Biomolecular dosimeter

Abstract

A novel dosimetry approach quantifies radiation damage potential due to linear energy transfer radiation (LET), dose, and dose rate factors. Here, the gaseous medium in ionization dosimetry is replaced with inanimate DNA molecules, or other appropriate biomolecules, under well-controlled environmental / chemical conditions. Radiation damage potential is then characterized as radiation-induced alterations in the DNA molecules as a function of spatial and temporal factors of the delivered radiation energy. A new Molecular Dosimetry Unit (MoD) is introduced based on the metrics of, clustered alteration size (CASi) denoting the ith number of alterations from i=0 to >60, and frequency of CASi (CAFi), in the DNA dosimeter as determinants of radiation dose, dose rate and LET. Treatment planning can be generated to optimize the MoD for a particular subject over a number of treatments, time, combination of treatment modalities, and re-treatment.

Description

BIOMOLECULAR DOSIMETERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 727.920, filed December 4, 2024. The content of the aforementioned application is herein incorporated by reference, in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates generally to radiation dosimetry. More particularly, the present invention relates to a biomolecular dosimeter for improved representation and measurement of delivered radiation and its biochemical effects.BACKGROUND OF THE INVENTION

[0003] The exposure of living organisms to ionizing radiation causes detrimental outcomes such as lethality, carcinogenesis and mutagenesis. Prediction of the biological outcome has always been imperative for a wide range of medical and industrial applications, such as radiation therapy of cancer patients and protection from radiation sources in medicine, nuclear reactors, and space travel. Energy7deposited in human tissues and other organisms has been an essential physical parameter for the evaluation of the radiation outcomes. Over many decades, radiation dosimetry7has been developed to characterize the beam output of radiation machines by measuring the radiation energy7transfer to a gaseous medium through ionization under well-controlled condition. This energy transfer is then used to calculate the energy absorbed in human tissues in radiation medicine and radiation protection applications. The long-standing unit derived from ionization energy7measurement and adopted by the International Commission of Radiation Units is Gray (Gy), which represents the energy deposition in the unit mass of a medium.

[0004] The Gy unit is a transformative standard to quantify output from various sources of radiation machines and model the radiation dose delivered to humans. However, it is also well known that Gy does not include fundamental information about the temporal (i.e., the rate of radiation energy' delivery ) and spatial (i.e., linear energy7transfer (LET) per ionization event) characteristics of the radiation modality. These factors have been shown to impart varying damages to living organisms as well as humans. For example, high LET particles, such as helium particles, inflict more lethal damage per unit Gy than electrons; while x-rays delivered at dose rate 1000 times that of clinical practice inflict less damage to normal tissues. The exclusion of this fundamental information led to the use of empirical factors known as Relative Biological Effectiveness (RBE) for the high-LET particles, and Dose Modifying Factor (DMF) for ultrahigh dose-rate irradiation to augment the Gy as the descriptor of radiation effects in radiation medicine and protection. The deficiency7of these factors has, unfortunately, limited their use as general guidelines in practice.

[0005] It would therefore be advantageous to provide a novel standard of biomedical dosimetry7for improved measurement and specification of delivered radiation and radiation effects.SUMMARY OF THE INVENTION

[0006] The foregoing needs are met, to a great extent, by the present invention, wherein one aspect is a system including a biomolecular sample. The system also includes a chamber configured for receiving a dose of radiation from a radiation source. The chamber defines an inner space for receiving the biomolecular sample. The biomolecular sample is contacted with the dose of radiation.

[0007] In accordance w ith an aspect of the present invention, the biomolecular sample includes inanimate DNA molecules. The system includes a sequencing component for sequencing the DNA sample and outputting a range of molecular alterations (e.g., based lesions, abasic sites, strand breaks, etc.) for the DNA sample. A processing device receives metrics related to alterations to the DNA sample, quantifying a level of alteration to the DNA sample from the received metrics, and generating an output quantifying alteration to the DNA. The processing device determines a molecular dosimetry7unit (MoD) for the alterations to the DNA. The MoD is calculated as a function of Clustered Alteration Size (CASi) for the ithnumber of alterations in the range of alterations and the corresponding frequency, or occurrence, of the CASi (CAFi).

