed genes between treatment groups, Limma package was used, which enabled a linear model fit of the data, followed by comparison of probes between the comparison groups using a mild T-test. To correct for multiple hypotheses, the test p-value using the Bonferroni-Hochberg method was adjusted and genes with adjusted p-values less than or equal to 0.05 were considered for downstream analysis (Benjamini and Hochberg, 1995).

Original pathway analysis (IPA): routine analysis of microarray data by generating a list of most regulated genes is not sufficient to understand the function of the regulated genes and their role in biological processes. Therefore, molecular pathway analysis was also performed using IPA to predict which typical pathways, upstream regulators, and biological functions have changed to identify molecular events and further reveal the molecular mechanisms that are regulated by changing the radiation dose rate from 1Gy/s to 40 Gy/s. IPA searches in a gene list and identifies genes associated with well-documented typical signaling or metabolic pathways.

IPA first queries the informaity Pathways knowledge base for interactions between focused genes, then all other gene objects stored in the knowledge base. For our data, genes that met the parameter P value of 0.05 were queried for IPA core analysis, followed by identification of the major canonical pathways modulated. The significance of the association between the data set and the representative path is determined based on two parameters: (1) the ratio of the number of genes mapped to a pathway in the dataset divided by the total number of genes mapped to a typical pathway; and (2) a P value calculated using a Fischer exact test followed by a Benjamini Hotchberg correction (FDR cutoff ≦ 0.1) that determines the probability that the association between a gene in the dataset and a typical pathway is due only to chance; 3) z scores cut off with an absolute value of 1.5 or higher to predict the enrichment and activation status of a particular pathway. Pathways meeting all of these criteria are predicted to be significantly regulated.

TUNEL method:

TUNEL fluorescent staining: apoptosis was determined by fluorescent staining of terminal deoxynucleotidyl transferase dUTP nick end marker (TUNEL) using the in situ cell death detection kit fluorescein (Sigma-Aldrich Corporation, Saint Louis, MO). The frozen lung tissue was cut to 5 μm thickness and then fixed, permeabilized and stained according to the manufacturer's instructions. DAPI (NucBlue) was also used^TM Fiexed Cell ReadyProbes^TMTissue sections were treated with reagent (Thermo Fisher Scientific, Waltham, MA), 2 drops per mL PBS, as counterstains. Images were taken at 100X magnification using DAPI and FITC channels on a Keyence BZ-X700 microscope (Keyence Corporation, osaka, japan) using LDF type immersion oil (Cargille-Sacher Laboratories inc., Cedar Grove, NJ).

And (3) analysis: multiple fields of view were imaged for each sample at 100X magnification on a nikon ECLIPSE Ni-E microscope (thousand daies, japan, nikon corporation) with oil immersion in DAPI and FITC fluorescence channels (nuclei and TUNEL, respectively). These images are imported into QuPath: (Bankhead.P.etal.OuPath:Opensource software for digital pathologyimage analysis.Sci.Rep.7,16878(2017)) And watershed cell detection plug-ins were used to segment and count nuclei in the DAPI channel (fig. 22). Finally, the Fiji distribution of ImageJ (Rueden, C.T.; Schindelin, J.&Hiner, M.C. et al (2017), "Image J2: Image J for the next generation of scientific Image data", BMC biologics 18; 529, doi:10.1186/sl 2859-017-; and Schindelin, j.; Arganda-Carreras, I.&Frise, E.et al (2012), "Fiji: an open-source platform for biological-image analysis". Nature methods 9(7):676 682, PMID 22743772, doi:10.1038/nmeth.2019) were used to quantify the number of TUNEL positive cells in each field. Gaussian blur is first applied to the image, and then the cells are segmented using a threshold (fig. 23A and 23B).

Lung function: lungs and penh were evaluated using a whole-body plethysmograph (Jackson et al Health Physics 2014). Baseline measurements of penh were performed and the animals were monitored for lung function every 2 weeks. The results are shown in fig. 6.

Pulmonary fibrosis: mice were untreated or treated with conventional, FLASH or split FLASH radiation. Mice were euthanized at various time points at

weeks

16, 24, and 36, and lung tissue was fixed in formalin and embedded in a sealing film. Lung sections were stained with masson triple stain and H & E stain and evaluated microscopically by a trained pathologist who scored fibrosis from 0-8 using an MFSS scoring protocol (Ashcroft Journal of Clinical Pathology 1988and Hubner et al biotechnology 2008), where 0 indicates no fibrosis and 8 corresponds to severe fibrosis with complete elimination of lung structure and complete loss of vacancy (occlusive fibrosis). The results are shown in fig. 10.

Rats were scored for dermatitis by using the douglas and fowler scales. The score was between 0 and 3.5, where 0 is normal and 3.5 is moist desquamation in most of the irradiated area and necrosis (Ryan et al Journal of invasive Dermatology 2012). The results are shown in fig. 9.

Survival: mice were treated with conventional or Flash radiation doses of 15, 17.5 and 20 Gy. Each radiation dose group consisted of 20 age-matched animals (50% male and 50% female). Animals were monitored three times a week and euthanized if weight loss exceeded 20% (no recovery within two days) or severe dermatitis was scored by veterinarian. The results are shown in fig. 7 and 8.

As a result:

use of FLASH radiation improves lung function and reduces fibrosis

Figure 6 shows that FLASH has improved lung function compared to conventional radiation therapy. This difference confirms the existence of dose rate dependence of the radiotoxicity. Figure 10 shows a 23% reduction in average pulmonary fibrosis severity at 17.5Gy for FLASH compared to the conventional group. This difference confirms the existence of dose rate dependence of the radiotoxicity. Figure 11 shows the conventional mean lung weight gain compared to FLASH treatment-0.46 g versus 0.40g (m); 0.37g and 0.32g (F). This study indicated that FLASH retained normal tissue.

Survival of mice after irradiation with FLASH was improved compared to conventional irradiation

Figure 7 shows that median survival of FLASH was improved compared to the conventional radiation treated group: 20.0 Gy: increase by 14%; 17.5 Gy: the increase is 18%. Figure 8 shows the probability of improved median survival of FLASH compared to the conventional radiation treated group, and a dose rate dependent treatment window: 17.5Gy FLASH >17.5Gy CON; 17.5Gy FLASH ═ 20.0Gy CON.

FLASH reduced dermatitis compared to conventional radiation

Figure 9 shows the average dermatitis reduction of FLASH compared to conventional radiation: FLASH: the reduction is 34%; Split-FLASH: the reduction was 52%. This difference confirms the existence of a dose rate dependence of the radiotoxicity associated with dermatitis.

Microarray:

flash radiotherapy (FLASH RT) modality synergizes with mitotic spindle inhibitors

Figure 12 shows a venn plot of differentially expressed genes at each time point. GSEA at 24 hours indicated that the G2M checkpoint and the E2F target were inhibited by all radiation therapy modalities (fig. 13A). These characteristics indicate that the cells stagnated at G1 and G2M after treatment. The E2F target includes various genes; however, cyclin is the primary target of this pathway { cite }. Thus, the use of Flash RT in combination with CDK4/6 inhibitors (Pablociclib) can maintain the cell cycle arrest induced by radiotherapy.

In both the Flash and split-Flash groups, mitotic spindles were specifically down-regulated (fig. 14A, 14B). Overlapping analysis of the core-enriched genes for each group indicated that key regulators of mitotic spindle genes, such as AURKA, Kinesin-like protein family genes (KIF11, KIF23, KIF23, KIF4A), and TPX2 (fig. 14C, fig. 14D) were down-regulated. There is evidence that AURKA and TPX2 play a major role in driving acquired resistance in the context of several targeted therapies (Donnella et al, 2018; Panicker et al, 2017). In these models AURKA expression is increased to drive resistance and thus blocks AURKA expression, which may be important for the efficacy of Flash and Split Flash therapies. AURKA is specifically targeted by using small molecule inhibitors (Alisertib, Tozasertib). TPX2 is a driver of AURKA and indicates the use of a consensus interferon (GSK1070916) to block TPX2-AURKA complex (Asteriti et al, 2015; Janecek et al, 2016). Finally, mitotic spindle integrity can be targeted by using taxanes (docetaxel, paclitaxel). At the end of 24 weeks, FLASH RT upregulated the mitotic spindle gene (fig. 13D) to support that adaptive gene expression changes may play a key role in the efficacy of radiotherapy.

