Methods for Identifying and Treating Breast Cancer

Abstract

Methods for the treatment and / or prognosis of breast cancer are provided. The methods include determining an immune score (IS) for a gene set selected from an immunological signature gene set (IS is based on an immunological model), determining a proliferation index (PI) for a gene set selected from an oncogenic signature gene set (PI is based on a tumor-intrinsic model), and integrating the IS and PI into an integrated model to stratify a subject and identify a high-risk clinical group. The immunological model is trained in basal and HER2+ tumors (IS), and the tumor-intrinsic model (PI) is trained in immunosuppressed tumors.

Description

Technical Field

[0001] Background The present technology generally relates to the quantification of tumor aggressiveness, and more specifically to the quantification of tumor aggressiveness, immune activity, and patient age for predicting prognosis and the benefit of radiotherapy (i.e., radiation therapy), which can then be used for individualized treatment as needed.

Background Art

[0002] Reference to Sequence Listing This application has been filed with a sequence listing in electronic format. The sequence listing is provided as a file entitled "2023-07-15_Sequence_Listing-PRLUD018WO.xml", which was created on July 15, 2023, last modified, and is approximately 13,712 bytes in size. The information in the electronic sequence listing is hereby incorporated by reference in its entirety.

Summary of the Invention

Means for Solving the Problems

[0003] Overview The methods disclosed herein each have several aspects, and none of them are the sole cause of their desirable attributes. Without limiting the claims, some of the prominent features are briefly considered here. Numerous other embodiments are also contemplated, including those with fewer, additional, and / or different components, steps, features, objectives, benefits, and advantages. The components, aspects, and steps can also be in different arrangements and orders. After considering this discussion, and particularly after reading the section entitled "Detailed Description", one will understand how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.

[0004] Disclosed herein are methods that include a final model based on quantification of tumor aggressiveness (measured by proliferation index), immune activity (measured by immune score), and patient age. In some embodiments, the final model is effective in predicting prognosis and radiation therapy (RT) benefit. However, the groups created are heterogeneous. For example, a "medium risk" score can include patients with aggressive tumors (high proliferation index) having strong immune activity (high immune score), or patients with low aggressive tumors (moderate proliferation index) who do not receive the benefit of the anti-tumor immune response. Further understanding of the specific biology of the tumor can be achieved by analyzing the proliferation index and immune score individually (which are always calculated for the final model), and healthcare providers / clinicians can further individualize treatment. This can also impact other types of therapies (e.g., immunotherapy; a medium final model score with high immune score and proliferation index is an excellent candidate for immunotherapy). Thus, models according to some non-limiting embodiments of the present disclosure are most likely to provide guidance for treatment by immunotherapy, chemotherapy, and other types of targeted therapies by providing information regarding prognosis and RT benefit and information regarding individual tumor biology. The analysis has also been shown to be highly prognostic for distant metastases, which is very useful in non-limiting examples for stratifying patients according to benefit from chemotherapy.

[0005] Also provided herein is a method for treating breast cancer, the method comprising determining an immune score (IS) based on an immune model composed of genes from an immunological gene set, determining a proliferation index (PI) based on a tumor-intrinsic model composed of genes from a tumor-intrinsic gene set, integrating the IS and the PI into an integrated model, integrating the integrated model and the age of the subject into a final model to identify whether the subject belongs to a specific-risk clinical group, and providing an appropriate therapy to the subject based on the specific-risk clinical group, wherein the immunological model is trained in basal tumors and HER2+ tumors, and the tumor-intrinsic model is trained in immune-exhausted tumors. In some embodiments, the immunological model comprises an immunological gene set. In some embodiments, the method further comprises performing a meta-analysis of the prognostic effect of each of the immunological gene sets.

[0006] In some embodiments, the immune gene set comprises C7 and any immune-related gene set from the H, C1, C2, C3, C4, C5, C6 or C8 category (e.g., identified using any of the keywords "LYMPHOCYTE|T_CELL|PD1|PD-1|PDL1|PD-L1|LAG3|CHECKPOINT_RECEPTOR|B_CELL|PERFORIN|GRANZYME|NK_CELL|CD8|CYTOTOXIC"). In some embodiments, the method further comprises collinearity filtering to remove highly correlated gene sets.

[0007] In some embodiments, the IS determination step comprises training an elastic net on the tumor, and optionally, in some embodiments, the PI determination step comprises training an elastic net on the tumor, wherein the tumor comprises immune-exhausted tumors. In some embodiments, the PI determination step comprises training an elastic net on the tumor, wherein the tumor comprises immune-exhausted tumors.

[0008] In some embodiments, the method further includes ranking genes in the gene set selected when determining the IS step based on Rho, and selecting one or more of the ranked genes. In some embodiments, the method is ranking genes in the gene set selected in the determination of the PI step based on a prognostic score, the prognostic score including Rho / Pmeta, and further excluding ranked genes having Rho from prognostic scores less than 0.2. In some embodiments, the method further includes combining the integrated model and the age of the subject into a final model to output a risk score, and recommending one or more treatment plans based on the risk score.

[0009] In some embodiments, the tumor-intrinsic model (referred to as the proliferation index) includes the H, C2, and / or C6 gene sets. In some embodiments, the final model includes extracting genes included in IS and PI, performing a meta-analysis of the prognostic effect of each of the extracted genes in a first cohort, selecting genes stably expressed across the core and tissue types in a second cohort, ranking the extracted genes, and selecting all of the genes extracted from IS and PI. In some embodiments, the number of genes extracted from IS is less than 23 per gene set. In some embodiments, the number of genes extracted from PI is less than 60 per gene set.

[0010] In some embodiments, the final model involves extracting the genes included in IS and PI, performing a meta-analysis of the prognostic effects of each of the extracted genes in a first cohort, selecting the genes stably expressed across the core and tissue types in a second cohort, ranking the extracted genes, selecting the top 23 genes extracted per gene set from IS, and selecting the top 60 genes extracted per gene set from PI. In some embodiments, the number of genes extracted per gene set from IS is 23 or more. In some embodiments, the number of genes extracted per gene set from PI is 60 or more.

[0011] In some embodiments, the first cohort includes a training cohort. In some embodiments, the second cohort includes the SweBCG91-RT cohort (or another similar cohort). In some embodiments, the SweBCG91-RT cohort passed RNA, cDNA, and microarray quality control. In some embodiments, the SweBCG91-RT cohort was treated with breast-conserving surgery and included in the multivariate analysis, or was treated with breast-conserving surgery and radiotherapy and included in the multivariate analysis.

[0012] In some embodiments, the risk score is a medium risk score that includes a high PI indicating an aggressive tumor and a high IS indicating strong immune activity. In some embodiments, the medium risk score corresponds to grade III tumors including 高 / PD-L1 高 / TIL 高 In some embodiments, the treatment plan includes immunotherapy. In some embodiments, the treatment plan includes de-intensifying one or more treatments for grade III tumors when IS and PI are high. In some embodiments, the risk score is a high risk score that includes medium / low IS and high PI. In some embodiments, the high risk score includes 低 / PD-L1 低 / TIL 低It is applicable to Grade III tumors including. In some embodiments, the high-risk score is PD-1 高 / PD-L1 高 / TIL 高 It is applicable to Grade I / II tumors including.

[0013] In some embodiments, the treatment plan is intensified. In some embodiments, the intensified treatment plan includes at least one of: when the standard recommended treatment does not include a boosting dose, adding a boosting dose to the standard recommended treatment for the subject, increasing the boosting dose beyond the standard amount for the subject, increasing the fractional dose per fraction above the standard amount for the subject, increasing the number of fractions of the recommended dose above the standard amount for the subject, and treating the subject with intensified radiotherapy including at least one of these doses. In some embodiments, the intensified treatment plan indicates at least one of intensified radiotherapy treatment, systemic therapy, mastectomy, additional use of a sensitizer for another therapy, therapy above the level set by at least one of the NCCN, ESMO, ESTRO, Clinical Practice Recommendations Australia, and / or NICE guidelines for the remaining indicators of the subject, or any combination thereof.

[0014] This specification also provides a method for treating breast cancer, the method comprising determining an IS based on an immunological model composed of genes from an immunological gene set, determining a PI based on a tumor-intrinsic model composed of genes from a tumor-intrinsic gene set, integrating the IS and the PI into an integrated model, integrating the integrated model and the age of the subject into a final model, identifying whether the subject is classified into a specific risk clinical group based on the risk score from the final model, and, when the risk score of the subject is a high risk score, intensifying the treatment plan for the subject, the treatment plan including adding a boost dose to the standard recommended treatment for the subject when the standard recommended treatment is less than 67 Gy and does not include a boost dose, increasing the boost dose beyond the standard amount for the subject, increasing the fraction dose per fraction beyond the standard amount for the subject, increasing the number of fractions of the recommended dose beyond the standard amount for the subject, and treating the subject with an intensified radiotherapy including at least one of the above doses. In some embodiments, the intensified treatment plan includes an intensified radiotherapy treatment, a systemic therapy, a mastectomy, an additional use of a sensitizer for another therapy, a therapy above the level set by at least one of the NCCN, ESMO, ESTRO, Clinical Practice Recommendations Australia, and / or NICE guidelines for the remaining indicators of the subject, or any combination thereof. In some embodiments, the immunological model is trained in basal tumors and HER2+ tumors. In some embodiments, the tumor-intrinsic model is trained in immune-exhausted tumors. In some embodiments, the high risk score groups the subject into a high risk clinical group.

[0015] Also provided herein is a method for treating breast cancer, the method comprising determining the expression level of a gene included in a model; adjusting the expression level of the gene by scaling or normalizing against a background population of representative tumors; summing the adjusted expression levels of the genes to determine an enrichment score for a gene set included in the model, the gene set comprising genes; normalizing the enrichment score by comparing the enrichment score to the background population; calculating IS and PI using the IS and PI models, respectively; normalizing the IS and PI scores by comparing the IS and PI scores to the background population; calculating an integrated score using an integrated model; normalizing the integrated score by comparing the score to a representative background population; determining a risk score for a subject using a final model, the final model comprising the age of the subject; determining whether the risk score of the subject is classified into a risk category by comparing the risk score of the subject to a representative background population, the risk category comprising a low-risk group, a medium-risk group, or a high-risk group; and providing treatment to the subject based on the risk category of the subject.

[0016] In some embodiments, the subject is part of the high-risk group and the therapy provided to the subject is RT intensification. In some embodiments, the subject is part of the low-risk group and the therapy provided to the subject is RT de-intensification. In some embodiments, the subject is part of the medium-risk group and the therapy provided to the subject is standard RT. In some embodiments, the background population is age-matched.

[0017] Also provided herein is a method for treating breast cancer, the method comprising determining an immune score (IS) from a tumor sample based on an immune model composed of genes from an immunological gene set, determining a proliferation index (PI) from the tumor sample based on a tumor-intrinsic model composed of genes from a tumor-intrinsic gene set, integrating the IS and the PI into an integrated model, integrating the integrated model and the age of the subject into a final model to identify whether the subject belongs to a specific risk clinical group, and providing an appropriate treatment to the subject based on the specific risk clinical group.

[0018] Also provided herein is a method for treating breast cancer, the method comprising identifying a relevant gene set from an immunological gene set and a tumor-intrinsic gene set, selecting a plurality of the relevant gene sets, creating an IS and a PI from the selected gene sets, integrating the IS and the PI into an integrated model, integrating the integrated model and the age of the subject to create a final model, identifying, using the final model, whether the subject belongs to a specific risk clinical group, and providing an appropriate treatment to the subject based on the specific risk clinical group. In some embodiments, the IS corresponds to the immunological gene set. In some embodiments, the PI corresponds to the tumor-intrinsic gene set.

[0019] Also provided herein is a method for treating breast cancer, the method comprising determining tumor aggressiveness, wherein tumor aggressiveness includes the histological grade of the tumor from at least a portion of a tumor sample provided by a subject, the histological grade including Grade I, Grade II, and Grade III, classifying the sample as low risk if determined to be a Grade I tumor, classifying the sample as high risk if determined to be a Grade III tumor, determining the tumor aggressiveness of the sample if determined to be a Grade II tumor, wherein the tumor aggressiveness further includes the proliferation index score of the sample, classifying a Grade II tumor as high risk if a) the proliferation index score is greater than or equal to the median of the background population of Grade III tumors, or b) low risk if the proliferation index score is less than the median of the background population of Grade III tumors, determining the immune score of the sample, the level of tumor infiltrating lymphocytes (TIL) in the sample, the level of checkpoint molecules in the sample, or any combination thereof to determine immunological activity, integrating tumor aggressiveness and immunological activity by training an elastic net having an interaction term between tumor aggressiveness and immunological activity, and determining a treatment plan based on the integration of tumor aggressiveness and immunological activity. In some embodiments, the proliferation index score is based on the expression of a first group of genes. In some embodiments, the immune score is based on the expression of a second group of genes. In some embodiments, the checkpoint molecules include programmed cell death protein-1 (PD-1) and programmed death ligand 1 (PD-L1). In some embodiments, immunological activity is determined to be active if i) the TIL score is 10% or greater and the checkpoint molecule score is 1% or greater of lymphocytes having positive staining for PD-1 or PD-L1, or ii) inactive if the TIL score is less than 10% and the checkpoint molecule score is less than 1% of lymphocytes having positive staining for both PD-1 and PD-L1. In some embodiments, active immunological activity indicates an activated immune infiltration. In some embodiments, inactive immunological activity indicates an inactive immune infiltration.In some embodiments, the first gene group comprises one or more genes listed in Table 6. In some embodiments, the second gene group comprises one or more genes listed in Table 4. In some embodiments, the treatment plan includes: c) omitting standard radiotherapy or radiotherapy if the sample is classified as a high-risk grade II tumor and the immunological activity is active; d) intensifying radiotherapy if the sample is classified as a high-risk grade II tumor and the immunological activity is inactive; e) intensifying radiotherapy if the sample is classified as a low-risk grade II tumor and the immunological activity is active; or f) de-intensifying radiotherapy or omitting radiotherapy if the sample is classified as a low-risk grade II tumor and the immunological activity is inactive.

[0020] Also provided herein is a method for treating breast cancer, the method comprising determining tumor aggressiveness, wherein tumor aggressiveness includes the histological grade of the tumor from at least a portion of a tumor sample provided by a subject, the histological grade including Grade I, Grade II, and Grade III, classifying the sample as low risk if determined to be a Grade I tumor, classifying the sample as high risk if determined to be a Grade III tumor, and determining the tumor aggressiveness of the sample if determined to be a Grade II tumor, wherein tumor aggressiveness further includes the proliferation index score of the sample, classifying the Grade II tumor as high risk if the proliferation index score is between the 60th and 95th percentiles compared to a background population of representative Grade II tumors, or classifying the Grade II tumor as low risk if the proliferation index score is below the 60th percentile compared to a background population of representative Grade II tumors, determining the immune score of the sample, the level of TILs in the sample, the level of checkpoint molecules in the sample, or any combination thereof to determine immunological activity, integrating tumor aggressiveness and immunological activity by training an elastic net having an interaction term between tumor aggressiveness and immunological activity, and determining a treatment plan based on tumor aggressiveness, immunological activity, and the interaction term. In some embodiments, the proliferation index score is based on the expression of a first group of genes. In some embodiments, the immune score is based on the expression of a second group of genes. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, immunological activity is determined to be active if i) the TIL score is 10% or more and the checkpoint molecule score is 1% or more of lymphocytes having positive staining for PD-1 or PD-L1; or ii) the TIL score is less than 10% and the checkpoint molecule score is less than 1% of lymphocytes having positive staining for both PD-1 and PD-L1, or the TIL score is 10% or more and the checkpoint molecule score is less than 1% of lymphocytes having positive staining for both PD-1 and PD-L1, and is determined to be inactive.In some embodiments, active immunological activity indicates an activated immune infiltration. In some embodiments, inactive immunological activity indicates an inactive immune infiltration. In some embodiments, the first gene group comprises one or more genes listed in Table 6. In some embodiments, the second gene group comprises one or more genes listed in Table 4. In some embodiments, the treatment plan is as follows: c) when the sample is classified as a high-risk grade II tumor and the immunological activity is active, omitting standard radiotherapy or radiotherapy; d) when the sample is classified as a high-risk grade II tumor and the immunological activity is inactive, intensifying radiotherapy; e) when the sample is classified as a low-risk grade II tumor and the immune activity is active, intensifying radiotherapy; f) when the sample is classified as a low-risk grade II tumor and the immune activity is inactive, de-intensifying radiotherapy or omitting radiotherapy; g) when the sample is classified as a grade III tumor and the immunological activity is inactive, intensifying radiotherapy; h) when the sample is classified as a grade III tumor and the immunological activity is active, de-intensifying or omitting radiotherapy; i) when the sample is classified as a grade I tumor and the immunological activity is active, intensifying radiotherapy; or j) when the sample is classified as a grade I tumor and the immunological activity is inactive, de-intensifying or omitting radiotherapy.

[0021] Also provided herein is a method for treating breast cancer, the method comprising subclassifying a tumor from at least a portion of a tumor sample provided by a subject, wherein the subtypes include luminal A, luminal B, HER2+, and triple negative, determining the tumor aggressiveness of the sample when the sample is a luminal A tumor or a luminal B tumor, wherein the tumor aggressiveness includes the proliferation index score of the tumor sample, when the sample is subclassified as a luminal A tumor, classifying the sample as a) high risk if the proliferation index score is in the 60-95th percentile compared to a background population of luminal A tumors with representative proliferation index scores, or b) low risk if the proliferation index score is below the 60th percentile compared to a background population of luminal A tumors with representative proliferation index scores, when the sample is subclassified as a luminal B tumor, classifying the sample as c) high risk if the proliferation index score is in the 60-95th percentile compared to a background population of luminal B tumors with representative proliferation index scores, or d) low risk if the proliferation index score is below the 60th percentile compared to a background population of luminal B tumors with representative proliferation index scores, scoring the immune score of the sample, scoring the level of TILs in the sample, scoring the level of checkpoint molecules in the sample, or determining immunological activity including any combination thereof, integrating tumor aggressiveness and immunological activity by training an elastic net having an interaction term between tumor aggressiveness and immunological activity, and determining a treatment plan based on the integrated tumor aggressiveness, immunological activity, and interaction term. In some embodiments, the scoring of the proliferation index is based on the expression of a first group of genes. In some embodiments, the scoring of the immune score is based on the expression of a second group of genes. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, the immunological activity is determined to be active if i) the TIL score is 10% or more and any of the checkpoint molecule scores is 1% or more, or ii) inactive if the TIL score is less than 10% and / or the checkpoint molecule score is less than 1%.In some embodiments, active immunological activity indicates an activated immune infiltration. In some embodiments, inactive immunological activity indicates an inactive immune infiltration. In some embodiments, the first gene group includes one or more genes listed in Table 6. In some embodiments, the second gene group includes one or more genes listed in Table 4. In some embodiments, the treatment plan includes: e) omitting radiotherapy when the tumor sample is classified as a low-risk luminal A tumor; f) omitting standard radiotherapy or radiotherapy when the sample is classified as a high-risk luminal B tumor and the immunological activity is active; or g) intensifying radiotherapy when the tumor sample is classified as a high-risk luminal B tumor and the immunological activity is inactive.

[0022] Also provided herein is a method for treating breast cancer, which comprises determining the tumor aggressiveness from at least a portion of a tumor sample provided by a subject, wherein the tumor aggressiveness includes a proliferation index that classifies the tumor as a) high-risk when the proliferation index score of the sample is in the 60-95th percentile compared to a background population of representative tumors, or b) low-risk when the proliferation index score of the sample is below the 60th percentile compared to a background population of representative tumors, determining the immunological activity of the tumor based on the immune score of the sample, integrating the tumor aggressiveness and the immunological activity based on an interaction term between the tumor aggressiveness and the immunological activity, and determining a treatment plan based on the tumor aggressiveness, the immunological activity, and the interaction term.

[0023] Also provided herein is a method for treating breast cancer, comprising determining the tumor aggressiveness from at least a portion of a tumor sample provided by a subject, wherein the tumor aggressiveness comprises a proliferation index score that classifies the tumor as a) high risk if the proliferation index score of the sample is greater than or equal to the median score of a background population of representative tumors, or b) low risk if the proliferation index score of the sample is less than the median score of a background population of representative tumors, determining the immunological activity of the tumor based on the immune score of the sample, integrating the tumor aggressiveness and the immunological activity based on an interaction term between the tumor aggressiveness and the immunological activity, and determining a treatment plan based on the tumor aggressiveness, the immunological activity, and the interaction term.