[0008] In accordance with another aspect of the present invention, the modeling of MoD comprises calculating CASi and CAFi using a relational method that integrates: (i) radiationtransport Monte Carlo simulations of ionization events and the resulting production of free radicals and low-energy7electrons in DNA with or without its hydration shells, and (ii) experimentally measured yields of DNA alterations induced by individual ionization events, free radicals, and low-energy electrons.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings provide visual representations, which will be used to more fully describe the representative embodiments disclosed herein and can be used by those skilled in the art to better understand them and their inherent advantages. In these drawings, like reference numerals identify corresponding elements and:

[0010] FIG. 1 A illustrates a schematic view of a dosimetry7measurement system according to an embodiment of the present invention, analogous to the conventional system shown in FIG. IB.

[0011] FIG. 2 illustrates a graphical view of radiation dose versus cell survivals for particle irradiation at different LET values.

[0012] FIGS. 3A and 3B illustrate graphical views of CASi versus CAFi to score the CASi and CAFi at different LETs.

[0013] FIGS. 4A and 4B are graphical views of CASi S and CAFi derived from LET and radiation dose.

[0014] FIGS. 5A-5E illustrate cell survival curves as a function of dose (Gy) of different cell lines at different LET irradiation.

[0015] FIGS. 6A illustrates the relationship of LET and dose at survival fractions of 75%, 50%, 10% and 1% for V79 cells. FIGS 6B shows the relationship of CAS; and CAFi at these same cell survival fractions in FIGS 6A for V79 cells. FIGS 6C shows the relationship of CASi and CAFi for these survival fractions for CHO cells.

[0016] FIG. 7 illustrates a graphical view of two cell survivals as a function of molecular dosimetry unit (MoD).

[0017] FIG. 8 illustrates a graphical view of dose-rate dependency of molecular dosimetry unit (MoD).

[0018] FIGS. 9A-9C illustrate, on the left side, graphical views of depth-dose and LET distributions modulated to deliver a uniform 2 Gy dose within a specified, spread-out Bragg peak (SOBP) and, on the right side, the corresponding calculated depth-MoD profiles in a water phantom.DETAILED DESCRIPTION

[0019] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Drawings, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Drawings. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0020] A novel dosimetry approach quantifies the range of radiation induced alterations of an irradiated sample of DNA molecules as Clustered Alteration Size (CASi) denoting ithnumber of alterations in the range due to LET, dose, and dose rate factors. Fundamentally, the gaseous medium in ionization dosimetry is replaced with inanimate DNA molecules, or other appropriate biomolecules, at well-controlled chemical conditions such as different hydration levels and chemical buffers. Radiation damage potential is then characterized as radiation-induced alterations in the DNA molecules as a function of physical parameters including spatial (LET and fluence) and temporal (fluence-rate or dose-rate) factors of the delivered radiation energy. The typical confounding effects of biological repair mechanisms for resolving DNA damage are avoided by using inanimate DNA molecules in a cell-free system. The use of inanimate DNA can differentiate the various types of radiation-induced molecular lesions including strand breaks, base release (abasic sites), base and sugar modifications. The novel concept of a new Molecular Dosimetry Unit (MoD) is introduced based on the metrics of, all i* CASi and the corresponding frequency of CASi (CAFi), in the DNA dosimeter as determinants of radiation dose / fluence, dose rate / fluence-rate and LET. The efficacy of MoD is demonstrated by its ability' to consolidate, for a particular cell line,the disparate cell survival data from different radiation modalities into a single, unified response curve. Furthermore, the applicability of the MoD in radiation treatment planning is demonstrated through the determination of depth-MoD curves, analogous to depth-dose curves, which indicates the variation of MoD as a function of particle penetration within a medium such as water. Importantly, analogous to the dissemination of ionization dosimetry, the MoD, expressed as a function, presently as a product, of all CASi and CAFi, can be measured using modem molecular biology' techniques such as DNA sequencing technologies, and can be modeled using Monte Carlo simulations of radiation transport and interaction relationally mapped to measured CAS;. The molecular-based MoD shows the potential of a new transformation in radiation dosimetry7standard for medicine and healthcare.

