Methods and kits for detecting radiotherapy-associated neuronal damage

Abstract

Methods and kits are disclosed to detect the early onset, determine the risk and / or severity, diagnose, and determine the progression or regression of radiotherapy-associated neuronal damage derived from samples of a subject by quantifying one or more biomarkers at concentrations indicative of the condition. The one or more biomarkers comprise neurofilament light chain (NfL).

Description

METHODS AND KITS FOR DETECTING RADIOTHERAPY-ASSOCIATED NEURONAL DAMAGECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 639,973, filed April 29, 2024, which is hereby incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates to the fields of molecular biology and health risks associated with elevated levels of neurofilament light chain protein.BACKGROUND

[0003] Cancers continue to be a very significant cause of death in humans. Cancer burden is projected to increase to 35 million new cancer cases by 2050 (World Health Organization, “Global Cancer Burden Growing, Amidst Mounting Need for Services,” (2024)). The leading cancer therapies today are surgery, cytostatic and / or cytotoxic chemotherapy, and radiotherapy. Overall, while radiotherapy, also called radiation therapy (“RT”) is an effective treatment modality for many types of cancer, it does not spare normal (or healthy) tissue which can lead to unintended damage to nearby nervous tissues, resulting in neuronal damage.

[0004] The incidence of neuronal damage from radiotherapy varies based on several factors including the type and location of the cancer, the radiotherapy dose and duration, the technique used for delivering radiation, and individual patient factors such as age, overall health, and any pre-existing neurological conditions. For example, for brain tumors or head and neck cancers, where radiotherapy is directed towards critical neural structures of the central nervous system, the incidence of neuronal damage is relatively high. In contrast, for cancers treated with radiation therapies in other parts of the body like breast, prostate, or colorectal cancers, where neural structures of the peripheral nervous system are less likely to be affected, the incidence of neuronal damage may be lower. Thus, during treatment planning, efforts are made to minimize radiation exposure to critical neural structures while maximizing tumor control.

[0005] Significant advances in cancer care have led to an increase in patient survival rates and longer life expectancy after treatment. Multimodal or combinational therapies such as radiotherapy and chemotherapy, or radiotherapy and immunotherapy, among others, have led to improved tumor control, overall treatment response, and patient quality of life.

[0006] Additionally, advancements in radiation technologies have led to improvements in some cancers where patients can expect the same treatment outcome using lower doses of radiation. While this is a significant improvement in the field, there is the risk of cancer recurrence or development of new cancers among cancer survivors with longer life expectancies. Reirradiation has risks. In the case of combinational therapies such as chemotherapy or immunotherapy, these treatments have their own associated risks. Patients who have undergone such treatments could become more radio sensitized afterwards, putting them at higher risk of neuronal damage. It is not yet clear what each individual’s cumulative radiation exposure threshold is, or how other treatment modalities impact those thresholds.

[0007] Knowing a patient’s neurological state at a particular time and understanding the risk of developing neuronal damage before the start of a radiotherapy -inclusive treatment intervention could greatly improve the current quality of care. For example, if a simple blood test were available, such a test may minimize the number of adverse neurological outcomes for cancer patients undergoing radiotherapy -inclusive interventions, and if such outcomes are detected would allow physicians to monitor neuronal damage progression or treat earlier with an intervention. Additionally, such a test could be used in the development of disease modifying treatments (DMTs) for radiotherapy -associated neuronal damage and better understanding cumulative dose tolerance thresholds for different cancer patient populations for RT-inclusive interv entions.

[0008] There is a need for a simple, fast method of detecting adverse consequences of radiotherapy, such as radiotherapy -associated neuronal damage. There is also a need for a process to assess radiotherapy-associated health risks before administering a particular radiotherapy as well as a process to monitor the progression or onset of radiotherapy-associated biomarkers during a patient’s treatment journey. Early detection of radiotherapy-associated risks or damage could allow for better control of RT delivery to limit, e.g., neuronal damage and allow for early therapeutic intervention to minimize potential neurotoxic side effects.

[0009] The present disclosure is directed to overcoming these and other deficiencies in the art.SUMMARY

[0010] One aspect of the present disclosure is a method for detecting early onset of radiotherapy -associated neuronal damage in a subject. This method involves measuring concentration of neurofilament light chain (“NfL”) protein in a sample obtained from a subject where the subject has undergone one or more therapeutic interventions, and where at least one ofthe therapeutic interventions is a radiation treatment. The method further involves detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control, where the control is a concentration or a range of concentrations of NfL protein from one or more individuals that have not been subject to radiation treatment, and where an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy-associated neuronal damage in the subject.

[0011] Another aspect of the present disclosure is a method for diagnosing radiotherapy- associated neuronal damage in a subject. The method involves measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject where the subject has undergone one or more therapeutic interventions, and where at least one of the therapeutic interventions is a radiation treatment. The method further involves detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control, where the control is a concentration or a range of concentrations of NfL protein from one or more individuals that have not been subject to radiation treatment, and where an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy- associated neuronal damage in the subject.

[0012] A further aspect of the present disclosure is a method for determining relative risk and / or severity of radiotherapy-associated neuronal damage in an intervention protocol. The method involves measuring concentration of neurofilament light chain (NfL) protein in a first sample obtained from a first subject or a first subject population, where the first subject or the first subject population has undergone one or more therapeutic interventions, and where at least one of the therapeutic interventions is a radiation treatment. The method further involves comparing the concentration of NfL protein in the first sample(s) to a concentration of NfL protein in a second sample obtained from a second subject or a second subject population having undergone one or more therapeutic interventions different from the one or more interventions of the first subject or the first subject population and determining whether the risk and / or severity of radiotherapy-associated neuronal damage of the first subject or the first subject population is higher or lower than that of the second subject or the second subject population based on an increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s), where a higher concentration of NfL protein in the first sample(s) or the second sample(s) indicates a higher risk and / or severity of radiotherapy-associated neuronal damage of the one or more interventions associated with that subject or subject population.

[0013] Yet another aspect of the present disclosure is a method for monitoring the progression or regression of radiotherapy-associated neuronal damage in a subject. The methodinvolves measuring concentration of neurofilament light chain (NfL) protein in a first sample obtained from a subject, where the subject has undergone a first therapeutic intervention involving a radiation treatment; measuring concentration of NfL protein in a second sample obtained from the subject optionally after a second therapeutic intervention; and determining the subject has a progression of radiotherapy-associated neuronal damage if the concentration of NfL protein in the second sample has increased relative to the concentration of NfL protein in the first sample, or determining the subject has a regression of radiotherapy-associated neuronal damage if the concentration of NfL protein in the second sample has decreased relative to the concentration of NfL protein in the first sample.

[0014] A further aspect of the present disclosure is a method for diagnosing a subject as having radiotherapy-associated neuronal damage. The method involves measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject and comparing the concentration of NfL protein in the sample to a predetermined concentration level of NfL protein, where an increased concentration of NfL protein in the sample relative to the predetermined concentration level is indicative of radiotherapy -associated neuronal damage in the subject.

[0015] Another aspect of the present disclosure is an in vitro method for detecting radiotherapy-associated neuronal damage in a subject. The method involves obtaining a sample from a subject having undergone one or more therapeutic interv entions, where at least one intervention is a radiation treatment; measuring concentration of neurofilament light chain (NfL) protein in the sample; and identifying the subject as having radiotherapy-associated neuronal damage if the concentration of the NfL protein in the sample is greater than a predetermined NfL protein concentration.

[0016] A further aspect of the present disclosure is a kit for detecting radiotherapy- associated neuronal damage. The kit includes reagents for determining expression level of neurofilament light chain (NfL) protein in a sample from a subject and instructions for comparing the expression level to a reference standard to detect radiotherapy-associated neuronal damage in the subject.

[0017] Neuronal cell related side effects are risks associated with increased tumor control / better treatment outcomes involving RT. which lead to some patients ending treatment abruptly. It is not clear which patients that go through treatment will experience delayed side effects since onset can take months to years after treatment and there are many other factors or conditions with similar features of neuronal cell damage, such as neuronal damage and / or neurotoxicity. If symptoms are presented early during treatment, radiation associated neuronaldamage is easier to diagnose. Currently, the diagnosis of late or delayed radiation induced neuropathies generally involves a comprehensive review of the patient’s medical history (e.g., prior radiation treatments or chemotherapy), physical examinations, and multiple non-specific tests such as imaging and / or blood tests to rule out underlying conditions or factors of nonirradiated origin (e.g., autoimmune diseases, infections, and / or localized tumor progression). This process could take weeks to months to diagnose, underlining the need for better approaches.

[0018] Described herein are methods and kits that address these issues. These methods and kits relate to biomarkers in biological samples that can be used, for example, to detect the early onset, diagnose, determine the relative risk and / or severity, and / or monitor the progression or regression of radiotherapy-associated neuronal damage, including peripheral neuropathy (PN). Biomarkers are typically measurable in blood, plasma, serum, or cerebral spinal fluid (CSF) by in vitro diagnostic methods, screening tests, or kits as described herein to detect radiotherapy- associated neuronal damage. These biomarkers may also be used to monitor subject response to treatment and inform clinical decision-making regarding disease management and treatment.

[0019] Specifically disclosed herein is the use of neurofilament light chain (NfL) protein as a biomarker of radiotherapy associated neuronal damage to better guide cancer treatment decisions. In some embodiments, biological specimens (blood or cerebral spinal fluid) are collected from a subject. The concentration of NfL in biological specimens is determined using an in vitro diagnostic assay developed to measure human NfL with high specificity and high sensitivity. See, e.g., Uzgiris et al., “Advances in Neurofilament Light Chain Analysis,” A dv. Clin. Chem. 126:31-71 (2025), which is hereby incorporated by reference in its entirety. NfL levels are measured before, during, and / or after a radiotherapy-inclusive treatment regimen. Interventions include radiotherapy, either alone or in combination with another treatment modality (e.g., chemotherapy, immunotherapy, hormone therapy, and / or surgery), where radiotherapy is administered before or after the other treatment modality . Treatment decisions can be made based on detection / analysis of NfL levels to provide comprehensive cancer care.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGs. 1A-B are schematic illustrations showing embodiments of detection of radiation-associated neuronal damage. The process of detecting radiotherapy-associated neuronal damage from direct and indirect damage to neurons is shown. In FIG. 1A, direct damage, neuronal injury could be due to events such as molecular damage to DNA or oxidative stress that can lead to cell dysfunction and cell death. Ionizing radiation can also directly damage axons which leads to the eventual breakdown and disintegration of damaged axons. In FIG. I B, indirectdamage, neuronal injury is due to radiation insult to neighboring non-neuron cell types / tissues such as endothelial cells. Vascular damage and inflammation can trigger an immune response that consequently damages nearby neurons. Additionally, neuroglia that support neurons could be directly affected by radiotherapy as well (e.g., unable to protect / support neurons due to dysfunction, glial activation). Both direct and indirect mechanisms lead to neurofilament release into extracellular fluid that can be collected from patients and quantified using assays.

[0021] FIG. 2 is a schematic illustration showing an example case of neuronal damage biomarkers in cancer interventions. An example of a subject’s RT-inclusive intervention and where NIL levels can be measured at multiple points in time to assess the risk and / or determine if a subject is developing a radiotherapy -associated neuronal damage is shown. In the embodiment illustrated, the first time point is the first patient visit where a diagnosis is given or follow up after a diagnosis. A baseline biological sample can be collected and used to assess the cunent neurological state of the subject before starting an intervention. For example, the patient may have had a history of other cancers that required interventions, which may have resulted in some neurotoxicity or other neuronal cell damage. In the embodiment illustrated, the subject may have elevated NfL above a set threshold or cutoff value where certain RT-inclusive interventions to the subject would not be advised, because the subject may be at high risk of developing neuronal cell damage. This baseline NfL level would be used to assess risk and to select different RT-inclusive interventions. Further along in the process is the start of the selected RT-inclusive interv ention (in the embodiment illustrated, an RT of 15 Gy in 3 fractions of 5 Gy each followed by systemic chemotherapy, abbreviated CT). NfL levels would be monitored over time after start of treatment to determine if there is an onset of neuronal damage. If there is an onset of neuronal damage, progression could be monitored until intervention is needed, whether it is a drug or pausing / stopping the intervention until NfL levels are reduced to normal or safe ranges. NfL levels may need to be assessed before starting another part of a regimen (in the illustrated case, chemotherapy) which might be known to be more neurotoxic. Lastly, after intervention, there is a chance there could be late, delayed neurotoxic effects. These could be monitored in follow up visits, if after completion of the RT-inclusive intervention, NfL levels are above thresholds or borderline thresholds. This is just one example, and there are many possible combinations of different treatment modalities, visits, and scenarios specific to different subjects.