[0024] In some embodiments, if the tumor is classified as high risk and the immunological activity is active, the treatment plan comprises standard radiotherapy or omission of radiotherapy. In some embodiments, if the tumor is classified as high risk and the immunological activity is inactive, the treatment plan comprises intensification of radiotherapy. In some embodiments, if the tumor is classified as low risk and the immunological activity is active, the treatment plan comprises intensification of radiotherapy. In some embodiments, if the tumor is classified as low risk and the immunological activity is inactive, the treatment plan comprises de-intensification of radiotherapy.

[0025] In some embodiments, the integration step comprises training an elastic net having an interaction term between the tumor aggressiveness and the immunological activity. In some embodiments, a high proliferation index score indicates an aggressive tumor. In some embodiments, if the sample is classified as an aggressive tumor and the immunological activity is inactive, the treatment comprises intensification of radiotherapy.

[0026] In some embodiments, a high proliferation index score includes a proliferation index of a sample that is at least at the 60th percentile compared to a background population of representative tumors. In some embodiments, a high proliferation index score indicates an aggressive tumor. In some embodiments, if the sample is classified as an aggressive tumor and the immunological activity is inactive, the treatment includes intensification of radiotherapy. In some embodiments, a high proliferation index score includes a proliferation index of a sample that is greater than or equal to the median score of a background population of representative tumors.

[0027] In some embodiments, the scoring of the proliferation index is based on the expression of a first group of genes. In some embodiments, the first group of genes includes one or more genes listed in Table 6. In some embodiments, the scoring of the immune score is based on the expression of a second group of genes. In some embodiments, the second group of genes includes one or more genes listed in Table 4.

[0028] In some embodiments, the determination of immunological activity further includes scoring the TILs in the sample. In some embodiments, activated immunological activity includes activated tumor infiltration. In some embodiments, activated tumor infiltration includes a TIL score of 10% or more. In some embodiments, the step of determining immunological activity further includes measuring the expression of one or more checkpoint molecules. In some embodiments, the step of determining immunological activity further includes measuring the expression of one or more checkpoint molecules. In some embodiments, checkpoint molecules include PD-1 and PD-L1.

[0029] In some embodiments, active immunological activity includes activated tumor infiltration. In some embodiments, activated tumor infiltration includes positive staining for the expression of one or more checkpoint molecules. In some embodiments, the step of determining immunological activity further includes scoring the TILs in the sample. In some embodiments, activated tumor infiltration further includes a TIL score of 10% or more. In some embodiments, inactive immunological activity includes a TIL score of less than 10%.

[0030] In some embodiments, the integration step is performed by training an elastic net having an interaction term between tumor aggressiveness and immunological activity. In some embodiments, tumor aggressiveness further includes the histological grade of a tumor sample. In some embodiments, the histological grade of the tumor sample is determined as a grade II tumor. In some embodiments, the method further includes determining the subtype of the tumor sample before determining tumor aggressiveness, the subtypes including luminal A, luminal B, HER2+, and triple negative. In some embodiments, the subtype is luminal A. In some embodiments, the subtype is luminal B.

[0031] Also provided herein is a method for treating breast cancer, the method comprising determining the histological grade of a tumor from at least a sample of the tumor provided by a subject, the histological grade of the tumor including grade I, grade II, or grade III, and assigning the tumor aggressiveness of the sample as a) low risk if the sample is determined as a grade I tumor; b) high risk if the sample is determined as a grade III tumor; c) low risk if the sample is determined as a grade II tumor and the proliferation index score of the sample is less than the median of the tumor background population; or d) high risk if the sample is determined as a grade II tumor and the proliferation index score of the sample is greater than or equal to the median of the tumor background population, and classifying the immunological activity as active or inactive, the classification including e) the immune score of the sample; f) the level of TILs in the sample; g) the level of checkpoint molecules in the sample; or h) any combination of e)-f), and integrating the tumor aggressiveness and the immunological activity using an interaction term to determine the benefit of a treatment plan.

[0032] In some embodiments, if the tumor is classified as high-risk and the immunological activity is active, the treatment plan includes standard radiotherapy or omission of radiotherapy. In some embodiments, if the tumor is classified as high-risk and the immunological activity is inactive, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low-risk and the immunological activity is active, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low-risk and the immunological activity is inactive, the treatment plan includes de-intensification of radiotherapy.

[0033] In some embodiments, the integration step includes training an elastic net having an interaction term between tumor aggressiveness and immunological activity. In some embodiments, a high proliferation index score indicates an aggressive tumor. In some embodiments, if the sample is classified as an aggressive tumor and the immunological activity is inactive, the treatment includes intensification of radiotherapy. In some embodiments, the score of the proliferation index is based on the expression of a first group of genes. In some embodiments, the first group of genes includes one or more genes listed in Table 6. In some embodiments, the immune score is based on the expression of a second group of genes. In some embodiments, the second group of genes includes one or more genes listed in Table 4. In some embodiments, the checkpoint molecules include PD-1 and PD-L1.

[0034] In some embodiments, the immunological activity is active when f) is 10% or more and g) at least one of the checkpoint molecules is 1% or more. In some embodiments, an active immunological activity indicates an activated tumor infiltration. In some embodiments, the activated tumor infiltration includes positive staining for the expression of one or more checkpoint molecules. In some embodiments, the immunological activity is inactive when f) is less than 10% and / or g) is less than 1% for both checkpoint molecules.

[0035] Also provided herein is a method for treating breast cancer, comprising supplying a sample of a tumor and receiving treatment based on an analysis of the sample, the analysis including determining the tumor aggressiveness of the tumor from the sample, determining the immunological activity of the tumor from the sample, and integrating the tumor aggressiveness and the immunological activity based on an interaction term. In some embodiments, the tumor aggressiveness includes a proliferation index score that classifies the tumor as high risk or low risk. In some embodiments, the immunological activity includes a) an immune score of the sample; b) the level of TILs in the sample; c) the level of checkpoint molecules in the sample; or d) any combination of a) to c).

[0036] In some embodiments, the tumor is classified as high risk if e) the proliferation index score of the sample exceeds a threshold of 60 to 95 percentile compared to a background population of representative tumors, or f) the proliferation index score of the sample is below a threshold below 60 percentile compared to a background population of representative tumors. In some embodiments, the tumor is classified as high risk if g) the proliferation index score of the sample is greater than or equal to the median of a background population of representative tumors, or h) the proliferation index score of the sample is less than the median of a background population of representative tumors. In some embodiments, if the tumor is classified as high risk and the immunological activity is active, the treatment plan includes standard radiotherapy or omission of radiotherapy. In some embodiments, if the tumor is classified as high risk and the immunological activity is inactive, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low risk and the immunological activity is active, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low risk and the immunological activity is inactive, the treatment plan includes de-intensification of radiotherapy.

[0037] In some embodiments, a high proliferation index score indicates that the tumor is aggressive. In some embodiments, when it is shown that the tumor is aggressive, treatment includes intensifying radiotherapy. In some embodiments, the activated immunological activity includes activated tumor infiltration. In some embodiments, the activated tumor infiltration includes a TIL score of 10% or more. In some embodiments, the inactivated tumor infiltration includes a TIL score of less than 10%. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, the activated tumor infiltrate further includes positive staining for the expression of one or more checkpoint molecules.

[0038] In some embodiments, tumor aggressiveness further includes the histological grade of a tissue sample of the tumor. In some embodiments, the histological grades include Grade I, Grade II, and Grade III. In some embodiments, tumor analysis further includes determining the subtype of the tumor sample before determining tumor aggressiveness, and the subtypes include luminal A, luminal B, HER2+, and triple-negative / basal. In some embodiments, the subtype is luminal A. In some embodiments, the subtype is luminal B.

[0039] In some embodiments, the proliferation index scoring is based on the expression of a first group of genes. In some embodiments, the first group of genes includes one or more genes listed in Table 6. In some embodiments, the immune score is based on the expression of a second group of genes. In some embodiments, the second group of genes includes one or more genes listed in Table 4. In some embodiments, the integration step includes training an elastic net having an interaction term between tumor aggressiveness and immunological activity. In some embodiments, the analysis further includes stratification by premenopausal and postmenopausal states. In some embodiments, the premenopausal state includes female subjects under 55 years old. In some embodiments, the postmenopausal state includes female subjects 55 years old or older. In some embodiments, the background population has a matching age.

[0040] In some embodiments, a method of treating a subject is provided, the method comprising determining a proliferation index based on one or more levels of the genes of Table 6, determining an immune score based on the expression of one or more of the genes of Table 4, combining the proliferation index and the immune score, optionally including the age of the subject as a factor, to determine whether the subject will respond to cancer treatment, and administering cancer treatment if the proliferation index, the immune score, and optionally the age of the subject indicate that the treatment will be successful. In some embodiments, a) if the proliferation index score of the sample exceeds the threshold of 60-95 percentile compared to a background population of representative tumors, it is high risk, or b) if the proliferation index score of the sample is below the threshold below the 60th percentile compared to a background population of representative tumors, it is low risk, c) combining the proliferation index and the immune score based on an interaction term between the two, and d) determining cancer treatment based on the proliferation index, the immune score, and the interaction term. In some embodiments, the background population has a matching age.

[0041] In some embodiments, a method of treating a subject is provided, the method comprising including as factors the levels of one or more genes from Table 6, including as factors the levels of one or more genes from Table 4, and including as a factor the age of the subject, thereby determining whether the subject should receive standard radiotherapy, intensified radiotherapy, de-intensified radiotherapy, or omission of radiotherapy.

[0042] In some embodiments, the expression of one or more genes for IS and / or PI is measured from a tissue sample derived from a subject. In some embodiments, the sample includes a formalin-fixed paraffin-embedded tissue sample. In some embodiments, the sample does not include TIL. In some embodiments, the analysis does not measure the TIL level. In some embodiments, the sample includes a formalin-fixed paraffin-embedded tissue sample. In some embodiments, the sample is collected prior to treatment of the subject for cancer. In some embodiments, the sample is collected prior to therapeutic surgery of the subject for cancer. In some embodiments, age is used as a factor in determining an appropriate therapy or treatment. In some embodiments, a subject less than 50 years old is considered young, and a subject 50 years old or older is considered elderly.

[0043] In some embodiments, a method for determining a therapy for treating cancer is provided, the method comprising determining an immune score of a core biopsy without determining the level of TIL surrounding the tumor from which the core biopsy was obtained, and using the immune score to determine the amount of radiation therapy to administer to the subject without including the level of TIL as a factor. In some embodiments, the subject is treated with neoadjuvant immunotherapy.

[0044] In some embodiments, the subject is 55 years old or older. In some embodiments, the subject is older than 55 years old. In some embodiments, the subject is 65 years old or older. In some embodiments, the subject is older than 65 years old. In some embodiments, the subject is 55 years old or less. In some embodiments, the subject is less than 55 years old. In some embodiments, the subject is less than 50 years old. In some embodiments, the subject is 50 to 65 years old. In some embodiments, stromal TILS around the tumor are not used in the analysis, and / or are not available for analysis, or are not used as part of the method.

[0045] In some embodiments, a method for predicting the effectiveness of cancer treatment is provided, the method including analyzing a sample for the presence of one or more of the genes of Table 4 and / or one or more of the genes of Table 6, wherein the variation at the gene level indicates the effectiveness of cancer treatment. In some embodiments, the method further includes administering an appropriate therapy to the subject based on the analysis of one or more genes of Table 4 and / or Table 6.

[0046] In some embodiments, a method for predicting the effectiveness of cancer treatment is provided, and a method for treating breast cancer is provided, the method comprising determining the histological grade of a tumor from at least a sample of the tumor provided by the subject, wherein the histological grade of the tumor includes Grade I, Grade II, or Grade III, and classifying the tumor aggressiveness of the sample as a) low risk if the sample is determined to be a Grade I tumor; b) low risk if the sample is determined to be a Grade II tumor and the proliferation index score of the sample is less than the median of the background population of high-risk tumors; c) high risk if the sample is determined to be a Grade II tumor and the proliferation index score of the sample is greater than or equal to the median of the background population of high-risk tumors; or d) high risk if the sample is determined to be i) a Grade III tumor, ii) the proliferation index score of the sample is greater than or equal to the median of the background population of high-risk tumors, iii) ER negative, iv) HER2 positive, v) the level of Ki67 is high, vi) the mutation load is high, or vii) any combination of i) - vi), and classifying the immunological activity including the level of TILs in the sample, integrating the tumor aggressiveness and the immunological activity using an interaction term, and determining a treatment plan.

[0047] In some embodiments, the level of Ki67 is 10% or more. In some embodiments, the level of Ki67 is 20% or more. In some embodiments, the level of Ki67 is 30% or more. In some embodiments, the mutation load is 5 mutations or more per genomic megabase (mutations / MB). In some embodiments, the mutation load is 7 mutations / MB or more. In some embodiments, the mutation load is 10 mutations / MB or more.

[0048] In some embodiments, a method for treating breast cancer is provided, the method comprising determining the histological grade of a tumor from at least a sample of a tumor provided by a subject, wherein the histological grade of the tumor comprises grade I, grade II, or grade III, assigning a tumor aggressiveness to the sample, classifying an immunological activity, including the level of TILs in the sample, and determining a treatment plan by integrating the tumor aggressiveness and the immunological activity using an interaction term.

[0049] In some embodiments, assigning a high risk to the tumor aggressiveness comprises determining the sample as i) a grade III tumor, ii) the proliferation index score of the sample being greater than or equal to the median score of the background population of the tumor, iii) ER negative, iv) HER2 positive, v) a high level of Ki67, vi) a high mutation load, or vii) any combination of i) to vi). In some embodiments, the level of Ki67 is 10% or more. In some embodiments, the level of Ki67 is 20% or more. In some embodiments, the level of Ki67 is 30% or more. In some embodiments, the mutation load is 5 mutations / MB or more. In some embodiments, the mutation load is 7 mutations / MB or more. In some embodiments, the mutation load is 10 mutations / MB or more.

[0050] In some embodiments, assigning a high risk of tumor aggressiveness includes determining the sample as a grade II tumor, and the proliferation index score of the sample is greater than or equal to the median score of the tumor background population. In some embodiments, the tumor aggressiveness is high risk. In some embodiments, the level of TILs in the sample ranges from 50% to 90%, and the treatment plan includes omission of radiation therapy. In some embodiments, the level of TILs in the sample ranges from 10% to 49%, and the treatment plan includes omission of boost.

[0051] In some embodiments, the classification of immunological activity further includes the level of checkpoint molecules in the sample. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, the level of PD-1 is 1% or more. In some embodiments, the level of PD-L1 is 1% or more. In some embodiments, the level of TILs is 10% or more. In some embodiments, the treatment plan includes omission of RT. In some embodiments, the level of PD-1 is less than 1%. In some embodiments, the level of PD-L1 is less than 1%. In some embodiments, the level of TILs is 10% or more. In some embodiments, the treatment plan includes omission of RT boost.

[0052] In some embodiments, the subject is 55 years of age or older. In some embodiments, the subject is older than 55 years. In some embodiments, the subject is 65 years of age or older. In some embodiments, the subject is older than 65 years. In some embodiments, the subject is 55 years of age or younger. In some embodiments, the subject is less than 55 years. In some embodiments, the subject is between 50 and 65 years of age. In some embodiments, the subject is less than 50 years of age. In some embodiments, the background population is age-matched. In some embodiments, the sample includes a core biopsy.

[0053] In some embodiments, a method for treating breast cancer is provided, the method comprising determining the tumor-intrinsic aggressiveness of a sample provided by a subject, wherein the tumor-intrinsic aggressiveness comprises the proliferation index of the sample, the histological grade of the sample, the level of Ki67 in the sample, the HER2 expression of the sample, the ER-status of the sample, or a combination thereof, and determining the immune infiltration of the sample, wherein the immune infiltration comprises the immune score of the sample, the level of TILs in the sample, the level of PD-1 in the sample, the level of PD-L1 in the sample, or a combination thereof, and providing a treatment based on the tumor-intrinsic aggressiveness and the immune infiltration. In some embodiments, the subject is 65 years of age or older.

[0054] In some embodiments, the tumor-intrinsic risk is high if the proliferation index is above the 60th percentile compared to a background population of representative patients and tumors, the histological grade is 3, the histological grade is 2 and the proliferation index is equal to or above the median of the background population of grade 3 tumors, the HER2 expression is amplified, the ER-status of the sample is negative, or a combination thereof.

[0055] In some embodiments, the immune infiltration is activated if the immune score is above a 60th percentile threshold compared to a background population of representative tumors, the level of TILs in the sample is greater than 10%, the level of PD-1 in the sample is greater than 1%, the level of PD-L1 in the sample is greater than 1%, or a combination thereof.

[0056] In some embodiments, the tumor-intrinsic risk is high, immune activation is inactive, and the treatment includes RT boost. In some embodiments, the tumor-intrinsic risk is high, immune activation is inactive, the subject has no comorbidities, and the treatment includes intensification of chemotherapy. In some embodiments, the treatment further includes omission of immunotherapy. In some embodiments, the sample is ER-positive, and the treatment further includes endocrine therapy. In some embodiments, the tumor-intrinsic risk is high, immune activation is inactive, the subject has no comorbidities, and the treatment includes immunotherapy. In some embodiments, the tumor-intrinsic risk is high, immune activation is inactive, the subject has no comorbidities, the sample is ER-positive, and the treatment includes endocrine therapy. In some embodiments, the tumor-intrinsic risk is not high, immune infiltration is not activated, the subject has no comorbidities, and the treatment includes omission of RT. In some embodiments, the treatment further includes omission or de-escalation of chemotherapy. In some embodiments, the treatment further includes endocrine therapy.

[0057] In some embodiments, the tumor-intrinsic risk is not high, immune infiltration is not activated, the subject has no comorbidities, and the treatment includes omission or de-escalation of chemotherapy. In some embodiments, the tumor-intrinsic risk is not high, immune infiltration has not been activated, the subject has no comorbidities, and the treatment includes endocrine therapy. In some embodiments, the tumor-intrinsic risk is not high, immune infiltration is activated, the subject has no comorbidities, and the treatment includes RT. In some embodiments, the treatment further includes chemotherapy. In some embodiments, the treatment further includes endocrine therapy.

[0058] In some embodiments, the tumor-intrinsic risk is not high, immune infiltration is activated, the subject has no comorbidities, and the treatment includes chemotherapy escalation. In some embodiments, the tumor-intrinsic risk is not high, immune infiltration is activated, the subject has no comorbidities, and the treatment includes endocrine therapy.

[0059] In some embodiments, the subject has a coexisting disease. In some embodiments, the coexisting disease includes a history of coronary artery disease, heart failure, chronic obstructive pulmonary disease, stroke (ischemic or hemorrhagic), uncontrolled hypertension, diabetes, osteoporosis, one or more other cancers, or a combination thereof. In some embodiments, the treatment includes omission of chemotherapy and de-escalation of RT.

[0060] In some embodiments, a method of treating breast cancer is provided, the method comprising constructing an integrated model from a sample provided by a subject to generate an integrated score, wherein the integrated model includes a proliferation index and an immune score; constructing a final model based on the integrated model and the age of the subject to generate a final score; and providing treatment based on the integrated / final score including the integrated score or the final score. In some embodiments, the subject is 65 years of age or older.

[0061] In some embodiments, the integrated / final score is high if it exceeds the 60th percentile of a representative background population of patients and tumors. In some embodiments, the background population is age-matched. In some embodiments, the background population is age-matched to the subject. In some embodiments, the method further includes determining the immune infiltration of the sample, the immune infiltration including the immune score of the sample. In some embodiments, the immune infiltration further includes the level of TILs in the sample, the level of PD-1 in the sample, the level of PD-L1 in the sample, or a combination thereof. In some embodiments, the immune infiltration is activated if the immune score exceeds the 60th percentile threshold compared to a representative tumor background population, the level of TILs in the sample exceeds 10%, the level of PD-1 in the sample exceeds 1%, the level of PD-L1 in the sample exceeds 1%; or a combination thereof. In some embodiments, the immune infiltration is activated if the immune score exceeds the 60th percentile threshold compared to a representative background population of patients and tumors. In some embodiments, the background population is age-matched. In some embodiments, the background population is age-matched to the subject.

[0062] In some embodiments, the integrated / final score is high and the treatment includes RT boost. In some embodiments, the integrated / final score is not high, the immune infiltration is active, and the treatment includes chemotherapy omission or chemotherapy de-escalation. In some embodiments, the treatment further includes immunotherapy. In some embodiments, the sample is ER positive and the treatment further includes endocrine therapy. In some embodiments, the integrated / final score is high, the immune infiltration is inactive, and the treatment includes immunotherapy omission. In some embodiments, the integrated / final score is not high, the immune infiltration is activated, and the treatment includes RT omission. In some embodiments, the treatment further includes chemotherapy omission or de-escalation. In some embodiments, the treatment further includes endocrine therapy. In some embodiments, the integrated / final score is not high, the immune infiltration is activated, and the treatment includes standard RT. In some embodiments, the treatment further includes chemotherapy escalation. In some embodiments, the integrated / final score is not high, the immune infiltration is activated, and the treatment includes chemotherapy escalation. In some embodiments, the treatment further includes endocrine therapy.