[0021] The present invention was motivated by7the lack of accurate quantification methodology7for radiation effects on living organisms, such as lethality7, carcinogenesis, mutagenesis, and prediction of the biological outcome of radiation dose for radiotherapy, radiation protection, etc. Currently, radiation descriptor includes dose with its unit of Gray (Gy), which was primarily developed to characterize radiation machines’ output. Gy represents the energy7deposition in the unit mass of a medium inferred from ionization measurement, as recommended in reports from AAPM TG21, AAPM TG51, IAEA TRS-398. Gy is also used for dose calculation. At present there is also no fundamental information about the temporal (dose-rate) and spatial (LET) characteristics of the radiation in ionizationbased radiation dose measurements.

[0022] Empirical factors have been introduced to compensate for inadequacy of the Gy. RBE is used for the high-LET particles and Dose Modifying Factor (DMF) for the ultrahigh doserate radiations. However, RBE and DMF are derived from specific irradiation measurements and have limited applications. The present invention is directed to producing a molecular metric (in terms of radiation induced molecular alteration), in an inanimate medium(independent of biological systems), which is better suited for biological response assessment compared to the Gy.

[0023] FIG. 1 A illustrates schematic views of a dosimetry measurement system according to an embodiment of the present invention, and an analogous system for measuring ionization shown in FIG. IB. The present invention is directed to a system where an inanimate biomolecule, such as a DNA molecule that has been treated to create cell-free DNA, is disposed in a water filled chamber configured to receive radiation from various sources of radiation. Radiation is applied, and the DNA is analyzed to assess alterations from the radiation. In an exemplary system of the present invention, parameters such as dose-rate, dose, LET, and source-tj pe can be manipulated to see the outcome on DNA. These parameters can be manipulated in any way known to or conceivable to one of skill in the art and in any combination that is known to or conceivable to one of skill in the art. The DNA is in a cell-free state to remove any biological effects of DNA disposed within a cell and to avoid complex biological processes. It should be noted that any source of radiation known to or conceivable to one of skill in the art can be assessed with the system and method of the present invention, including but not limited to proton radiation, x-ray radiation, electron radiation, helium, carbon and light ion radiations, cosmic radiation etc.

[0024] The system of FIG. 1A can measure a number of metrics including clustered alteration size (CASi) and frequency of CASi (CAFi). A molecular dosimetry unit (MoD) is calculated as a function of all CASi and the corresponding CAFi, as will be described further herein. In some exemplary systems, the DNA can be disposed in an aqueous medium. The DNA sample in some embodiments can be hydrated, and the hydration level can be manipulated to measure direct and indirect actions of radiation. It should be noted that CASi represents the specific i* number (typically from 0 to 90) of alterations in a nanometric volume of DNA (e g., 15 bp) and its frequency CAFi represents the number of the ithalterations in the dosimeter containing the collection of nanometric volumes of DNA. CASi and CAFi are determinants of radiation dose, dose rate and LET. In some embodiments theDNA sample takes the form of cell-free DNA to remove any biological effects of DNA disposed within a cell and to avoid complex biological processes.