[0022] FIGs. 3A-B are graphs showing serum NfL (sNfL) changes from baseline after spinal cord irradiation in mini pigs. FIG. 3A is a graph showing the average baseline level of sNfL. FIG. 3B is a graph showing the mean sNfL fold change (NfL at post-treatment time over baseline NfL) after 28 (± 3 days) was 2.42-fold increased from baseline. In most of the pigs(n= 12), sNfL fold changes were between 2- to 5-fold after 4 weeks. For a subset of pigs (n=5), NfL fold changes peaked at the terminal time point, > 5-fold above baseline, with the highest change > 60-fold.DETAILED DESCRIPTION

[0023] Disclosed herein are methods and kits to detect the early onset, diagnose, determine the relative risk and / or severity, and / or monitor the progression or regression of nerve cell damage, such as neuronal damage, radiotherapy-associated neurotoxicity (RAN), radiation- induced peripheral neuropathy (PN), or other nerve cell damage, by leveraging the detection of specific biomarkers in biological samples such as blood or cerebral spinal fluid (CSF). These biomarkers, identifiable through in vitro diagnostic or screening tests, serve as indicators of the risk, onset, and progression of nerve cell damage. Additionally, they can be used to monitor a subject’s response to treatment. Based on the levels and changes in these biomarkers, clinicians can make informed decisions regarding disease management and treatment. This may include adjustments to radiotherapy protocols to minimize nerve cell damage, the initiation of neuroprotective measures, or the implementation of specific therapeutic interventions aimed at mitigating the effects of nerve cell damage. Thus, the present disclosure provides a comprehensive approach to managing radiotherapy -associated neuronal damage, enhancing patient care, and potentially improving treatment outcomes.

[0024] Unless otherwise indicated, the definitions and embodiments descnbed in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person of ordinary' skill in the art.

[0025] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to a person of ordinary skill in the art upon reading this disclosure. In another example, reference to “a cell” includes both a single cell and a plurality of cells.

[0026] The term “about” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 10% or within 5% of a given value or range.

[0027] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0028] In understanding the scope of the present disclosure, the term ‘'comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting of’ and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers, and / or steps.

[0029] The terms “patient”, “individual”, “subject”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc ). In some embodiments, the subject is a human.

[0030] The term “biomarker” is used herein to refer to an entity whose presence, level, or form, correlates with a particular biological event or state of interest, so that it is considered to be a '‘marker” of that event or state. To give but a few examples, in some embodiments, a biomarker may be or include a marker for a particular disease state, or for likelihood that a particular disease, disorder, or condition may develop, occur, or reoccur. In some embodiments, a biomarker may be or include a marker for a particular disease or therapeutic outcome, or likelihood thereof. Thus, in some embodiments, a biomarker is predictive, in some embodiments, a biomarker is prognostic, in some embodiments, a biomarker is diagnostic, of the relevant biological event or state of interest. In some embodiments, a biomarker is a possible biomarker of the relevant biological event or state of interest. A biomarker may be an entity of any chemical class. For example, in some embodiments, a biomarker may be or include a nucleic acid, a polypeptide, a small molecule, or a combination thereof. In some embodiments, a biomarker is a cell surface marker. In some embodiments, a biomarker is intracellular. In some embodiments, a biomarker is found in a particular tissue (e.g., neuronal tissue). In some embodiments, a biomarker is found outside of cells (e.g., is secreted or is otherwise generated or present outside of cells, e.g., in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc.).

[0031] As used herein, “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms ■‘cancer”, “cancerous”, and “tumor” are not mutually exclusive as referred to herein.

[0032] The terms “treat” or “treatment” of a state, disorder, or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at leastone clinical or sub-clinical symptom of the state, disorder, or condition developing in a subject that may be afflicted with or predisposed to the state, disorder, or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder, or condition; or (2) inhibiting the state, disorder, or condition, i.e.. arresting, reducing, or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. “Treating” can also mean prolonging survival of a patient beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of disease temporarily, or in some embodiments halting the progression of disease permanently in a subject. The benefit to a subject to be treated may be significant or at least perceptible to the subject or to the physician. As used herein the term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof.Radiotherapy-Associated Neuronal Damage

[0033] One aspect of the present disclosure is a method for detecting early onset of radiotherapy-associated neuronal damage in a subject. The method involves measuring concentration of neurofilament light chain (NIL) protein in a sample obtained from a subject where the subject has undergone one or more therapeutic interventions, and where at least one of the therapeutic interventions is a radiation treatment. The method further involves detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control, where the control is a concentration or a range of concentrations of NfL protein from one or more individuals that have not been subject to radiation treatment, and where an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy-associated neuronal damage in the subject.

[0034] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0035] As used herein “neuronal damage” refers to degeneration of the long, thread-like nerve fibers called axons. The term may be used herein synonymously with other nerve cell damage associated with radiation therapy, including “radiotherapy-associated neurotoxicity” (“RAN”), also known as “radiation-induced neurotoxicity,” which refers to the harmful effects that radiotherapy can have on the nervous system, including cognitive impairment, memory loss, and other neurological deficits. RAN is a significant concern in the medical field, particularly in the treatment of brain tumors and other central nervous system disorders as well as the effects of radiotherapy on the peripheral nervous system. The effect of RAN can be acute, occurringimmediately after treatment, or it can be delayed, manifesting months or even years later. Whether RAN is acute or chronic can depend on factors such as dose, duration, and location of radiation exposure, as well as individual patient characteristics. Acute neurotoxic effects may include symptoms such as headache, nausea, vomiting, fatigue, and cognitive changes, which typically occur shortly after treatment. Chronic neurotoxic effects may develop over time and include symptoms of neuropathy, cognitive dysfunction, radiation necrosis, leukoencephalopathy, and secondary' malignancies within the nervous system. In some embodiments, neuronal damage and / or RAN is acute. In some embodiments, neuronal damage and / or RAN is chronic.

[0036] In some embodiments, early onset of radiotherapy-associated neuronal damage in a subject is detected. Early onset of radiotherapy-associated neuronal damage occurs when increased levels of a biomarker are detected relative to a control, indicating damage to neurons. See, e.g., Uzgiris et al.. “Advances in Neurofilament Light Chain Analysis,” A dv. Clin. Chem. 126:31-71 (2025), which is hereby incorporated by reference in its entirety. For example, as shown in FIG. 2, the biomarker levels w ould be monitored over time after start of a first radiation treatment to determine if there is an onset of neuronal damage or neurotoxicity', and additionally, could be monitored after each radiation treatment. If neuronal damage and / or RAN is detected, this monitoring can be used to monitor progression until intervention is needed, whether it is a drug or pausing / stopping the intervention until biomarker levels reduce to normal or safe ranges. Biomarker levels may also be assessed before starting another part of a therapeutic regimen (in the embodiment shown in FIG. 2, chemotherapy), which might be known to increase the risk of neuronal damage. In some embodiments, the subject has been identified to have neuronal damage and / or RAN.

[0037] In some embodiments, a neuronal biomarker such as neurofilament light chain (NfL) protein is detected in a sample obtained from a subject, where the subject has undergone one or more therapeutic interventions, and where at least one of the therapeutic interventions is a radiation treatment in comparison to the amount of NfL protein in a control sample. In some embodiments, early onset of radiotherapy-associated neuronal damage and / or neurotoxicity is determined before symptoms of neuronal damage and / or neurotoxicity in a subject are observed or diagnosed.

[0038] In some embodiments, RAN is a risk of neuronal damage. The exact mechanisms of RAN are not fully understood. Without being bound by any particular theory', RAN is believed to involve a complex interplay of factors such as radiation-induced damage to neural cells, inflammation, and changes in the neurons' microenvironment. Despite advancements inradiotherapy techniques aimed at minimizing damage to healthy tissue, RAN remains a significant challenge, underscoring the need for novel strategies to detect early onset, diagnose, determine the relative risk and / or severity, prevent or mitigate these effects, and / or monitor the progression or regression of RAN.

[0039] Neurological complications affecting the nerves outside the brain and spinal cord as a result of radiotherapy are known as radiation induced peripheral neuropathy (“PN”).Radiation induced PN refers to damage or dysfunction of peripheral nerves resulting in sensory, motor, or autonomic symptoms. Neuropathic symptoms may include numbness, tingling, weakness, pain, or loss of sensation in the affected area. Similar to the general terms of ■‘neuronal damage" and “neurotoxicity” above, neuropathy can occur as an acute or delayed complication and also depends on the same factors. In some embodiments, the radiotherapy- associated neuronal damage is peripheral neuropathy.

[0040] In some embodiments, one or more interventions cause direct damage to neurons in the subject. As shown in FIG. 1A. radiation intervention can result in direct damage to the neurons themselves. In some embodiments, one or more interventions cause indirect damage to neurons in a subject. As shown in FIG. IB, radiation intervention can result in indirect neuronal injury by affecting neighboring non-neuron cell types and tissues such as endothelial cells. Damage to the vascular system and inflammation can also trigger an immune response that consequently damages nearby neurons. Additionally, neuroglia that support neurons could be directly affected by radiotherapy as well (e.g., unable to protect / support neurons due to dysfunction, glial activation).

[0041] The term “radiation” includes certain types of energy’ that can decrease the proliferation of an aberrantly proliferating cell (e.g., ionizing radiation). The type of radiation referred to herein can vary. For example, radiation can be electromagnetic or particulate in nature. Electromagnetic radiation includes, but is not limited to, x-rays and gamma rays. Particulate radiation includes, but is not limited to, electron beams (beta particles), proton beams, neutron beams, alpha particles, and negative pi mesons. In some embodiments, the source of radiation for radiation therapy is X-rays, gamma rays, alpha particles, beta particles, proton beams, neutron beams, or negative Pi mesons. Common radioisotopes used include, but are not limited to, cesium (137Cs), cobalt (60Co), iodine (' '’l) phosphorus-32 (32P), gold-198 (198Au), iridium-192 (192Ir). yttrium-90 (90Y). and palladium-109 (109Pd).

[0042] The radiation can be delivered using conventional radiological treatment apparatuses and methods, and by intraoperative and stereotactic methods. Additional discussion regarding radiation treatments can be found in Leibel and Phillips, Textbook of RadiationOncology (1997) (publ. W. B. Saunders Company; ISBN 0-72165-336-7, which is hereby incorporated by reference in its entirety) and particularly in Chapters 13 and 14. Radiation can also be delivered by other methods such as targeted delivery, for example by radioactive “seeds,'’ or by systemic delivery of targeted radioactive conjugates (e.g., see Padawer et al., “Combined Treatment with Radioestradiol-Lucanthone in Mouse C3HBA Mammary Adenocarcinoma and with Estradiol -Lucanthone in an Estrogen Bioassay,” Int. J. Radiat. Oncol. Biol. Phys. 7:347-357 (1981), which is hereby incorporated by reference in its entirety). Other radiation delivery methods can be used in the various embodiments of the disclosure.

[0043] The terms “radiation therapy.” “radiotherapy,” and “radiation treatment” (“RT”) are used interchangeably herein and generally refer to the use of doses of radiation to kill cancer cells and / or shrink tumors. In some embodiments, the radiation treatment comprises exposing cell(s) to radiation. RT can be delivered externally, where a machine outside the body directs radiation towards cancer, or internally, where radioactive material is placed inside the body near cancer cells, also known as brachytherapy. The specific type and delivery method of RT is determined based on the type, size, location of the cancer, and the patient’s overall health. RT is a widely used treatment modality for the management of primary and metastatic cancers. RT can be used either alone (as a primary treatment), in combination with surgery either before (neoadjuvant therapy) or after (adjuvant therapy), or in combination with other treatment modalities (e g., chemotherapy, molecularly targeted therapy, hormone therapy, and / or immunotherapy) to achieve better treatment response. It is estimated that at least 50% of cancer patients will receive radiation therapy at some point during their care (Chaput and Regnier, “Radiotherapy: Clinical Pearls for Primary Care”, Can Fam Physician 67(10):753-757 (2021). which is hereby incorporated by reference in its entirety).