[0063] In some embodiments, a method for treating breast cancer is provided, the method comprising determining the tumor-intrinsic aggressiveness of a sample provided by a subject, wherein the tumor-intrinsic aggressiveness includes the proliferation index of the sample, the histological grade of the sample, the level of Ki67 of the sample, the HER2 expression of the sample, the ER-status of the sample, or a combination thereof, determining the immune infiltration of the sample, wherein the immune infiltration includes the immune score of the sample, the level of TIL of the sample, the level of PD-1 of the sample, the level of PD-L1 of the sample, or a combination thereof, and providing a treatment based on the tumor-intrinsic aggressiveness and the immune infiltration, wherein the subject is less than 55 years old.

[0064] In some embodiments, a method for treating breast cancer is provided, the method comprising constructing an integrated model from a sample provided by a subject to generate an integrated score, the integrated model including a proliferation index and an immune score, constructing a final model based on the integrated model and the age of the subject to generate a final score, and providing treatment based on the integrated / final score including the integrated score or the final score, wherein the subject is less than 55 years old.

[0065] In some embodiments, the integrated / final score is less than the 5th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes de-escalation of RT. In some embodiments, the integrated / final score is greater than the 50th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes escalation of RT compared to standard RT. In some embodiments, the RT escalation includes RT boost. In some embodiments, the integrated / final score is greater than the 95th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes surgical escalation. In some embodiments, the surgical escalation includes mastectomy. In some embodiments, the surgical escalation further includes a wider resection margin. In some embodiments, the treatment further includes escalation of systemic therapy. In some embodiments, the treatment includes escalation of systemic therapy. In some embodiments, the background population is age-matched. In some embodiments, the background population is age-matched to the subject.

[0066] In some embodiments, a method for treating breast cancer is provided, the method comprising constructing an integrated model from a sample provided by a subject to generate an integrated score, the integrated model including a proliferation index and an immune score, constructing a final model based on the integrated model and the age of the subject to generate a final score, and providing treatment based on the integrated / final score including the integrated score or the final score, wherein the subject is over 55 years old. In some embodiments, the integrated / final score is above the 70th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes RT escalation. In some embodiments, the RT escalation includes an RT boost. In some embodiments, the integrated / final score is above the 85th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes mastectomy. In some embodiments, the treatment includes systemic therapy escalation. In some embodiments, the background population is age-matched. In some embodiments, the background population is age-matched to the subject.

[0067] The features of the embodiments of the present disclosure will become apparent by referring to the following detailed description and the drawings. In the detailed description and the drawings, like reference numerals, which may not be the same, correspond to similar components. For the sake of brevity, reference numerals or features having functions described above may or may not be described in connection with other drawings in which they appear.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0100] All patents, applications, published applications, and other publications referred to herein are hereby incorporated by reference in their entirety.

[0101] The impact of local immune infiltration in tumor progression is closely related to tumor-intrinsic factors. Genomically unstable tumors, or highly proliferative tumors with high tumor mutation burden, benefit more from immune infiltration. Provided herein are methods for integrating immunological factors and tumor-intrinsic factors to identify clinically high-risk patients who may be candidates for radiotherapy (RT) de-escalation. In some embodiments, the methods also enable upgrading clinically low / medium-risk tumors to high-risk tumors (administering appropriate treatment as desired). In some embodiments, the sample to be analyzed can be obtained from a core biopsy (and / or, lacking analysis of stromal TILs around the tumor, and / or, the sample is obtained prior to neoadjuvant therapy and / or adjuvant therapy). In some embodiments, provided are methods that employ one or more of the genes provided herein (such as one or more of Tables 4 and / or 6), where variation in the gene (e.g., at the transcript level) indicates the efficacy of potential cancer treatment, and optionally, subsequently administer an appropriate treatment tailored to the disposition of a particular gene to a subject.

[0102] Definitions The term “and / or” is intended to provide explicit support for both meanings or either meaning.

[0103] Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, is meant to include the stated element, integer or step, or group of elements, integers or steps, but not to exclude other elements, integers or steps, or group of elements, integers or steps.

[0104] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those skilled in the art in practicing the present disclosure. The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a nucleic acid molecule" is considered to include a single or multiple nucleic acid molecules and is equivalent to the phrase "comprising at least one nucleic acid molecule." The term "or" refers to a single element of the recited alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B" means "including A, B, or A and B" without excluding additional elements. Unless otherwise defined, the definitions provided herein are controlling when the definitions herein may be different from other possible definitions.

[0105] As used herein, the term "gene set" refers to a collection of genes where the common denominator of the genes of a particular gene set is involvement in a particular biological process. Gene sets can be accessed at the Molecular Signatures Database (www.gsea-msigdb.org / gsea / msigdb / ). Without being bound by theory, gene sets can be considered surrogates for particular biological processes. All gene sets and their associated genes mentioned herein are hereby incorporated by reference in their entirety. Any of the gene sets and their associated genes can be modified or even discarded based on the feature selection criteria employed by any of the methods disclosed herein.

[0106] As used herein, the term "gene" means a nucleic acid in the genome of a subject that can be expressed to produce mRNA, in addition to intervening intron sequences, and in addition to regulatory regions that control the expression of the gene, such as a promoter or a fragment thereof.

[0107] As used herein, the term "immune score" (IS) refers to the name of a model that captures immune activity according to some embodiments. As used herein, the immune score is compatible with the "cytotoxicity score" (CS).

[0108] As used herein, the term "proliferation index" (PI) refers to the name of a model that captures aggressive tumor-intrinsic properties according to some embodiments. As used herein, the proliferation index is compatible with the term "genomic index" (GI).

[0109] As used herein, the term "integrated model" refers to a model that combines an immune score model and a proliferation index model according to some embodiments.

[0110] As used herein, the term "final model" refers to a model that combines an integrated model and the age of a patient according to some embodiments. In some embodiments, age is included as a continuous variable in the final model.

[0111] As used herein, the term "subject" encompasses any animal, preferably a mammal, including humans. Exemplary subjects include, but are not limited to, humans, primates, livestock (e.g., sheep, cows, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). Preferably, the mammal is a human or a primate. More preferably, the mammal is a human. The terms "subject" and "patient" are used interchangeably herein.

[0112] As used herein, the term "diagnosis", and variations thereof such as, but not limited to, "diagnose" or "diagnosing", includes, but is not limited to, a primary diagnosis of a clinical condition or any primary diagnosis of a clinical condition. Further, the term refers to the process of identifying a disease based on signs, symptoms, and various test results. Also, the conclusion reached through that process is also referred to as a "diagnosis". Forms of tests commonly performed include biopsies to retrieve tumors. In some embodiments, the prognosis may be the likelihood of subsequent (within 10 years, 15 years, or 20 years) invasive breast cancer events.

[0113] As used herein, the term "prognosis" refers to the outcome or course of a disease. In some embodiments provided herein, when this phrase is used in the context of a human already having invasive breast cancer, it refers to the likelihood that a subject having invasive breast cancer will subsequently (within a period of 10, 15, or 20 years) develop a contralateral invasive breast cancer event following surgical removal of the primary tumor. This outcome may include a) the likelihood of a contralateral breast event, b) the likelihood of a contralateral breast event occurring within a specific time (e.g., 1, 2, 3, or 5 years), c) the likelihood that a specific therapy (e.g., radiation) will prevent a contralateral breast event, d) the optimal treatment useful for preventing a contralateral event that matches the severity of the most likely event, or e) combinations thereof.

[0114] As used herein, the term "breast tumor" refers to a tumorous condition of breast tissue that can be either benign or malignant. The term "tumor" is synonymous with "neoplasm" and "lesion". Exemplary breast tumors include invasive breast cancer, ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and atypical ductal hyperplasia (ADH).

[0115] As used herein, the term "cancer" refers to a malignant neoplasm characterized by a characteristic lack of formation accompanied by loss of differentiation, an increased growth rate, invasion of surrounding tissues, and the ability to metastasize. The term "cancer" is intended to include diseases characterized by uncontrolled growth of cells within the subject, including but not limited to invasive breast cancer. In some embodiments, invasion of surrounding tissues is invasion of the basement membrane.

[0116] As used herein, the term "intraductal lesion" refers to a tumor that is confined to the interior of the duct and is thus not invasive breast cancer. Exemplary intraductal lesions include ADH and DCIS.

[0117] As used herein, ADH is a neoplastic intraductal (non-invasive) lesion characterized by the proliferation of uniformly distributed monomorphic breast epithelial cells.

[0118] As used herein, DCIS is a neoplastic intraductal (non-invasive) lesion characterized by an increase in breast epithelial proliferation with mild to marked cellular atypia. DCIS is graded (low, intermediate, and high) based on factors such as nuclear atypia, intraluminal necrosis, and mitotic activity. Low-grade DCIS and ADH are morphologically identical, and ADH is distinguished from DCIS based on the extent of the lesion when determined by the size of the lesion and / or the number of ducts involved. DCIS is typically first diagnosed from a tissue biopsy induced by suspicious findings (e.g., microcalcifications, abnormal masses, tissue distortion or asymmetry, etc.) on mammography and / or ultrasound imaging. It may also be diagnosed from routine screening images or, more rarely, from an imaging diagnosis induced by a positive physical examination (e.g., a palpable mass, nipple discharge, skin changes, etc.) or a significant change in a previously identified tumor.

[0119] Cell proliferation in DCIS is confined within the duct. If the proliferating cells penetrate the basement membrane of the myoepithelial cell (MEC) layer lining the duct and thus appear in the surrounding stroma, even if DCIS is present, the lesion is considered an invasive breast cancer. In some cases, the invasion is minimal (microinvasion) or the only evidence of invasion is disruption of the MEC layer (e.g., as observed by discontinuity in MEC-specific protein marker staining such as SMMHC and / or p63). Typically, these microinvasive cases are treated as invasive breast cancers, but there is debate about the treatment of these cases.

[0120] It is difficult to estimate the recurrence rate of DCIS with current treatments. However, it is thought that approximately 20% of patients who undergo breast tumor excision without any further treatment experience recurrence within 10 years, which is approximately equal between DCIS and invasive events, while less than 2% of patients who undergo mastectomy experience recurrence. The standard treatment for breast tumor excision is postoperative adjuvant radiotherapy (RT). Several randomized clinical trials provide evidence that postoperative adjuvant radiotherapy after breast tumor excision reduces the recurrence risk by about half in both types of DCIS and invasive events, and that current clinical and pathological evaluation techniques are unable to identify a low-risk subgroup in which the benefits of radiotherapy are not recognized.

[0121] As used herein, LCIS is a non-invasive lesion that occurs in the terminal duct-lobular unit of the breast and is generally small and often composed of loosely aggregated cells. When it spreads within the duct, it can be distinguished from DCIS based on morphology and / or marker staining.

[0122] As used herein, the term "invasive breast cancer" indicates that neoplastic (tumor) cells have penetrated and invaded through the epithelial basement membrane. This distinguishes invasive breast cancer from other hyperplastic (ductal hyperplasia) or dysplastic (atypical ductal hyperplasia, ADH) or non-invasive neoplastic (DCIS, LCIS) breast lesions characterized by an intact (non-invasive) basement membrane. It can be divided into stages (I, IIA, IIB, IIIA, IIIB, and IV). In some embodiments, any of the methods provided herein can be applied to invasive breast cancer to determine the success of radiotherapy for preventing recurrence of invasive breast cancer. In some embodiments, any of the methods provided herein can be applied to DCIS to determine the success of radiotherapy for preventing recurrence of DCIS cancer.

[0123] As used herein, the term "radiotherapy" (RT) refers to a therapy that involves or includes a therapeutic amount of radiation in any form to a subject.

[0124] As used herein, the term "RT boost" or "boost", when the latter is used in the context of RT, includes an additional / extra dose of radiation applied to a site. The site can be the tumor bed. The site can be the initial tumor site. The site can be any tissue or location that requires an additional or extra dose of radiation. The additional / extra dose can be integrated / used concomitantly (i.e., performed together with standard radiotherapy) or sequential (i.e., performed after standard radiotherapy). The additional / extra dose of radiation can be at least 10 Gy if performed sequentially or at least 5 Gy if integrated / used concomitantly. The additional / extra dose of radiation can be 16 Gy or less. The additional / extra dose can be provided using external radiotherapy, internal radiotherapy / sealed source brachytherapy, stereotactic radiotherapy, or intraoperative radiotherapy.

[0125] Surgery is the treatment of breast tumors and is frequently involved in diagnosis. The type of surgery depends on the extent of the tumor (tumor stage) at the time of diagnosis, as well as the type and grade of the tumor.

[0126] As used herein, the terms "treatment" and "therapy" are used interchangeably and do not require the subject to be completely or 100% cured. Instead, it encompasses a broader concept, or delaying the onset of one or more symptoms, extending the life and / or quality of life of the subject, reducing the severity of one or more symptoms, and the like.

[0127] As used herein, "standard treatment" or "standard" has the meaning customarily used by those skilled in the art in view of the present disclosure with respect to treatment or therapy. In some embodiments, standard treatment refers to treatment or treatment options recommended to patients under guidelines such as those provided by NCCN, ESMO, ESTRO, ASTRO, Clinical Practice Recommendations Australia, or NICE guidelines, and optionally any one or more of their respective guidelines as of June 2022. In some embodiments, the therapy recommended under standard treatment for a patient does not take into account the guidance provided by the analysis of the markers disclosed herein.

[0128] The terms "standard radiotherapy" and "standard radiation therapy" are used interchangeably herein and refer to a therapy that involves or includes some form of radiation in an amount that is therapeutic for the subject under the current standard treatment for breast cancer. In some embodiments, the standard treatment is any one defined by NCCN, ESMO, ESTRO, ASTRO, Clinical Practice Recommendations Australia, or NICE guidelines, and optionally any one or more of their respective guidelines as of June 2022. In some embodiments, the standard treatment is any one of those provided in Tables 14 and 15.

[0129] As used herein, the term "de-escalation of radiotherapy" includes omission of radiotherapy (i.e., not performing radiotherapy at all), or simply reducing the radiation dose or the number of fractions of a given dose fractionation scheme.

[0130] As used herein, the term "local recurrence" indicates that the recurrence is in the breast that has been surgically treated. As used herein, local recurrence is interchangeable with ipsilateral breast tumor recurrence (IBTR).

[0131] As used herein, the term "regional recurrence" indicates that the recurrence is in regional lymph nodes (axillary, supraclavicular, infraclavicular, within the pectoralis major muscle, or internal mammary lymph nodes).

[0132] As used herein, the term "distant metastasis" refers to all recurrences other than the above types of recurrence (local or regional). In other words, distant metastasis refers to recurrence in all other tissues of the body.

[0133] Method An enormous amount (about 10,000) of existing gene sets were tested in an extensive training cohort to identify and carefully select biological processes related to anti-tumor immune activation and tumor aggressiveness. This was done by calculating the enrichment of each gene set in each tumor according to the method by Barbie, D.A. et al., Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature, 2009, Vol. 462 (No. 7269): 108-112. This means that the expression levels of the genes included in a given gene set are ranked by a specific formula (empirical cumulative distribution function) in relation to all other genes. In this way, it is possible to determine whether a specific gene set is enriched in a tumor. Next, the gene sets were analyzed in a meta-analysis of the training cohort of breast cancer, and the gene sets that most influence the prognosis were selected.

[0134] To further improve the above method, two models were created by combining different gene sets that most influence prognosis. One is related to immune activation and was named the immune score (an example is shown in Table 4); the other is related to tumor aggressiveness and was named the proliferation index (an example is shown in Table 6). This was done by a method known as elastic net, i.e., the penalized linear regression method. Without being bound by theory, the immune score and the proliferation index are thought to strongly influence each other, and thus, they were integrated to create a new model called the integrated model. This was done by calculating, centering, and scaling the immune score and the proliferation index respectively within each training cohort, and then training a new elastic net that includes the interaction term between the immune score and the proliferation index. Thereafter, the cohorts were merged and the integrated model was fitted using the formula (immune score + proliferation index)^2.

[0135] This model (also called the "final model") is based on the quantification of tumor aggressiveness (measured by the proliferation index), immune activity (measured by the immune score), and patient age. The final model is effective in predicting prognosis and RT benefit. However, the groups created are heterogeneous; for example, the "intermediate risk" score can include patients with aggressive tumors (high proliferation index) with strong immune activity (high immune score), or patients with low aggressive tumors (moderate proliferation index) who do not benefit from the anti-tumor immune response. Aggressive tumors with abundant immune infiltration (high IS) and high PI correspond to an intermediate risk score in terms of the absolute risk of recurrence (i.e., when compared to all tumors including low-risk tumors). However, when compared to high-risk tumors based on clinical characteristics [e.g., histological grade III and / or ER-negative tumors and / or young patients (e.g., age less than 55 years)], this is considered a low-risk score. Similarly, PD-1 高 / PD-L1 高 / TIL 高Grade III tumors with [the given condition] also correspond to a low-risk score when compared to other Grade III tumors. However, when compared to all tumors (including low-risk tumors), they are probably considered to have a medium-risk score (although Grade III itself is an inherent marker of poor prognosis, this risk is downgraded from high risk to medium / low risk of recurrence due to an active immune response). Tumors with high IS, a medium risk score in the final model, and [the given marker] 高 / PD-L1 高 / TIL 高 Grade I / II tumors with [the given marker] are considered high-risk tumors when compared to other Grade I / II tumors. However, when compared to all tumors (including Grade III tumors which are of higher risk), they are considered to have a medium recurrence risk. Table 13 provides additional disclosure regarding methods other than tumor grade that can be used to define risk groups.

[0136] Also, by analyzing the proliferation index and immune score individually (which are always calculated for the final model), the specific biology of the tumor can be understood, and further individualization of treatment can be enabled. This can also affect other types of therapies (e.g., immunotherapy; when the final model score is medium and the immune score and proliferation score are high, it becomes an excellent candidate for immunotherapy). Therefore, this model provides information regarding prognosis and RT usefulness, and also provides information regarding the individual tumor biology, and is likely to serve as a guide for treatment with immunotherapy, chemotherapy, and other types of targeted therapies. A high recurrence risk may also indicate that mastectomy is more beneficial than breast-conserving surgery. Since this model has a very significant impact on the prognosis of distant metastasis, it is very useful, for example, in stratifying patients according to the benefit of chemotherapy. The proliferation index can be used to quantify tumor aggressiveness and to estimate the benefit from chemotherapy in patients with clinically low-risk tumors (e.g., luminal A tumors).

[0137] To further improve the integrated model, one final model including the patient's age with local recurrence as the endpoint was trained and named the final model. This was done in a public cohort called Servant. Figure 1 illustrates the overall method up to this step. In some embodiments, Figure 1 shows that all redundant biological information is filtered, and the distillate of the most important biological processes (represented by gene sets) is isolated, thereby enabling the creation of a model based on the interaction between genomic instability and immune activity. Figure 2 can be said to be a continuation of Figure 1 (i.e., the remainder of this part of Figure 1 is shown in Figure 2). Figure 2 shows how the most useful genes in each gene set included in the model (shown in Figure 1) were identified, and Figure 2 also shows the process of removing all redundant genes considering the correlation between different tissue types (fresh frozen vs. formalin-fixed paraffin-embedded), the heterogeneity of expression across different tumor cores, and the prognostic effect of individual genes.

[0138] Therefore, the above model uses all the genes included in each gene set in their calculations (a total of up to approximately 3,000 genes). To create a tool that works effectively clinically, it is necessary to significantly reduce the number of genes and exclude approximately 90 - 95% of all genes. Since reducing the number of genes makes the model unstable, high precision is required for this. In this tool, the following genes were selected.

[0139] Point 1. In formalin-fixed paraffin-embedded tissue (which is the type of tumor tissue preparation used clinically), this tissue type introduces significant non-biological dispersion, so the profile is good.

[0140] Point 2. Indicates low heterogeneity between different tumor cores (high heterogeneity makes the tool unstable and requires sampling multiple cores to increase reliability).

[0141] Point 3. Provide the most biological information (e.g., some genes have a better prognosis than others; these are the genes with the best prognosis that medical professionals / clinicians would like to find).