Flash RT mode and DNA damage repair and reaction inhibitor combination

Flash RT specifically inhibits DNA repair pathway genes. At the pathway level, Flash RT down-regulates the Hallmark DNA repair pathway (FIGS. 13A and 15A). Core enrichment analysis revealed down-regulation of key homology-directed repair (HR) genes, such as LIG1, RAD51, and BRCA2 (see figure 15B for a complete list). The direct implications of these down-regulated genes indicate that FLASH therapy induces the BRCAness phenotype and therefore can indicate that PARP inhibitors (Talazoparib, rucapaparib, olaparib) bind to FLASH RT to target tumors (Lord and Ashworth, 2016; Murai et al, 2012).

In addition, in the case of inhibited DNA repair (i.e., BRCA1 mutation or brcasting), the DNA damage response is blocked by blocking a key regulator of this pathway. For example, RAD51 is required to repair double strand breaks, so the combination of FLASH RT and RAD51 inhibitor (RI-1) can act synergistically. In addition, other DNA damage response kinases such as CHCK1(AZD7762), ATM (KU-55933, KU-60019, NU7026, VE-821), ATR (NU7026) (Ashworth and Lord, 2018; Lord and Ashworth, 2016) targeted to Flash RT-induced BRCAness were also effective.

Combination of Flash RT and MAPK pathway inhibitors

After 24 hours, expression of many pathways increased in all treatment groups (fig. 13B-13D), and in particular KRAS signal was upregulated over time in all treatment groups, and in particular "KRAS signal" was enriched in the conventional and FLASH groups between 8-24 weeks (fig. 16A-16C). KRAS signaling is the major driver of cancer and drives many biological programs through the MAPK pathway, including proliferation, cell cycle progression and pro-survival signaling (Sun et al, 2015). There is evidence that conventional radiation therapy depends on blocking EGF/EGFR signaling (Wang et al, 2011). Although direct inhibitors of RAS are still in the early stages, the goal is to block upstream and downstream RAS signals using a combination of FLASH RT and inhibitors upstream and downstream of KRAS (Downward, 2003). As an example, upstream inhibitors such as EGFR (erlotinib, gefitinib, lapatinib, afatinib) and SHP2(RMC-4550) may be used in combination with FLASH RT. As another example, downstream inhibitors such as MEK (trametinib, cerulorninib, PD0325901), BRAF (dabrafenib, virafenib, PLX-4720) or ERK (SCH772984, LY3214996, GDC-0994) may be combined with FLASH therapy.

Radiotherapy induced EMT

Over the course of 8-10 weeks, epithelial to mesenchymal (EMT) conversion was an upregulated pathway in Flash between 8-16 weeks (fig. 13B-13C); however, at the end of 24 weeks, EMT was up-regulated in both conventional and Flash treatments (fig. 17A-17B). EMT is known to drive resistance to targeted and non-targeted therapies, and thus may pose challenges to the efficacy of radiotherapy (Kitai and Ebi, 2016; Liang et al, 2015). EMT is the result of changes in gene expression that drive cells from one state to another, and targeting EMT may therefore pose a challenge. Overlay analysis of the conventional and Flash EMT signatures at 24 weeks revealed a total of 56 gene overlaps (fig. 17C, table 5). In both the overlapping TGFB1 and TGFBI, both were upregulated in both conventional and flash treated mice. TGFB is a known driver of EMT, and therefore TGFB inhibitors (SD-208, LY2109761, LY21157299) can prevent EMT from developing and cause radiation resistant tumors (Foroutan et al, 2017)

Differential gene expression analysis:

in order to obtain an assessment of the early response of Flash to conventional therapy, microarray assessment was performed on irradiated mice that were euthanized 24 hours after irradiation. Preliminary clustering analysis revealed that for earlier time points, the Flash and sham samples clustered together, whereas conventionally processed mice clustered with the Split Flash group. This follows the pattern originally observed in survival and dermatitis, i.e. Flash is at first much better than conventional treatment, but later more closely to conventional treatment. Based on gene expression analysis, conventional irradiation (1Gy/s) regulated 2131 genes most significantly at 24 hours (p value 0.05), whereas Flash regulated 257 genes only at the 24 hour time point, 175 of which were common, all regulated by both irradiation types (fig. 5).

IPA analysis:

IPA analysis reveals that these different radiation treatment regimes alter many of the classical pathways. Major changes were observed at the initial time point of 24 hours, where most of the regulation of RNA levels occurred, which was captured by the whole genome microarray. Most of the significantly upregulated pathways in conventional radiation (p-value ≦ I0.05, Z-score ≧ 1.5, and FDR of 0.1) are involved in inflammatory responses, such as interferon signaling, IL-8 signaling, STAT3 signaling, GP6 signaling pathway, and phospholipase C signaling. Some cancer-related pathways are also upregulated, such as pancreatic adenocarcinoma signals and colorectal cancer metastasis signals. It is predicted that the cyclin and cell cycle regulatory pathways are inhibited after conventional irradiation. (FIG. 18 and tables 6A-C). At 24 hours of Flash irradiation, most pathways were down-regulated, including the mitosis of polo-like kinases, estrogen-mediated S-phase entry, Ary1 hydrocarbon receptor signaling, cyclin and cell cycle regulation, TH1 helper T cell pathway, dendritic cell maturation, and calcium-induced T lymphocyte apoptosis. In split-Flash, the regulated pathway is primarily involved in the cell cycle, where different phases of the cell cycle are regulated differently together with the p53 pathway. The common pathway regulated by all types of treatment is cyclin and cell cycle regulation, which are known to be disrupted following radiation therapy.

When a comparison between conventional radiation and Flash radiation was performed (overlapping p-value >0.05, Z-score of 1.5), there was little path between these treatments that was adjusted by the difference (fig. 18). Dendritic cell maturation, PKC signaling in lymphocytes, TH1 pathway, and calcium-induced T lymphocyte apoptosis are shown to be elevated following conventional irradiation and reduced following Flash treatment. Although many pathways are significantly upregulated after conventional irradiation, they are not affected after Flash irradiation, e.g. IL-8, ROS and MO production in macrophages, interferon signaling, Rho family GTPases, GP6 signaling, colon cancer metastasis signaling, sphingosine-1-phosphate signaling, etc.

TH1 pathway: the T helper type1 (Th1) cell is CD4⁺A subset of effector T cells, and play a key role in immune responses as well as cancer immunotherapy. The Th1 subtype is involved in activation of Antigen Presenting Cells (APC) and recruitment of macrophages or mast cells to the tumor site [1]. They are also involved in the direct activation of cytokines in the tumor microenvironment, which can lead to enhanced tumor cell killing [2]. This pathway was found to be Flash inhibited compared to untreated mice, and therefore the combination of Flash radiation and immunotherapy would be more effective when combined with an adjuvant that induces Th1 activity. Adjuvants used in conjunction with FLASH delivery include, but are not limited to: 1) cytokines associated with Th1 activation, such as IL-12, IFN-alpha, beta or gamma, IL-2, IL-18, IL-27, CD80 (drug targets abatacept, beletacept, glaximab), ICAM1 and TNF-alpha [3](ii) a 2) Toll-like receptor agonists directed against TLR4 and TLR9, such as monophosphoryl lipid a and bacillus calmette-guerin, Agatolimod, ISS-1018, HYB2055 and MGN 1703; 3) STAT3 modulators, such as danvatissen and OPB-31121; 4) live bacteria (e.g., Listeria monocytogenes) or intracellular parasites (e.g., Leishmania major and Toxoplasma) or compounds derived therefrom that trigger interferon gamma or IL-12 production, bacterial lipopolysaccharides, killed Mycobacterium tuberculosis, bacterial superantigens that activate Th1 responses in vivo, such as staphylococcal enterotoxin B and unmethylated CpG nucleotides [4 ]]。

Calcium-mediated T cell apoptosis: one important pathway for T cells to undergo apoptosis is through the calcium channel, which involves the binding of MHC class II complexes on antigen presenting cells to TCR-CD3 complexes on T cells, which results in the activation of PLC-gamma, which in turn activates the accumulation of PKC and calcium in the cytoplasm. Free calcium triggers apoptotic pathways through interaction with calcineurin, CABIN and NFAT. After Flash, the path is turned down and activated by conventional illumination. Thus, Flash radiation can retain (spare) T lymphocytes by means of a faster treatment regime (higher dose rate) which avoids irradiating more blood and by down-regulation of this pathway, resulting in molecular retention of T lymphocytes. Lymphocytes are considered high risk organs during radiation therapy, and many organs with high blood flow (such as the lungs and brain) must be dose-limited to reduce lymphocyte killing [5 ]. Therefore, Flash radiation in combination with treatment planning algorithms can be used to treat organ sites with high lymphocyte counts. Lymphocyte retention will also have an impact on providing an effective immunotherapeutic combination with radiation.