[0044] In the various methods and embodiments described herein various methods of delivering radiotherapy to a subject may be employed. In some embodiments, the radiation treatment is external radiation therapy, internal radiotherapy, systemic radiation therapy, stereotactic radiotherapy (or stereotactic radiosurgery), three-dimensional (3-D) conformal radiation therapy, intensity modulated radiotherapy (IMRT), intraoperative radiation therapy (IORT), image-guided radiation therapy (IGRT), or combinations thereof. In some embodiments, external radiation therapy includes, without limitation, intraoperative radiation therapy and prophylactic cranial irradiation (PC). In some embodiments, internal radiotherapy includes, without limitation, brachiotherapy, interstitial radiation therapy, intracavitary radiation therapy, or intraluminal radiation therapy, among others. Brachytherapy involves placing radioactive material inside the body near the cancer cells, which can be done through implantable devices orthrough injectable radioactive substances. Systemic radiation therapy involves introducing a radioactive substance (such as a radiolabeled monoclonal antibody) into the bloodstream or a body cavity, which then travels and treats the entire body. Three-dimensional (3-D) conformal radiation therapy is an external radiation therapy that shapes the radiation beams to match the shape of the tumor. Intraoperative radiation therapy (IORT) is a type of radiation therapy that is administered during surgery directly to the area where the tumor was, before the surgical wound is closed. The choice of radiotherapy method depends on various factors, including the type, size, and location of the cancer, the patient’s overall health, and the intended therapeutic outcome. See for example, Hellman. “Principles of Radiation Therapy, Cancer”, in Principles and Practice of Oncology, Devita et al., Eds., 4th Edition, Vol. 1, pp. 248-275 (1993), which is hereby incorporated by reference in its entirety.

[0045] Radiation is generally measured in Gray units (Gy), where 1 Gy=100 rads. In some embodiments, the radiation dose per radiation treatment is about 0. 1 Gy, 1 Gy, 1.8 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy, 12 Gy, 13 Gy, 14 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, 20 Gy, 21 Gy, 22 Gy, 23 Gy, 24 Gy, 25 Gy, 30 Gy, 40 Gy, 50 Gy, or 60 Gy or any number or range therein, or more than 60 Gy. In some embodiments, at least one radiation treatment is carried out at a dose of at least 0.1 Gy, 1 Gy, 1.8 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy. 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy, 12 Gy, 13 Gy, 14 Gy. 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, or 20 Gy or any number or range therein, or more than 20 Gy. In some embodiments, at least one of the radiation treatments is carried out at a dose of 1.8 Gy or higher. In some embodiments, at least one of the radiation treatments is carried out at a dose of 5 Gy or higher.

[0046] In some embodiments, radiation therapy is administered through multiple sessions. The cumulative radiation exposure from these multiple sessions constitutes the total radiation dose. In some embodiments, the total radiation dose is about 1 to 10 Gy, 10 to 40 Gy, 8 to 60 Gy, 20 to 50 Gy, 20 Gy to 70 Gy, 60 to 80 Gy, 1 Gy to about 75 Gy. In some embodiments, the total radiation dose is about 1 Gy to about 60 Gy. about 1 Gy to about 50 Gy, about 1 Gy to about 40 Gy, about 1 Gy to about 30 Gy, about 1 Gy to about 25 Gy. about I Gy to about 20 Gy, about 1 Gy to about 15 Gy, about 1 Gy to about 10 Gy, about 1 Gy to about 8 Gy, about 1 Gy to about 6 Gy, about 4 Gy to about 75 Gy, about 4 Gy to about 60 Gy, about 4 Gy to about 50 Gy, about 4 Gy to about 40 Gy, about 4 Gy to about 30 Gy, about 4 Gy to about 25 Gy, about 4 Gy to about 20 Gy, about 4 Gy to about 15 Gy, about 4 Gy to about 10 Gy. about 4 Gy to about 8 Gy, about 5 Gy to about 75 Gy, about 5 Gy to about 60 Gy, about 5 Gy to about 50 Gy, about 5 Gy to about 40 Gy, about 5 Gy to about 30 Gy, about 5 Gy to about 25 Gy, about 5 Gy to about 20 Gy, about 5 Gy to about 15 Gy, about 5 Gy to about 10 Gy, about 5 Gy to about 8 Gy,about 2 Gy, about 3 Gy, about 4 Gy, about 5 Gy, about 6 Gy, about 7 Gy, about 8 Gy, about 9 Gy, about 10 Gy, about 11 Gy, about 12 Gy, about 13 Gy, about 14 Gy, about 15 Gy, about 16Gy, about 17 Gy, about 18 Gy, about 19 Gy, about 20 Gy, about 21 Gy, about 22 Gy, about 23Gy. about 24 Gy. about 25 Gy. about 30 Gy. about 35 Gy, about 40 Gy, about 45 Gy, about 50Gy. about 55 Gy. about 60 Gy. about 65 Gy. about 70 Gy. about 75 Gy or more than 75 Gy, or any number or range therein.

[0047] Despite advances in radiation technology , collateral damage to surrounding healthy tissues is inevitable. Radiation-induced injury to normal tissues can lead to acute (weeks to months) or late (months to years) toxicities (or side effects”) (Az“zam et al., “Radiation- Induced Neuropathies in Head and Neck Cancer: Prevention and Treatment Modalities,” Ecancermedicalscience 14 (2020), which is hereby incorporated by reference in its entirety). Depending on the severity, some side effects during treatment can interrupt treatment schedules or a patient’s ability to continue with a particular regimen, ultimately putting the intervention’s therapeutic potential and the patient’s health at risk.

[0048] The terms “intervention” and “therapeutic intervention" are used interchangeably herein and refer to any measure or action taken to alter the course of a disease, improve the patient's quality of life, or manage the symptoms and side effects of the disease or its treatment. In some embodiments, an intervention is a radiation treatment. In some embodiments, one or more interventions include multiple radiation treatments. In some embodiments, an intervention such as one or more radiation treatment(s) can be used alone or in combination with other interventions, such as surgery or chemotherapy, for example, to treat various types of cancer. In some embodiments, an intervention includes, without limitation, surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplant, precision medicine, or any combination thereof. Additionally, interventions can also encompass palliative care measures, supportive treatments, lifestyle modifications, and psychological support aimed at improving the patient’s well-being and comfort. The choice of intervention depends on various factors including the type, stage, and location of cancer, the patient’s overall health, and the potential benefits and risks of the intervention. In some embodiments, the method involves selecting a subject having undergone one or more interventions. In some embodiments, method described herein involve selecting a subject having undergone one or more radiation treatments. In some embodiments, a subject has undergone more than one radiation treatments. In some embodiments, the subject has undergone at least one radiation treatment and at least one other intervention. In some embodiments, methods described herein involve obtaining a sample from a subject having undergone one or more intervention. In some embodiments, methodsdescribed herein involve administering to a subject one or more interventions. In some embodiments, a selected subject has undergone more than one radiation treatment.

[0049] Specific treatments for the initiation of neuroprotective measures or the implementation of therapeutic interventions aimed at mitigating the effects of neuronal damage may include pharmacological and non-pharmacological approaches. Pharmacological interventions can involve the use of drugs such as antioxidants, anti-inflammatory agents, or neurotrophic factors. Antioxidants, such as vitamin E and coenzyme Q10, can help mitigate oxidative stress, a key contributor to neuronal damage. Anti-inflammatory agents, such as corticosteroids, can help reduce inflammation, another factor implicated in neuronal damage. Neurotrophic factors, such as nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), can support the survival and growth of neurons. Non-pharmacological interventions can include lifesty le modifications, such as a balanced diet, regular exercise, and adequate sleep, which can help enhance overall brain health and resilience. Additionally, cognitive rehabilitation therapies, such as cognitive training or cognitive behavioral therapy, can help manage cognitive symptoms associated with axonal damage. The specific choice of neuroprotective measures or therapeutic interventions would be tailored to the individual patient’s needs and circumstances, taking into account factors such as the severity of the neuronal damage, the patient’s overall health status, and the patient’s personal preferences.

[0050] In some embodiments, an intervention involves treatment with a “radiosensitizer” or “radiosensitizing agent”, which is a molecule that makes cells more sensitive to radiation and / or to promote the treatment of diseases which are treatable with radiation. In certain embodiments, the radiosensitizer or radiosensitizing agent when present during irradiation, enhances the cytotoxic effects of radiation.

[0051] As described above, it is also contemplated that when used to treat various diseases / disorders, the methods and kits of the present disclosure can be utilized with interventions in addition to radiation treatment such as use of additional therapeutic methods / agents suitable for the same or similar diseases / disorders. Such other therapeutic methods / agents can be co-administered (simultaneously or sequentially) to generate additive or synergistic effects. Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0052] In some embodiments, at least one of the interventions is a systemic therapy. Non-limiting examples of systemic therapy include chemotherapy or combinatorial therapy. Exemplary non-limiting examples of chemotherapeutic compounds which can be used in an intervention include, for example, aminoglutethimide, amsacrine, anastrozole, arsenic trioxide,asparaginase, azacitidine, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramnustine, etoposide, exemestane. filgrastim, fludarabine. fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nelarabine, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0053] These chemotherapeutic compounds may be categorized by their mechanism of action into, for example, the following groups: anti-metabolites / anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxy adenosine (cladribine)): antiproliferative / antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracy clines, bleomycin, busulfan, camptothecin. carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, tri ethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D). daunorubicin, doxorubicin (adnamycin), idarubicin. anthracy clines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan. chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes- dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, my cophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone. prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0054] In some embodiments, an intervention includes therapeutic agents such as anti- angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art. including, e.g., TNP-470, platelet factor 4, thrombospondin- 1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain, '‘Angiogenesis in Cancer and Other Diseases,” Nature 407(6801):249-57 (2000), which is hereby incorporated by reference in its entirety. In some embodiments, the intervention includes a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof (e g., anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).

[0055] In some embodiments, the intervention includes an immunomodulatory treatment such as, e.g., therapeutic vaccines (including but not limited to GV AX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 4-1BB, 0X40, etc.). The methods of the disclosure can be also combined with other treatments that possess the ability tomodulate NKT function or stability, including but not limited to CD Id, CD Id-fusion proteins, CD Id dimers or larger polymers of CD Id either unloaded or loaded with antigens, CDld- chimeric antigen receptors (CDld-CAR), or any other of the five known CD1 isomers existing in humans (CDla, CDlb, CDlc, CDle). The intervention may also include other treatments such as midostaurin, enasidenib, or a combination thereof.

[0056] In some embodiments, the intervention comprises one or more compounds selected from immuno-suppressives, biologicals, probiotics, prebiotics, cytokines (e.g., IFN or IL-2), and therapies that block inflammation (e.g., via blockage of IL1, INFα / β, IL6, TNF, IL23, etc.).

[0057] Another non-limiting example of a systemic therapy is a combinatorial therapy. As used herein, a combinatorial therapy is a therapy that uses a combination of more than one medication or modality to treat the disease. The goal of this strategy' is to use different treatments that work in different ways to get a more effective overall response. This approach can be particularly beneficial in treating complex and heterogeneous diseases like cancer, where a single therapy may not be sufficient. Combinatorial therapy leverages the unique mechanisms of action of different therapeutic agents to enhance treatment efficacy, circumvent drug resistance, and potentially reduce side effects. The therapeutic agents employed in combinatorial therapy can span a broad spectrum, including but not limited to, chemotherapeutic agents (e.g.. cisplatin, doxorubicin), targeted therapies (e.g., trastuzumab, imatinib), immunotherapies (e g., pembrolizumab, nivolumab), hormone therapies (e.g., tamoxifen, leuprolide), and biological therapies (e.g., interferons, interleukins). The specific combination of agents is tailored based on factors such as the type and stage of the disease, patient's overall health, and the genetic profile of the disease cells. In some embodiments, the intervention is a combinatorial therapy.

[0058] The methods of the present disclosure can be used in subjects suffering from a broad range of cancers, which may be treated with radiotherapy. Non-limiting examples of relevant cancers include, e.g., breast cancer, prostate cancer, multiple myeloma, transitional cell carcinoma, lung cancer (e.g.. non-small cell lung cancer (NSCLC)). renal cancer, thyroid cancer, leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia), lymphoma (e.g., B cell lymphoma, T cell lymphoma, non-Hodgkin’s lymphoma. Hodgkin’s lymphoma), head and neck cancer, esophageal cancer, stomach cancer, colon cancer, intestinal cancer, colorectal cancer, rectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct, cancer of the gall bladder, ovarian cancer, uterine endometrial cancer, vaginal cancer, cervical cancer, bladder cancer, neuroblastoma, sarcoma, osteosarcoma, malignant melanoma, squamous cell cancer, bone cancer, including both primarybone cancers (e.g., osteosarcoma, chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, malignant fibrous histiocytoma, adamantinoma, giant cell tumor, and chordoma) and secondary (metastatic) bone cancers, soft tissue sarcoma, basal cell carcinoma, angiosarcoma, hemangiosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, gastrointestinal cancer, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenstroom's macroglobulinemia, papillary' adenocarcinomas, cystadenocarcinoma, bronchogenic carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, medullary carcinoma, thymoma, sarcoma, etc.Neurofilament Light Chain and Other Biomarkers

[0059] The mammalian nervous system is divided into the central (CNS) and peripheral (PNS) nervous systems where the CNS is comprised of the brain and spinal cord and the PNS is comprised of the nerves that connect the brain and spinal cord to the rest of the body. There are two types of cells in the nervous system: neurons and neuroglia. Neurons are responsible for transmitting nerve impulses and include a cell body, dendrites, and an axon. Dendrites receive signals from other neurons while myelinated axons transmit impulses away from the cell body. The methods and kits of the present disclosure involve measuring one or more biomarkers for detection of radiotherapy-associated neuronal damage. In some embodiments, the biomarker is a neuronal protein.