[0142] Points 1 and 2. In formalin-fixed paraffin-embedded (FFPE), mRNA is fragmented compared to fresh frozen (FF), so gene profiling is very poor. Also, cores are less reliable than, for example, whole sections (which evaluate a wider range of the tumor). To overcome this problem, 31 tumor samples were collected with cores from both fresh frozen samples and FFPE samples. By analyzing how genes correlate between two different cores and tissue types from the same tumor, it was possible to determine which genes showed sufficient reproducibility in FFPE tissue of another core. All genes were ranked by their rho value, which is a measure of this correlation. The analysis was performed on tumors from the SweBCG91RT cohort (Table 1) for which additional fresh frozen samples with gene expression information were available.

[0143] Point 3. To identify the genes that provide the most information, a meta-analysis of all genes (included in different gene sets included in the final model) in the training cohort was performed as shown in Table 3. Subsequently, the genes were ranked by their prognostic values. For genes from the gene set of the immune score, an analysis was performed for HER2 and basal tumors because the prognostic signal from immunological biomarkers is the most robust among these tumors (samples of these genes are listed in Table 4). All tumors were used to rank the genes of the proliferation index based on prognosis (samples of these genes are listed in Table 6).

Table 1

[0144] Gene Selection To determine the optimal method for gene selection and identify the optimal number of genes for the model to function, different methods for gene selection were evaluated. These methods are shown in FIGS. 3A - B and FIGS. 4A - B. The methods are as follows.

[0145] Rho: Genes were ranked according to the correlation between fresh frozen tissue and FFPE tissue, and the number of genes included was varied (from the highest correlation downwards).

[0146] P: Genes were ranked according to the p - values from a meta - analysis of public datasets (e.g., training cohorts), and values were imputed if the gene was missing in the dataset. For immune signature genes, the prognostic effects in HER2 and basal tumors were tested, and for PI - signature genes, the prognostic effects in whole tumors were tested. Genes were selected from the lowest p - values and increased.

[0147] Prognostic score: By calculating Rho / P, a value was created that considered both the FFPE - to - fresh - frozen correlation and the prognostic effect in the public dataset.

[0148] Prognostic score + rho > X: Here, first a correlation threshold (Rho) (e.g., 0.2, 0.3, 0.4) was set, and then the remaining genes were ranked based on that score.

[0149] To evaluate the method, instead of using all genes to calculate the enrichment of gene sets by the GSEA method (Barbie et al., 2009), the genes were normalized and the average value of the genes for each gene set was calculated. Further, unlike the GSEA method, gene sets are no longer used, and a smaller subset of genes is used instead. Next, the average value of the genes is multiplied by each coefficient in the original model. In some embodiments, the gene values are first normalized to the values of the housekeeping genes, and then the average value is calculated for the genes corresponding to each coefficient. In some embodiments, the housekeeping genes include one or more genes shown in Table 2 below. In some embodiments, the housekeeping genes include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more of the following genes) of the genes shown in Table 2 below.

Table 2

[0150] Figures 3A and 3B show different methods of genes for the immune score. The x-axis (N genes) indicates how many of the top-ranked genes from each gene set are selected. The y-axis in Figure 3A shows the p-value and the direction of association (negative value = favorable prognosis, positive value = unfavorable prognosis) of the method for grade III tumors (a model expected to be prognostically unfavorable). The y-axis in Figure 3B shows the p-value and direction of the interaction test with the original growth index for different methods (a negative value as close to zero as possible is preferred). In both Figures 3A and 3B, the Rho method tends to function best, and it can be seen that the usefulness drops sharply when there are less than 5 genes per gene set. 23 genes are included per signature (i.e., N genes = 23), and if the gene set contains >23 genes, the top-ranked 23 are selected, and otherwise, all are selected. Further, these methods can evaluate hundreds of genes for each gene set. In some embodiments, the genes are ranked based on the combination of the prognostic effect in the meta-analysis of the first cohort and Rho in the second cohort.

[0151] In some embodiments, genes with insufficiently predictable prognosis are removed, and then the remaining genes are ranked based on Rho (a measure of correlation). In the immune score, this is achieved by removing genes from the immunological gene set that have a coefficient with the opposite sign to the gene set coefficient of the corresponding gene set in the immune score model (for example, when the coefficient of the gene set in the immune score model is negative while the coefficient of the gene in the same gene set is positive), and by removing genes with p-values above a certain threshold (e.g., 0.05 or 0.1 or 0.2 or 0.3) in the meta-analysis of HER2+ tumors and basal cell tumors. In the proliferation index, this is achieved by removing genes from the tumor-intrinsic gene set that have a coefficient with the opposite sign to the gene set coefficient of the corresponding gene set in the proliferation index model, and by removing genes with p-values above a certain threshold (e.g., 0.05 or 0.1 or 0.2 or 0.3) in the meta-analysis of all subtypes. Subsequently, the genes are ranked from high to low Rho, and the top number of genes (denoted as N) are selected from each gene set.

[0152] In some embodiments, a composite score integrating the prognostic effect and Rho value is generated, and then all genes are ranked based on this score. This can be achieved as follows: 1) by multiplying the sign of the meta-analysis coefficient (negative if the gene is associated with a good prognosis and positive if associated with an unfavorable prognosis) by the p-value of the meta-analysis. This analysis is performed between HER2+ tumors and basal-type tumors for genes included in the gene set contained in the immune score, and between all subtypes for genes included in the gene set contained in the proliferation index; 2) by scaling and transforming the above variables while maintaining the sign. This can be achieved by normalizing the variables to have a mean of 0 and a standard deviation of 1; 3) by multiplying the above variables by the rho value. Optionally, the rho value can be scaled and transformed to have a mean of 0 and a standard deviation of 1; 4) for each gene set, by multiplying the variables generated above by the sign of the gene set coefficient in the model (if the coefficient is negative, multiply the variable by -1, and if the coefficient is positive, multiply the variable by 1). Then, the variables are ranked from high to low, and the top number of genes (denoted as N) is selected from each gene set.

[0153] In some embodiments, genes with insufficient correlation (i.e., Rho values below a certain threshold, e.g., 0.3) are removed, and then the remaining genes are ranked based on the prognostic effect. This is achieved as follows: 1) by multiplying the sign of the meta-analysis coefficient (negative if the gene is associated with a good prognosis and positive if it is associated with a poor prognosis) by the p-value of the meta-analysis. The analysis is performed between HER2+ tumors and basal-type tumors for genes included in the gene set contained in the immune score, and between all subtypes for genes included in the gene set contained in the proliferation index; 2) by scaling and transforming the above variables while maintaining the sign. This could be achieved by normalizing the variables such that the mean is 0 and the standard deviation is 1; 3) for each gene set, by multiplying the variable created above by the sign of the gene set coefficient in the model (if the coefficient is negative, multiply the variable by -1, and if the coefficient is positive, multiply the variable by 1). Then, rank the variables from high to low and select the top number of genes (denoted as N) from each gene set.

[0154] Figures 4A and 4B show different methods for genes of the proliferation index. The x-axis (N gene) indicates, as in Figures 3A and 3B, how many of the top-ranked genes are selected from each gene set. The y-axis in Figure 4A shows the p-value and the direction of the association of the method (negative value = prognostically favorable, positive value = prognostically unfavorable) (the model is expected to be prognostically unfavorable; a positive value as close to 0 as possible is desired). The Y-axis in Figure 4B shows the direction between the p-value of the interaction test and the original immune score of different methods (a negative value as close to 0 as possible is desired). In both Figure 4A and Figure 4B, the method called "Rho >= 0.2 + prognostic score" or the Rho method tends to function best, and it can be seen that the usability drops sharply when there are fewer than 25 genes per gene set. As shown in Figure 2, the prognostic score indicates Rho / Pmeta. Here, 60 genes per signature (i.e., N gene = 60) were selected. If the gene set contains >60 genes, the top-ranked 60 genes are selected, and if not, all are selected. Pmeta is a score based on a meta-analysis of the prognostic effects of each gene. In some embodiments, the top-ranked 5 genes were selected from each gene set. In some embodiments, 100 top-ranked genes were selected from each gene set. In some embodiments, any number of top-ranked genes from 5 to 100 were selected from each gene set.

[0155] Two main points from Figures 4A and 4B are that without the present disclosure, it is not clear which method is best (determined by extensive experiments), and not all gene combinations perform equally well, as shown by the sharp drop in performance as the number of genes included is severely restricted.

[0156] Final model Using the calculated and designated genes (shown in Tables 4, 5, 6, and 7) and the methods outlined above, a model can be created that includes 552 genes integrating immunological, tumor-intrinsic, and clinical (patient age) information.

[0157] Figure 5 shows the usage process of this tool. Generally speaking, it can be said that the calculation of the model functions best when the values of the constituent genes are normalized and scaled. This means that, for example, the values are related to a representative background population with a mean value of 0 and a standard deviation of 1. This step is preferably performed for each gene and for the models leading to the final model (immune score, proliferation index, integrated model).

[0158] As shown in Figure 5, in some embodiments, the expression levels of the genes included in the model are determined. Various methods are available for determining these expression levels, including, but not limited to, microarray, RNA-seq, PCR, qPCR (with normalization), Nanostring, etc. Preferably, the normalization of the expression levels uses either the housekeeping genes shown in Table 2 or a combination thereof. The normalization of the expression levels is not limited to using housekeeping genes as other normalization methods may be contemplated. In some embodiments, the genes are scaled relative to a representative tumor background population. The enrichment of the gene sets included in the model (representing different biological pathways) is calculated by summing the (normalized and standardized) values of the genes of each included gene set. The calculated enrichment score is standardized by comparing the patient's sample to a representative background population. Then, using the immunological model and the tumor-intrinsic model, IS and PI are calculated respectively. The scores of IS and PI are standardized by comparing them to a representative background population. The integrated model is employed to calculate the integrated score (as shown in Figure 11), and then it is further standardized by comparing it to a representative background population. To determine the final risk score, the final model including the patient's age is used. The patient's score is compared to a representative background population, and it is determined whether the patient is classified into the low / medium / high risk group, and appropriate treatment is provided based on the patient's risk group.

[0159] In some embodiments, the method can be described as in FIG. 12 and can include one or more steps being repeated or additional steps being added between each of the indicated steps. In some embodiments, the method can be described as in FIG. 13 and can include one or more steps being repeated or additional steps being added between each of the indicated steps.

[0160] General method In some embodiments, as shown in FIG. 14, the general steps for each tumor-intrinsic model and immunological model include identifying the relevant gene sets (e.g., the immunological gene set for IS and the tumor-intrinsic gene set for PI); selecting the most important gene sets; creating the IS model and the PI model; integrating the IS model and the PI model into an integrated model; and integrating the integrated model with the patient age to create a final model. When these steps are completed, the genes with the best performance among each selected gene set are filtered and become the list of finally selected genes. This reduces the number of genes in each gene set in the final model and improves the clinical utility of the method. After selection, the final model can be used to identify into which risk group the subject is classified using the final list of genes selected from each respective gene set. The gene set score can be calculated using the average value of the selected genes and is preferably normalized against one or more housekeeping genes shown in Table 2. In some embodiments, the final model does not vary according to the subject (i.e., the process of creating this model is not repeated for each subject). One or more of the method steps can be repeated, and it is contemplated that additional steps from any other disclosed method can be added between each of the described steps. Also, in creating the final model, it is contemplated that useful results can be obtained by performing minor rearrangements.

[0161] Although not limited, several permutations are possible, including adjusting the number of selected gene sets, adjusting the threshold for correlation filtering, adjusting the percentage of patients in which the PI has the lowest IS trained, adjusting the number of top-ranked genes from each gene set, and using housekeeping genes as a control. In some embodiments, the number of selected gene sets is adjusted. In some embodiments, the top 5 gene sets are selected. In some embodiments, the top 50 gene sets are selected. In some embodiments, the top 100 gene sets are selected. In some embodiments, the top 200 gene sets are selected. In some embodiments, the gene sets selected can range from the top 5 gene sets to the top 200 gene sets. In some embodiments, a Spearman Rho of 0.7 is used as the threshold for correlation / collinearity filtering. In some embodiments, a Spearman Rho of 0.5 is used as the threshold for correlation / collinearity filtering. In some embodiments, a Spearman Rho of 0.9 is used as the threshold for correlation / collinearity filtering. In some embodiments, a Spearman Rho in the range of 0.5 to 0.9 is used as the threshold for correlation / collinearity filtering. In some embodiments, the percentage of patients includes patients having the lowest 33% of the IS in which the PI was trained. In some embodiments, the percentage of patients includes patients having the lowest 5% of the IS in which the PI was trained. In some embodiments, the percentage of patients includes patients having the lowest 50% of the IS in which the PI was trained. In some embodiments, the percentage of patients includes patients having a range from the lowest 5% IS to the lowest 50% IS in which the PI was trained.

[0162] Regarding the number of top genes from each gene set, in some embodiments, the top 23 genes from each gene set for IS were used in a common method. In some embodiments, the top 5 genes from each gene set for IS were used in a common method. In some embodiments, the top 100 genes from each gene set for IS were used in a common method. In some embodiments, the top genes in the range from the top 5 genes to the top 100 genes from each gene set for IS were used in a common method. In some embodiments, the top 60 genes from each gene set for PI were used in a common method. In some embodiments, the top 5 genes from each gene set for PI were used in a common method. In some embodiments, the top 100 genes from each gene set for PI were used in a common method. In some embodiments, the top genes in the range from the top 5 genes to the top 100 genes from each gene set for PI were used in a common method. Different groups

[0163] The groups produced using this method correspond to the groups that were attempted to be identified using histological grade and PD-1 / PD-L1 / TIL. TIL refers to tumor-infiltrating lymphocytes.

[0164] "Programmed death protein 1", "PD-1", "PD1", "PDCD1" are used interchangeably herein and refer to the gene or gene product of PDCD1. In some embodiments, PD-1 is human PD-1. In some embodiments, the PD-1 protein has the amino acid sequence shown in SEQ ID NO: 1, regardless of the presence or absence of a signal peptide (underlined). [Chemical formula]

[0165] "PD-L1" and "PDL1" are used interchangeably herein and refer to the gene or gene product of CD274. In some embodiments, PD-L1 is human PD-L1. In some embodiments, the PD-L1 protein has the amino acid sequence set forth in SEQ ID NO: 2, regardless of the presence or absence of a signal peptide (underlined).

Chemical Structure

[0166] In some embodiments, the PD-L1 protein has the amino acid sequence set forth in SEQ ID NO: 3, regardless of the presence or absence of a signal peptide (underlined).

Chemical Structure

[0167] In some embodiments, the PD-L1 mRNA has the nucleotide sequence set forth in SEQ ID NO: 4, or a processed form thereof:

Chemical Structure

[0168] In some embodiments, the PD-L1 mRNA has the nucleotide sequence set forth in SEQ ID NO: 5, or a processed form thereof:

Chemical Structure

[0169] One group has grade III PD-1 低 / PD-L1 低 / TIL 低A high-risk non-responsive group corresponding to the group is included, which reflects the high-risk score of the final model. In one way, the high-risk score of the final model is independent of other clinical features. In another way, tumors can be more effectively identified by combining the histological grade (or other clinical markers of tumor aggressiveness such as ER-negative or pre-menopausal age) with the model. For example, tumors with histological grade III will have a high-risk score. In some embodiments, the high-risk score in the final model is combined with a medium / low immune score and a high proliferation index score. In some embodiments, the high-risk score in the final model is combined with a medium / low immune score and a high proliferation index score. This method is completely dependent on the in-house development model.

[0170] Another group includes grade III PD-1 高 / PD-L1 高 / TIL 高 An immune activation group corresponding to the group is included, which reflects the low / medium-risk score of the final model with a high proliferation index and a high immune score. In one way, the risk score of the final model is moderate. These tumors are most effectively identified by combining the histological grade (or other clinical markers of tumor aggressiveness) with the model. For example, tumors with histological grade III are combined with a low / medium score. In another way, they have a high immune score and a high proliferation index. These tumors are shown as medium / low scores in the final model. Looking at the proliferation index and the immune score separately is useful because low-risk tumors may also show medium / low scores in the final model. For low-risk tumors, instead, the score of the proliferation index is low / medium. This affects what treatment options should be selected for medium-score tumors.

[0171] Yet another group includes grade I / II PD-1 高 / PD-L1 高 / TIL 高A low-risk non-responsive group corresponding to the group is included, which reflects the medium / low-risk score of the final model. Humans grouped by the medium / low-risk score are those in the prior disclosure (International Application No. PCT / US2021 / 032080 filed on May 21, 2022 and International Application No. PCT / US2022 / 022934 filed on March 31, 2022, all of which are incorporated herein by reference) Grade I / II PD-1 高 / PD-L1 高 / TIL 高 Similar enhanced treatment as the group is required. In one method, this group includes the medium-risk score of the final model and a medium proliferation index score. This group can be distinguished from the "immune activation group" by having a low proliferation index score. Usually, this group also has a medium or high immune score. In another method, the histological grade (or other clinical markers of tumors with low tumor aggressiveness) is combined with the model. For example, tumors with a medium histological grade I / II in the final model, or tumors with a medium / high immune score and histological grade I / II.

[0172] In yet another group, there is a low-risk immunological group corresponding to the Grade I / II PD-1 高 / PD-L1 高 / TIL 高 group, which reflects the medium / low-risk score of the final model. These tumors require similar enhanced treatment as the prior application's Grade I / II PD-1 高 / PD-L1 高 / TIL 高 group. In one method, this method obtains a low / medium-risk score with a low / medium proliferation index and a medium / high immune score from the final model. In another method, this method combines the histological grade (or other clinical markers of low tumor invasiveness of the tumor) with the model. For example, tumors with histological grade I / II will have a medium-risk score in the final model and preferably a high immune score.

[0173] The intensified treatment is more aggressive than the standard radiotherapy provided herein. In some embodiments, the intensified treatment includes a radiotherapy treatment (e.g., intensified radiotherapy). In some embodiments, the radiotherapy treatment is whole breast external beam radiotherapy, partial breast radiotherapy or brachytherapy, or a combination thereof. In some embodiments, the radiotherapy treatment (e.g., intensified radiotherapy) includes using a biological effective dose (BED) of about 73 Gy or more, such as about 78 Gy or more, about 83 Gy or more, about 87 Gy or more, about 93 Gy or more, about 97 Gy or more, about 100 Gy or more, or about 104 Gy or more, about 111 Gy or more, or about 133 Gy or more. In some embodiments, the radiotherapy treatment includes using an appropriate biological effective dose (BED) where the tumor alpha / beta ratio is 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the radiotherapy treatment includes using a BED of 73 Gy or more where the tumor alpha / beta ratio is 5. In some embodiments, the radiotherapy treatment includes using a BED of 78 Gy or more where the tumor alpha / beta ratio is 4. In some embodiments, the radiotherapy treatment includes using a BED of 87 Gy or more where the tumor alpha / beta ratio is 3. In some embodiments, the radiotherapy treatment includes using a BED of 104 Gy or more where the tumor alpha / beta ratio is 2. In some embodiments, the radiotherapy treatment uses a BED of 93 Gy or more where the tumor alpha / beta ratio is 5. In some embodiments, the radiotherapy treatment uses a BED of 100 Gy or more where the tumor alpha / beta ratio is 4. In some embodiments, the radiotherapy treatment uses a BED of 111 Gy or more where the tumor alpha / beta ratio is 3. In some embodiments, the radiotherapy treatment uses a BED of 133 Gy or more where the tumor alpha / beta ratio is 2 when the patient is recommended a boosting dose in accordance with the guidelines.In some embodiments, BED is defined as a measure of the true biological dose delivered to tissue characterized by a particular radiosensitivity (alpha / beta ratio) as a function of the combination of dose per fraction (d) and number of fractions (n): BED = n × d (1 + d / (α / β)), where α (alpha) is the linear dose damage response and β (beta) is the secondary dose response in the tissue. Without being bound by theory, the alpha / beta ratio generally indicates how resistant a cell or tissue is to radiation damage.

[0174] In some embodiments, an enhanced treatment plan comprises treating a subject with an enhanced radiotherapy that includes at least one dose of: adding a boosting dose to the standard recommended treatment for the subject when the standard recommended treatment does not include a boosting dose and the dose is 67 Gy or greater, increasing the boosting dose beyond the standard amount for the subject, increasing the dose per fraction beyond the standard amount for the subject, or increasing the number of fractions of the recommended dose beyond the standard amount for the subject. In some embodiments, an enhanced treatment plan indicates at least one of: an enhanced radiotherapy treatment, a systemic therapy, a mastectomy, an additional use of a sensitizer for another therapy, a therapy above the level set by at least one of the NCCN, ESMO, ESTRO, Clinical Practice Recommendations Australia, and / or NICE guidelines for the remaining indicators of the subject, or any combination thereof provided in Tables 14 and 15.