For the 16-week analysis, it was found that the two classical pathways were significantly regulated in the Flash group. The cyclin and cell cycle regulatory pathways are activated, while the NFAT pathway in cardiac hypertrophy is down-regulated (p value ≦ I0.05, Z score ≧ 1.5 and FDR 0.1). In a comparative analysis between regular 15Gy and Flash15Gy radiation using Fischer exact statistical analysis (p-value ≦ I0.05 and Z-score ≧ 1.5), it was found that both treatments regulated many pathways. Interferon signals were found to be up-regulated in both groups, but were found to be more activated in the conventional group (fig. 18). Flash was found to down-regulate the BMP signaling pathway compared to conventional.

And (4) PTEN: phosphatase and tensin homolog (PTEN) pathways that protect against cancer are activated in Flash, and thus Flash is predicted to have a potential for tissue protection as compared to conventional. Loss of PTEN expression in mouse fibroblasts was shown to lead to pulmonary fibrosis (paraura et al, Matrix Biol 2015). PTEN knockout mice treated with bleomycin (radiation mimicking drug) showed increased pulmonary fibrosis. Because reduced pulmonary fibrosis was observed in Flash compared to conventional radiation therapy (fig. 10), one of the mechanisms by which Flash caused reduced pulmonary fibrosis may be activation through the PTEN pathway. Activators/agonists of the PTEN pathway are described in US 2011/0189169a1 and include mTOR inhibitors such as: rapamycin (I)

Sirolimus, ATC code L04AA10, commercially available from Whitberg) and chemical analogs thereof, such as CCI-779 (sirolimus, anatomically therapeutic Chemicals)(ATC) code L01XE09, commercially available from Wyeth; RAD-001 (everolimus, ATC code L04AA18, commercially available from Nowa pharmaceutical Co.) and AP-2357 (Clin. cancer Res.12: 679,2006, Granville et al)), which are incorporated herein by reference. Additional activators of PTEN activity include Ublituximab, Rituximab, Sunitinib (inducing PTEN), Trastuzumab and Pertuzumab (increasing PTEN through Src inhibition), resistins (p38MAPK modulators, increasing PTEN), simvastatin (NFO-kB inhibitors), lovastatin (rosuvastatin and rosiglitazone) PPAR- γ modulators), NVP-AEW541 (IGF-1R modulators that increase PTEN), and PP1 herbimycin (Src inhibitor) (see Expert OpinTher Pat 2013 May of bosani et al: 23(5): 569-580).

BMP pathway: bone Morphogenetic Proteins (BMPs) are members of the TGF- β superfamily. TGF-. beta.s are proteins known to be involved in a variety of normal tissue toxic effects, including pulmonary fibrosis. BMP pathways were down-regulated at a 16 week time point in the Flash treatment group. The Flash treated samples also exhibited reduced pulmonary fibrosis at the 16 and 24 week time points. Thus, inhibition of the TGF- β pathway may reduce toxicity in normal tissues.

Interferon signaling:

type1 Interferons (IFNs) affect the development of innate and adaptive immune responses. In contrast to Conventional (CONV), the IFN signaling pathway, one of the most aberrantly regulated classical pathways, is down-regulated in FLASH. Type1 interferons modulate the innate immune response in a balanced manner that promotes antigen presentation and natural killer cell function, while inhibiting pro-inflammatory pathways and cytokine production. Type1 interferons also activate the adaptive immune system, thereby promoting the development of high affinity antigen-specific T and B cell responses and immunological memory. Type1 interferon in the microenvironment promotes maturation and antigen presentation of DCs and enhances endogenous NK or CD8+ T cell-mediated anti-tumor immune responses. Thus, type1 interferon activators may be used in conjunction with FLASH delivery to create an environment in which immune cells, including T cells, may thrive to eradicate tumor cells.

One of the key pathways involved in the production of type1 interferons is the STING/cGAS pathway. Stimulator of interferon genes (STING) is an intracellular signaling molecule that senses Cyclic Dinucleotides (CDNs). CDN is exogenously derived from infectious agents, or produced by the mammalian dsDNA sensor cGAS (cyclic guanosine monophosphate adenosine monophosphate; cyclic GMP-AMP synthase). The STING/cGAS sensing mechanism induces an innate immune response, including the production and release of type1 interferons. Thus, activators of the STING/cGAS pathway can be used in conjunction with FLASH delivery to create an environment in which immune cells can thrive to eradicate tumor cells. These activators include, inter alia, synthetic CDN STING agonists, small molecule STING pathway agonists, virally-encoded STING pathway agonists, bacterially-encoded STING pathway agonists, or STING agonist encapsulated nanoparticles and liposomes. Other type1 interferon activators include compounds that bind to and activate Toll-like receptors (TLRs), such as TLR4 and TLR9, as well as compounds that activate the MAVS pathway.

Maturation of dendritic cells:

dendritic cell maturation is down-regulated in FLASH. The regulation of phagocytic function in dendritic cells, and thus antigen processing and presentation by innate signals, represents a critical level of adaptation and integration of the innate immune system. The combination of FLASH and synthetic peptide vaccines may be beneficial in inducing a sustained immune response.

In addition, inhibitors of CD 47/SIRP-a may be used to enhance antigen cross-presentation by dendritic cells and increased T cell priming. These include, inter alia, antibodies, antibody derivatives, and small molecules that inhibit the CD 47/SIRP-alpha interaction.

Nano-particles:

nanoparticles are small particles with a size between 1nm and 100 nm. They may have different shapes such as spherical, rod-shaped or star-shaped. They can be targeted to tumors passively through enhanced permeability and retention, but also actively, such as by conjugating antibodies or peptides to nanoparticles. In addition, they may be encapsulated in vesicles or inserted into gel matrices to increase tumor targeting.

It has been previously shown that nanoparticles with high effective atomic numbers (e.g., gold or gadolinium) can have a synergistic effect when administered prior to radiation therapy by providing dose enhancement (Hainfeld et al, 2004). This is because when struck by a photon, a photoelectric effect occurs in the nanoparticle, thereby emitting electrons and additional x-rays. Furthermore, the irradiation of the nanoparticles may also cause hyperthermia in the tumor, thereby increasing the therapeutic effect. It has also been shown that dose enhancement can be achieved when nanoparticles are used in combination with proton therapy (Lin et al, 2014). Finally, radiation can also be used to modulate and enhance nanoparticle delivery to tumors by altering the tumor microenvironment (e.g., by reducing tumor interstitial pressure) (Stapleton et al, 2017).

Flash RT can be used in combination with nanoparticles

One of the major obstacles to the use of nanoparticles in conjunction with radiation therapy is the short half-life of the nanoparticles in vivo. Delivering nanoparticles before each of several treatments in conventional partitioning arrangements is also challenging and impractical. In addition, some nanoparticles may still be toxic at sufficiently high concentrations, especially when not rapidly cleared from the body. These challenges can be overcome by using nanoparticles in conjunction with Flash radiation therapy. By treating one or more tumors in only a single fraction, the concentration of nanoparticles in one or more tumors can remain sufficiently high throughout the treatment process, even if the nanoparticles have a relatively short half-life. Furthermore, since nanoparticles with a short half-life are still effective, the possible toxicity will be reduced since the nanoparticles will be rapidly cleared from the body. One example of a workflow where Flash radiation therapy is combined with nanoparticles is the administration of nanoparticles followed by imaging, such as by CT or MRI. These imaging data are used to create a Flash radiation therapy treatment plan, taking into account the distribution of the nanoparticles. Then, prior to treatment, the nanoparticles were re-injected and the planned Flash treatment was delivered.

The arene acceptor pathway:

in the Flash treated lung, the aryl hydrocarbon receptor (ahR) signaling pathway was down-regulated. The general function of this pathway is to detect aromatic hydrocarbons (aryls) and activate a set of xenogeneic metabolic genes, particularly cytochrome P450 enzymes. This is of great importance for drug combination strategies, as down-regulation of this pathway can alter clearance of other drugs in the patient. In addition, activation of this pathway may also facilitate resistance to small molecules to pan-drugs or the ability of cells to handle toxic substances as a result of radiotherapy. AhR pathway activation is also associated with cancer immunotherapy, where AhR regulates both innate and adaptive immunity, activating anti-inflammatory Treg cells and M2 macrophages. Thus, the combination of radiation therapy with ahR inhibitors (such as SR1, CH-223191, UM729, galangin) may inhibit the ability of tumors to eliminate toxic substances and activate immune cells.