[0025] In some embodiments, DNA molecular alterations can be measured via DNA sequencing, digital PCR, or other DNA assessment methodology known to or conceivable to one of skill in the art. More particularly, the DNA subject to the radiation source can be processed by, but not limited to, : (1) DNA sequencing to determine the bases alterations and their spatial distribution along the DNA molecule, (2) enzymatic treatment followed by quantitative or digital PCR to determine an accounting of broken ends of the DNA, and (3) other analytical techniques based on mass spectrometry such as LC-MS / MS or GC-MS to identify and quantify specific DNA alterations. This processing and analytical assessments can be done either within the system or outside of the system, as is known to or conceivable to one of skill in the art. Ultimately, the output of the device can in some embodiments be a breakdown or accounting of these DNA alterations, base sequencing, and broken ends, along with all CASi, CAFi, and MoD for the input radiation and other manipulatable criteria of the system.

[0026] FIG. 2 illustrates a graphical view of radiation dose versus cell survival for the extraction of a Molecular Dosimetry Unit (MoD) from dose-response and RBE data.Where as Dose in Gy oc Fluence X Stopping Power « Fluence x LET MoD is represented as a function = f(CAS , CAF , ... )Where, CAS: The range of Clustered Alteration SizeCAF: The Frequency, or occurrence, of CASA computational experiment was performed to find a functional form of MoD. FIG. 2 shows published cell survival data as a function of dose (Gy) for different LETs. For a given cellline, the goal of the present invention is to consolidate all the survival curves at different LETs with a resultant MoD unit replacing Gy.

[0027] FIGS. 3A and 3B illustrate graphical views of CASi versus CAFi to score the CASi and CAFi at different LETs. Further FIGS. 3A and 3B show the relationship of CASi vs CAFi. Hydrated DNA is used as the biomolecular dosimeter. CASi represents the clustered alteration size = no. of (any) molecular alterations within scoring nanometric volume, each, in this configuration, contained 15 base-pair DNA hydrated with 2O H2O molecules per nucleotide. A Monte-Carlo radiation transport simulation provides the quantification of ionization events and secondary reactive species (free radicals and sub-ionization-energy' electrons) that are relationally mapped with the experimentally measured yields of damage to generate the CASi and CAFi in the irradiated DNA volume (8.0 / rm3) at different LETs of Alpha particles and Protons.

[0028] FIGS. 4A and 4B are graphical views of CASi and CAFi derived from LET and radiation dose. CASi is proportional to LET. CAFi is a function of dose and CAS;.

[0029] FIGS. A-5E illustrate different cell survival rates as a function of radiation dose (Gy) at different LET particles. Here, Chinese hamster CHO-K1 cells and V79 cells were used.

[0030] FIG. 6A indicates four survival fractions data can be represented as functions of LET and dose (Gy). FIGS. 6B-6C illustrate these cell survival fractions can be represented as functions of CASi and CAFi.

[0031] As illustrated in FIGS. 6A-6C, survival fractions for each cell line have similar behavior with respect to LET and Dose and with respect to CASi and CAFi.

[0032] FIG. 7 illustrates a graphical view of cell survival as a function of molecular dosimetry unit (MoD) calculated as follows. All cell survival curves at different LETs are consolidated into one curve using MoD for each cell line.Where: CASi the i* number of molecular alterations in a nanometric volume of DNA CAFi: number of CASi in the dosimeter sensitive volumeAs an example of a simplified approach for calculation, mean values of CASi (CASj) and CAFtfcAs,) are employed such that the above equation is reduced toMoD = CASi ■ CAFiThe curves shown in FIG. 7 are fitted to the data based on the following exponential equation:SF = a. eb MoD+ c. ed MoDSF : survival fractionMoD'. molecular dosimetry unit

[0033] FIG. 8 illustrates a graphical view of dose-rate dependency of molecular dosimetry unit (MoD). The results are for the delivery of 100 Gy at various dose rates by kV x-rays to an aqueous solution of cell-free plasmid DNA in ambient conditions (21% O2).

[0034] Plasmid, linear, and synthetic DNA can be candidate biomolecules for the dosimeter. The use of MoD = CASt. CAFtas the determinant unit consolidates both LET and dose rate. MoD offers a new molecular metric for radiation dosimetry that can be measured with advancing DNA sequencing and other analytic technologies and modeled as shown above. Measurement techniques can be developed to improve sensitivity and specificity, ensure reproducibility.