[0060] Neurofilaments ( “NF” “NFs”) are structural proteins expressed exclusively in neurons that play a vital role in cytoskeletal support, helping to maintain neuronal integrity and stability (see Yuan et al., “Neurofilaments and Neurofilament Proteins in Health andDisease,” Cold Spring Harb. Per sped. Biol. 9: a018309 (2017). which is hereby incorporated by reference in its entirety ). Thus, NFs are abundant within axons. There are five NF subunits, neurofilament heavy, medium, light polypeptides (abbreviated as NfH, NfM, and NfL respectively), peripherin. and α-intemexin, that can be differentiated by their molecular masses of approximately 70. 160, 200. 56 and 66 kilo Dalton (kDa), respectively (see Yuan et al., “Neurofilaments at a glance. J. Cell Sci. 2012 ;125(14) : 3257-3263 (2012) ; Yuan et al., “Alpha-Intemexin is Structurally and Functionally Associated with The Neurofilament Triplet Proteins in the Mature CNS,” J. Neurosci. 26(39): 10,006-10,019 (2006); Yuan et al.. “Peripherinis a Subunit of Peripheral Nerve Neurofilaments: Implications for Differential Vulnerability of CNS and Peripheral Nervous System Axons,” J. Neurosci. 32(25): 8501-8508 (2012), each of which is hereby incorporated by reference in its entirety). Neuronal injury (e.g., neuronal damage) or cell death results in NF leakage into extracellular fluid.

[0061] Of the NF subunits. NfL is the most widely studied due to elevated levels in numerous neurological disorders and neurodegenerative diseases. It is also highly abundant and soluble in CSF and blood (e.g., see Narayanan, “Neurofilament light: A Narrative Review on Biomarker Utility,” Fac Rev. 10:46 (2021), which is hereby incorporated by reference in its entirety), relatively stable, and resistant to most types of clinically relevant variation in sampling (see Linnemann et al., “NfL Reliability Across Laboratories, Stage-Dependent Diagnostic Performance And Matrix Comparability in Genetic FTD: a large GENFI study,” Journal of Neurology, Neurosurgery & Psychiatry' doi: 10.1136 / jnnp-2023-332464 (2024), which is hereby- incorporated by reference in its entirety). See, also, Uzgiris et al., “Advances in Neurofilament Light Chain Analysis,” Adv. Clin. Chem. 126:31-71 (2025). which is hereby incorporated by reference in its entirety . In some embodiments, the biomarker is neurofilament light chain (NfL) protein.

[0062] The present disclosure provides that certain biomarkers (e.g., NfL) are useful in detecting radiation-associated neuronal damage with improved sensitivity and / or selectivity. The increased sensitivity and / or selectivity achieved with biomarkers (e g., NfL) described herein allows the detection of early onset of radiation-associated neuronal damage, a diagnosis of radiation-associated neuronal damage, an assessment of the relative risk and / or severity of radiation-associated neuronal damage, and monitoring the progression or regression of radiotherapy-associated neuronal damage. Such detection helps to ensure that patients receive critical care for preventing further symptoms, and conditions associated with radiation-associated neuronal damage.

[0063] In some embodiments, methods described herein involve measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject. As used herein, the term “sample” refers to a biological sample obtained or derived from a subject. In some embodiments, a biological sample includes biological tissue or fluid. In some embodiments, a biological sample may include blood, blood cells, serum, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, cerebrospinal fluid, lymph, tissue biopsy specimens, surgical specimens, other body fluids, secretions, and / or excretions, and / or cells therefrom. In some embodiments, a biological sample includes cells obtained from an individual (e.g., from a human or animal subject). In some embodiments, obtained cells are or include cellsfrom an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, or collection of body fluid (e.g., blood). In some embodiments, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. As another example of sample processing, the sample may be a plasma sample that is treated with an anticoagulant (e.g., EDTA. heparin, or citrate). As another example of sample processing, the sample may be processed to isolate one or more proteins (e.g., by capturing proteins with one or more antibodies). A “processed sample” may include, for example, nucleic acids or polypeptides extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA. isolation and / or purification of certain components. In some embodiments, the sample is from serum, plasma, whole blood, or cerebral spinal fluid.

[0064] In some embodiments, measuring a biomarker (e.g., NfL), as described herein, encompasses measuring the presence of the biomarker, the absence of the biomarker, an amount of the biomarker, an absolute amount of the biomarker, a relative amount of the biomarker, or a concentration of the biomarker.

[0065] Biomarkers such as NfL can be measured and quantified in various biological samples using techniques like enzy me-linked polymerase chain reaction (PCR), mass spectrometry, and immunoassays. See. e.g., Uzgiris et al., “Advances in Neurofilament Light Chain Analysis,” Adv. Clin. Chem. 126:31-71 (2025), which is hereby incorporated by reference in its entirety .

[0066] In some embodiments, measuring biomarkers such as NfL include methods for measuring biomarkers as nucleic acids. Nucleic acid-based methods of measuring a biomarker include performing nucleic acid amplification methods, such as polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). In some embodiments, a nucleic acidbased method of measuring biomarkers includes detecting hybridization between one or more nucleic acid probes and one or more nucleotides that encode the biomarker. In some embodiments, the nucleic acid probes are each complementary to at least a portion of one of the one or more nucleotides that encode the biomarker. In some embodiments, the nucleotides thatencode the biomarker include DNA (e.g., cDNA). In some embodiments, the nucleotides that encode the biomarker include RNA (e.g., mRNA).

[0067] In some embodiments, the biomarker protein concentration in a sample is measured using mass spectrometry. In some embodiments, mass spectrometry includes MS, MS / MS, MALDI-TOF, electrospray ionization mass spectrometry (ESIMS), ESI-MS / MS, ESI- MS / (MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS), tandem liquid chromatography-mass spectrometry' (LC- MS / MS) mass spectrometry, desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry' (APCI-MS), APCI-MS / MS, APCI-(MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS)n, quadrupole mass spectrometry. Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry. In some embodiments, the MS approach quantifies a fragment of a biomarker rather than the full-length protein. MS approaches, however, can be sufficient to determine the protein level of the biomarker to an accuracy sufficient for methods and / or measurements as disclosed.

[0068] In some embodiments, the biomarker protein concentration in a sample is measured using an immunoassay. Immunoassays are a broad category' of methods that use the specific binding of an antibody to its antigen to measure the concentration of the antigen. Various immunoassay technologies include, without limitation, enzyme-linked immunoassay (ELISA), single molecule array (SiMoA), electrochemiluminescence immunoassay (ECLIA), fluorescence (FL), nucleic acid-linked immunosorbent assay (NULISA), lateral flow assay (LFA), immunomagnetic reduction (IMR), and proximity' extension assay (PEA). See, e.g., Uzgiris et al., “Advances in Neurofilament Light Chain Analysis,” Adv. Clin. Chem. 126: 31-71 (2025), which is hereby incorporated by reference in its entirety. In some embodiments, the biomarker is a protein (e.g., NIL), and is detected by' detection of nucleotides that encode NfL in whole or in part (e.g., anti- NfL nucleotide sequence antibody' agents, probes, complementary nucleic acids, etc.). In some embodiments, an immunoassay can be a high-throughput and / or an automated immunoassay platform. For example, a high-throughput and / or automated immunoassay platform can be used to analyze at least 240 tests per hour or at least 440 tests per hour, although any other number of tests per hour can also be used in other examples.

[0069] In some embodiments, an immunoassay uses one or more antibodies reactive with the biomarker. The term antibody refers to a polyclonal antibody, a monoclonal antibody,antigen binding fragments of such antibodies, single chain antibodies as well as to genetic constructs comprising the binding domain of an antibody.

[0070] In some embodiments, methods of measuring NfL in a sample include contacting a sample with one or more antibody agents directed to NfL. In some embodiments, the antibody agent directed to NfL binds only to fragments of NfL that are caused by neuroneuronal degeneration. In some embodiments, the epitope bound by the antibody to NfL is not exposed in the intact full-sized NfL protein (see, e.g., Shaw et al., “Uman-Type Neurofilament Light Antibodies Are Effective Reagents for the Imaging of Neurodegeneration,’' Brain Commun. 5:fcad067 (2023) and Uzgiris et al., Advances “ in Neurofilament Light Chain Analysis,” Adv. Clin. Chem. 126: 31-71 (2025), each of which is hereby incorporated by reference in its entirety). In some embodiments, such methods also include contacting the sample with a first set of one or more detection agents. In some embodiments, the antibody agents are labeled with the first set of one or more detection agents. In some embodiments, the first set of one or more detection agents includes one or more acridinium ester (“AE”) molecules.

[0071] AE molecules can be used to label proteins and nucleic acids. Acridinium-labeled proteins can be used for detection in immunoassays. Exposing AE to an alkaline H2O2 (hydrogen peroxide) produces chemiluminescence. Light is emitted at a wavelength maximum in the range of 430 to 480 nm, depending on the specific AE variant. Such light can be detected, for example, by high-efficiency photomultiplier tubes. The tight emission is rapid and completes within 1 to 5 seconds. Diversity in AE forms contributes to better assay performance, including improved sensitivity and robustness. AE molecules can be used to label small molecules, large analytes, and antibodies.

[0072] One commercially available immunoassay to measure biomarkers (e.g., NfL protein) is the Atellica IM Serum Neurofilament Light Chain (sNfL) assay (Siemens Healthineers). The Atellica IM Serum Neurofilament Light Chain (sNfL) assay can be used in the quantitative measurement of NfL in human serum and plasma (EDTA and lithium heparin) using the Atellica® IM Analyzer. The Atellica IM sNfL assay is a fully automated 2-step sandwich immunoassay using acridinium ester chemiluminescent (CL) technology. The assay employs two anti-NfL antibodies. The first antibody, in the Lite Reagent, is a mouse monoclonal anti-NfL antibody labeled with acridinium ester. The second antibody is a biotinylated mouse monoclonal anti-NfL antibody that is bound to streptavidin-coated paramagnetic microparticles in the Solid Phase. A direct relationship exists between the amount of NfL present in the patient sample and the amount of relative light units (RLUs) detected by the system. See, e.g., theimmunoassay described in PCT Publication No. WO 2018 / 087229, which is hereby incorporated by reference in its entirety.

[0073] In some embodiments, the biomarker protein concentration in a sample is measured using aptamers. A non-limiting example of aptamers includes SOMAmer® (Slow Off- rate Modified Aptamer) (see. e.g., Timsina et al. “Comparative Analysis of Alzheimer’s Disease Cerebrospinal Fluid Biomarkers Measurement by Multiplex Somascan Platform and Immuno- Based Approach,” J. Alzheimers Dis. 89: 193-207 (2023), which is hereby incorporated by reference in its entirety). In some embodiments, the biomarker protein concentration in a sample is measured using label-free detection technologies. A non-limiting example of label-free detection is Surface Plasmon Resonance (SPR) (see, e.g., Jucknischke et al., “Antibody Profiling: Kinetics with Native Biomarkers for Diagnostic Assay and Drug Development,” Biosensors (Basel) 13: 12 (2023), which is hereby incorporated by reference in its entirety ).

[0074] In some embodiments, the methods of the present disclosure involve detecting an increased concentration of NfL protein in a sample compared to a concentration of NIL protein in a control.

[0075] In some embodiments, the NfL is measured in comparison to a control. In some embodiments, a control is the concentration of NfL protein from a subject that has not been subject to radiation treatment. In some embodiments, an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy-associated neuronal damage in the subject. In some embodiments, the control is obtained from a healthy subject, a healthy cohort, or from the selected subject prior to radiation treatment. In some embodiments, the control is the concentration of NfL protein in the subject prior to radiation treatment. In some embodiments, the control reference value is the concentration of NfL protein in other patients and / or control subjects (e.g., normal healthy individuals or individuals of a similar age, sex, and / or physical condition). For example, NfL levels in serum from over 2,000 individuals representing 212 million US residents aged 20-75 ranged from 4.7 pg / mL for the 10thpercentile of 20 to 29-year-olds up to 45.4 pg / mL for the 90thpercentile of individuals aged 70 years and up, with an overall mean of 16.76 pg / mL and a 95% confidence interval of 14.45 to 19.07 pg / mL (see, e.g., Beltran, “Normative Values for Serum Neurofilament Light Chain in US Adults,” J. Clin. Neurol. 20:46-49 (2024) and Uzgiris et al.. “Advances in Neurofilament Light Chain Analysis.” Adv. Clin. Chem. 126: 31-71 (2025), each of which is hereby incorporated by reference in its entirety). In some embodiments, the control reference value for NfL is 14.45 to 19.07 pg / mL.