Apoptosis in lung tissue

Fig. 24 is a graph showing TUNEL results. The figure shows the average number of counts per animal for approximately 20 animals per group (male and female pooled). Quantification of TUNEL cells constitutes a counting problem, where the number of TUNEL positive cells is counted for each group of animal samples, and thus poisson statistics should be followed. The vertical lines on the graph indicate the 95% confidence intervals calculated using the accurate estimation method proposed by Ulm et al. (Ulm, K., "A simple method to calculate the confidence interval of a stabilized reliability ratio (SMR)", Am J Epidemiol, 1990, 131 (2): 373-5). The overlap of the 95% confidence intervals indicates that SplitFlash (pulsed FLASH) and FLASH are more similar to Sham than conventional, indicating a lower incidence of apoptosis for these novel therapeutic techniques.

Table 2: 24 hours minimum enriched pathway

Table 2A conventional and Sham:

table 2B Flash and Sham:

TABLE 2C Split Flash and Sham

NES ═ normalized enrichment score, Nom p value ═ normalized value, FDR q value ═ false discovery rate value

Table 3: most enriched pathway for 8 weeks

Table 3A conventional and Sham:

table 3B Flash and Sham:

symbolic path	NES	NOM p value	FDR q value
				Marked coagulation	1.406174	0.045714	0.225907
Indicative TNFA signalling via NFKB	1.382228	0.036329	0.225861
				Symbolic top knot	1.3685	0.016	0.231629
Marker WNT beta catenin signal	1.361896	0.02924	0.227167
				Signatory KRAS signal rise	1.351275	0.043561	0.228315

Table 4: 16 weeks most enriched pathway

Table 4A conventional and Sham:

symbolic path	NES	NOMP value	FDRq value
				Marked coagulation	1.5755655	0.001	0.17153585
Marker hedgehog signal	-1.6175224	0.0056926	0.032724753

Table 4B Flash and Sham:

table 4C Flash and Sham:

symbolic path	NES	NOM p value	FDR q value
				Pathway of symbolic P53	1.5119545	0.0087912	0.24653515

Table 5: 24 weeks most enriched pathway

Table 5A conventional and Sham:

table 5B Flash and Sham:

symbolic path	NES	NOMP value	FDRq value
				Marked coagulation	1.754068	0.001	0.030982
Epithelial-mesenchymal transition	1.743082	0.002066	0.017006
				Symbolic MYC target V2	1.66075	0.014056	0.036597
Marker glycolysis	1.598933	0.004193	0.076556
				Marker IL6 JAK STAT3 signal	1.580787	0.006098	0.078151
Marker IL2 STAT5 Signal	1.571376	0.011858	0.07368
				Symbolic mitotic spindle	1.553222	0.001927	0.075656
Marker inflammatory response	1.522947	0.029528	0.093476
				Marked hypoxia	1.468816	0.001972	0.130024
Marker apoptosis	1.416187	0.03937	0.141715
				Marked xenobiotic metabolism	1.390655	0.030181	0.151785
Marked delayed estrogenic response	1.348313	0.023715	0.158576
				Marked estrogen response early	1.327884	0.02449	0.175084

NES is normalized enrichment score, Nom p value is normalized value, FDR q value is false discovery rate value.

Table 6: 24 hour best typical access by IPA

Table 6A conventional and Sham:

table 6B Flash and Sham:

TABLE 6C Split Flash and Sham

Example 2

This example demonstrates that Flash radiation causes altered activation of the hedgehog (Hh) signal.

The Hh signaling pathway has recently been identified as an important signaling pathway and therapeutic target in cancer. In adults, mutations or dysregulation of this pathway play a critical role in both proliferation and differentiation, leading to tumorigenesis or accelerated tumor growth in a variety of tissues. Inappropriate activation of the Hh signaling pathway has been implicated in the development of several other cancers, including lung, prostate, breast and pancreatic cancers. In addition, some recent findings suggest that Hh may also promote tumorigenesis by being transmitted in a paracrine fashion from the tumor into the surrounding stroma or Cancer Stem Cells (CSCs). As shown in fig. 25 and table 7, flash irradiation resulted in reduced activation of the hedgehog signaling pathway compared to conventional proton irradiation. Cumulative enrichment scores indicate down-regulation of the gene. The components of the Hh/GLI signaling pathway involved in signaling to GLI transcription factors include hedgehog ligands (Sonic Hh (SHh), Indian Hh (IHh), and Desert Hh (DHh)), repair receptors (Ptchl, Ptch2), smoothing receptors (Smo), inhibitors of fusion homologues (Sufu), kinesin Kif7, protein kinase a (pka), and cyclic adenosine monophosphate (cAMP). The activator form of GLI reaches the nucleus and stimulates transcription of target genes by binding to the promoter of the target gene. The major target genes for the Hh signaling pathway are PTCH1, PTCH2, and GLI family zinc finger 1(GLI 1).

Several hedgehog antagonists, including but not limited to the hedgehog antagonists Smo antagonist, PTCH1 inhibitor (such as RU-SKI 43), cyclopamine, vismodegib, LDE 225, saridegib, BMS 833923, LEQ 506, PF-04449913, PF-5274857, GANT61, SANT-1, Glabra (PF-04449913), Taladegib (LY2940680), and TAK-441 can work in conjunction with Flash therapy.

TABLE 7 hedgehog pathway genes down-regulated by Flash irradiation compared to conventional irradiation

Reference documents:

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it is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, patent applications, sequence accession numbers, and UniProt numbers cited herein are hereby incorporated by reference in their entirety for all purposes.

Form sequence List

SEQ ID NO:1 sonic hedgehog protein (SHH) (HHG-1) (SHH untreated N-terminal signal and C-terminal autoprocessing domain) (ShhNC) [ split into sonic hedgehog protein N-product (ShhN) (SHH N-terminal processing signal domain) (ShhNp) ] (UniProt ID Q15465) > sp | Q154651| SHH _ HUMAN sonic hedgehog protein OS ═ Chiense OX ═ 9606 GN ═ SHH PE ═ l SV ═ l

SEQ ID NO:2 India hedgehog protein (IHH) (HHG-2) [ split into: indian hedgehog protein N-product; indian hedgehog protein C-product (UniProt ID Q14623) > sp | Q14623| IHH _ HUMAN indian hedgehog protein OS ═ homo sapiens OX ═ 9606 GN ═ IHH PE ═ 1 SV ═ 4

3 desert hedgehog protein (DHH) (HHG-3) [ split into: desert hedgehog protein N-product; desert hedgehog protein C-product (UniProt ID 043323) > sp |043323| DHH _ HUMAN desert hedgehog protein OS ═ homo sapiens OX ═ 9606 GN ═ DHH PE ═ 1 SV ═ 1

Protein Patch homolog (PTC)1(PTC1) 4 (SEQ ID NO). UniProt ID q13635 isoform L (UniProt Q13635-1) > sp | Q13635| PTC1_ HUMAN protein prosthetic homolog 1 OS homo sapiens OX 9606 GN ═ PTCH1 PE ═ 1 SV ═ 2

SEQ ID NO:5 isoform L'. UniProt ID Q13635-2.> sp | Q13635-2| PTCl _ HUMAN protein patchwork isoform 1L' OS ═ homo sapiens OX ═ 9606 GN ═ PTCH1

SEQ ID NO 6 isoform M. UniProt ID Q13635-3.> sp | Q13635-3| PTCl _ HUMAN protein patchwork isoform 1M OS ═ homo sapiens OX ═ 9606 GN ═ PTCH1

SEQ ID NO 7 isoform S. UniProt ID Q13635-4.> sp | Q13635-4| PTCl _ HUMAN protein patchwork isoform 1S OS ═ homo sapiens OX ═ 9606 GN ═ PTCH1

Protein Patch homolog 2(PTC2) of SEQ ID NO: 8. UniProt ID q9y6c5. isoform 1(Q9Y6C5-1) > sp | Q9Y6C5| PTC2_ HUMAN protein prosthetic homolog 2 OS ═ homo sapiens OX ═ 9606 GN ═ PTCH2 PE ═ SV ═ 2

Protein Patch homolog 2(PTC2) of SEQ ID NO 9. UniProt ID q9y6c5. isoform 2(Q9Y6C5-2) > sp | Q9Y6C5-2| PTC2_ HUMAN protein prosthetic homolog 2 isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ PTCH2