[0175] In any method of the present disclosure, in some embodiments, the method is for a subject for whom boost is not otherwise recommended (e.g., not recommended as standard treatment or without guidance provided by biomarker analysis herein), and the intensified radiation treatment regimen includes whole breast external radiation therapy, partial breast radiation therapy, or brachytherapy, or a combination thereof, with a tumor alpha / beta ratio of 5 and a biological effective dose (BED) of 73 Gy or more, or a tumor alpha / beta ratio of 4 and a BED of 78 Gy or more, or a tumor alpha / beta ratio of 3 and a BED of 87 Gy or more, or a tumor alpha / beta ratio of 2 and a BED of 104 Gy or more. In some embodiments, for a subject with a recommended boost otherwise, the intensified radiation treatment regimen is one or more of whole breast external radiation therapy, partial breast radiation therapy, brachytherapy, or a combination thereof, and for patients for whom boost is recommended according to current guidelines, with a tumor alpha / beta ratio of 5 and a biological effective dose (BED) of 93 Gy or more, a tumor alpha / beta ratio of 4 and a BED of 100 Gy or more, or a tumor alpha / beta ratio of 3 and a BED of 111 Gy or more, or a tumor alpha / beta ratio of 2 and a BED of 133 Gy or more.

[0176] In some embodiments, an enhanced treatment, e.g., enhanced radiation therapy, or a more aggressive treatment, is enhanced in one or more relevant aspects of the treatment as compared to a standard treatment therapy. In some embodiments, the exposure of a subject to a therapeutic agent (e.g., radiation, antibody, cytotoxic agent, etc.) is increased in an enhanced treatment or a more aggressive treatment as compared to a standard treatment therapy in a meaningful way, e.g., to achieve a better prognosis. In some embodiments, the exposure period to a therapeutic agent (e.g., radiation, antibody, cytotoxic agent, etc.) is increased as compared to a standard treatment in an enhanced treatment or a more aggressive treatment. In some embodiments, the exposure amount to a therapeutic agent (e.g., radiation, antibody, cytotoxic agent, etc.) increases as compared to a standard treatment therapy in an enhanced treatment or a more aggressive treatment. In some embodiments, the number and / or frequency of exposure to a therapeutic agent (e.g., radiation, antibody, cytotoxic agent, etc.) increases as compared to a standard treatment therapy in an enhanced treatment or a more aggressive treatment. In some embodiments, the enhanced treatment or the more aggressive treatment increases by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, or any percentage within a range defined by any two of the preceding values, in at least one aspect (e.g., at least one of the length, amount, number, frequency of exposure to the treatment agent) as compared to a standard treatment therapy.

[0177] Yet another group includes grade I / II PD-1 低 / PD-L1 低 / TIL 低A low-risk favorable group corresponding to the group is included, which reflects the low-risk score of the final model. In some embodiments, the low-risk score of the final model is independent of other clinical features. In some embodiments, the low-risk score of the final model is combined with a low proliferation index score and a low immune score. In some embodiments, grade I / II tumors (and / or having other markers of good prognosis such as ER / PgR positive, elderly (>60 / 70 years old), low Ki67, HER2 negative, etc.) and the low-risk score from the final model are combined with a low immune score and a low proliferation index score.

[0178] Performance in the SweBCG91RT cohort As shown in FIG. 6, the final model can be used for all tumor types, including all patients from the SweBCG91RT cohort. Patients are grouped based on quartiles (<25 th means less than the 25th percentile, etc.). A high-risk score (preferably in relation to the background population) leads to treatment intensification (grade III PD-1 低 / PD-L1 低 / TIL 低 group, grade I / II PD-1 高 / PD-L1 高 / TIL 高 group, with a prescription similar to that of). The high-risk score is a score compared to a representative tumor and the background population of patients. The threshold for the high-risk score can be any above the 60th percentile (e.g., 67th percentile, 75th percentile, 80th percentile, etc.). The low-risk score is (grade III PD-1 高 / PD-L1 高 / TIL 高 group, and grade I / II PD-1 低 / PD-L1 低 / TIL 低Treatment according to a similar prescription as the group results in de-intensification. The low-risk score is the score compared to a representative tumor and patient background population. The threshold for the low-risk score can be any below the 40th percentile (e.g., 5th percentile, 15th percentile, 25th percentile, etc.). The medium-risk score results in standard treatment or, under special circumstances, treatment according to the description in the above paragraph. The medium-risk score is the score compared to a representative tumor and patient background population. The threshold for the medium-risk score can be any interval between the 30th and 70th percentiles (e.g., 30 - 70th percentile, 40 - 60th percentile, 45 - 65th percentile, etc.). The high, medium, and low risk scores are also summarized in Table 13.

[0179] This model can also be used for high-risk tumor types, as shown in Figure 7, which includes tumor patients under 70 years old with grade III tumors from the SweBCG91RT cohort and patients under 60 years old. Patients are grouped based on tertiles. High-risk patients can be defined as having grade III tumors, or being under 60 years old, or having estrogen receptor-negative tumors, or having a high recurrence risk according to other prognostic tests. For high-risk patients, the following can be said: The high-risk score (preferably in relation to the background population) leads to treatment intensification (grade III PD-1 低 / PD-L1 低 / TIL 低 group with a similar prescription); the low-risk score leads to treatment de-intensification (grade III PD-1 高 / PD-L1 高 / TIL 高 group with the same prescription). The thresholds for the high-risk score and the low-risk score are the same as those for the high-risk score and the low-risk score summarized in Table 13.

[0180] Furthermore, different groups Also provided herein is a method for treating breast cancer, the method comprising determining tumor aggressiveness, wherein tumor aggressiveness includes the histological grade of the tumor from at least a portion of a tumor sample provided by a subject, the histological grade including Grade I, Grade II, and Grade III, classifying the sample as low risk if determined to be a Grade I tumor, classifying the sample as high risk if determined to be a Grade III tumor, and determining the tumor aggressiveness of the sample if determined to be a Grade II tumor, wherein tumor aggressiveness further includes the proliferation index score of the sample. In some embodiments, the proliferation index score is based on the expression of a first group of genes. In some embodiments, the first group of genes includes one or more genes listed in Table 6. In some embodiments, the method further includes classifying a Grade II tumor as a) high risk if the proliferation index score is greater than or equal to the median score of the background population of Grade III tumors, or b) low risk if the proliferation index score is less than the median score of the background population of Grade III tumors, and determining the immune score of the sample, the level of tumor infiltrating lymphocytes (TILs) in the sample, the level of checkpoint molecules in the sample, or any combination thereof to determine immunological activity. In some embodiments, the immune score is based on the expression of a second group of genes. In some embodiments, the second group of genes includes one or more genes listed in Table 4. In some embodiments, the checkpoint molecules include programmed cell death protein-1 (PD-1) and programmed death ligand 1 (PD-L1). In some embodiments, immunological activity is determined to be active if i) the TIL score is 10% or greater and the checkpoint molecule score is 1% or greater (e.g., 1% or more of lymphocytes with positive staining for PD-1 or PD-L1), or ii) inactive if the TIL score is less than 10% and / or the checkpoint molecule score is less than 1% (e.g., less than 1% of lymphocytes with positive staining for both PD-1 and PD-L1).In some embodiments, the method further includes integrating tumor aggressiveness and immunological activity by training an elastic net having an interaction term between tumor aggressiveness and immunological activity, and determining a treatment plan based on the integration of tumor aggressiveness and immunological activity. In some embodiments, the method further includes classifying a grade II tumor as high risk if the proliferation index score is greater than or equal to the median score of the background population of grade II tumors. In some embodiments, the method further includes classifying a grade II tumor as high risk if the proliferation index score is greater than or equal to a cutoff value that exceeds the 67th percentile of the background population of grade II tumors. In some embodiments, the method further includes classifying a grade II tumor as low risk if the proliferation index is less than the median score of the background population of grade II tumors. In some embodiments, the method further includes classifying a grade II tumor as low risk if the proliferation index is less than a cutoff value that is below the 67th percentile (e.g., 67th percentile, 50th percentile, 33rd percentile, etc.) of the background population of grade II tumors.

[0181] In some embodiments, active immunological activity indicates an activated immune infiltration. In some embodiments, inactive immunological activity indicates an inactive immune infiltration. In some embodiments, the treatment plan includes standard radiotherapy or omission of radiotherapy if the sample is classified as a high-risk grade II tumor and the immunological activity is active. In some embodiments, the treatment plan includes intensified radiotherapy if the sample is classified as a high-risk grade II tumor and the immunological activity is inactive. In some embodiments, the treatment plan includes intensified radiotherapy if the sample is classified as a low-risk grade II tumor and the immunological activity is active. In some embodiments, the treatment plan includes de-intensified radiotherapy or omission of radiotherapy if the sample is classified as a low-risk grade II tumor and the immunological activity is inactive.

[0182] Also provided herein is a method for treating breast cancer, the method comprising determining tumor aggressiveness, wherein tumor aggressiveness includes the histological grade of the tumor from at least a portion of a tumor sample provided by a subject, the histological grade including Grade I, Grade II, and Grade III, classifying the sample as low risk if determined to be a Grade I tumor, classifying the sample as high risk if determined to be a Grade III tumor, and determining the tumor aggressiveness of the sample if determined to be a Grade II tumor, wherein tumor aggressiveness further includes the proliferation index score of the sample, and classifying a Grade II tumor as high risk if the proliferation index score exceeds the threshold of 60-95 percentile compared to a background population of representative Grade II tumors, or classifying as low risk if the proliferation index score is below the threshold of 30-60 percentile compared to a background population of representative Grade II tumors. In some embodiments, a Grade II tumor is classified as high risk if the proliferation index score exceeds the threshold of 60 percentile or more compared to a background population of representative Grade II tumors. In some embodiments, the method further comprises determining the immune score of the sample, the level of TILs in the sample, the level of checkpoint molecules in the sample, or any combination thereof, to determine immunological activity, integrating tumor aggressiveness and immunological activity by training an elastic net having an interaction term between tumor aggressiveness and immunological activity, and determining a treatment plan based on tumor aggressiveness, immunological activity, and the interaction term. In some embodiments, the proliferation index score is based on the expression of a first group of genes. In some embodiments, the immune score is based on the expression of a second group of genes. In some embodiments, the checkpoint molecules include PD-1 and PD-L1.In some embodiments, the immunological activity is active if i) the TIL score is 10% or more and the checkpoint molecule score is 1% or more of lymphocytes having positive staining for PD-1 or PD-L1, or ii) the TIL score is less than 10% and the checkpoint molecule score is less than 1% of lymphocytes having positive staining for both PD-1 and PD-L1. In some embodiments, an active immunological activity indicates an activated immune infiltration. In some embodiments, an inactive immunological activity indicates an inactive immune infiltration. In some embodiments, the first gene group includes one or more genes listed in Table 6. In some embodiments, the second gene group includes one or more genes listed in Table 4. In some embodiments, the treatment plan is as follows: c) if the sample is classified as a high-risk grade II tumor and the immunological activity is active, standard radiotherapy or omission of radiotherapy; d) if the sample is classified as a high-risk grade II tumor and the immunological activity is inactive, intensification of radiotherapy; e) if the sample is classified as a low-risk grade II tumor and the immunological activity is active, intensification of radiotherapy; f) if the sample is classified as a low-risk grade II tumor and the immunological activity is inactive, de-intensification of radiotherapy or omission of radiotherapy; g) if the sample is classified as a grade III tumor and the immunological activity is inactive, intensification of radiotherapy; h) if the sample is classified as a grade III tumor and the immunological activity is active, de-intensification of radiotherapy or omission of radiotherapy; i) if the sample is classified as a grade I tumor and the immunological activity is active, intensification of radiotherapy; or j) if the sample is classified as a grade I tumor and the immunological activity is inactive, de-intensification of radiotherapy or omission of radiotherapy.

[0183] Also provided herein is a method for treating breast cancer, the method comprising subclassifying a tumor from at least a portion of a tumor sample provided by a subject, the subtypes being luminal A, luminal B, HER2 +and comprising triple-negative / basal, determining the tumor aggressiveness of a sample if the sample is a luminal A tumor or a luminal B tumor, (wherein the tumor aggressiveness includes the proliferation index of the tumor sample), if the sample is subclassified as a luminal A tumor, classifying the sample as a) high risk if the proliferation index score exceeds the threshold of 60-95 percentile compared to the background population of representative luminal A tumors, or b) low risk if the proliferation index score is below the threshold below the 60th percentile compared to the background population of representative luminal A tumors, if the sample is subclassified as a luminal B tumor, classifying the sample as c) high risk if the proliferation index score exceeds the threshold of 60-95 percentile compared to the background population of representative luminal B tumors, or d) low risk if the proliferation index score is below the threshold below the 60th percentile compared to the background population of representative luminal B tumors, scoring the immune score of the sample, scoring the level of TIL in the sample, scoring the level of checkpoint molecules in the sample, or determining immunological activity including any combination thereof, integrating tumor aggressiveness and immunological activity by training an elastic net having an interaction term between tumor aggressiveness and immunological activity, and determining a treatment plan based on the integrated tumor aggressiveness, immunological activity, and interaction term. In some embodiments, the scoring of the proliferation index is based on the expression of a first group of genes. In some embodiments, the scoring of the immune score is based on the expression of a second group of genes. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, the immunological activity is determined to be active if i) the TIL score is 10% or more and any of the checkpoint molecule scores is 1% or more, or ii) the TIL score is less than 10% and / or the checkpoint molecule score is less than 1%. In some embodiments, the active immunological activity indicates an activated immune infiltration. In some embodiments, the inactive immunological activity indicates an inactive immune infiltration.In some embodiments, the first gene group comprises one or more genes listed in Table 6. In some embodiments, the second gene group comprises one or more genes listed in Table 4. In some embodiments, the treatment plan is as follows: e) omission of radiotherapy if the tumor sample is classified as a low-risk luminal A tumor; f) standard radiotherapy, de-escalation of radiotherapy, or omission of radiotherapy if the sample is classified as a high-risk luminal B tumor and the immunological activity is active; or g) intensification of radiotherapy if the tumor sample is classified as a high-risk luminal B tumor and the immunological activity is inactive.

[0184] In some embodiments, when classified as a subtype of luminal A tumor, the sample is classified as high-risk if the proliferation index score exceeds a threshold of 60th percentile or higher compared to the background population of representative luminal A tumors. In some embodiments, when the sample is classified as a subtype of luminal B tumor, the sample is classified as high-risk if the proliferation index score exceeds a threshold of 60th percentile or higher compared to the background population of representative luminal B tumors.

[0185] Also provided herein is a method for treating breast cancer, which includes determining tumor aggressiveness from at least a portion of a tumor sample provided by a subject, wherein the tumor aggressiveness includes a proliferation index that classifies the tumor as a) high-risk if the proliferation index score of the sample exceeds a threshold of 60-95th percentile compared to the background population of representative tumors, or b) low-risk if the proliferation index score of the sample is below a threshold below the 60th percentile compared to the background population of representative tumors, determining the immunological activity of the tumor based on the immune score of the sample, integrating the tumor aggressiveness and the immunological activity based on the interaction term between the tumor aggressiveness and the immunological activity, and determining a treatment plan based on the tumor aggressiveness, the immunological activity, and the interaction term. In some embodiments, the tumor is classified as high-risk if the proliferation index score of the sample exceeds a threshold of 60th percentile or higher compared to the background population of representative tumors.

[0186] Also provided herein is a method for treating breast cancer, which includes determining the tumor aggressiveness from at least a part of a tumor sample provided by a subject, where the tumor aggressiveness includes a proliferation index score for classifying the tumor as a) high risk if the proliferation index score of the sample is greater than or equal to the median score of a representative tumor background population, or b) low risk if the proliferation index score of the sample is less than the median score of a representative tumor background population, determining the immunological activity of the tumor based on the immune score of the sample, integrating the tumor aggressiveness and the immunological activity based on an interaction term between the tumor aggressiveness and the immunological activity, and determining a treatment plan based on the tumor aggressiveness, the immunological activity, and the interaction term.

[0187] In some embodiments, if the tumor is classified as high risk and the immunological activity is active, the treatment plan includes standard radiotherapy, de-escalation of radiotherapy, or omission of radiotherapy. In some embodiments, if the tumor is classified as high risk and the immunological activity is inactive, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low risk and the immunological activity is active, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low risk and the immunological activity is inactive, the treatment plan includes de-escalation of radiotherapy or omission of radiotherapy.

[0188] In some embodiments, the integration step includes training an elastic net having an interaction term between the tumor aggressiveness and the immunological activity. In some embodiments, a high proliferation index score indicates an aggressive tumor. In some embodiments, if the sample is classified as an aggressive tumor and the immunological activity is inactive, the treatment includes intensification of radiotherapy.

[0189] In some embodiments, a high proliferation index score includes a proliferation index of a sample that is at least at the 60th percentile compared to a background population of representative tumors. In some embodiments, a high proliferation index score indicates an aggressive tumor. In some embodiments, if the sample is classified as an aggressive tumor and the immunological activity is inactive, the treatment includes intensification of radiotherapy. In some embodiments, a high proliferation index score includes a proliferation index of a sample that is greater than or equal to the median score of a background population of representative tumors.

[0190] In some embodiments, the scoring of the proliferation index is based on the expression of a first gene group. In some embodiments, the first gene group includes one or more genes listed in Table 6. In some embodiments, the scoring of the immune score is based on the expression of a second gene group. In some embodiments, the second gene group includes one or more genes listed in Table 4.

[0191] In some embodiments, the determination of immunological activity further includes scoring TILs in the sample. In some embodiments, active immunological activity includes activated tumor infiltration. In some embodiments, activated tumor infiltration includes a TIL score of 10% or more. In some embodiments, the step of determining immunological activity further includes measuring the expression of one or more checkpoint molecules. In some embodiments, the step of determining immunological activity further includes measuring the expression of one or more checkpoint molecules. In some embodiments, the checkpoint molecules include PD-1 and PD-L1.

[0192] In some embodiments, the activated immunological activity includes activated tumor infiltration. In some embodiments, the activated tumor infiltration includes positive staining for the expression of one or more checkpoint molecules. In some embodiments, the step of determining the immunological activity further includes scoring the TILs in the sample. In some embodiments, the activated tumor infiltration further includes a TIL score of 10% or more. In some embodiments, the activated tumor infiltration further includes a TIL score of 10% or more and a checkpoint molecule score of more than 1%. In some embodiments, the inactive immunological activity includes a TIL score of less than 10%.

[0193] In some embodiments, the integration step is performed by training an elastic net having an interaction term between tumor aggressiveness and immunological activity. In some embodiments, the tumor aggressiveness further includes the histological grade of the tumor sample. In some embodiments, the histological grade of the tumor sample is determined as a grade II tumor. In some embodiments, the method further includes determining the subtype of the tumor sample before determining the tumor aggressiveness, and the subtype includes luminal A, luminal B, HER2 + , and triple-negative / basal. In some embodiments, the subtype is luminal A. In some embodiments, the subtype is luminal B.

[0194] Also provided herein is a method for treating breast cancer, the method comprising determining the histological grade of a tumor from at least a sample of the tumor provided by a subject, the histological grade of the tumor including Grade I, Grade II, or Grade III, and assigning the tumor aggressiveness of the sample as a) low risk if the sample is determined to be a Grade I tumor; b) high risk if the sample is determined to be a Grade III tumor; c) low risk if the sample is determined to be a Grade II tumor and the proliferation index score of the sample is compared to the background population of the tumor; or d) high risk if the sample is determined to be a Grade II tumor and the proliferation index score of the sample is compared to the background population of the tumor, and classifying the immunological activity as active or inactive, the classification including determining e) the immune score of the sample; f) the level of TILs in the sample; g) the level of checkpoint molecules in the sample; or h) any combination of e) - f), and integrating the tumor aggressiveness and the immunological activity using an interaction term to determine the benefit of the treatment plan. In some embodiments, the immunological activity is active when f) is 10% or more and g) at least one of the checkpoint molecules is 1% or more. In some embodiments, an active immunological activity exhibits activated tumor infiltration. In some embodiments, the activated tumor infiltration includes positive staining for the expression of one or more checkpoint molecules. In some embodiments, the immunological activity is inactive when f) is less than 10% and / or g) is less than 1% for both checkpoint molecules. In some embodiments, in case c), the proliferation index score of the sample is below the threshold between the 20th and 60th percentiles of the representative background population of the tumor. In some embodiments, in case c), the proliferation index score of the sample is below the 60th percentile of the representative background population of the tumor. In some embodiments, in case c), the proliferation index score of the sample is below the median of the background population of the tumor. In some embodiments, in case d), the proliferation index score of the sample is greater than or equal to the 60th - 95th percentile of the background population of the tumor.In some embodiments, in case d), the growth index score of the sample is at or above the 60th percentile of the tumor background population. In some embodiments, in case d), the growth index score of the sample is greater than or equal to the median of the tumor background population. In some embodiments, for c) and / or d), the tumor background population is the background population of grade II tumors. In some embodiments, for c) and / or d), the tumor background population is the background population of grade III tumors. In some embodiments, for c) and / or d), the tumor background population is the background population of grade I tumors.