[0076] In some embodiments, the concentration of NfL protein in a sample is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any number or range therein higher, or more than 100% higher than the concentration of NfL protein in a control sample. In some embodiments, the concentration of NfL protein in the sample is at least 10% higher than the concentration of NfL protein in the control.

[0077] In some embodiments, the concentration of NfL protein in a sample is at least 5 pg / mL. 6 pg / mL. 7 pg / mL. 8 pg / mL. 9 pg / mL. 10 pg / mL, 11 pg / mL, 12 pg / mL, 13 pg / mL. 14 pg / mL, 15 pg / mL, 16 pg / mL, 17 pg / mL, 18 pg / mL, 19 pg / mL, 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 26 pg / mL, 27 pg / mL, 28 pg / mL, 29 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, 50 pg / mL, 55 pg / mL, 60 pg / mL, 65 pg / mL, 70 pg / mL, 75 pg / mL, 80 pg / mL, 85 pg / mL, 90 pg / mL, 95 pg / mL, 100 pg / mL or any number or range therein higher, or more than lOOpg / mL higher than the concentration of NfL protein in a control sample. In some embodiments, the control sample value for NfL is 14.45-19.07 pg / mL.

[0078] In some embodiments, an increase in NfL concentration of at least 5%-10% compared to a control is indicative of an early onset of neuronal damage and / or RAN. In some embodiments, an increase in NfL concentration of at least 10% compared to a control is indicative of an early onset of neuronal damage and / or RAN. In some embodiments, an increase in NfL concentration of at least 10%-20% compared to a control is indicative of early onset of neuronal damage and / or RAN. In some embodiments, an increase in NfL concentration of at least 5%, 6%, 7%, 8%. 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any number or range therein higher, or more than 100% higher is indicative of early onset of neuronal damage and / or RAN.

[0079] In some embodiments, a first sample is obtained from a first subject or a first subject population, where the first subject has undergone one or more therapeutic interventions, and the concentration of NfL protein in the first sample is compared to a concentration of NfL protein in a second sample obtained from a second subject or a second subject population having undergone one or more therapeutic interventions different from the one or more interventions of the first subject or the first subject population.

[0080] In some embodiments, measuring the NfL protein concentration is repeated at different times. For example, measurements of NfL concentration can be taken before radiation treatment, and again after each radiation treatment (e.g., at each step indicated with a box in FIG.2), and also after chemotherapy or some other non-radiation treatment. In some embodiments, measurements of NfL concentration are taken after each radiation treatment. In some embodiments, measurements of NfL concentration are taken after multiple radiation treatments. In some embodiments, measurements of NfL concentration are taken after each intervention. In some embodiments, measurements of NfL concentration are taken after multiple interventions.

[0081] In some embodiments, the NfL protein concentration is measured immediately after at least one of the radiation treatments. In some embodiments, the NfL protein concentration is measured 1-14 days, 15-30 days, 1-3 months, 3-12 months and / or 1 month-2 years, or more than 2 years, or any number or range therein, after at least one of the radiation treatments. In some embodiments, the NfL protein concentration is measured at 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 50 weeks, 100 weeks, 200 weeks, 300 weeks, 400 weeks, 500 weeks, or any number or range therein. In some embodiments, the NfL protein concentration is measured 2 weeks, 6 weeks, and / or 12 weeks after at least one of the radiation treatments. In some embodiments, the NfL protein concentration is measured 1 month - 2 years after at least one of the radiation treatments. In some embodiments, the NfL protein concentration is measured after the systemic therapy.

[0082] In some embodiments, the measuring further comprises measuring one or more biomarkers of inflammation and / or one or more biomarkers of fibrosis in the sample. In some embodiments, the measuring further comprises measuring one or more biomarkers of inflammation in the sample. In some embodiments, the one or more biomarkers of inflammation comprises Interleukin-6 (IL-6) and the one or more biomarkers of fibrosis comprises hyaluronic acid (HA), Tissue Inhibitor of Metalloproteinase 1 (TIMP-1), and / or procollagen III, N-terminal pro-peptide (PIIINP). Additional biomarkers of inflammation may include, but are not limited to, C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-a), and other cytokines known to be elevated during inflammatory responses. In some embodiments, the measuring further comprises measuring one or more biomarkers of fibrosis in the sample. Biomarkers of fibrosis may encompass, but are not limited to, tissue inhibitor of metalloproteinases- 1 (TIMP-1), procollagen III, N-terminal pro-peptide (PIIINP), alpha-smooth muscle actin (a-SMA), transforming growth factor-beta (TGF-J3), and other molecules associated with the fibrotic process. The levels of these biomarkers can provide valuable information about the presence, severity, or progression of inflammation and fibrosis in the subject, thereby informing diagnosis, prognosis, and treatmentstrategies. Similar methods can be used to measure the concentration of these biomarkers, such as the methods described above for NfL protein concentration (e.g., immunoassays).

[0083] In some embodiments, the methods of the present disclosure further comprise determining a subject has radiotherapy-associated neuronal damage if (i) the concentration of NfL protein in the subject is increased relative to the control and (ii) the concentration of the one or more biomarkers of inflammation and / or fibrosis is higher than the concentration of each of the one or more biomarkers of inflammation and / or fibrosis in the control.

[0084] In some embodiments, the methods of the present disclosure include stopping or delaying further radiation treatment and / or other therapeutic interventions in the subject if the sample has an increased concentration of NfL protein compared to the control and, optionally, an increased concentration of the biomarkers of inflammation and / or fibrosis relative to the control.Additional Methods & Kits

[0085] Another aspect of the present disclosure is a method for diagnosing radiotherapy- associated neuronal damage in a subject. The method involves measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject where the subject has undergone one or more therapeutic interventions, and where at least one of the therapeutic interventions is a radiation treatment. The method further involves detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control, where the control is a concentration or a range of concentrations of NfL protein from one or more individuals that have not been subject to radiation treatment, and where an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy- associated neuronal damage in the subject.

[0086] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0087] In some embodiments, diagnosing radiotherapy-associated neuronal damage in a subject involves detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control. In some embodiments, the control used for diagnosis is a concentration of NfL protein from an individual that has not been subject to radiation treatment.

[0088] In some embodiments, an increase in NfL concentration of at least 5%-l 0% compared to a control is indicative of a diagnosis of neuronal damage and / or RAN. In some embodiments, an increase in NfL concentration of at least 10% compared to a control is indicative of a diagnosis of neuronal damage and / or RAN. In some embodiments, an increase inNfL concentration of at least 10%-20% compared to a control is indicative of a diagnosis of neuronal damage and / or RAN. In some embodiments, an increase in NfL concentration of at least 1%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%. 70%. 75%. 80%. 85%. 90%. 95%. 100% or any number or range therein higher, or more than 100% higher compared to a control is indicative of a diagnosis of neuronal damage and / or RAN. In some embodiments, an increase in NfL concentration of 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, 200-fold, 500-fold. 1000-fold, or any number or range therein, or more than 1000-fold is indicative of a diagnosis of neuronal damage and / or RAN.

[0089] A further aspect of the present disclosure is another method for diagnosing a subject as having radiotherapy-associated neuronal damage. The method involves measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject and comparing the concentration of NfL protein in the sample to a predetermined concentration level of NfL protein, where an increased concentration of NfL protein in the sample relative to the predetermined concentration level is indicative of radiotherapy-associated neuronal damage in the subject.

[0090] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0091] In some embodiments, predetermined concentration level of NfL protein is determined using a concentration that is indicative of radiotherapy-associated neuronal damage in a subject. In some embodiments, the predetermined concentration is a threshold value, which refers to a value (or a range of values) that is used as a reference to classify the results of a measurement, for example, the results of a measurement attained m an assay. A threshold value can be determined based on one or more control samples. A threshold value can be determined prior to. concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold value can be a range of values In some embodiments, the predetermined concentration level of NfL protein is the threshold value for NfL protein is the average value of NfL in a healthy cohort of subjects. In some embodiments, the threshold value is 14.45 to 19.07 pg / mL. In some embodiments, the threshold value is 16.76 pg / mL (see, e.g., Beltran, "Normative Values for Serum Neurofilament Light Chain in US Adults / ’ J. Clin. Neurol. 20:46-49 (2024) and Uzgiris et al., “Advances in Neurofilament Light Chain Analysis,” Adv. Clin. ChemA26: 31-71 (2025), each of which is hereby incorporated by reference in its entirety ). In some embodiments, the threshold value is the concentration level of NfL protein in normal healthy individuals,individuals of a similar age, individuals of a similar sex, and / or individuals of a similar physical condition. In some embodiments, the predetermined concentration level of NfL protein is based on the level of NfL in the subject prior to radiation treatment.

[0092] In some embodiments, the predetermined concentration level of NfL protein is 5%-10% higher than a control. In some embodiments, the predetermined concentration level of NfL protein is 10% higher than a control. In some embodiments, the predetermined concentration level of NfL protein is 10%-20% higher than a control. In some embodiments, the predetermined concentration level of NfL protein is 1%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. 21%. 22%. 23%. 24%. 25%. 26%. 27%. 28%. 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any number or range therein higher, or more than 100% higher than a control. In some embodiments, the predetermined concentration level of NfL protein is 1-fold, 2-fold, 3 -fold, 4- fold, 5-fold, 10-fold, 20-fold, 40-fold, 60-fold. 80-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or any number or range therein, or more than 1000-fold higher than a control. In some embodiments, the control value for NfL is 14.45-19.07 pg / mL.

[0093] A further aspect of the present disclosure is a method for determining relative risk and / or severity of radiotherapy-associated neuronal damage in an intervention protocol. The method involves measuring concentration of neurofilament light chain (NfL) protein in a first sample obtained from a first subject or a first subject population, where the first subject or the first subject population has undergone one or more therapeutic interventions, and where at least one of the therapeutic interventions is a radiation treatment. The method further involves comparing the concentration of NfL protein in the first sample(s) to a concentration of NfL protein in a second sample obtained from a second subject or a second subject population having undergone one or more therapeutic interventions different from the one or more interventions of the first subject or the first subject population, and determining whether the risk and / or severity of radiotherapy-associated neuronal damage of the first subject or the first subject population is higher or lower than that of the second subject or the second subject population based on an increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s), where a higher concentration of NfL protein in the first sample(s) or the second sample(s) indicates a higher risk and / or severity of radiotherapy-associated neuronal damage of the one or more interventions associated with that subject or subject population.

[0094] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0095] After quantification of the NfL level in a biological sample, the NfL result can be compared to a cutoff value that stratifies the relative risk and / or severity of the subject developing radiotherapy-associated neuronal damage. For example, if the NfL levels are elevated above a cutoff value, the subject is determined to be at a higher risk of developing radiotherapy- associated neuronal damage or in need of treatment for radiotherapy-associated neuronal damage or other associated risk if another intervention was used prior and there is no history of radiotherapy. Alternatively, if NfL levels are normal or below a cutoff value, the subject is determined to be at a lower risk of developing radiotherapy-associated neuronal damage. In some embodiments, the concentration of NfL protein correlates with risk and / or severity of radiotherapy-associated neuronal damage. For example, as shown in FIG. 2, NfL levels can be measured at multiple points in time to assess the risk and / or determine if the subject is developing a radiotherapy -associated neuronal damage. The first time point is the first patient visit where a diagnosis is given or a follow up after a diagnosis. A baseline biological sample can be collected and used to assess the current neurological state of the subject. For example, the patient may have had a history of other cancers that required interventions and may have resulted in some neuronal damage. In this case, the subject may have elevated NfL above a set threshold or cutoff value where certain RT-inclusive interventions would not be advised to the subject or at high risk of developing neuronal damage.

[0096] In some embodiments, the increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s) is at least 5-10%. In some embodiments, the increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s) is at least 10%. In some embodiments, the increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s) is at least 10-20%. In some embodiments, the increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s) is at least 1%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%. 28%. 29%. 30%. 35%. 40%. 45%. 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95%, 100% or any number or range therein higher or lower, or more than 100% higher. In some embodiments, the increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s) is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20- fold, 40-fold. 60-fold, 80-fold, 100-fold. 200-fold. 500-fold, 1000-fold, or any number or range therein, or more than 1000-fold higher.