[0035] The utility of MoD for radiation treatment prescription and planning overcomes the shortcomings of using ionization-based Gy in clinical applications. The left panels on FIGS. 9A-9C illustrate graphical views of conventional ionization-based depth-dose and LET distributions delivered wi th modulated particle beams to attain a uniform 2 Gy dose, knownas spread-out Bragg peak (SOBP) in a water phantom, over a span of 7 cm in FIGS 9A and 9B, and 4 cm in FIG 9C. The right panels on FIGS. 9A-9C show the corresponding results where the Gy profiles are replaced with MoD profiles. FIG. 9A illustrates the results of a SOBP region of 7 cm for a 135 MeV proton beam. FIG. 9B illustrates the corresponding results for a 180 MeV proton beam. FIG. 9C illustrates the results of a 4 cm SOBP for a 240 MeV / u carbon ion beam.

[0036] FIGS. 9A-9C show that the MoD of the present invention can be used to supplant the unit Gy for clinical treatment prescription and planning with the significant advantages to (1) accommodate mixed modality treatments, such as using x-rays, electrons, and particles, in an on-going treatment session or in a re-treatment scenario in combination with previous treatments; and (2) without the use of empirical dose modifying factors. It should be noted that MoD can be applied to external beam radiation therapy as well as to treatments employing internalized radiation sources, including sealed sources (e.g., brachytherapy) and unsealed sources (e.g., radiopharmaceutical therapy). Application of MoD to any treatment methodology known to or conceivable to one of skill in the art is considered included herein.

[0037] A computer processing device can be used to receive metrics related to alterations to the DNA sample. The computer processor quantifies the range of ithalterations, that from ith= 0 to > 60 alterations in each nanometric volume, to the DNA sample from the received metrics and generates an output of the DNA alterations. The computer processor therefore determines the MoD for the alterations to the DNA. as described herein, as a function of clustered alteration size (CASi) and frequency of the alterations (CAFi).

[0038] Further, a computer processor can also be used for determining an amount of radiation being delivered to a subject and quantifying the levels of alterations, or a mean level of alteration, to the subject from the amount of radiation being delivered to the subject. The computer processor generates a metric of quantification of alteration to the subject generatedby the amount of radiation being delivered to the sample. A treatment plan is then determined with the computer processor, or another computer processor. The treatment plan is determined based on maintaining the metric of quantification of alteration to the subject within a predetermined limit.

[0039] The need to develop new metrics for radiation descriptors is pressing now. High-LET particles, light ions, and FLASH in cancer treatment can be unified with existing x-ray and electron modalities and would benefit greatly from improved quantitative radiation prescription and prediction of biological outcome. The technology of the present invention is also beneficial for assessing and mitigating radiation hazards from cosmic radiations on earth or during space travel. The present invention can be used for radiation protection, replacing empirical factors such as RBE, DMF, and Quality Factors, which are overly simplified. The present invention acts as a biomolecular dosimeter. It allows for accurate quantification of molecular alternations. The new metrics of CASi and CAFi are determinants of LET, doserate, and dose. MoD, described herein, can also be used to consolidate the effects of LET, dose, and dose-rate. The present invention provides more sensitive metrics for quantifying the biological effects of radiation. The present invention allows for the resolution of disparate cell survival curves without RBE factor, while adapting modem technologies to measure CASi and CAFi. While several examples for applications of the present invention are described herein, it should be noted that these are included simply to further illustrate the invention. The present invention can be used or applied for the quantification of radiation damage in any implementation known to or conceivable to one of skill in the art.

[0040] It should be noted that aspects of the system and method, its control, and calculations can be executed with a program(s) fixed on one or more non-transitory computer readable medium. The non-transitory computer readable medium can be loaded onto a computingdevice, microprocessor, servo, server, actuator, device processor, smartphone, tablet, phablet, a Control Box, or any other suitable device known to or conceivable by one of skill in the art.