[0195] In some embodiments, the tumor is classified as high-risk, the immunological activity is active, and the treatment plan includes standard radiotherapy, de-escalation of radiotherapy, or omission of radiotherapy. In some embodiments, if the tumor is classified as high-risk and the immunological activity is inactive, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low-risk and the immunological activity is active, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low-risk and the immunological activity is inactive, the treatment plan includes de-escalation of radiotherapy (including omission of radiotherapy).

[0196] In some embodiments, the integration step includes training an elastic net having an interaction term between tumor aggressiveness and immunological activity. In some embodiments, a high growth index score indicates an aggressive tumor. In some embodiments, if the sample is classified as an aggressive tumor and the immunological activity is inactive, the treatment includes intensification of radiotherapy. In some embodiments, the growth index score is based on the expression of a first gene group. In some embodiments, the first gene group includes one or more genes listed in Table 6. In some embodiments, the immune score is based on the expression of a second gene group. In some embodiments, the second gene group includes one or more genes listed in Table 4. In some embodiments, the checkpoint molecules include PD-1 and PD-L1.

[0197] Also provided herein is a method for treating breast cancer, which comprises supplying a sample of a tumor and receiving treatment based on an analysis of the sample, the analysis including determining the tumor aggressiveness of the tumor from the sample, determining the immunological activity of the tumor from the sample, and integrating the tumor aggressiveness and the immunological activity based on an interaction term. In some embodiments, the tumor aggressiveness includes a proliferation index score that classifies the tumor as high risk or low risk. In some embodiments, the immunological activity includes a) an immune score of the sample; b) the level of TILs in the sample; c) the level of checkpoint molecules in the sample; or d) any combination of a) to c).

[0198] In some embodiments, the tumor is classified as high risk if the proliferation index score of the sample exceeds a threshold of 60-95 percentile compared to a background population of representative tumors, or as low risk if the proliferation index score of the sample is below a threshold below 60 percentile compared to a background population of representative tumors. In some embodiments, the tumor is classified as high risk if the proliferation index score of the sample is greater than or equal to the median of a background population of representative tumors, or as low risk if the proliferation index score of the sample is less than the median of a background population of representative tumors. In some embodiments, if the proliferation index score of the sample exceeds a threshold of 60 percentile or higher compared to a background population of representative tumors, the tumor is classified as high risk. In some embodiments, if the tumor is classified as high risk and the immunological activity is active, the treatment plan includes standard radiotherapy, de-intensification of radiotherapy, or omission of radiotherapy. In some embodiments, if the tumor is classified as high risk and the immunological activity is inactive, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low risk and the immunological activity is active, the treatment plan includes intensification of radiotherapy. In some embodiments, if the tumor is classified as low risk and the immunological activity is inactive, the treatment plan includes de-intensification or omission of radiotherapy.

[0199] In some embodiments, a high proliferation index score indicates that the tumor is aggressive. In some embodiments, when the tumor is indicated to be aggressive, the treatment includes intensification of radiation therapy. In some embodiments, the activated immunological activity includes activated tumor infiltration. In some embodiments, the activated tumor infiltration includes a TIL score of 10% or more. In some embodiments, the inactivated tumor infiltration includes a TIL score of less than 10%. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, the activated tumor infiltration further includes positive staining of at least 1% of lymphocytes for the expression of one or more checkpoint molecules.

[0200] In some embodiments, the tumor aggressiveness further includes the histological grade of a sample of the tumor. In some embodiments, the histological grades include Grade I, Grade II, and Grade III. In some embodiments, the tumor analysis further includes determining the subtype of the tumor sample before determining the tumor aggressiveness, and the subtypes include luminal A, luminal B, HER2 + , and triple-negative / basal. In some embodiments, the subtype is luminal A. In some embodiments, the subtype is luminal B.

[0201] In some embodiments, the proliferation index scoring is based on the expression of a first gene group. In some embodiments, the first gene group includes one or more genes listed in Table 6. In some embodiments, the immune score is based on the expression of a second gene group. In some embodiments, the second gene group includes one or more genes listed in Table 4. In some embodiments, the integration step includes training an elastic net, and the elastic net includes an interaction term between tumor aggressiveness and immunological activity. In some embodiments, the elastic net further includes age information of the subject who provided the sample. In some embodiments, the analysis further includes stratification by pre-menopausal and post-menopausal status. In some embodiments, the pre-menopausal status includes female subjects under 55 years old. In some embodiments, the post-menopausal status includes female subjects 55 years old and above.

[0202] In some embodiments, a method of treating a subject is provided, the method comprising determining a proliferation index based on one or more levels of genes from Table 6, determining an immune score based on the expression of one or more genes from Table 4, combining the proliferation index and the immune score, optionally including the age of the subject as a factor, to determine whether the subject will respond to cancer treatment, and administering cancer treatment when the proliferation index, the immune score, and optionally the age of the subject indicate that the therapy will be successful. In some embodiments, a) if the proliferation index score of the sample exceeds a threshold between the 60th and 95th percentiles compared to a background population of representative tumors, it is high risk, or b) if the proliferation index score of the sample is below a threshold below the 60th percentile compared to a background population of representative tumors, it is low risk. In some embodiments, c) the proliferation index and the immune score are combined based on an interaction term therebetween, and d) cancer treatment is determined based on the proliferation index, the immune score, and the interaction term.

[0203] In some embodiments, a method of treating a subject is provided, the method including including the level of one or more genes from Table 6 as a factor, including the level of one or more genes from Table 4 as a factor, and including the age of the subject as a factor, thereby determining whether the subject should receive standard radiotherapy, intensified radiotherapy, de-intensified radiotherapy, or omission of radiotherapy. More specifically, this information must be extracted from Tables 4 and 6. In some embodiments, a method of treating a subject is provided, the method including including the level of one or more genes for each gene set in Table 6 (the "gene set" column) as a factor and multiplying by each coefficient in Table 6 (the "coefficient" column), and including the level of one or more genes for each gene set in Table 4 (the "gene set" column) as a factor and multiplying by each coefficient in Table 4 (the "coefficient" column). In some embodiments, the level of one or more genes in Table 6 includes one or more levels of one or more genes in Table 6, and the level of one or more genes in Table 4 includes one or more levels of one or more genes in Table 4.

[0204] In some embodiments, the expression of one or more genes related to IS and / or PI is measured from a sample derived from the subject. In some embodiments, the sample includes a tissue sample. In some embodiments, the tissue sample includes a formalin-fixed paraffin-embedded tissue sample. In some embodiments, the tissue sample includes or essentially consists of a core biopsy. In some embodiments, the sample does not include TIL. In some embodiments, the analysis does not measure the TIL level. In some embodiments, the sample consists of a core biopsy. In some embodiments, the sample is collected prior to treatment of the subject for cancer. In some embodiments, the sample is collected prior to the subject's therapeutic surgery for cancer.

[0205] In some embodiments, age is used as a factor in determining an appropriate therapy or treatment. In some embodiments, a subject under 50 years of age is considered young and a subject 50 years of age or older is considered elderly. Age is preferably weighted as a continuous variable. It is desirable to use a continuous variable so as not to lose information by dichotomization.

[0206] Age is used as a continuous variable. The final model includes the patient's age and the integrated score, and a predicted risk is calculated. For each one-year increase in age, the risk score decreases by 0.03471772. This is in contrast to the weight of the integrated score, where for each one-standard-deviation increase (compared to a representative background population), the risk score increases by 0.19541174. These numerical values are all arbitrary and thus do not represent absolute risks. However, it can be concluded that a 0.19541174 / 0.03471772 = 5.63-year decrease in age has the same effect on the predicted risk as a one-standard-deviation increase in the integrated score. Absolute thresholds / cutoffs for defining high-risk and low-risk groups are obtained by comparing scores to a representative background population as described herein.

[0207] Age is used as an ordinal / categorical variable. It is also possible to use the integrated score / model or the proliferation index in combination with a variable representing age groups. In some embodiments, the age groups are estimated, for example, as pre-menopausal / pre-menopausal and post-menopausal (less than 55 years old), 55 - 65 years old, and 65 years old and above. The integrated model score or the proliferation index can have different interpretations depending on the age category of the subject. For example, a low integrated model score and / or proliferation index in women under 50 years old may result in a recommendation to omit RT boost (current guidelines recommend RT boost for young women), while a low integrated model score and / or proliferation index in women 65 years old and above may result in a recommendation to omit RT completely.

[0208] In some embodiments, the integrated model score functions well for young women (less than 65 years old, less than 60 years old, or even less than 55 years old), while the proliferation index functions well for older women (over 65 years old). This is because the integrated model takes into account the immune response, and without being bound by theory, it is generally considered that young women are more likely to have the conditions for an immune response.

[0209] In some embodiments, a method for determining a therapy for treating cancer is provided, the method comprising determining an immune score of a core biopsy without determining the level of tumor-infiltrating lymphocytes (TILs) surrounding the tumor from which the core biopsy was obtained, and using the immune score to determine the amount of radiation therapy to administer to a subject without including the level of TILs as a factor. In some embodiments, the subject is treated with neoadjuvant immunotherapy. Neoadjuvant immunotherapy is administered when the subject is thought to benefit therefrom, not when RT is inadequate. The clinical problem is to identify patients who will benefit from immunotherapy. Candidates for neoadjuvant immunotherapy are defined as any of the following: the subject shows benefit from this treatment: high proliferation index; high proliferation index + high immune score; moderate proliferation index + high immune score; low / moderate integrated model score and high proliferation index and high immune score; low / moderate integrated model score and high proliferation index, high immune score, and young age (e.g., less than 55 years old); low / moderate final model score and high proliferation index and high immune score; low / moderate final model score and high proliferation index, high immune score, and young age (e.g., less than 55 years old).

[0210] In some embodiments, one of ordinary skill in the art can examine the levels of gene expression to determine what types of cancer treatments are effective and appropriate for a subject. The genes to be examined can be any one or more of Tables 4 and / or 6. In some embodiments, the gene levels can be compared to the standard levels of the genes in healthy subjects.

[0211] In some embodiments, the subject is 55 years old or younger. In some embodiments, the subject is older than 55 years old. In some embodiments, the subject is 55 years old or older. In some embodiments, the subject is 65 years old or older. In some embodiments, the subject is older than 65 years old. In some embodiments, the subject is 55 years old or younger. In some embodiments, the subject is younger than 55 years old. In some embodiments, the subject is younger than 50 years old. In some embodiments, the subject is between 50 and 65 years old. In some embodiments, the age cutoffs pointed out herein are used as additional variables to define which treatment pathway to take (in any of the embodiments provided herein, including any of the flowcharts depicted in the figures). In some embodiments, the cutoffs are 55 years old and / or younger and 65 years old and / or older.

[0212] In some embodiments, an immune score (IS) can be used when a surgical resection specimen is not available and / or when the sample for analysis is by core biopsy. In some embodiments, this can be the case, for example, when stromal TILs around the tumor are not available for analysis. In some embodiments, the tumor can be sampled before neoadjuvant or adjuvant therapy for accurate prognosis and prediction.

[0213] In some embodiments, a method for treating breast cancer is provided, the method comprising determining the histological grade of a tumor from at least a sample provided by a subject, wherein the histological grade of the tumor includes grade I, grade II, or grade III, and assigning a tumor aggressiveness to the sample. In some embodiments, the tumor aggressiveness is assigned as low risk when the sample is determined to be a grade I tumor. In some embodiments, the tumor aggressiveness is assigned as low risk when the sample is determined to be a grade II tumor and the proliferation index score of the sample is less than the median score of the background population of the tumor. In some embodiments, the tumor aggressiveness is assigned as high risk when the sample is determined to be a grade II tumor and the proliferation index score of the sample is greater than or equal to the median of the background population of the tumor. In some embodiments, the tumor aggressiveness is assigned as high risk when the sample is determined to be a grade III tumor. In some embodiments, the tumor aggressiveness is assigned as high risk when the proliferation index score of the sample is greater than or equal to the median of the background population of the tumor. In some embodiments, the tumor aggressiveness is assigned as high risk when the sample is determined to be ER negative. In some embodiments, the tumor aggressiveness is assigned as high risk when the sample is determined to be HER2 positive. In some embodiments, the tumor aggressiveness is assigned as high risk when the level of Ki67 in the sample is high. In some embodiments, when the level of Ki67 is determined from a TMA, a level of Ki67 ≥ 10% is considered high. More preferably, in some embodiments, when the level of Ki67 is determined from a TMA, a level of Ki67 ≥ 20% is considered high. Most preferably, in some embodiments, when the level of Ki67 is determined from a TMA, a level of Ki67 ≥ 30% is considered high. In some embodiments, when the level of Ki67 is determined from a whole slide, a level of Ki67 ≥ 20% is considered high. More preferably, in some embodiments, when the level of Ki67 is determined from a whole slide, a level of Ki67 ≥ 25% is considered high.Most preferably, in some embodiments, when the Ki67 level is determined from the whole slide, a level of Ki67 ≥ 30% is considered high. In some embodiments, when the Ki67 level is determined from the whole slide, a level of Ki67 ≤ 10% is considered low. More preferably, in some embodiments, when the Ki67 level is determined from the whole slide, a level of Ki67 ≤ 5% is considered low. When scoring on the whole slide, Ki67 is preferably scored as a global assessment, i.e., the percentage of tumor cells with positive staining, using automated scoring. In some embodiments, hot spots are used and the values of different hot spots are averaged. In some embodiments, a hot spot includes the percentage of tumor cells with a positive Ki67 score in the region with the strongest staining.

[0214] In some embodiments, tumor aggressiveness is assigned a high risk when the mutation load is high. In some embodiments, a mutation load of 5 mutations or more per genomic megabase (mutations / MB) is considered a high mutation load, more preferably 7 mutations / MB or more, and most preferably 10 mutations / MB or more. The mutation load threshold may be different when the mutation load assay is used for cancers of other organs. In some embodiments, tumor aggressiveness is assigned a high risk when the sample is determined to be a grade III tumor; when the proliferation index score of the sample is greater than or equal to the median score of the tumor background population; when it is ER negative; when it is HER2 positive; when the level of Ki67 is high; when the mutation load is high; or in any combination thereof. Preferably, the treatment of the high-risk group is determined based on immunological factors. High risk + activated immune infiltration (determined by, for example, high TIL and / or high immune score and / or high PD-1 and / or high PD-L1, etc.) leads to de-escalation of radiotherapy. This can be radiotherapy boost omission (when the boost is otherwise indicated and immune activation above a moderate threshold is seen), or complete radiotherapy omission (suitable when immunological values exceed a high threshold; for example, any of the following, or a combination of the following (preferred): TIL of 50% or more, high PD-1 / PD-L1, TIL higher than 75%, immune score exceeding a high threshold (e.g., 70th percentile of a representative background population)). In addition, in these tumors, certain forms of systemic therapy (immunotherapy, anti-HER2 therapy, chemotherapy, etc.) may be more effective, and when radiotherapy is provided, its necessity can be further reduced. High risk + non-activated immune infiltration may lead to radiotherapy boost and / or intensified systemic therapy.

[0215] In some embodiments, the method further includes classifying immunological activity based on the level of TILs in the sample. In some embodiments, the level of TILs in the sample is in the range of 50% to 90%. In some embodiments, the level of TILs in the sample is in the range of 10% to 49%. In some embodiments, tumor aggressiveness is assigned as high risk. In some embodiments, a treatment plan is determined by integrating high-risk tumor aggressiveness and immunological activity using an interaction term. In some embodiments, the treatment plan includes RT omission when the level of TILs in the sample is in the range of 50% to 90%. In some embodiments, the treatment plan includes RT boost omission when the level of TILs in the sample is in the range of 10% to 49%. Additional factors such as whether other forms of targeted therapy are available (e.g., anti-HER2 treatment), additional clinical variables (e.g., tumor size, lymph node status, patient age, etc.), and patient preferences may be considered. Additionally, accuracy is improved by integrating different metrics. For example, RT boost omission is possible if TIL is 10 - 49%, while RT omission is justified if TIL is 10 - 49% + PD-1 / PD-L1 is 1% or more. Additional measurements (PD-1 / PD-L1 / immune score) can upgrade or downgrade treatment for borderline TIL values (e.g., TIL 10 - 49%). In a non-limiting example, tumors with PD-1 / PD-L1 of 1% or more and TIL of 10% or more had a better prognosis than tumors with TIL of 50% or more without considering PD-1 or PD-L1; furthermore, tumors with TIL of 50% or more had a better prognosis than tumors with TIL of 10 - 49%. These results indicate that there is a dose-response relationship between TIL level and local recurrence risk, and that PD-1 / PD-L1 provides information beyond TIL. The latter is particularly useful in borderline cases. For RT treatment of high-risk tumors, an increase in TIL is utilized for RT boost omission or complete RT omission. The optimal threshold for boost omission is the level of TIL between 10 - 49%, while the threshold for RT omission is somewhere above the TIL level of 50%.In some embodiments, RT boost omission: It may be anywhere as long as the level of TILs exceeds 10%; RT omission: It may be anywhere as long as the level of TIL exceeds 50%.

[0216] In some embodiments, the classification of immunological activity further includes the level of checkpoint molecules in the sample. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, the level of PD-1 is 1% or more. In some embodiments, the level of PD-L1 is 1% or more. In some embodiments, the level of TIL is 10% or more. In some embodiments, when the level of PD-1 is 1% or more, the level of PD-L1 is 1% or more, and the level of TIL is 10% or more, the treatment plan includes RT omission. In some embodiments, the level of PD-1 is less than 1%. In some embodiments, when the level of PD-L1 is less than 1%, PDL-1 is less than 1%, and TIL is 10% or more, the treatment plan includes RT boost omission. For PD-1, the level represents the proportion of lymphocytes having positive staining for PD-1. For PD-L1, the level represents the proportion of lymphocytes having positive staining for PD-L1. The staining assay for obtaining these levels can be performed by a pathologist using TMA / core or whole sections (the latter is preferred), or using automated image analysis.

[0217] In some embodiments, the sample includes a core biopsy. In some embodiments, the level of TIL is obtained by automated scoring of whole tissue sections of the sample under a high-resolution microscope.

[0218] In some embodiments, the method is combined with clinical variables. The principles for integrating additional clinical variables include the following: (1) The presence of high-risk clinical variables can, optionally, upgrade the recommendation for treatment de-escalation to standard treatment or upgrade the recommendation for treatment omission (e.g., RT omission or chemotherapy omission) to treatment de-escalation (e.g., RT de-escalation (e.g., reduction in dose or fractionation) or chemotherapy de-escalation). As used herein, chemotherapy de-escalation includes any of the following: a) switching from a high-dose density (shortening the interval between each treatment and more than 8 treatments, e.g., for more than 8 treatments, the interval is changed from 3 weeks to 1 - 2 weeks; a common regimen is to administer epirubicin + cyclophosphamide every other week for 4 times, and then paclitaxel once a week for 12 weeks) to a standard period (6 - 8 treatments with an interval of 3 weeks between each treatment); b) switching from a treatment longer than the standard period (e.g., more than 8 treatments) to a standard treatment period (6 - 8 treatments with an interval of 3 weeks); c) switching from a standard treatment period (6 - 8 treatments with an interval of 3 weeks) to a shorter treatment period (4 - 6 treatments with an interval of 3 weeks). As used herein, chemotherapy escalation also includes the reverse switch as described in the definition of "chemotherapy de-escalation". As used herein, "chemotherapy omission" means switching from any treatment by chemotherapy to complete omission of chemotherapy. As used herein, RT de-escalation includes any of the following: a) switching from whole breast radiotherapy (WBRT) to intraoperative radiotherapy (IORT) or accelerated partial breast irradiation (APBI); b) reduction in dose or fractionation; c) switching from standard RT + boost to standard RT without boost; and d) a significant reduction in biologically effective dose (BED) compared to the recommended guidelines. As used herein, RT escalation also includes the reverse switch as described in the definition of "RT de-escalation". As used herein, RT omission includes switching from any treatment by RT to complete omission of RT.It should be noted that the indicators of tumor aggressiveness related to proliferation (histological grade, Ki67, ER-status) are not included in this concept. This is because this method has surprisingly shown that such variables predict a good prognosis when related to an activated immune infiltration, and (2) the absence of high-risk clinical variables does not downgrade the treatment recommendations for high-risk groups without an activated immune infiltration. If high-risk clinical variables are present in the low tumor-intrinsic risk group without an activated immune infiltration, de-escalation therapy can be upgraded to standard therapy. Non-limiting examples of such variables include a tumor diameter of 5 cm or more, no implementation of endocrine therapy, and extensive lymphatic invasion. Non-limiting examples of various scenarios are shown in Table 29.