[0097] Y et another aspect of the present disclosure is a method for monitoring the progression or regression of radiotherapy-associated neuronal damage in a subject. The methodinvolves measuring concentration of neurofilament light chain (NfL) protein in a first sample obtained from a subject, where the subject has undergone a first therapeutic intervention involving a radiation treatment, measuring concentration of NfL protein in a second sample obtained from the subject optionally after a second therapeutic intervention, and determining the subject has a progression of radiotherapy-associated neuronal damage if the concentration of NfL protein in the second sample has increased relative to the concentration of NfL protein in the first sample, or determining the subject has a regression of radiotherapy-associated neuronal damage if the concentration of NfL protein in the second sample has decreased relative to the concentration of NfL protein in the first sample.

[0098] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0099] In some embodiments, the methods described herein provide the requisite resolution to track progression of neuronal damage and / or RAN. For example, the concentration of the analyte may be measured from a first sample taken from a subject. After a time period, a second biological sample may be taken from the subject and the concentration may be determined. Changes in the biomarker level may be correlated with disease progression. In some embodiments, measuring is repeated at different times (e.g.. as indicated in FIG. 2). For example, NfL results can also be compared to prior NfL results from the same subject to monitor whether the subject is developing radiotherapy-associated neuronal damage and if the condition progresses. Similarly, a subject may be determined to have a regression of radiotherapy- associated neuronal damage if the concentration of NfL protein in the second sample or in additional samples have decreased relative to the concentration of NfL protein in the first sample.

[0100] In some embodiments, the concentration of the biomarker may be measured from a first sample taken from a subject. After a time period, a second biological sample may be taken from the subject and the concentration may be determined. In some embodiments, additional samples can be taken after additional time periods. In some embodiments, additional samples may coincide with additional treatment interventions. In some embodiments, additional samples may be taken after additional treatment interventions. In some embodiments, additional samples may be taken after additional time periods with no additional treatment interventions. Changes in the biomarker level are correlated with neuronal damage and / or RAN progression or regression.

[0101] Another aspect of the present disclosure is an in vitro method for detecting radiotherapy-associated neuronal damage in a subject. The method involves obtaining a sample from a subject having undergone one or more therapeutic interventions, where at least oneintervention is a radiation treatment, measuring concentration of neurofilament light chain (NfL) protein in the sample, and identifying the subject as having radiotherapy-associated neuronal damage if the concentration of the NfL protein in the sample is greater than a predetermined NfL protein concentration.

[0102] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0103] In some embodiments, NfL can be used for an in-vitro diagnostic (IVD) or screening test for the condition of radiation-associated neuronal damage. In some embodiments, a diagnostic test detects whether NfL is present in a sample obtained from a subject. In some embodiments, a diagnostic test as disclosed herein can assist in the detection or diagnosis of radiation-associated neuronal damage in a subject.

[0104] In some embodiments, a diagnostic test as disclosed herein is adapted to an immunoassay platform. In some embodiments, such an immunoassay platform includes a semiautomated or automated immunoassay platform. In some embodiments, a diagnostic test as disclosed herein is adapted for semi-automated testing of one or more biomarkers.

[0105] In some embodiments, a diagnostic test as disclosed herein is an improved diagnostic for radiation-associated neuronal damage as compared to standard techniques in that the diagnostic test of the present disclosure includes one or more of the following benefits: improved sensitivity for identifying radiation-associated neuronal damage, improved specificity for identifying radiation-associated neuronal damage, improved accuracy for identifying radiation-associated neuronal damage, reduced time to diagnosis for radiation-associated neuronal damage, and / or reduced cost of screening patients for radiation-associated neuronal damage.

[0106] In some embodiments, a diagnostic test as disclosed herein can be a plasma-based screening assay. In some embodiments, a diagnostic test is adapted for the Siemens Atellica® system or the Siemens Advia Centaur® system, by way of example only.

[0107] In some embodiments, methods provided herein include detecting a level of NfL present in a sample to obtain a biomarker profile and using the biomarker profile to compute a biomarker score. In some embodiments, methods provided herein include detecting a level of NfL in a sample to obtain a biomarker profile and using the biomarker profile and demographic factors to compute a biomarker score. In some embodiments, methods provided herein include detecting a level of NfL in a sample to obtain a biomarker profile and using the biomarker profile and imaging-based biomarkers to compute a biomarker score. In some embodiments, methods provided herein include detecting a level of NfL in a sample to obtain a biomarker profile, andusing the biomarker profile, demographic factors, and imaging-based biomarkers to compute a biomarker score. In some embodiments, NfL levels, such as those disclosed in Beltran, “Normative Values for Serum Neurofilament Light Chain in US Adults,’' Clin. Neurol. 20:46- 49 (2024) and Uzgiris et al., “Advances in Neurofilament Light Chain Analysis," Adv. Clin. Chem. 126: 31-71 (2025), each of which is hereby incorporated by reference in its entirety, are used to compute a biomarker score.

[0108] In some embodiments, methods provided herein including receiving a level of NfL, demographic factors, and / or imaging-based biomarkers in a sample. In some embodiments, receiving includes electronically receiving. In some embodiments, demographic factors include one or more of age, weight, biological sex, ethnicity, BMI, medical history, risk factors, family history, or geographic location.

[0109] In some embodiments, methods described herein include using a biomarker score to select a subject for further neurological tests. In some embodiments, methods described herein include using a biomarker score to select a subject to receive one or more doses of a therapeutically effective medication. In some embodiments, methods described herein include using a biomarker score to identify a subject as having or being at risk of having radiation- associated neuronal damage.

[0110] In some embodiments, methods described herein include comparing a biomarker score to a reference biomarker score. In some embodiments, methods described herein include administering one or more doses of a therapeutically effective medication to a subject. In some embodiments, methods described herein include performing one or more neurological tests on a subject.

[0111] In some embodiments, measuring is carried out with an immunoassay. In some embodiments, measuring is carried out with an acridinium ester-based immunoassay. In some embodiments, measuring is carried out over several time points.

[0112] A further aspect of the present disclosure is a kit for detecting radiotherapy- associated neuronal damage. The kit includes reagents for determining expression level of neurofilament light chain (NfL) protein in a sample from a subject and instructions for comparing the expression level to a reference standard to detect radiotherapy-associated neuronal damage in the subject.

[0113] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0114] In some embodiments, a kit includes one or more or two or more anti-NfL biomarker agents and instructions for use (e.g., treatment, prophylactic, or diagnostic use). Insome embodiments, the kit is used for an in vitro diagnostic assay to detect early onset of radiotherapy-associated neuronal damage. In some embodiments, the kit is used for an in vitro diagnostic assay to detect early onset of radiotherapy-associated neurotoxicity'. In some embodiments, the kit is used for an in vitro diagnostic assay to detect early onset of peripheral neuropathy. In some embodiments, the kit is used for an in vitro diagnostic assay to diagnose radiotherapy-associated neuronal damage. In some embodiments, the kit is used for an in vitro diagnostic assay to diagnose radiotherapy-associated neurotoxicity. In some embodiments, the kit is used for an in vitro diagnostic assay to diagnose peripheral neuropathy. In some embodiments, the kit is used for an in vitro diagnostic assay to determine relative risk and / or severity- of radiotherapy-associated neuronal damage. In some embodiments, the kit is used for an in vitro diagnostic assay to determine relative risk and / or severity of radiotherapy-associated neurotoxicity. In some embodiments, the kit is used for an in vitro diagnostic assay to determine relative risk and / or severity of peripheral neuropathy. In some embodiments, the kit is used for an in vitro diagnostic assay to monitor the progression or regression of radiotherapy-associated neuronal damage. In some embodiments, the kit is used for an in vitro diagnostic assay to monitor the progression or regression of radiotherapy-associated neurotoxicity. In some embodiments, the kit is used for an in vitro diagnostic assay to monitor the progression or regression of peripheral neuropathy. In some embodiments, the kit is used for an in vitro diagnostic assay to detect radiotherapy-associated neuronal damage. In some embodiments, the kit is used for an in vitro diagnostic assay to detect radiotherapy-associated neurotoxicity. In some embodiments, the kit is used for an in vitro diagnostic assay to detect peripheral neuropathy.

[0115] In some embodiments, the one or more or two or more anti-NfL biomarker agents include antibody agents. In some embodiments, two or more of the antibody agents are labeled with a detectable moiety. In some embodiments, the kit further includes a detection agent (e.g., one or more acridinium ester molecules). In some embodiments, one or more of the antibody agents are labeled with one or more of the acridinium ester molecules such as HEGAE. In some embodiments, the kit further includes one or more secondary antibody agents that specifically bind to one or more of the anti-NfL biomarker antibody agents.

[0116] In some embodiments, the kit further includes one or more control samples. In some embodiments, the control samples include one or more NfL biomarker standards.

[0117] In addition to the above, a kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, tergitol, and / or an agent for treating a condition or disorder described herein. Alternatively, other ingredients can be included in a kit, but in differentcompositions or containers than the anti-NfL biomarker agents. In such embodiments, a kit can include instructions for admixing the anti-NfL biomarker agents and the other ingredients, or for using the anti-NfL biomarker together with the other ingredients.

[0118] In certain embodiments, kits for use in accordance with the present disclosure may include, a reference or control sample(s), instructions for processing samples, performing tests on samples, instructions for interpreting the results, buffers and / or other reagents necessary for performing tests.

[0119] In some embodiments, the instructions comprise instructions for the methods of the present disclosure described herein.

[0120] Additionally, non-transitory computer readable media containing executable instructions that when executed cause a processor to perform operations including a method as provided herein are provided. For example, anon-transitory computer readable medium containing executable instructions that when executed cause a processor to perform operations including a method of the present disclosure, including but not limited to: a method for detecting early onset of radiotherapy-associated neuronal damage in a subject; a method for diagnosing radiotherapy-associated neuronal damage in a subject; a method for determining relative risk and / or severity of radiotherapy-associated neuronal damage in an intervention protocol, and a method for monitoring the progression or regression of radiotherapy-associated neuronal damage in a subject, each method as described herein. Illustrative embodiments of systems and methods disclosed herein may include computations performed locally by a computing device. However, computations performed over a network are also contemplated, e.g. an illustrative network environment for use in the methods and systems described herein. Suitable systems for non- transitory computer readable medium are described in WO2024227045A1 entitled High- Sensitivity assay for serum neurofilament light change cross reference to related applications having international application number PCT / US2024 / 026599 to Merabet et al. (herein entirely incorporated by reference). See e.g. FIG. 4 in WO2024227045A1, depicting a block diagram of an illustrative cloud computing environment suitable for use in methods of the present disclosure is shown and described. Computer devices including processor, memory, storage device, interface suitable for use in providing a non-transitory computer readable media containing executable instructions that when executed cause a processor to perform operations including a method as provided herein are disclosed.

[0121] In embodiments, of the present disclosure the memory stores information within the computing device. In some implementations, the memory is a volatile memory unit or units. In some implementations, the memory is a non-volatile memory unit or units. In embodiments,the memory may also be another form of computer-readable medium, such as a magnetic or optical disk.

[0122] In embodiments, the storage device is capable of providing mass storage for the computing device. In some implementations, the storage device may be or contain a computer- readable medium, such as a hard disk device, an optical disk device, a flash memory or other similar solid state memory device, or an array of devices, including devices in a storage area network or other configurations. Instructions can be stored in an information carrier. The instructions, when executed by one or more processing devices, perform one or more methods, such as those described above. The instructions can also be stored by one or more storage devices such as computer- or machine-readable mediums (for example, the memory, the storage device, or memory on the processor).

[0123] In embodiments, a high-speed interface manages bandwidth-intensive operations for the computing device, while the low-speed interface manages lower bandwidth-intensive operations. Such allocation of functions is an example only. In some implementations, the highspeed interface is coupled to the memory, the display (e.g., through a graphics processor or accelerator), and to the high-speed expansion ports, which may accept various expansion cards (not shown). In the implementation, the low-speed interface is coupled to the storage device and the low-speed expansion port. The low-speed expansion port, which may include various communication ports (e g., USB, Bluetooth®, Ethernet, wireless Ethernet) may be coupled to one or more input / output devices, such as a keyboard, a pointing device, a scanner, or a networking device such as a switch or router, e.g., through a network adapter.

[0124] In embodiments, the computing device may be implemented in a number of different forms, as shown in the figure. For example, it may be implemented as a standard server, or multiple times in a group of such servers. In addition, it may be implemented in a personal computer such as a laptop computer. It may also be implemented as part of a rack server system. Alternatively, components from the computing device may be combined with other components in a mobile device (not shown), such as a mobile computing device. Each of such devices may contain one or more of the computing device and the mobile computing device, and an entire system may be made up of multiple computing devices communicating with each other.