SEQ ID NO 10 smoothed homolog (SMO) (protein Gx). UniProt ID Q99835.> sp | Q99835|, SMO _ HUMAN smoothed homolog OS ═ homo ═ 9606 GN ═ SMO PE ═ 1 SV ═ 1

11: inhibitor of fusion homolog (SUFUH). UniProt ID q9umx1. isoform 1.> sp | Q9UMXl | SUFU _ HUMAN fusion homolog inhibitor OS ═ homo sapien ═ 9606 GN ═ SUFU PE ═ 1 SV ═ 2

12: inhibitor of fusion homolog (SUFUH). UniProt ID Q9UMX1-2. isoform 2.> sp | Q9UMXl-2| SUFU _ HUMAN fusion homolog of inhibitor isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ SUFU ═

13: inhibitor of fusion homolog (SUFUH). UniProt ID Q9UMX1-3, isoform 3. 3 OS homo sapiens OX 9606 GN SUFU for inhibitors of > sp | Q9UMXl-3| SUFU HUMAN fusion homologues

Kinesin-like protein KIF7, SEQ ID NO 14. UniProt ID Q2M1P5.> sp | Q2MlP5| KIF7_ HUMAN kinesin-like protein KIF7 OS. homo sapiens OX. 9606 GN. KIF7 PE. l SV 2. cndot

15 cadherin EGF LAG seven G receptor type1 CELR 1. UniProt ID q9nyq6. isoform 1.> sp | Q9NYQ6| CELRl _ HUMAN cadherin EGF LAG seven G-type receptor 1 OS ═ homo sapiens OX ═ 9606 GN ═ CELSR1 PE ═ SV ═ l

16 cadherin EGF LAG seven G receptor type1 CELR 1. UniProt ID Q9NYQ6-2 isoform 2.> sp | Q9NYQ6-2| CELRl _ HUMAN cadherin EGF LAG septenary G receptor 1 isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ CELSR1

17 transducin-like enhancer protein 3TLE 3. UniProt ID q04726. isoform 1.> sp | Q04726| TLE3_ HUMAN transducin-like enhancer protein 3 OS ═ homo sapiens OX ═ 9606 GN ═ TLE3 PE ═ 1 SV ═ 2

18 transducin-like enhancer protein 3TLE 3. UniProt ID Q04726-2 isoform 2.> sp | Q04726-2| TLE3_ HUMAN transducin-like enhancer protein 3 isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ TLE3

19 transducin-like enhancer protein 3TLE 3. UniProt ID Q04726-3 isoform 3.> sp | Q04726-3| TLE3_ HUMAN transducin-like enhancer protein 3 isoform 3 OS ═ homo sapiens OX ═ 9606 GN ═ TLE3

20 transducin-like enhancer protein 3TLE 3. UniProt ID Q04726-4 isoform 4.> sp | Q04726-4| TLE3_ HUMAN transducin-like enhancer protein 3 isoform 4 OS ═ homo sapiens OX ═ 9606 GN ═ TLE3

21 transducin-like enhancer protein 3TLE 3. UniProt ID Q04726-5 isoform 5.> sp | Q04726-5| TLE3_ HUMAN transducin-like enhancer protein 3 isoform 5 OS ═ homo sapiens OX ═ 9606 GN ═ TLE3

22 transducin-like enhancer protein 3TLE 3. UniProt ID Q04726-6 isoform 6.> sp | Q04726-6| TLE3_ HUMAN transducin-like enhancer protein 3 isoform 6 OS ═ homo sapiens OX ═ 9606 GN ═ TLE3

23 transducin-like enhancer protein 3TLE 3. UniProt ID Q04726-7 isoform 7.> sp | Q04726-7| TLE3_ HUMAN transducin-like enhancer protein 3 isoform 7 OS ═ homo sapiens OX ═ 9606 GN ═ TLE3

24: lecithin-1 OPHN 1. UniProt ID O60890, isoform 1> sp | O60890| OPHNl _ HUMAN lecithin-l OS ═ homo sapiens OX ═ 9606 GN ═ OPHN1 PE ═ 1 SV ═ 1

SEQ ID NO:25 lecithin-1 OPHN 1. UniProt ID O60890-2 isoform 2> sp | O60890-2| OPHNl _ HUMAN lecithin-1 isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ OPHN1

26 adhesion G-protein coupled receptor G1 AGRG 1. UniProt ID q9y653, isoform 1.> sp | Q9Y653| AGRGl _ HUMAN adhesin G-protein coupled receptor G1 OS ═ homo sapiens OX ═ 9606 GN ═ ADGRG1 PE ═ 1 SV ═ 2

27 adhesion G-protein coupled receptor G1 AGRG 1. UniProt ID Q9Y653-2 isoform 2.> sp | Q9Y653-2| AGRGl _ HUMAN adhesion G-protein coupled receptor G1 isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ ADGRG1

28 of the adhesion G-protein coupled receptor G1 AGRG 1. UniProt ID Q9Y653-3 isoform 3.> sp | Q9Y653-3| AGRGl _ HUMAN adhesion G-protein coupled receptor G1 isoform 3 OS ═ homo sapiens OX ═ 9606 GN ═ ADGRG1

29 adhesion G-protein coupled receptor G1 AGRG 1. UniProt ID Q9Y653-4 isoform 4.> sp | Q9Y653-4| AGRGl _ HUMAN adhesion G-protein coupled receptor G1 isoform 4 OS ═ homo sapiens OX ═ 9606 GN ═ ADGRG1

30 of the adhesion G-protein coupled receptor G1 AGRG 1. UniProt ID Q9Y653-5 isoform 5.> sp | Q9Y653-5| AGRGl _ HUMAN adhesion G-protein coupled receptor G1 isoform 5 OS ═ homo sapiens OX ═ 9606 GN ═ ADGRG1

31 protein Patch homolog 1PTC 1. UniProt ID q13635. isoform L. > sp | Q13635| PTCl _ HUMAN protein prosthetic homolog 1 OS ═ homo sapien OX ═ 9606 GN ═ PTCHl PE ═ SV ═ 2

32 protein Patch homolog 1PTC 1. UniProt ID Q13635-2 isoform L '> sp | Q13635-2| PTCl _ HUMAN protein prosthetic homolog 1 isoform L' OS ═ homo sapiens OX ═ 9606 GN ═ PTCH1

33 protein Patch homolog 1PTC 1. UniProt ID Q13635-3 isoform M. > sp | Q13635-3| PTCl _ HUMAN protein prosthetic homolog 1 isoform M OS ═ homo sapiens OX ═ 9606 GN ═ PTCH1

Protein Patch homolog 1PTC1, SEQ ID NO: 34. UniProt ID Q13635-4. isoform S. > sp | Q13635-4| PTCl _ HUMAN protein repair homolog 1 isoform S OS ═ homo sapiens OX ═ 9606 GN ═ PTCHl

35 transducin-like enhancer protein 1TLE 1. UniProt ID Q04724.> sp | Q04724| TLEl _ HUMAN transducin-like enhancer protein 1 OS ═ homo sapiens OX ═ 9606 GN ═ TLEl PE ═ SV ═ 2

SEQ ID NO:36 myosin-9 MYH 9. UniProt ID P35579, isoform 1.> sp | P355791| MYH9_ HUMAN myosin-9 OS ═ homo sapiens OX ═ 9606 GN ═ MYH9 PE ═ 1 SV ═ 4

SEQ ID NO:37 myosin-9 MYH 9. UniProt ID P35579-2 isoform 2.> sp | P35579-2| MYH9_ HUMAN myosin-9 isoform 3 OS ═ homo sapiens OX ═ 9606 GN ═ MYH9

38: Lass GTPase activating protein 1RASA 1. UniProt ID P20936. isoform 1.> sp | P20936| RASAl _ HUMAN GTP enzyme activator protein 1 OS ═ homo sapiens OX ═ 9606 GN ═ RASA1 PE ═ SV ═ l

SEQ ID NO:39 Lass GTPase activating protein 1RASA 1. UniProt ID P20936-2 isoform 2 ≧ sp | P20936-2| RASAl _ HUMAN gtpase activator protein 1 isoform 2 OS ═ homo sapien OX ═ 9606 GN ═ RASA1

SEQ ID NO 40 Las GTPase activating protein 1RASA 1. UniProt ID P20936-3 isoform 3 ≧ sp | P20936-3| RASAl _ HUMAN gtpase activator protein 1 isoform 3 OS ═ homo sapien OX ═ 9606 GN ═ RASAl