[0219] In some embodiments, the general flowchart shown in FIG. 29 shows a method using clinical variables. A tumor biopsy is performed and / or obtained, different analyses of the biopsy are performed, and tumor-intrinsic aggressiveness, immune infiltration, and age (in the case of the final model) are determined. By integrating additional clinical variables (e.g., age, tumor size, lymph node status, lymphatic invasion) with the analysis, the tumor biopsy is classified as high risk, medium risk, or low risk as shown, each leading to treatment escalation, standard treatment, and treatment de-escalation, respectively. With respect to age, in some embodiments, among young subjects with high-risk tumors (determined by tumor-intrinsic aggressiveness, point 1) and abundant immune infiltration (immune infiltration analysis, point 2), young age becomes less of a significant risk-increasing factor. Here, the risk increase due to young age (e.g., less than 50 years old) may be partially or completely offset by a better immune response in such patients. Further, in elderly subjects (e.g., 65 years old and above) with high-risk tumors (point 1), the importance of the immune response (point 2) is reduced because the immune response declines with age. For elderly subjects, it may be preferable to use only point 1 (e.g., proliferation index) associated with aging to determine appropriate treatment methods (systemic therapy and radiotherapy shown in FIGS. 30 and 31, respectively). In some embodiments, only elderly subjects, tumor-intrinsic aggressiveness, and age are included in the model. In some embodiments, the method can be, in whole or in part, the method described in FIG. 29 and includes passing through one of the decision lines, two of the decision lines, or all of the decision lines.

[0220] In the case of the systemic therapy shown in FIG. 30, after obtaining or performing a tumor biopsy, one or more evaluations are performed to determine the risk level of the tumor. In some embodiments, the evaluations include tumor intrinsic aggressiveness, immune infiltration, an integrated score or a final score, or combinations thereof. After determining the risk level of the tumor, various treatments exemplified in FIG. 30 can be performed based on the determined risk level of the tumor. In some embodiments, the method can be, in whole or in part, the method described in FIG. 30 and includes passing one, two, or all of the decision lines.

[0221] Additional aspects of the systemic therapy shown in FIG. 30 are provided. The tumor aggressiveness assessment ( * ") includes a proliferation index, histological grade, Ki67, HER2-status, ER-status. The evaluation of immune infiltration ( ** ") includes an immune score, TIL, PD-1, PD-L1. The evaluation of the integrated score or final score ( *** ") includes an integrated model combining the proliferation index and the immune score, and the final model is a combination of the integrated model and age. Regarding the remaining indicators shown in FIG. 30, **** " - can be omitted if there are no high-risk features: extensive lymphatic invasion, positive lymph node metastasis, large tumor (e.g., 50 mm or more). Otherwise, de-escalate chemotherapy. *****」 - Can be omitted if there are low-risk features: negative lymph node metastasis, advanced age (e.g., 65 years or older), small tumor (e.g., less than 20 mm), endocrine therapy. 「^」 - Any one or combination of the following: 1. Exceeds the threshold of the 60th percentile of the proliferation index compared to the representative tumor and the background population of patients. 2. Histological grade 3 or histological grade 2 with a proliferation index equal to or exceeding the median of the background population of grade 3 tumors. 3. HER2 amplification. 4. High Ki67 (e.g., exceeding the threshold higher than 20% scored as a global assessment). 5. ER-negative tumors. 「^^」 - 1. Exceeds the threshold of the 60th percentile of the immune score compared to the representative tumor and the background population of patients. 2. Exceeds the threshold of TIL exceeding 10%. 3. PD-1 or PD-L1 exceeds 1%. 「^^^」 - An integrated score or final score exceeding the threshold of the 60th percentile of the representative background population of patients and tumors. 「^^^^」 - Any one or combination of the following: 1. The proliferation index is below the threshold below the 30th percentile compared to the representative tumor and the background population of patients. 2. Histological grade 1 or histological grade 2 with a proliferation index below the median of the background population of grade 3 tumors. 3. HER2-negative. 4. Low Ki67 (e.g., exceeding the threshold less than 20% scored as a global assessment). 5. ER-positive tumors. 6. The integrated score or final score is below the threshold below the 40th percentile of the representative background population of patients and tumors, and no immune infiltration is observed. 「†」 - Examples in the case of borderline: The integrated / final score / proliferation index / global Ki67 score is intermediate (somewhere between the 30th and 70th percentiles of the representative background population of tumors and patients), and / or histological grade 2. If the intermediate score is low (e.g., 40th percentile) and no high-risk features (positive lymph node metastasis, tumor diameter 20 mm or more, age less than 50 years) are observed, de-escalation or standard treatment is performed. If the intermediate score is high (e.g., 60th percentile) and there are high-risk features, standard treatment or treatment escalation. Otherwise, standard treatment."††" can be performed preoperatively together with anti-HER2 therapy and / or endocrine therapy. If a pathological complete response is obtained, de-escalation of RT is possible. Alternatively, it can also be administered preoperatively together with neoadjuvant RT that indicates that de-escalation or omission of postoperative systemic therapy (e.g., chemotherapy) is possible if a pathological complete response is obtained.

[0222] In the radiotherapy shown in FIG. 31, after performing and / or obtaining a tumor biopsy from a patient, one or more evaluations are performed to determine the risk level of the tumor. In some embodiments, the evaluation includes intratumoral aggressiveness, immune infiltration, an integrated score or a final score, or a combination thereof. After determining the risk level of the tumor, various radiotherapy shown in FIG. 31, or an adjustment thereof, can be performed based on the determined risk level of the tumor. In some embodiments, the method can be, in whole or in part, the method described in FIG. 31 and includes passing through one of the decision lines, two of the decision lines, or all of the decision lines.

[0223] An additional aspect of the radiotherapy shown in FIG. 31 is provided. The tumor aggressiveness assessment (" * ") includes a proliferation index, histological grade, Ki67, ER-status, and HER2-status. The tumor aggressiveness assessment preferably combines some of the listed assessments. In some embodiments, at least the proliferation index and histological grade are evaluated. The evaluation of immune infiltration (" ** ") includes an immune score, TIL, PD-1, PD-L1, or a combination thereof. The evaluation of the integrated score or final score (" *** ") includes an integrated model that combines the proliferation index and the immune score, and the final model is a combination of the integrated model and age. Regarding the remaining indicators shown in FIG. 31, " ****」 - Low-risk characteristics: negative lymph node metastasis, advanced age (e.g., 65 years or older), small tumor (e.g., less than 20 mm), endocrine therapy. 「^」 - Any one or a combination of the following: 1. Exceeds a threshold above the 60th percentile of the proliferation index compared to a background population consisting of representative tumors and patients. 2. Histological grade 3 or histological grade 2 with a proliferation index equal to or exceeding the median of the background population of grade 3 tumors. 3. HER2-amplified tumors. 4. High Ki67 (e.g., exceeds a threshold higher than 20% scored as a global assessment). 5. ER-negative tumors. 「^^」 - Is activated (or does not exist if not) in the case of one or more (preferably a combination of two or more) of the following: 1. When the immune score exceeds a threshold above the 60th percentile compared to the background population of representative tumors and patients. 2. TILs exceeding a threshold above 10%. 3. PD-1 and / or PD-L1 exceeding 1%. 「^^^」 - An integrated score or final score below a threshold below the 40th percentile of the representative background population of patients and tumors. 「†」 Intermediate integrated / final score; a score somewhere between the 30th and 70th percentiles of the representative background population of tumors and patients. If the intermediate score is low (e.g., 40th percentile) and no high-risk characteristics (positive lymph node metastasis, tumor diameter of 20 mm or more, age less than 50 years) are observed, de-escalation or standard treatment; the indication for performing standard treatment rather than de-escalation increases as the tumor diameter increases when the tumor diameter is 20 - 50 mm. If the intermediate score is high (e.g., 60th percentile) and there are high-risk characteristics, standard treatment or treatment escalation is performed. Otherwise, standard treatment. 「††」 - RT de-escalation (e.g., omission of RT boost, reduction of total RT dose, switching from whole breast radiotherapy to intraoperative radiotherapy or accelerated partial breast irradiation) or omission of RT depends on the degree of activation of immune infiltration and the presence or absence of high-risk clinical characteristics.For example, when the TIL is 50% or more (preferably in combination with PD-1 / PD-L1 being 1% or more and / or the immune score being above the threshold value exceeding the 75th percentile of the representative background population of the tumor and the patient), and there are no high-risk clinical features, RT omission is justified. When the TIL is 10% or more and less than 50% or there are high-risk clinical features, RT de-escalation is justified. Combining with preoperative systemic therapy (anti-HER2 therapy and / or immunotherapy and / or chemotherapy and / or endocrine therapy) can further induce RT de-escalation. Pathological complete response to preoperative systemic therapy strengthens the indication for RT omission. "†††" - Preoperative systemic therapy can be used as a substitute for postoperative RT. If pathological complete response is obtained, the indication for RT omission is further strengthened. "††††" - All of the following: low proliferation index score (e.g., below the threshold value below the 40th percentile of the representative background population of the tumor and the patient), low histological grade (e.g., grade 1 or grade 2 with a low / moderate proliferation index score (e.g., below the median of the representative background population of grade 3 or grade 2 tumors)), low Ki67 score (e.g., below the threshold value with a global score below 10%), ER-positive tumor, HER2-negative tumor, low final score or integrated score (e.g., below the threshold value below the 40th percentile of the representative background population of the tumor and the patient), patients receiving endocrine therapy. "‘’" - Preoperative RT can be used as an alternative to de-escalation or omission of postoperative RT. Preferably, it is combined with preoperative systemic therapy (chemotherapy and / or anti-HER2 therapy and / or immunotherapy and / or endocrine therapy). If pathological complete response is obtained, postoperative systemic therapy can be de-escalated. "‘‘’’" - In the case of standard RT according to the guidelines, an RT boost is added. If an RT boost is already indicated according to the guidelines, systemic therapy (mainly chemotherapy) is escalated. "‘‘’’’’" - "When there are 1 to 3 positive lymph node metastases, regional lymph node irradiation is omitted. When there are 4 or more positive lymph node metastases, regional lymph node irradiation is omitted or the intensity is reduced.

[0224] In some embodiments, the method can be any one or a combination of the methods described in any of the methods shown in FIGS. 29, 30, and / or 31. In some embodiments, the method can include passing through one of the decision lines of FIGS. 29, 30, and / or 31, two of the decision lines, three of the decision lines, four of the decision lines, five of the decision lines, six of the decision lines, seven of the decision lines, eight of the decision lines, or nine of the decision lines. In some embodiments, the sample is provided by or obtained from a patient. In some embodiments, the sample includes a tumor biopsy. In some embodiments, the tumor biopsy is evaluated as to which risk level it corresponds to. In some embodiments, the evaluation is performed by a trained practitioner. In some embodiments, the evaluation is performed by an electronic device. In some embodiments, the evaluation is performed partly by a trained practitioner and partly by a machine. In some embodiments, the evaluation includes determining tumor intrinsic aggressiveness. In some embodiments, the evaluation further includes determining immune infiltration. In some embodiments, the evaluation includes determining a risk level by an integrated model to generate an integrated score. In some embodiments, the evaluation further includes determining a risk level by a final model that includes the integrated score and the patient's age to generate a final score. In some embodiments, the determination of tumor intrinsic aggressiveness includes a proliferation index, histological grade, Ki67, ER-status, HER2-status, or a combination thereof. In some embodiments, the determination of immune infiltration includes determining an immune score, level of TIL, level of PD-1, level of PD-L1, or a combination thereof. In some embodiments, the integrated model combines a proliferation index and an immune score. In some embodiments, the final model combines the integrated model and the patient's age. In some embodiments, if the proliferation index exceeds a threshold above the 60th percentile compared to a representative tumor and the patient's background population, the histological grade is 3 or histological grade 2, and if the proliferation index is equal to or exceeds the median of the background population of grade 3 tumors, is high Ki67, is an ER-negative tumor, is a HER2-amplified tumor, or is a combination thereof, the biopsy risk level is a high tumor intrinsic risk. In some embodiments, high Ki67 exceeds a threshold higher than 20% scored as a global assessment.

[0225] In some embodiments, if the immune score exceeds a threshold above the 60th percentile compared to a representative tumor and the patient's background population, if the level of TILs exceeds a threshold above 10%, if the level of PD-1 or PD-L1 exceeds 1%, or is a combination thereof, there is an activated immune infiltration of the biopsy. In some embodiments, if the threshold of the integrated score or the final score exceeds the 60th percentile compared to a representative background population of patients and tumors, the integrated / final score is high.

[0226] In some embodiments, compared to a representative tumor and the patient's background population, if the immune score is below a threshold below the 60th percentile, if the level of TILs is below a threshold below 10%, if the level of PD-1 or PD-L1 is below 1%, or is a combination thereof, the activated immune infiltration of the biopsy is considered not to exist. Preferably, to arrive at a determination of the absence of activated immune infiltration, all of these conditions related to the determination of the absence of activated immune infiltration are present. A dose-dependent relationship may be seen such that the lower the threshold, the stronger the fit to classify the patient as having no immune response.

[0227] As shown in FIG. 30, in some embodiments, if the biopsy has a high tumor intrinsic risk and lacks an activated immune infiltration, or has a high integration / final score, intensified chemotherapy and / or omission of immunotherapy are provided to the patient. In some embodiments, if the biopsy has a high tumor intrinsic risk and an activated immune infiltration, or has a high tumor intrinsic risk and a low integration / final score, immunotherapy is administered and / or chemotherapy is de-escalated / omitted. In some embodiments, the method further includes an evaluation of immune infiltration; if the biopsy has a low tumor intrinsic risk and lacks an activated immune infiltration, or has a high integration / final score. In some embodiments, if there is no immune infiltration, de-escalation / omission of chemotherapy is provided to the patient. In some embodiments, if the immune infiltration is activated, chemotherapy is administered to the patient.

[0228] As shown in FIG. 31, in some embodiments, if the biopsy has a high tumor intrinsic risk and lacks an activated immune infiltration, or has a high integration / final score, radiotherapy is intensified for the patient. In some embodiments, if the biopsy has a high tumor intrinsic risk and an activated immune infiltration, or has a high tumor intrinsic risk and a low integration / final score, radiotherapy is de-escalated or omitted, and / or preoperative radiotherapy is provided to the patient, and / or preoperative systemic therapy is provided to the patient, and / or omission or de-escalation of regional nodal irradiation is performed regardless of the previous lymph node status. In some embodiments, the method further includes an evaluation of immune infiltration. In some embodiments, if the biopsy has a low tumor intrinsic risk and lacks an activated immune infiltration, and other low-risk clinical features are present, radiotherapy is omitted from the patient. In some embodiments, the low-risk clinical features include negative lymph node metastasis, advanced age (e.g., 65 years or older), small tumor (e.g., <20 mm), and endocrine therapy. In some embodiments, if it is shown that the immune infiltration is activated, standard radiotherapy is provided to the patient.

[0229] In some embodiments, a method for predicting the effectiveness of cancer treatment is provided, and a method for treating breast cancer is provided. The method includes determining the histological grade of a tumor from at least a sample of the tumor provided by a subject, wherein the histological grade of the tumor includes grade I, grade II, or grade III, and classifying the tumor aggressiveness of the sample as a) low risk if the sample is determined to be a grade I tumor; b) low risk if the sample is determined to be a grade II tumor and the proliferation index score of the sample is less than the median of the background population of high-risk tumors; c) high risk if the sample is determined to be a grade II tumor and the proliferation index score of the sample is greater than or equal to the median of the background population of high-risk tumors; or d) high risk if the sample is determined to be i) a grade III tumor, ii) the proliferation index score of the sample is greater than or equal to the median of the background population of high-risk tumors, iii) ER negative, iv) HER2 positive, v) the level of Ki67 is high, vi) the mutation load is high, or vii) any combination of i) to vi), and classifying the immunological activity including the level of TILs in the sample, and integrating the tumor aggressiveness and the immunological activity using interaction terms to determine a treatment plan.

[0230] In some embodiments, the level of Ki67 is 10% or more. In some embodiments, the level of Ki67 is 20% or more. In some embodiments, the level of Ki67 is 30% or more. In some embodiments, the mutation load is 5 mutations or more per genomic megabase (mutations / MB). In some embodiments, the mutation load is 7 mutations / MB or more. In some embodiments, the mutation load is 10 mutations / MB or more.

[0231] In some embodiments, a method for treating breast cancer is provided, the method comprising determining a histological grade of a tumor from at least a sample of the tumor provided by a subject, wherein the histological grade of the tumor comprises grade I, grade II, or grade III, assigning a tumor aggressiveness to the sample, classifying an immunological activity including a level of TILs in the sample, and integrating the tumor aggressiveness and the immunological activity using an interaction term to determine a treatment plan.

[0232] In some embodiments, assigning a high-risk tumor aggressiveness comprises determining the sample as i) a grade III tumor, ii) a proliferation index score of the sample being greater than or equal to the median of the tumor background population, iii) ER negative, iv) HER2 positive, v) a high level of Ki67, vi) a high mutation burden, or vii) any combination of i) - vi). In some embodiments, the level of Ki67 is 10% or more. In some embodiments, the level of Ki67 is 20% or more. In some embodiments, the level of Ki67 is 30% or more. In some embodiments, the mutation burden is 5 mutations / MB or more. In some embodiments, the mutation burden is 7 mutations / MB or more. In some embodiments, the mutation burden is 10 mutations / MB or more.

[0233] In some embodiments, assigning a high-risk tumor aggressiveness comprises determining the sample as a grade II tumor, wherein the proliferation index score of the sample is greater than or equal to the median score of the tumor background population. In some embodiments, the tumor aggressiveness is high risk. In some embodiments, the level of TILs in the sample is in the range of 50% - 90%, and the treatment plan includes omission of radiotherapy. In some embodiments, the level of TILs in the sample is in the range of 10% - 49%, and the treatment plan includes omission of boost.

[0234] In some embodiments, the classification of immunological activity further includes the level of checkpoint molecules in the sample. In some embodiments, the checkpoint molecules include PD-1 and PD-L1. In some embodiments, the level of PD-1 is 1% or more. In some embodiments, the level of PD-L1 is 1% or more. In some embodiments, the level of TIL is 10% or more. In some embodiments, the treatment plan includes RT omission. In some embodiments, the level of PD-1 is less than 1%. In some embodiments, the level of PD-L1 is less than 1%. In some embodiments, the level of TIL is 10% or more. In some embodiments, the treatment plan includes RT boost omission.

[0235] In some embodiments, the subject is 55 years of age or older. In some embodiments, the subject is over 55 years of age. In some embodiments, the subject is 65 years of age or older. In some embodiments, the subject is over 65 years of age. In some embodiments, the subject is 55 years of age or younger. In some embodiments, the subject is less than 55 years of age. In some embodiments, the subject is between 50 and 65 years of age. In some embodiments, the subject is less than 50 years of age. In some embodiments, the background population has a consistent age. In some embodiments, the sample includes a core biopsy.

[0236] In some embodiments, a method for treating breast cancer is provided, the method comprising determining the tumor-intrinsic aggressiveness of a sample provided by a subject, wherein the tumor-intrinsic aggressiveness includes the proliferation index of the sample, the histological grade of the sample, the level of Ki67 in the sample, the HER2 expression of the sample, the ER-status of the sample, or a combination thereof, and determining the immune infiltration of the sample, wherein the immune infiltration includes the immune score of the sample, the level of TIL in the sample, the level of PD-1 in the sample, the level of PD-L1 in the sample, or a combination thereof, and providing a treatment based on the tumor-intrinsic aggressiveness and the immune infiltration. In some embodiments, the subject is 65 years of age or older.

[0237] In some embodiments, the tumor intrinsic risk is high if the proliferation index is above the 60th percentile compared to a representative patient and tumor background population; the histological grade is 3; the histological grade is 2 and the proliferation index is equal to or greater than the median of the grade 3 tumor background population; the HER2 expression is amplified; the ER-status of the sample is negative; or a combination thereof.