[0125] In embodiments, a mobile computing device includes a processor, a memory', an input / output device such as a display, a communication interface, and a transceiver, among other components. The mobile computing device may also be provided with a storage device, such as a micro-drive or other device, to provide additional storage. Each of the processor, the memory', the display, the communication interface, and the transceiver, are interconnected using various buses,and several of the components may be mounted on a common motherboard or in other manners as appropriate.

[0126] The processor can execute instructions within the mobile computing device, including instructions stored in the memory. The processor may be implemented as a chipset of chips that include separate and multiple analog and digital processors. The processor may provide, for example, for coordination of the other components of the mobile computing device, such as control of user interfaces, applications run by the mobile computing device, and wireless communication by the mobile computing device.

[0127] In embodiments, the processor may communicate with a user through a control interface and a display interface coupled to the display. The display may be, for example, a TFT (Thin-Film-Transistor Liquid Cry stal Display) display or an OLED (Organic Light Emitting Diode) display, or other appropriate display technology. The display interface may include appropriate circuitry for driving the display to present graphical and other information to a user. The control interface may receive commands from a user and convert them for submission to the processor. In addition, an external interface may provide communication with the processor, so as to enable near area communication of the mobile computing device with other devices. The external interface may provide, for example, for wired communication in some implementations, or for wireless communication in other implementations, and multiple interfaces may also be used.

[0128] The memory stores information within the mobile computing device. The memory can be implemented as one or more of a computer-readable medium or media, a volatile memory unit or units, or a non-volatile memory unit or units. An expansion memory may also be provided and connected to the mobile computing device through an expansion interface, which may include, for example, a SIMM (Single In Line Memory Module) card interface. The expansion memory may provide extra storage space for the mobile computing device, or may also store applications or other information for the mobile computing device. Specifically, the expansion memory may include instructions to carry out or supplement the processes described above, and may include secure information also. Thus, for example, the expansion memory may be provided as a security module for the mobile computing device, and may be programmed with instructions that permit secure use of the mobile computing device. In addition, secure applications may be provided via the SIMM cards, along with additional information, such as placing identifying information on the SIMM card in a non-hackable manner.

[0129] The memory may include, for example, flash memory and / or NVRAM memory (non-volatile random access memory), as discussed below. In some implementations, instructions are stored in an information carrier and, when executed by one or more processing devices (forexample, processor), perform one or more methods, such as those described above. The instructions can also be stored by one or more storage devices, such as one or more computer- or machine-readable mediums (for example, the memory, the expansion memory, or memory on the processor). In some implementations, the instructions can be received in a propagated signal, for example, over the transceiver or the external interface.

[0130] Various implementations of the systems and techniques described here can be realized in digital electronic circuitry, integrated circuitry7, specially designed ASICs (application specific integrated circuits), computer hardware, firmware, software, and / or combinations thereof. These various implementations can include implementation in one or more computer programs that are executable and / or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device.

[0131] These computer programs (also known as programs, software, software applications or code) include machine instructions for a programmable processor, and can be implemented in a high-level procedural and / or object-oriented programming language, and / or in assembly / machine language. As used herein, the terms machine-readable medium and computer- readable medium refer to any computer program product, apparatus and / or device (e.g., magnetic discs, optical disks, memory, Programmable Logic Devices (PLDs)) used to provide machine instructions and / or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term machine-readable signal refers to any signal used to provide machine instructions and / or data to a programmable processor.

[0132] To provide for interaction with a user, the systems and techniques described here can be implemented on a computer having a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) for displaying information to the user and a keyboard and a pointing device (e.g.. a mouse or a trackball) by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well; for example, feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback); and input from the user can be received in any form, including acoustic, speech, or tactile input.

[0133] The systems and techniques described here can be implemented in a computing system that includes a back end component (e.g., as a data server), or that includes a middleware component (e.g., an application server), or that includes a front end component (e.g., a client computer having a graphical user interface or a Web browser through which a user can interactwith an implementation of the systems and techniques described here), or any combination of such back end, middleware, or front end components. The components of the system can be interconnected by any form or medium of digital data communication (e.g., a communication network). Examples of communication networks include a local area network (LAN), a wide area network (WAN), and the Internet.

[0134] The computing system can include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[0135] Methods described herein can be implemented in a computer system having a processor that executes specific instructions in a computer program. In some embodiments, a computer system may be arranged to execute methods of the present disclosure. Particularly, a computer program may include instructions for the system to select appropriate next steps.

[0136] The present disclosure also provides a system including a non-transitory computer readable medium and a processor, wherein the non-transitory computer readable medium has executable instructions stored thereon that, when executed by the processor, perform operations including a method for detecting early onset of radiotherapy-associated neuronal damage in a subject of the present disclosure.

[0137] The present disclosure also provides a system including a non-transitory computer readable medium and a processor, wherein the non-transitory' computer readable medium has executable instructions stored thereon that, when executed by the processor, perform operations including a method for diagnosing radiotherapy-associated neuronal damage in a subject as described herein.

[0138] The present disclosure also provides a system including a non-transitory' computer readable medium and a processor, wherein the non-transitory computer readable medium has executable instructions stored thereon that, when executed by the processor, perform operations including a method for diagnosing radiotherapy-associated neuronal damage in a subject as described herein.

[0139] The present disclosure also provides a system including a non-transitory' computer readable medium and a processor, wherein the non-transitory computer readable medium has executable instructions stored thereon that, when executed by the processor, perform operations including a method for determining relative risk and / or severity of radiotherapy-associated neuronal damage in an interv ention protocol as described herein.

[0140] The present disclosure also provides a system including a non-transitory computer readable medium and a processor, wherein the non-transitory computer readable medium has executable instructions stored thereon that, when executed by the processor, perform operations including a method for monitoring the progression or regression of radiotherapy-associated neuronal damage in a subject, each method as described herein.CERTAIN EMBODIMENTS OF THE PRESENT DISCLOSURE

[0141] Embodiment 1.A method for detecting early onset of radiotherapy-associated neuronal damage in a subject, the method comprising: measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject, wherein the subject has undergone one or more therapeutic interventions, and wherein at least one of the therapeutic interventions is a radiation treatment; and detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control, wherein the control is a concentration or a range of concentrations of NfL protein from one or more individuals that have not been subject to radiation treatment, and wherein an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy-associated neuronal damage in the subject.

[0142] Embodiment 2. The method of Embodiment 1, wherein the sample is from serum, plasma, whole blood, or cerebral spinal fluid.

[0143] Embodiment 3. The method of Embodiments 1-2, wherein the control is obtained from a healthy individual, a healthy cohort, or from the subject prior to the radiation treatment.

[0144] Embodiment 4. Embodiments 1-3, wherein said measuring is repeated at different times.

[0145] Embodiment 5. Embodiments 1-4, wherein the subject has undergone more than one radiation treatment.

[0146] Embodiment 6. Embodiments 1-5, wherein said measuring is carried out after each radiation treatment.

[0147] Embodiment 7. Embodiments 1-6. wherein said measuring is carried out 2 weeks, 6 weeks, and / or 12 weeks after at least one of the radiation treatments.

[0148] Embodiment 8. Embodiments 1-7, wherein said measuring is carried out 1 month to 2 years after at least one of the radiation treatments.

[0149] Embodiment 9. Embodiments 1-8. wherein the concentration of NfL protein in the sample is at least 10% higher than the concentration of NfL protein in the control.

[0150] Embodiment 10. Embodiments 1 -9, wherein the one or more interventions caused direct damage to neurons in the subject.

[0151] Embodiments 11. Embodiments, 1-10, wherein the one or more interventions cause indirect damage to neurons in the subject.

[0152] Embodiment 12. Embodiments 1-11, wherein at least one of the radiation treatments was carried out at a dose of at least 1.8 Gy.

[0153] Embodiment 13. Embodiments 1-12, wherein at least one of the interventions is a systemic therapy.

[0154] Embodiment 14. Embodiments 1-13, wherein the systemic therapy is chemotherapy or a combinatorial therapy.

[0155] Embodiment 15. Embodiments 1-14, wherein said measuring is carried out after the systemic therapy.

[0156] Embodiment 16. Embodiments 1-15, further comprising or consisting of: measuring one or more biomarkers of inflammation and / or one or more biomarkers of fibrosis in the sample.

[0157] Embodiment 17. Embodiments 1-16, wherein the one or more biomarkers of inflammation comprises Interleukin-6 (IL-6) and the one or more biomarkers of fibrosis comprises hyaluronic acid (HA), Tissue Inhibitor of Metalloproteinase 1 (TIMP-1), and / or procollagen III, N-terminal pro-peptide (PIIINP).

[0158] Embodiment 18. Embodiments 1-17, further comprising or consisting of: determining the subject has radiotherapy-associated neuronal damage if (i) the concentration of NfL protein in the subject is increased relative to the control and (ii) the concentration of the oneor more biomarkers of inflammation and / or fibrosis is higher than the concentration of each of the one or more biomarkers of inflammation and / or fibrosis in the control.

[0159]

[0160] Embodiment 19. Embodiments 1-18, further comprising or consisting of: stopping or delaying further radiation treatment and / or other therapeutic interventions in the subject if the sample has an increased concentration of NfL protein compared to the control and, optionally, an increased concentration of the biomarkers of inflammation and / or fibrosis relative to the control.

[0161] Embodiment 20. Embodiments 1-19, wherein said measuring is performed with an immunoassay.

[0162] Embodiment 21. Embodiments 1-20, wherein the neuronal damage or neurotoxicity is peripheral neuropathy.

[0163] Embodiment 22. A method for diagnosing radiotherapy-associated neuronal damage in a subject, the method comprising or consisting of: measuring concentration of neuro filament light chain (NfL) protein in a sample obtained from a subject, wherein the subject has undergone one or more therapeutic interventions, and wherein at least one of the therapeutic interventions is a radiation treatment; and detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control, wherein the control is a concentration or a range of concentrations of NfL protein from one or more individuals that have not been subject to radiation treatment, and wherein an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy-associated neuronal damage in the subject.

[0164] Embodiment 23. A method for determining relative risk and / or severity of radiotherapy-associated neuronal damage in an intervention protocol, the method comprising or consisting of: measuring concentration of neurofilament light chain (NfL) protein in a first sample obtained from a first subject or a first subject population, wherein the first subject or the first subject population has undergone one or more therapeutic interventions, and wherein at least one of the therapeutic interventions is a radiation treatment; comparing the concentration of NfL protein in the first sample(s) to a concentration of NfL protein in a second sample obtained from a second subject or a second subject population having undergone one or more therapeutic interventions different from the one or more interventions of the first subject or the first subjectpopulation; and determining whether the risk and / or severity of radiotherapy-associated neuronal damage of the first subject or the first subject population is higher or lower than that of the second subject or the second subject population based on an increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s), wherein a higher concentration of NfL protein in the first sample(s) or the second sample(s) indicates a higher risk and / or severity of radiotherapy-associated neuronal damage of the one or more interventions associated with that subject or subject population.

[0165] Embodiment 24. The method of Embodiment 23. wherein the concentration of NfL protein correlates with risk and / or severity of radiotherapy-associated neuronal damage.

[0166] Embodiment 25, The method of claim Embodiments 23-24, wherein the increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s) is at least 10%.

[0167] Embodiment 26. A method for monitoring the progression or regression of radiotherapy-associated neuronal damage in a subject, the method comprising or consisting of measuring concentration of neurofilament light chain (NfL) protein in a first sample obtained from a subject, wherein the subject has undergone a first therapeutic intervention involving a radiation treatment; measuring concentration of NfL protein in a second sample obtained from the subject optionally after a second therapeutic intervention; and determining the subject has a progression of radiotherapy-associated neuronal damage if the concentration of NfL protein in the second sample has increased relative to the concentration of NfL protein in the first sample, or determining the subject has a regression of radiotherapy-associated neuronal damage if the concentration of NfL protein in the second sample has decreased relative to the concentration of NfL protein in the first sample.

[0168] Embodiment 27. A method for diagnosing a subject as having radiotherapy- associated neuronal damage, the method comprising or consisting of: measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject and comparing the concentration of NfL protein in the sample to a predetermined concentration level of NfL protein, wherein an increased concentration of NfL protein in the sample relative to the predetermined concentration level is indicative of radiotherapy -associated neuronal damage in the subject.

[0169] Embodiment 28. The method of Embodiment 27, wherein the predetermined concentration level of NfL protein is derived from a healthy individual or a healthy cohort.

[0170] Embodiment 29. An in vitro method for detecting radiotherapy-associated neuronal damage in a subject, the method comprising or consisting of: obtaining a sample from a subject having undergone one or more therapeutic interventions, wherein at least one intervention is a radiation treatment; measuring concentration of neurofilament light chain (NfL) protein in the sample; and identifying the subject as having radiotherapy-associated neuronal damage if the concentration of the NfL protein in the sample is greater than a predetermined NfL protein concentration.