41 SEQ ID NO. Lass GTPase activating protein 1RASA 1. UniProt ID P20936-4. isoform 4.> sp | P20936-4| RASAl _ HUMAN gtpase activator protein 1 isoform 4 OS ═ homo sapien OX ═ 9606 GN ═ RASAl

42 hairiness/division enhancer 1 HEY1 related to YRPW motif protein. UniProt ID q9y5j3. isoform 1.> sp | Q9Y5J3| HEY1_ HUMAN and YRPW motif protein related hairy/split enhancer 1 OS ═ homo sapiens OX ═ 9606 GN ═ HEY1 PE ═ 1 SV ═ 1

43 hairiness/division enhancer 1 HEY1 associated with the YRPW motif protein. UniProt ID Q9Y5J3-2 isoform 2.> sp | Q9Y5J3-2| HEYl _ HUMAN associated with YRPW motif protein hairy/split enhancer 1 isoform 2 OS ═ homo sapien OX ═ 9606 GN ═ HEY1

44 protein C-ETS-2ETS 2. UniProt ID P15036.> sp | P15036| ETS2_ HUMAN protein C-ETS-2 OS (homo sapiens OX 9606 GN ═ ETS2 PE ═ 1 SV ═ 1-

45: hairiness/division enhancer 2 HEY2 associated with the YRPW motif protein. UniProt ID Q9UBP5.> sp | Q9UBP5| HEY2_ HUMAN associated with YRPW motif protein hairy/split enhancer 2 OS ═ homo sapien OX ═ 9606 GN ═ HEY2 PE ═ 1 SV ═ 1

46 SEQ ID NO LIM Domain binding protein 1LDB 1. UniProt ID q86u70 isoform 1.> sp | Q86U70| LDBl _ HUMAN LIM domain binding protein 1LDB1 OS ═ homo sapiens OX ═ 9606 GN ═ LDB1 PE ═ 1 SV ═ 2

47 SEQ ID NO: LIM Domain binding protein 1LDB 1. UniProt ID Q86U70-3 isoform 2.> sp | Q86U70-3| LDBl _ HUMAN LIM domain binding protein 1 isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ LDB1

48 SEQ ID NO LIM Domain binding protein 1LDB 1. UniProt ID Q86U70-2 isoform 3.> sp | Q86U70-2| LDBl _ HUMAN LIM domain binding protein 1 isoform 3 OS ═ homo sapiens OX ═ 9606 GN ═ LDB1

49 of spindle protein receptor UNC 5C. UniProt ID O95185, isoform 1.> sp | O95185| UNC5C _ HUMAN spindle protein receptor UNC5C isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ UNC5C

50: spindle protein receptor UNC 5C. UniProt ID O95185-2 isoform 2.> sp | O95185-2| UNC5C _ HUMAN spindle protein receptor UNC5C isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ UNC5C

51 parts of SEQ ID NO. neurofibrillary protein NF 1. UniProt ID P21359, isoform 2.> sp | P21359| NFl _ HUMAN neuropilin OS ═ homo sapiens OX ═ 9606 GN ═ NF1 PE ═ 1 SV ═ 2

52 parts of SEQ ID NO. neurofibrillary protein NF 1. UniProt ID P21359-2 isoform 1.> sp | P21359-2| NF1_ HUMAN neurofibrin isoform 1 OS ═ homo sapiens OX ═ 9606 GN ═ NF1

53 Neurofibrin NF1, SEQ ID NO. UniProt ID P21359-3 isoform 3.> sp | P21359-3| NFl _ HUMAN neurofibrin isoform 3 OS ═ homo sapiens OX ═ 9606 GN ═ NF1

54. Neurofibrin NF 1. UniProt ID P21359-4 isoform 4.> sp | P21359-4| NFl _ HUMAN neurofibrin isoform 4 OS ═ homo sapiens OX ═ 9606 GN ═ NF1

55 parts of SEQ ID NO. neurofibrillary protein NF 1. UniProt ID P21359-5 isoform 5.> sp | P21359-5| NFl _ HUMAN neurofibrin isoform 5 OS ═ homo sapiens OX ═ 9606 GN ═ NF1

56: neurofibrillary protein NF 1. UniProt ID P21359-6 isoform 6.> sp | P21359-6| NFl _ HUMAN neurofibrin isoform 6 OS ═ homo sapiens OX ═ 9606 GN ═ NF1

57 cyclin-dependent kinase 6CDK 6. UniProt ID Q00534.> sp | Q00534| CDK6_ HUMAN cyclin-dependent kinase 6 OS ═ homo sapiens OX ═ 9606 GN ═ CDK6 PE ═ SV ═ 1

58, cyclin very low density lipoprotein receptor VLDLR. UniProt ID P98155. long isoform > sp | P981551 VLDLR HUMAN ultra low density lipoprotein receptor. OS ═ homo sapiens OX ═ 9606 GN ═ VLDLR PE ═ 1 SV ═ 1

VLDLR as cyclin receptor of very low density lipoprotein of cyclin of protein of SEQ ID NO. 59. UniProt ID P98155-2. short isoform > sp | P98155-2| VLDLR _ HUMAN ultra low density lipoprotein receptor short isoform OS ═ homo ═ 9606 GN ═ VLDLR

60 parts of SEQ ID NO of neuropilin-2 NRP 2. UniProt ID O60462, isoform a22.> sp | O60462| NRP2_ HUMAN neuropilin-2 OS ═ homo sapiens OX ═ 9606 GN ═ NRP2 PE ═ 1 SV ═ 3

61 Neuropilin-2 NRP2 in SEQ ID NO. UniProt ID O60462-2 isoform a0.> sp | O60462-2| NRP2_ HUMAN neuropilin-2 isoform a0 OS ═ homo sapiens OX ═ 9606 GN ═ NRP2

62 of the sequence shown in SEQ ID NO. neuropilin-2 NRP 2. UniProt ID O60462-3 isoform a17.> sp | O60462-3| NRP2_ HUMAN neuropilin-2 isoform a17 OS ═ homo sapiens OX ═ 9606 GN ═ NRP2

63 of the sequence shown in SEQ ID NO. neuropilin-2 NRP 2. UniProt ID O60462-4. isoform B0.> sp | O60462-4| NRP2_ HUMAN neuropilin-2 isoform B0 OS ═ homo sapiens OX ═ 9606 GN ═ NRP2

64, Neuropilin-2 NRP 2. UniProt ID O60462-5 isoform B5.> sp | O60462-5| NRP2_ HUMAN neuropilin-2 isoform B5 OS ═ homo sapiens OX ═ 9606 GN ═ NRP2

65 parts of SEQ ID NO. neuropilin-2 NRP 2. UniProt ID O60462-6. isoform s9.> sp | O60462-6| NRP2_ HUMAN neuropilin-2 isoform s9 OS ═ homo sapiens OX ═ 9606 GN ═ NRP2

66 SEQ ID NO. dihydropyrimidinase associated protein 2 DPYL 2. UniProt ID q16555 isoform 1.> sp | Q16555| DPYL2_ HUMAN dihydropyrimidinase associated protein 2 OS ═ homo sapiens OX ═ 9606 GN ═ DPYSL2 PE ═ 1 SV ═ 1

67 dihydropyrimidinase-related protein 2 DPYL 2. UniProt ID Q16555-2 isoform 2.> sp | Q16555-2| DPYL2_ HUMAN dihydropyrimidinase associated protein 2 isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ DPYSL2

68 of Neuropilin-1 NRP 1. UniProt ID O14786. isoform 1.> sp | O14786| NRPl _ HUMAN neuropilin-1 OS ═ homo sapiens OX ═ 9606 GN ═ NRP1 PE ═ SV ═ 3 ═ f

69 parts of the sequence shown in SEQ ID NO: neuropilin-1 NRP 1. UniProt ID O14786-2 isoform 2.> sp | O14786-2| NRPl _ HUMAN neuropilin-1 isoform 2 OS ═ homo sapiens OX ═ 9606 GN ═ NRP1

70 of neuropilin-1 NRP 1. UniProt ID O14786-3. isoform 3.> sp | Ol4786-3| NRPl _ HUMAN neuropilin-1 isoform 3 OS ═ homo sapiens OX ═ 9606 GN ═ NRPl ═ n

Claims

1. A method for treating a tumor in a subject having cancer, the method comprising administering to the tumor an effective amount of ultra-high dose rate (FLASH) radiation and a therapeutic agent.