[0041] It should also be noted that herein the steps of the method described can be carried out using a computer, non-transitory computer readable medium, or alternately a computing device, microprocessor, or other computer type device independent of or incorporated with the radiation detection device. The computing device for executing the present invention can be a completely unique computer designed especially for the implementation of this method. Indeed, any suitable method of analysis known to or conceivable by one of skill in the art could be used. It should also be noted that while specific equations are detailed herein, variations on these equations can also be derived, and this application includes any such equation known to or conceivable by one of skill in the art.

[0042] A non-transitory computer readable medium is understood to mean any article of manufacture that can be read by a computer. Such non-transitory computer readable media includes, but is not limited to, magnetic media, such as a floppy disk, flexible disk, hard disk, reel-to-reel tape, cartridge tape, cassette tape or cards, optical media such as CD-ROM, writable compact disc, magneto-optical media in disc, tape or card form, and paper media, such as punched cards and paper tape.

[0043] It should be noted that the software associated with the present invention is programmed onto a non-transitory computer readable medium that can be read and executed by any of the computing devices mentioned in this application. The non-transitory computer readable medium can take any suitable form know n to one of skill in the art. The non- transitory computer readable medium is understood to be any article of manufacture readable by a computer. Such non-transitory computer readable media includes, but is not limited to,magnetic media, such as floppy disk, flexible disk, hard disk, reel-to-reel tape, cartridge tape, cassette tapes or cards, optical media such as CD-ROM, DVD, Blu-ray, writable compact discs, magneto-optical media in disc, tape, or card form, and paper media such as punch cards or paper tape. Alternately, the program for executing the method and algorithms of the present invention can reside on a remote server or other networked device. Any databases associated with the present invention can be housed on a central computing device, server(s), in cloud storage, or any other suitable means known to or conceivable by one of skill in the art. All of the information associated with the application is transmitted either wired or wirelessly over a network, via the internet, cellular telephone network, RFID, or any other suitable data transmission means known to or conceivable by one of skill in the art.

[0044] The many features and advantages of the invention are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the invention which fall within the true spirit and scope of the invention.Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation illustrated and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.

Claims

What is claimed is:

1. A system comprising: a chamber configured for receiving a dose of radiation from a radiation source, wherein the chamber defines an inner space for receiving a biomolecular sample, such that the biomolecular sample is contacted with the dose of radiation.

2. The system of claim 1 wherein the biomolecular sample comprises inanimate DNA molecules of desirable number of base-pair and structural configuration.

3. The system of claim 2 further comprising a sequencing component for sequencing the DNA molecules in the biomolecular sample and outputting a number of molecular alterations for the biomolecular sample.

4. The system of claim 1 further comprising a processing device for receiving metrics related to damage to the biomolecular sample, quantifying the levels of alterations to the biomolecular sample from the received metrics, and generating an output quantifying alterations to DNA within the biomolecular sample.

5. The system of claim 4 wherein the processing device determines a molecular dosimetry unit (MoD) for the alterations to the DNA.

6. The system of claim 5 wherein the MoD is calculated as a function of the range of ithnumber of Clustered Alterations Size (CASi) from i=0 to >60, and frequency of all ithalterations (CAFi) for each biomolecular sample of the DNA.

7. The system of claim 4 wherein the biomolecular sample comprises cell-free DNA.

8. A method of quantifying radiation-induced alterations comprising: receiving with a computer processor metrics related to alterations to DNA; quantifying with the computer processor a level of alterations to the DNA from the received metrics; and generating an output quantifying alterations to the DNA.

9. The method of claim 7 further comprising determining with the computer processor a molecular dosimetry unit (MoD) for the total alterations to the DNA.10 . The method of claim 8 further comprising calculating the MoD as a function of all molecular alterations (CASi) and frequency of the alterations (CAFi).

11. The method of claim 7 further comprising applying radiation to the DNA to generate the alterations to the DNA.

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