[0171] Embodiment 30. The method of Embodiment 29, wherein said measuring is carried out with an immunoassay.

[0172] Embodiment 31. The method of Embodiments 29-30, wherein said measuring is carried out with an acridinium ester-based immunoassay.

[0173] Embodiment 32. The method of any one of Embodiments 1-31, wherein said measuring is carried out over several time points.

[0174] Embodiment 33. The method of any one of claims 1-32, wherein the sample is from serum, plasma, whole blood, or cerebral spinal fluid.

[0175] Embodiment 34. The method of any one of Embodiments 1-33, wherein the subject has undergone one or more rounds of radiation treatment.

[0176] Embodiment 35. The method of any one of Embodiments 1-23, wherein the predetermined NfL protein concentration is based on NfL protein concentration in a sample obtained from a healthy individual, a healthy cohort, or the subject prior to the radiation treatment.

[0177] Embodiment 36. The method of any one of Embodiments 1-35, wherein the concentration of NfL protein in the sample correlates with severity of radiotherapy-associated neuronal damage in the subject.

[0178] Embodiment 37. The method of Embodiments 1-36, wherein the radiation treatment comprises a radiation dose of 5 Gy or higher.

[0179] Embodiment 38. The method of any one of Embodiments 1-37, wherein at least one therapeutic intervention is a systemic therapy.

[0180] Embodiment 39. The method of Embodiments 1-38, wherein the systemic therapy is chemotherapy or a combinatorial therapy.

[0181] Embodiment 40. The method of Embodiments 1-39, wherein the sample is obtained after the systemic therapy.

[0182] Embodiment 41. The method of any one of Embodiments 1-40, wherein the subject has undergone multiple rounds of radiation.

[0183] Embodiment 42. The method of any one of Embodiments 1-41, wherein the neuronal damage is associated with peripheral neuropathy.

[0184] Embodiment 43. A kit for detecting radiotherapy-associated neuronal damage, comprising or consisting of: reagents for determining expression level of neurofilament light chain (NfL) protein in a sample from a subject and instructions for comparing the expression level to a reference standard to detect radiotherapy-associated neuronal damage in the subject.

[0185] Embodiment 44. A system including a non-transitory computer readable medium and a processor, wherein the non-transitory computer readable medium has executable instructions stored thereon that, when executed by the processor, perform operations including a method for diagnosing radiotherapy-associated neuronal damage in a subject as described herein.

[0186] Embodiment 45. A system including a non-transitory computer readable medium and a processor, wherein the non-transitory computer readable medium has executable instructions stored thereon that, when executed by the processor, perform operations including a method for detecting early onset of radiotherapy-associated neuronal damage in a subject of the present disclosure.

[0187] A system including a non-transitory computer readable medium and a processor, wherein the non-transitory computer readable medium has executable instructions stored thereon that, when executed by the processor, perform operations for a method for detecting early onset of radiotherapy-associated neuronal damage in a subject, the operations comprising: measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject, wherein the subject has undergone one or more therapeutic interventions, and wherein at leastone of the therapeutic interventions is a radiation treatment; and detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control, wherein the control is a concentration or a range of concentrations of NfL protein from one or more individuals that have not been subject to radiation treatment, and wherein an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy-associated neuronal damage in the subject.

[0188] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.EXAMPLESExample 1 - NfL Biomarker Study for Neuronal Damage in Pigs

[0189] Materials and Methods. Blood samples were obtained from 18 Yucatan mini pigs subjected to single fraction irradiation treatment targeting the cervical spinal cord, with doses equal to or exceeding 17.5 Gy. The collection occurred at pre-treatment (baseline) and various post-treatment intervals. Serum NfL levels were measured utilizing research-use-only kits of the Atellica IM Serum Neurofilament Light Chain (sNfL) on an Atellica Solution Immunoassay Module analyzer (Siemens Healthineers, Tarrytown. NY, USA). This study employed a customized assay dilution and diluent that had been previously optimized with the Atellica IM sNfL assay for porcine specimens. Each sample was tested in duplicate.

[0190] Results. The majority of the diluted serum measurements from pre-treatment and various post-treatment intervals (baseline. 28 ± 3 days, and terminal endpoint) fell within the assay's measurement range. Of the baseline NfL measurements, 1 out of 18 were below the lower limit of the assay's measuring range (< 2.5 pg / mL). The mean baseline NfL level (n=17) was 8.0 pg / mL, with values ranging from 5.2 to 12.5 pg / mL (FIG. 3A). After 28 ± 3 days, the mean sNfL fold change (NfL at post-treatment time over baseline NfL) showed a 2.42 -fold increase from baseline (FIG. 3B). In most pigs (n=12), sNfL fold changes ranged from 2- to 5-fold after 4 weeks. For a subset of pigs (n=5), NfL fold changes peaked at the terminal time point, exceeding 5-fold above baseline, with the highest change being greater than 60-fold.

[0191] Conclusions. Baseline NfL levels in treated pigs were comparable to and within the expected normative range for humans. Most mini pigs exhibited peak NfL values 4 weeks post-irradiation treatment of the spinal cord. Some pigs continued to show an increase in NfL until termination. Neurofilament light chain is a marker for neuronal damage, and the observed post-treatment increase in NfL suggests that it may serve as a response biomarker toradiotherapy. These observations in mini pigs are translatable to humans, with similar effects anticipated in human subjects.PROPHETIC Example 2 - NfL Biomarker Study for Neuronal Damage

[0192] A biomarker sub-study of a clinical study investigating therapeutic response to different neoadjuvant therapies / interv entions is performed. The number of patients with reported neuropathies during treatment out of approximately 120 total cancer patients is collected.

[0193] Patients will undergo 1 of 3 interventions before surgery: short course radiotherapy (SCRT), short course radiotherapy followed by chemotherapy (SCRT + chemo), or long course chemoradiotherapy (LCCR) (X % SCRT, Y% SCRT + chemo, Z% LCCR).

[0194] Fractions of 1.8 Gy and 5 Gy will be used for different RT regimens. By 2 weeks, patients in different groups will have had different total doses of 25 Gy and 21 .8 Gy.

[0195] The levels of serum biomarkers will be measured over time including concentration of neurofilament light chain (NfL) protein.

[0196] Serum is collected at baseline (pretreatment) and up to 3 time points after intervention. For example, sample specimens are collected at 2 weeks, 6 weeks, and 12 weeks post radiation (day 0).

[0197] NfL levels are measured with the Siemens Healthineers Atellica IM sNfL assay.

[0198] Biomarker levels are analyzed, and biomarkers are correlated with clinical outcomes using modeling. Outcomes include adverse events (e.g., peripheral neuropathy).

[0199] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the disclosure and these are therefore considered to be within the scope of the disclosure as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting early onset of radiotherapy-associated neuronal damage in a subject, the method comprising: measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject, wherein the subject has undergone one or more therapeutic interventions, and wherein at least one of the therapeutic interventions is a radiation treatment; and detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control, wherein the control is a concentration or a range of concentrations of NfL protein from one or more individuals that have not been subject to radiation treatment, and wherein an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy-associated neuronal damage in the subject.

2. The method of claim 1, wherein the sample is from serum, plasma, whole blood, or cerebral spinal fluid.

3. The method of claim 1 or claim 2, wherein the control is obtained from a healthy individual, a healthy cohort, or from the subject prior to the radiation treatment.

4. The method of any one of the preceding claims, wherein said measuring is repeated at different times.

5. The method of any one of the preceding claims, wherein the subject has undergone more than one radiation treatment.

6. The method of claim 5, wherein said measuring is carried out after each radiation treatment.

7. The method of any one of the preceding claims, wherein said measuring is carried out 2 weeks, 6 weeks, and / or 12 weeks after at least one of the radiation treatments.

8. The method of any one of the preceding claims, wherein said measuring is carried out 1 month to 2 years after at least one of the radiation treatments.

9. The method of any one of the preceding claims, wherein the concentration of NfL protein in the sample is at least 10% higher than the concentration of NfL protein in the control.

10. The method of any one of the preceding claims, wherein the one or more interventions caused direct damage to neurons in the subject.

11. The method of any one of the preceding claims, wherein the one or more interventions cause indirect damage to neurons in the subject.

12. The method of any one of the preceding claims, wherein at least one of the radiation treatments was carried out at a dose of at least 1.8 Gy.

13. The method of any one of the preceding claims, wherein at least one of the interventions is a systemic therapy.

14. The method of claim 13, wherein the systemic therapy is chemotherapy or a combinatorial therapy.

15. The method of claim 13 or claim 14, wherein said measuring is carried out after the systemic therapy.

16. The method of any one of the preceding claims further comprising: measuring one or more biomarkers of inflammation and / or one or more biomarkers of fibrosis in the sample.

17. The method of claim 16, wherein the one or more biomarkers of inflammation comprises Interleukin-6 (IL-6) and the one or more biomarkers of fibrosis comprises hyaluronic acid (HA). Tissue Inhibitor of Metalloproteinase 1 (TIMP-1), and / or procollagen III, N-terminal pro-peptide (PIIINP).

18. The method of claim 16 or claim 17 further comprising: determining the subject has radiotherapy-associated neuronal damage if(i) the concentration of NfL protein in the subject is increased relative to the control and(ii) the concentration of the one or more biomarkers of inflammation and / or fibrosis is higher than the concentration of each of the one or more biomarkers of inflammation and / or fibrosis in the control.

19. A method for diagnosing radiotherapy-associated neuronal damage in a subject, the method comprising: measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject, wherein the subject has undergone one or more therapeutic interventions, and wherein at least one of the therapeutic interventions is a radiation treatment; and detecting an increased concentration of NfL protein in the sample compared to a concentration of NfL protein in a control, wherein the control is a concentration or a range of concentrations of NfL protein from one or more individuals that have not been subject to radiation treatment, and wherein an increased concentration of NfL protein in the sample relative to the control is indicative of radiotherapy-associated neuronal damage in the subject.

20. A method for determining relative risk and / or severity of radiotherapy- associated neuronal damage in an intervention protocol, the method comprising: measuring concentration of neurofilament light chain (NfL) protein in a first sample obtained from a first subject or a first subject population, wherein the first subject or the first subject population has undergone one or more therapeutic interventions, and wherein at least one of the therapeutic interventions is a radiation treatment; comparing the concentration of NfL protein in the first sample(s) to a concentration of NfL protein in a second sample obtained from a second subject or a second subject population having undergone one or more therapeutic interventions different from the one or more interventions of the first subject or the first subject population; and determining whether the risk and / or severity of radiotherapy-associated neuronal damage of the first subject or the first subject population is higher or lower than that of the second subject or the second subject population based on an increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s), wherein a higher concentration of NfL protein in the first sample(s) or the second sample(s) indicates a higher risk and / or severity of radiotherapy-associated neuronal damage of the one or more interventions associated with that subject or subject population.

21. The method of claim 20, wherein the concentration of NfL protein correlates with risk and / or severity of radiotherapy-associated neuronal damage.

22. The method of claim 21, wherein the increased or decreased concentration of NfL protein in the first sample(s) compared to the second sample(s) is at least 10%.

23. A method for monitoring the progression or regression of radiotherapy- associated neuronal damage in a subject, the method comprising: measuring concentration of neurofilament light chain (NfL) protein in a first sample obtained from a subject, wherein the subject has undergone a first therapeutic intervention involving a radiation treatment; measuring concentration of NfL protein in a second sample obtained from the subject optionally after a second therapeutic intervention; and determining the subject has a progression of radiotherapy-associated neuronal damage if the concentration of NfL protein in the second sample has increased relative to the concentration of NfL protein in the first sample, or determining the subject has a regression of radiotherapy-associated neuronal damage if the concentration of NfL protein in the second sample has decreased relative to the concentration of NfL protein in the first sample.

24. A method for diagnosing a subject as having radiotherapy-associated neuronal damage, the method comprising: measuring concentration of neurofilament light chain (NfL) protein in a sample obtained from a subject and comparing the concentration of NfL protein in the sample to a predetermined concentration level of NfL protein, wherein an increased concentration of NfL protein in the sample relative to the predetermined concentration level is indicative of radiotherapy -associated neuronal damage in the subject.

25. The method of claim 24, wherein the predetermined concentration level of NfL protein is derived from a healthy individual or a healthy cohort.

26. A kit for detecting radiotherapy-associated neuronal damage, comprising: reagents for determining expression level of neurofilament light chain (NfL) protein in a sample from a subject and instructions for comparing the expression level to a reference standard to detect radiotherapy -associated neuronal damage in the subject.

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