2. The method of claim 1, wherein the method reduces damage to normal tissue as compared to administering conventional radiation to the tumor at a dose of 0.5 Gy/sec.

3. The method of claim 1, wherein the radiation is administered at a dose rate equal to or greater than 40Gy/sec, or the dose is administered in 1 second or less, and the radiation is administered in a single pulse or in multiple pulses.

4. The method of claim 1, wherein the radiation comprises or consists of protons.

5. The method of claim 1, wherein the therapeutic agent is an immunomodulator, an anti-aging agent, a radiosensitizer, or a nanoparticle.

6. The method of claim 1, wherein the therapeutic agent is a mitotic spindle inhibitor, a DNA damage repair and response inhibitor, a MAPK pathway inhibitor, an epithelial-to-mesenchymal (EMT) inhibitor, an activator of T helper type1 (TH1) lymphocytes, an activator of the PTEN pathway and an inhibitor of the TGF- β pathway, an activator of the type1 interferon signaling pathway, an activator of dendritic cell maturation, an inhibitor of CD47/SIRP- α, or an inhibitor of arene receptor (ahR).

7. The method of claim 6, wherein the mitotic spindle inhibitor is selected from a CDK4/6 inhibitor, an AURKA inhibitor, a TPX2-AURKA complex inhibitor, or a taxane.

8. The method of claim 6, wherein the inhibitor of DNA damage repair and response is selected from the group consisting of: a PARP inhibitor, a RAD51 inhibitor, or an inhibitor of a DNA damage response kinase selected from CHCK1, ATM or ATR.

9. The method of claim 6, wherein said MAPK pathway inhibitor is an inhibitor of EGFR, MEK, BRAF, or ERK.

10. The method of claim 6, wherein the EMT inhibitor is a TGF- β pathway inhibitor selected from a compound, small molecule, antibody, or fragment thereof that binds TGF- β.

11. The method of claim 6, wherein the activator of T helper type1 (TH1) lymphocytes is a cytokine, toll-like receptor agonist, STAT3 modulator, compound derived from an inactivated bacterium or parasite or derivatives thereof that trigger interferon gamma or IL-12 production, staphylococcal enterotoxin B, unmethylated CpG nucleotides, or a bacterial or viral based gene expression system that results in the production of IL2, IL-12 and IFN-gamma when injected at a tumor site.

12. The method of claim 6, wherein the activator of the PTEN pathway is an mTOR inhibitor selected from: rapamycin, temsirolimus, everolimus, sirolimus or AP-2357, ubuliximab, rituximab, sunitinib, trastuzumab, pertuzumab, resistin, simvastatin, lovastatin, rosiglitazone, NVP-AEW541, Src inhibitors or PP1 herbimycin.

13. The method of claim 6, wherein the activator of the type1 interferon signaling pathway is a STING agonist, a Toll-like receptor (TLR) agonist, or a MAVS agonist.

14. The method of claim 6, wherein the activator of dendritic cell maturation is a synthetic peptide vaccine and the inhibitor of CD 47/SIRP-a is selected from an antibody or fragment of an antibody, or a small molecule compound that inhibits the CD 47/DSIRP-a interaction.

15. The method of claim 6, wherein the nanoparticles have a high effective atomic number or comprise gold or gadolinium.

16. The method of claim 6, wherein the ahR inhibitor is SR1, CH-223191, UM729, or galangin.

17. The method of claim 2, wherein dermatitis, pulmonary fibrosis, or lymphocyte apoptosis is reduced as compared to administration of conventional radiation.

18. A therapeutic agent for use in a method of treating a tumor in a subject having cancer, the method comprising administering FLASH radiation and the therapeutic agent to the tumor.

19. A therapeutic agent for use in a method of treating a tumor in a subject having cancer, wherein the therapeutic agent is administered in combination with FLASH radiation.

20. The therapeutic agent for use in a method of treating a tumor in a subject having cancer according to claim 19, wherein the therapeutic agent is administered to the immunocompetent tumor environment caused by the FLASH radiation.

21. A hedgehog antagonist for use in a method of treating a tumor in a subject having cancer, wherein the hedgehog antagonist is administered in combination with FLASH radiation.

22. A non-transitory computer-readable storage medium having computer-executable instructions for causing a computing system to perform a method of an ultra-high dose rate (FLASH) radiation therapy plan for treating a tumor in combination with a therapeutic agent; wherein the method comprises: the information about treating the tumor with the therapeutic agent is used to adjust beam parameters.

23. The non-transitory computer-readable storage medium of claim 22, the method comprising:

accessing values of parameters from a memory of the computing system, wherein the parameters include a direction of a beam to be directed into a sub-volume in a target and a beam energy of the beam;

accessing information specifying limits for the radiation therapy plan, wherein the limits are based on a dose threshold and include limits on exposure time for each sub-volume outside the target;

wherein the information specifying a limit comprises information about treating the tumor with a therapeutic agent; and

adjusting the values of the parameters that affect the calculated amount of dose to be delivered by the beam until a difference between the respective total values for the sub-volumes satisfies a threshold.

24. The non-transitory computer-readable storage medium of claim 23, wherein each portion of the beam that is in the target is represented as a respective set of longitudinal beam regions, and wherein the method further comprises:

for each of the beam regions, calculating an amount of dose to be delivered by the beam region and assigning a value corresponding to the amount to the beam region; and

for each of the sub-volumes, calculating a total value for the sub-volume by adding together the values of each beam region of each beam that reaches the sub-volume;

wherein the adjusting further comprises adjusting the parameters that affect the calculated amount of dose to be delivered by the beam region until a difference between respective total values for the sub-volumes satisfies the threshold.

25. The non-transitory computer-readable medium of claim 24, wherein the adjusting further comprises:

determining whether a beam overlaps any other beam outside the target; and

weighting beam intensities for beam segments of the beam according to how many other beams overlap the beam outside the target.

26. The non-transitory computer-readable medium of claim 25, wherein the method further comprises: performing dose calculations for the target outer sub-volume, wherein the performing dose calculations comprises:

accessing a value of a dose calculation factor for the target outer sub-volume, wherein the value of the dose calculation factor is determined according to how many beams reach the target outer sub-volume;

calculating a dose for the outer sub-volume of the target; and

applying the value of the dose calculation factor to the dose calculated for the target outer sub-volume.

27. The non-transitory computer readable medium of claim 26, wherein the dose calculation factor reduces the dose calculated for the target outer sub-volume if only one beam reaches the target outer sub-volume.

28. The non-transitory computer-readable medium of claim 27, wherein the restriction is selected from the group consisting of: a limit on illumination time for each sub-volume in the target; a limit on dose rate for each sub-volume in the target; and a limit on dose rate for each sub-volume outside the target.

29. The non-transitory computer readable medium of claim 28, wherein the dose threshold is further dependent on a tissue type.

30. A computer-implemented method of radiation therapy planning for treating a tumor in combination with an immunomodulatory agent, the method comprising: beam parameters for ultra-high dose rate (FLASH) radiation are determined, the method using a prescribed dose based on the tumor's response to a therapeutic agent.

31. The computer-implemented method of claim 30, the method comprising: determining a prescribed dose of ultra-high dose rate (FLASH) radiation to be delivered into and across a tumor target, wherein the prescribed dose is determined based on a response of the tumor to a therapeutic agent;

accessing values of parameters including a number of beams in a plurality of beams to be directed into a sub-volume in the target, directions of the plurality of beams, and beam energies of the plurality of beams, wherein each beam in the beams comprises a plurality of beam segments;

for each beam of the plurality of beams, determining a maximum beam energy of the beam and determining a beam energy of the beam segment of the beam as a percentage of the maximum beam energy of the beam; and

for each of the overlapping beams that overlap outside the target, reducing a beam intensity of the beam segment of the overlapping beam by a dose calculation factor, wherein the beam intensities of the beam segments of the plurality of beams are determined such that a cumulative dose delivered to the target satisfies the specified dose.

32. The computer-implemented method of claim 31, wherein the method further comprises:

representing each beam of the beams that is in the target as a respective set of longitudinal beam regions, wherein each beam region of the set has a value corresponding to a calculated amount of dose to be delivered by the beam region;

for each sub-volume in the target, adding together the values for each beam region of each beam that reaches the sub-volume to determine a total value for the sub-volume to produce a respective total value for the sub-volume in the target; and

adjusting the values of the parameters that affect the calculated amount of dose to be delivered by the beam region until a difference between the total values for the sub-volumes satisfies a threshold.

33. The computer-implemented method of claim 32, wherein the method further comprises:

accessing a value of the dose calculation factor for an outer sub-volume of a target, wherein the value of the dose calculation factor is determined according to how many beams reach the outer sub-volume of the target;

calculating a dose for the outer sub-volume of the target; and

applying the value of the dose calculation factor to the dose calculated for the outer target sub-volume, wherein the dose calculation factor reduces the dose calculated for the outer target sub-volume if only one beam reaches the outer target sub-volume.

34. The computer-implemented method of claim 33, wherein the method includes using a dose threshold to specify limits for the radiation therapy plan, wherein the limits are selected from the group consisting of: a limit on illumination time for each sub-volume in the target; a limit on illumination time for each sub-volume outside the target; a limit on dose rate for each sub-volume in the target; and a limit on dose rate for each sub-volume outside the target.

35. An in vitro method of identifying a subject with cancer as a candidate for a treatment comprising ionizing FLASH radiation and an immunomodulator, the method comprising:

(a) determining the expression level of one or more biomarkers in a tumor sample from the subject;

(c) classifying the subject as a candidate for treatment comprising ionizing FLASH radiation and the immunomodulator if the expression level of the one or more biomarkers in the tumor sample is modified compared to the expression level in the normal tissue sample.

36. An in vitro method of selecting a treatment for a subject having cancer, the method comprising:

(c) selecting a treatment comprising ionizing FLASH radiation and an immunomodulator if the expression level of the one or more biomarkers in the tumor sample is modified compared to the expression level in the normal tissue sample.

37. The in vitro method of claim 36, wherein:

a treatment comprising ionizing FLASH radiation and an immunomodulator is selected if the expression level of CD44 is increased and/or the expression level of MFG-E8 is decreased relative to the expression level in a normal or control sample;

(ii) an increase in the amount of selected ionizing FLASH radiation and/or an increase in the amount of immunomodulator if the expression level of CD44 is increased and/or the expression level of MFG-E8 is decreased relative to the expression level in a normal or control sample; and

the amount of selected ionizing FLASH radiation and/or the amount of immunomodulator is reduced if the expression level of CD44 is reduced and/or the expression level of MFG-E8 is increased relative to the expression level in a normal or control tissue sample.

38. The in vitro method of claim 37, wherein:

a treatment comprising ionizing FLASH radiation and an immunomodulator is selected if the expression level of CD68 is elevated relative to the expression level in a normal or control tissue sample;

(ii) if the expression level of CD68 is increased relative to the expression level in a normal or control tissue sample, the amount of selected ionizing FLASH radiation and/or the amount of immunomodulator is increased;

if the expression level of CD68 is decreased relative to the expression level in a normal or control tissue sample, the amount of ionizing FLASH radiation and/or the amount of immunomodulator selected can be decreased.

39. The in vitro method of claim 38, wherein:

the treatment is selected using a computer-implemented algorithm that analyzes the expression level of the biomarker in the tumor sample relative to levels in the normal sample;

wherein the algorithm comprises one or more of:

a linear regression algorithm comprising biomarker expression levels and coefficients for combining the expression levels;

a least squares fit for calculating the coefficients; and

a non-parametric regression tree;

wherein a treatment comprising FLASH radiation and an immunomodulator is selected depending on the algorithm determining that the expression level of the biomarker in the tumor sample is increased or decreased relative to the normal sample.

40. The in vitro method of claims 35 to 39, wherein:

the biomarker is one or more of: CD44, MMP9, ALDH1a1, vimentin, hyaluronic acid, beta catenin, MFG-E8, CD68, TGF β pathway-associated biomarkers; and/or

The expression levels of one or more biomarkers are ranked or weighted.

41. Use claims 18-21, medium claims 22-29 and method claims 30-40 wherein said radiation is administered at a dose rate equal to or greater than 40Gy/sec or said dose is administered in 1 second or less and said radiation is administered in a single pulse or in multiple pulses.

42. Use claims 18-21, medium claims 22-29 and method claims 30-40, wherein said radiation comprises or consists of protons.

43. The use claims 18-21, the mediator claims 22-29, and the method claims 30-40, wherein the therapeutic agent is an immunomodulator, an anti-aging agent, a radiosensitizer, or a nanoparticle.

44. The use claims 18-21, media claims 22-29, and method claims 30-40, wherein the therapeutic agent is a mitotic spindle inhibitor, a DNA damage repair and response inhibitor, a MAPK pathway inhibitor, an epithelial-to-mesenchymal (EMT) inhibitor, an activator of T helper type1 (TH1) lymphocytes, an activator of PTEN pathway and an inhibitor of TGF-beta pathway, an activator of type1 interferon signaling pathway, an activator of dendritic cell maturation, an inhibitor of CD 47/SIRP-alpha, or an inhibitor of arene receptor (ahR).

45. The use, medium and method according to claim 44 wherein the mitotic spindle inhibitor is selected from CDK4/6 inhibitors, AURKA inhibitors, TPX2-AURKA complex inhibitors, or taxanes.

46. The use, medium and method according to claim 44, wherein the inhibitor of DNA damage repair and response is selected from the group consisting of: a PARP inhibitor, a RAD51 inhibitor, or an inhibitor of a DNA damage response kinase selected from CHCK1, ATM or ATR.

47. The use, medium and method according to claim 44, wherein the MAPK pathway inhibitor is an inhibitor of EGFR, MEK, BRAF or ERK.

48. The use, medium and method according to claim 44, wherein the EMT inhibitor is a TGF- β pathway inhibitor selected from a compound, small molecule, antibody or fragment thereof that binds TGF- β.

49. The use, medium and method of claim 44 wherein the activator of T helper type1 (TH1) lymphocytes is a cytokine, toll-like receptor agonist, STAT3 modulator, compound derived from inactivated bacteria or parasites or their derivatives that trigger interferon gamma or IL-12 production, staphylococcal enterotoxin B, unmethylated CpG nucleotides, or a bacterial or viral based gene expression system that results in the production of IL2, IL-12 and IFN-gamma when injected at a tumor site.

50. The use, medium and method of claim 44 wherein the activator of the PTEN pathway is an mTOR inhibitor selected from: rapamycin, temsirolimus, everolimus, sirolimus or AP-2357, ubuliximab, rituximab, sunitinib, trastuzumab, pertuzumab, resistin, simvastatin, lovastatin, rosiglitazone, NVP-AEW541, Src inhibitors or PP1 herbimycin.

51. The use, medium and method according to claim 44, wherein the activator of the type1 interferon signaling pathway is a STING agonist, a Toll-like receptor (TLR) agonist, or a MAVS agonist.

52. The use, medium and method of claim 44, wherein the activator of dendritic cell maturation is a synthetic peptide vaccine and the inhibitor of CD 47/SIRP-a is selected from an antibody or fragment of an antibody, or a small molecule compound that inhibits the CD 47/DSIRP-a interaction.

53. The use, medium or method according to claim 44, wherein the nanoparticles have a high effective atomic number or comprise gold or gadolinium.

54. The use, medium and method of claim 44, wherein the ahR inhibitor is SR1, CH-223191, UM729 or galangin.

55. A method for reducing activation of the hedgehog signaling pathway in a subject, comprising administering to the subject an effective amount of ultra-high dose rate (FLASH) radiation, wherein the activation of the hedgehog signaling pathway is reduced as compared to administration of conventional proton radiation to the subject.

56. The method of claim 55, wherein the expression of a gene activated by the hedgehog signaling pathway is reduced when FLASH radiation is administered to the subject as compared to administration of conventional proton radiation therapy.

57. The method of claim 56, wherein the expression of one or more genes selected from the following is reduced when FLASH radiation is administered to the subject as compared to administration of conventional proton radiation therapy: CELSR1, TLE3, OPHN1, GPR56, PTCH1, TLE1, MYH9, RASA1, HEY1, ETS2, HEY2, LDB1, UNC5C, NF1, CDK6, VLDLR, NRP2, DPYSL2, and NRP 1.

58. The method of claim 55, further comprising administering a hedgehog antagonist to the subject.

59. The method of claim 58, wherein the hedgehog antagonist is selected from the group consisting of: smo antagonists, PTCH1 inhibitors, cyclopamine, vismodegib, LDE 225, saridegib, BMS 833923, LEQ 506, PF-04449913, PF-5274857, GANT61, SANT-1, Glabra (PF-04449913), Taladegib (LY2940680), or TAK-441.

60. The method of claims 55-59 wherein said FLASH radiation comprises or consists of protons.