[0238] In some embodiments, the immune infiltration is activated if the immune score is above the 60th percentile threshold compared to a representative tumor background population; the level of TILs in the sample is greater than 10%; the level of PD-1 in the sample is greater than 1%; the level of PD-L1 in the sample is greater than 1%; or a combination thereof.

[0239] In some embodiments, where the tumor intrinsic risk is high and the immune activation is inactive, the treatment includes RT boost. In some embodiments, where the tumor intrinsic risk is high and the immune activation is inactive, the subject has no comorbidities, and the treatment includes intensification of chemotherapy. In some embodiments, the treatment further includes immunotherapy. In some embodiments, the sample is ER positive, and the treatment further includes endocrine therapy. In some embodiments, the tumor intrinsic risk is high, the immune activation is inactive, the subject has no comorbidities, and the treatment includes immunotherapy. In some embodiments, the tumor intrinsic risk is high, the immune activation is inactive, the subject has no comorbidities, the sample is ER positive, and the treatment includes endocrine therapy. In some embodiments, where the tumor intrinsic risk is not high and the immune infiltration is not activated, the subject has no comorbidities, and the treatment includes RT omission. In some embodiments, the treatment further includes omission or de-escalation of chemotherapy. In some embodiments, the treatment further includes endocrine therapy.

[0240] In some embodiments, the tumor intrinsic risk is not high, the immune infiltration is not activated, the subject has no comorbidities, and the treatment includes omission or de-escalation of chemotherapy. In some embodiments, the tumor intrinsic risk is not high, the immune infiltration is not activated, the subject has no comorbidities, and the treatment includes endocrine therapy. In some embodiments, the tumor intrinsic risk is not high, the immune infiltration is activated, the subject has no comorbidities, and the treatment includes RT. In some embodiments, the treatment further includes chemotherapy. In some embodiments, the treatment further includes endocrine therapy.

[0241] In some embodiments, the tumor intrinsic risk is not high, the immune infiltration is activated, the subject has no comorbidities, and the treatment includes escalation of chemotherapy. In some embodiments, the tumor intrinsic risk is not high, the immune infiltration is activated, the subject has no comorbidities, and the treatment includes endocrine therapy.

[0242] In some embodiments, the subject has comorbidities. Some non-limiting examples of comorbidities include, as comorbidities, coronary artery disease, heart failure, chronic obstructive pulmonary disease, a history of stroke (ischemic or hemorrhagic), uncontrolled hypertension, diabetes, osteoporosis, one or more other cancers, or combinations thereof. In some embodiments, the treatment includes omission of chemotherapy and de-escalation of RT.

[0243] In some embodiments, a method for treating breast cancer is provided, the method comprising constructing an integrated model from a sample provided by a subject to generate an integrated score, wherein the integrated model includes a proliferation index and an immune score, constructing a final model based on the integrated model and the age of the subject to generate a final score, and providing treatment based on the integrated / final score including the integrated score or the final score. In some embodiments, the subject is 65 years of age or older.

[0244] In some embodiments, the integrated / final score is high if the integrated score or the final score exceeds the 60th percentile of a representative background population of patients and tumors. In some embodiments, the background population has a matching age. In some embodiments, the background population has a matching age to the subject. In some embodiments, the method further comprises determining the immune infiltration of the sample, wherein the immune infiltration comprises the immune score of the sample. In some embodiments, the immune infiltration further comprises the level of TILs in the sample, the level of PD-1 in the sample, the level of PD-L1 in the sample, or a combination thereof. In some embodiments, the immune infiltration is activated if the immune score exceeds a 60th percentile threshold compared to a representative tumor background population; the level of TILs in the sample is greater than 10%; the level of PD-1 in the sample is greater than 1%; the level of PD-L1 in the sample is greater than 1%; or a combination thereof. In some embodiments, the immune infiltration is activated if the immune score exceeds a 60th percentile threshold compared to a representative background population of patients and tumors. In some embodiments, the background population has a matching age. In some embodiments, the background population has a matching age to the subject.

[0245] In some embodiments, the integrated / final score is high and the treatment includes RT boost. In some embodiments, the integrated / final score is not high, the immune infiltration is active, and the treatment includes chemotherapy omission or chemotherapy de-escalation. In some embodiments, the treatment further includes immunotherapy. In some embodiments, the sample is ER positive and the treatment further includes endocrine therapy. In some embodiments, the integrated / final score is high, the immune infiltration is activated, and the treatment includes immunotherapy. In some embodiments, the integrated / final score is not high, the immune infiltration is not activated, and the treatment includes RT omission. In some embodiments, the treatment further includes chemotherapy omission or de-escalation. In some embodiments, the treatment further includes endocrine therapy. In some embodiments, the integrated / final score is not high, the immune infiltration is activated, and the treatment includes standard RT. In some embodiments, the treatment further includes chemotherapy escalation. In some embodiments, the integrated / final score is not high, the immune infiltration is activated, and the treatment includes chemotherapy escalation. In some embodiments, the treatment further includes endocrine therapy.

[0246] In some embodiments, a method for treating breast cancer is provided, the method comprising determining the tumor-intrinsic aggressiveness of a sample provided by a subject, the tumor-intrinsic aggressiveness including the proliferation index of the sample, the histological grade of the sample, the level of Ki67 of the sample, the HER2 expression of the sample, the ER-status of the sample, or a combination thereof, determining the immune infiltration of the sample, the immune infiltration including the immune score of the sample, the level of TIL of the sample, the level of PD-1 of the sample, the level of PD-L1 of the sample, or a combination thereof, and providing a treatment based on the tumor-intrinsic aggressiveness and the immune infiltration, wherein the subject is less than 55 years old.

[0247] In some embodiments, a method for treating breast cancer is provided, the method comprising constructing an integrated model from a sample provided by a subject to generate an integrated score, wherein the integrated model includes a proliferation index and an immune score; constructing a final model based on the integrated model and the age of the subject to generate a final score; and providing treatment based on the integrated / final score including the integrated score or the final score, wherein the subject is less than 55 years old.

[0248] In some embodiments, the integrated / final score is less than the 5th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes RT de-escalation. In some embodiments, the integrated / final score is greater than the 50th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes RT escalation compared to standard RT. In some embodiments, the RT escalation includes an RT boost. In some embodiments, the integrated / final score is greater than the 95th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes surgical escalation. In some embodiments, the surgical escalation includes mastectomy. In some embodiments, the surgical escalation consists of a wider resection margin. In some embodiments, the treatment further includes escalation of systemic therapy. In some embodiments, the treatment includes escalation of systemic therapy. In some embodiments, the background population is age-matched. In some embodiments, the background population is age-matched to the subject.

[0249] In some embodiments, a method for treating breast cancer is provided, the method comprising constructing an integrated model from a sample provided by a subject to generate an integrated score, the integrated model including a proliferation index and an immune score; constructing a final model based on the integrated model and the subject's age to generate a final score; and providing treatment based on the integrated / final score including the integrated score or the final score, wherein the subject is over 55 years old. In some embodiments, the integrated / final score is above the 70th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes RT escalation. In some embodiments, RT escalation includes an RT boost. In some embodiments, the integrated / final score is above the 85th percentile of a representative background population of patients and tumors. In some embodiments, the treatment includes mastectomy. In some embodiments, the treatment includes escalation of systemic therapy. In some embodiments, the background population has a matching age. In some embodiments, the background population has an age matching the subject. For high-risk tumor patients with high tumor-intrinsic risk (e.g., high proliferation index, high tumor grade, young age, etc.) and high immune score / low integrated score, the prognosis is surprisingly good. Therefore, these patients require de-escalation therapy compared to current guidelines. Some non-limiting examples of de-escalation therapy include de-escalation of chemotherapy (e.g., omission of chemotherapy, non-implementation of high-dose-density chemotherapy, shortening of chemotherapy duration, etc.), omission of RT boost, immunotherapy only, and long-term non-implementation of endocrine therapy. In some embodiments, for high-risk tumor patients with high tumor-intrinsic risk (e.g., high proliferation index, high tumor grade, young age, etc.) and low immune score / high integrated score, the prognosis is surprisingly poor. Therefore, these patients require escalation therapy. Some non-limiting examples of escalation therapy include high-dose-density chemotherapy, antibody-drug conjugates, combined boost RT, and long-term endocrine therapy.

[0250] Chemotherapeutic agents used in cancer treatment are divided into several groups according to their mechanism of action. Some chemotherapeutic agents directly damage DNA and RNA. Such chemotherapeutic agents inhibit DNA replication, thereby completely stopping replication or resulting in the production of nonsense DNA or RNA. This category includes, for example, cisplatin (Platinol (registered trademark)), daunorubicin (Cerubidine (registered trademark)), doxorubicin (Adriamycin (registered trademark)), and etoposide (VePesid (registered trademark)). Another group of cancer chemotherapeutic agents interferes with the formation of nucleotides or deoxyribonucleotides and thus blocks RNA synthesis and cell replication. Examples of drugs in this class include methotrexate (Abitrexate (registered trademark)), mercaptopurine (Purinethol (registered trademark)), fluorouracil (Adrucil (registered trademark)), and hydroxyurea (Hydrea (registered trademark)). A third class of chemotherapeutic agents acts on the synthesis or breakdown of the mitotic spindle and, as a result, interrupts cell division. Examples of drugs in this class include vinblastine (Velban (registered trademark)), vincristine (Oncovin (registered trademark)) and taxanes, for example, paclitaxel (Taxol (registered trademark)), and docetaxel (Taxotere (registered trademark)). Docetaxel is currently approved in the United States for the treatment of patients with locally advanced or metastatic breast cancer after the failure of prior chemotherapy, and patients with locally advanced or metastatic non-small cell lung cancer after the failure of prior platinum-based chemotherapy. Predicting the patient response to all of these and other chemotherapeutic agents is particularly within the scope of the present invention.

[0251] In some embodiments, chemotherapy includes treatment with a taxane derivative. Taxane derivatives include, but are not limited to, paclitaxel (Taxol®) and docetaxel (Taxotere®), which are widely used in the treatment of cancer. As discussed above, taxanes act on a cellular structure called a microtubule and play an important role in cell function. In normal cell growth, microtubules are formed when a cell begins to divide. When cell division stops, the microtubules are disassembled or destroyed. Taxanes prevent the destruction of microtubules and block cell growth.

[0252] In some embodiments, chemotherapy includes treatment with an anthracycline derivative such as, for example, doxorubicin, daunorubicin, and aclacinomycin.

[0253] In some embodiments, chemotherapy includes treatment with a topoisomerase inhibitor such as, for example, camptothecin, topotecan, irinotecan, 20-S-camptothecin, 9-nitrocamptothecin, 9-aminocamptothecin, or GI147211.

[0254] Treatment with any combination of these and other chemotherapy drugs is specifically contemplated.

[0255] Most patients receive chemotherapy immediately after surgical resection of the tumor. This approach is generally called adjuvant postoperative therapy. However, chemotherapy can also be performed before surgery as so-called neoadjuvant therapy. Neoadjuvant chemotherapy originated in the treatment of inoperable advanced breast cancer, but has also been accepted for the treatment of other cancers. The effectiveness of neoadjuvant chemotherapy has been verified in several clinical trials. In the neoadjuvant therapy of the multi-institutional collaborative National Surgical Adjuvant Breast and Bowel Project B-18 (NSAB B-18) trial (Fisher et al., J. Clin. Oncology 15: 2002-2004 (1997); Fisher et al., J. Clin. Oncology 16: 2672-2685 (1998)), combination therapy of adriamycin and cyclophosphamide ("AC regimen") was performed. In another clinical trial, neoadjuvant therapy was performed using a combination of 5-fluorouracil, epirubicin and cyclophosphamide ("FEC regimen") (van Der Hage et al., J. Clin. Oncol. 19: 4224-4237 (2001)). In new clinical trials, neoadjuvant treatment regimens containing taxanes are also being used. See, for example, Holmes et al., J. Natl. Cancer Inst. 83: 1797-1805 (1991) and Moliterni et al., Seminars in Oncology, 24: S17-10-S-17-14 (1999). For further information on neoadjuvant chemotherapy for breast cancer, see Cleator et al., Endocrine-Related Cancer 9: 183-195 (2002).

[0256] Regimen Additional non-limiting embodiments of the present disclosure are provided in the following numbered arrangements. 1. A method of treating breast cancer, comprising determining an immune score (IS) based on an immune model composed of genes from an immunological gene set, Determining a proliferation index (PI) based on a tumor-intrinsic model composed of genes from a tumor-intrinsic gene set, Integrating the IS and PI into an integrated model, Integrating the integrated model and the age of the subject into a final model to identify whether the subject belongs to a specific risk clinical group, Providing an appropriate treatment to the subject based on the specific risk clinical group including The immunological model is trained in basal tumors and HER2 + tumors, The tumor-intrinsic model is a method trained in immune-exhausted tumors. 2. The method according to arrangement 1, wherein the immunological model includes an immunological gene set. 3. The method according to arrangement 2, further comprising performing a meta-analysis on the prognostic effect of each of the immunological gene sets. 4. The method according to arrangement 2 or 3, wherein the immune gene set includes C7 and any immune-related gene set from the H, C1, C2, C3, C4, C5, C6 or C8 category (e.g., identified using any of the keywords "LYMPHOCYTE|T_CELL|PD1|PD-1|PDL1|PD-L1|LAG3|CHECKPOINT_RECEPTOR|B_CELL|PERFORIN|GRANZYME|NK_CELL|CD8|CYTOTOXIC"). 5. The method according to any one of arrangements 1 to 4, further comprising collinearity filtering to remove highly correlated gene sets. 6. The IS determination step includes training an elastic net for the tumor, and optionally, The PI determination step includes training an elastic net for tumors including immune-exhausted tumors. The method according to any one of arrangements 1 to 5. 7. The method according to arrangement 6, wherein the tumor includes HER2 + tumors and basal tumors. 8. The method according to any one of arrangements 1 to 5, wherein the PI determination step includes training an elastic net for tumors including immune-depleted tumors. 9. The method according to arrangement 6, wherein the IS determination step is performed before the PI determination step. 10. Ranking the genes in the gene set selected in the step of determining IS based on Rho, and selecting one or more of the ranked genes The method according to any one of arrangements 1 to 8, further comprising. 11. Ranking the genes in the gene set selected in the step of determining PI based on Rho or a prognostic score, wherein the prognostic score is Rho / Pmeta including, and excluding the ranked genes with a Rho of the prognostic score less than 0.2 The method according to any one of arrangements 1 to 10, further comprising. 12. Combining the integrated model and the age of the subject into the final model to output a risk score, and recommending one or more treatment plans based on the risk score The method according to any one of arrangements 1 to 11, further comprising. 13. The method according to any one of arrangements 1 to 12, wherein the tumor-intrinsic model includes the H, C2, and / or C6 gene sets. 14. The final model has the following steps: Extracting the genes included in IS and PI; Performing a meta-analysis on the prognostic effect of each extracted gene in the first cohort; Selecting genes stably expressed across the core and tissue types in the second cohort; Ranking the extracted genes; and Selecting all the genes extracted from IS and PI including, The number of genes extracted from IS is less than 23, The method according to arrangement 12, wherein the number of genes extracted from PI is less than 60. 15. The final model has the following steps: Extraction of genes contained in IS and PI; Performing a meta-analysis on the prognostic effect of each extracted gene in the first cohort; Selecting genes stably expressed across cores and tissue types in the second cohort; Ranking the extracted genes; Selecting the top 23 genes extracted from IS; and Selecting the top 60 genes extracted from PI and The number of genes extracted from IS is 23 or more, The method according to arrangement 12, wherein the number of genes extracted from PI is 60 or more. 16. The method according to arrangement 14 or 15, wherein the first cohort includes a training cohort. 17. The second cohort includes the SweBCG91-RT cohort or another similar cohort, The SweBCG91-RT cohort passes quality control for RNA, cDNA, and microarrays, The SweBCG91-RT cohort Is treated with breast-conserving surgery and included in the multivariate analysis; or The method according to any one of arrangements 14 to 16, which is treated with breast-conserving surgery and radiotherapy and included in the multivariate analysis. 18. The method according to arrangement 12, wherein the risk score is a medium risk score including a high PI indicating an aggressive tumor and a high IS indicating strong immune activity. 19. The medium risk score corresponds to a grade III tumor including PD-1 高 / PD-L1 高 / TIL 高 The method according to arrangement 12. 20. The method according to arrangement 18 or 19, wherein the treatment plan includes immunotherapy. 21. The method according to arrangement 19 or 20, wherein IS and PI are high. 22. The method according to arrangement 21, wherein high IS is a value exceeding the threshold in the range of 60-90 percentile, and high PI is a value exceeding the threshold in the range of 60-90 percentile. 23. The method according to arrangement 19, wherein the treatment plan includes de-escalating one or more treatments for grade III tumors when IS and PI are high. 24. The method according to arrangement 23, wherein high IS is a value exceeding the threshold in the range of 67-90 percentile, and high PI is a value exceeding the threshold in the range of 67-90 percentile. 25. The method according to arrangement 12, wherein PI is high, being a value exceeding the threshold in the range of 67-90 percentile. 26. The method according to arrangement 25, further comprising scoring the level of TIL. 27. The method according to arrangement 26, wherein the score level of TIL is in the range of 50%-90%, and the scored level of TIL indicates high-risk tumors that are candidates for omission of radiotherapy. 28. The method according to arrangement 26, wherein the scored level of TIL is in the range of 10%-49%, and the scored level of TIL indicates high-risk tumors that are candidates for omission of boost. 29. The method according to arrangement 12, wherein the risk score is a high-risk score including medium / low IS and high PI. 30. The method according to arrangement 29, wherein the high-risk score corresponds to grade III tumors including PD-1 低 / PD-L1 低 / TILs 低 . 31. The method according to arrangement 29, wherein the high-risk score corresponds to grade III tumors including PD-1 高 / PD-L1 高 / TIL 高The method according to arrangement 29 corresponding to grade I / II tumors including 32. Moderate IS is a value below the threshold in the range of less than 10-40 percentiles, Low IS is a value below the threshold in the range of less than 30-70 percentiles, High PI is a value above the threshold in the range of 60-90 percentiles, the method according to any one of arrangements 29-31. 33. The treatment plan is enhanced, the method according to arrangement 30 or 31. 34. The enhanced treatment plan includes at least one of adding a boosting dose to the standard recommended treatment for the subject when the standard recommended treatment does not include a boosting dose and is 67 Gy or more, increasing the boosting dose beyond the standard amount for the subject, increasing the dose per fraction above the standard amount for the subject, increasing the number of fractions of the recommended dose above the standard amount for the subject, and treating the subject with enhanced radiotherapy including at least one of these doses, the method according to arrangement 33. 35. The enhanced treatment plan includes at least one of enhanced radiotherapy treatment, systemic therapy, mastectomy, additional use of a sensitizer for another therapy, therapy above the level set by at least one of the NCCN, ESMO, ESTRO, Clinical Practice Recommendations Australia, and / or NICE guidelines for the remaining indicators of the subject, or any combination thereof, the method according to arrangement 33. 36. A method for treating breast cancer, determining IS based on an immunological model composed of genes of an immunological gene set, determining PI based on a tumor-intrinsic model composed of genes of a tumor-intrinsic gene set, integrating IS and PI into an integrated model, integrating the integrated model and the age of the subject into a final model to identify whether the subject belongs to a specific risk clinical group based on the risk score from the final model, When the risk score of the subject is a high risk score, strengthening the treatment plan for the subject, the treatment plan including adding a boosting dose to the standard recommended treatment for the subject when the standard recommended treatment does not include a boosting dose and is 67 Gy or more, increasing the boosting dose beyond the standard amount for the subject, increasing the dose per fraction beyond the standard amount for the subject, increasing the number of fractions of the recommended dose beyond the standard amount for the subject, and treating the subject with an intensified radiotherapy including at least one of the above doses including The intensified treatment plan indicates at least one of an intensified radiotherapy treatment, a systemic therapy, a mastectomy, an additional use of a sensitizer for another therapy, a therapy exceeding the level set by at least one of the NCCN, ESMO, ESTRO, Clinical Practice Recommendations Australia, and / or NICE guidelines for the remaining indicators of the subject, or any combination thereof The immunological model is trained in basal tumors and HER2+ tumors The tumor-intrinsic model is trained in immune-exhausted tumors The high risk score is a method of grouping the subject into a high risk clinical group 37. A method for treating breast cancer, determining the expression level of the genes included in the model adjusting the gene expression level by scaling or normalizing it against a representative tumor background population summing the adjusted expression levels of the genes to determine a enrichment score for the gene set included in the model, the gene set including genes normalizing the enrichment score by comparing it with the background population calculating IS and PI respectively using the IS model and the PI model normalizing the IS score and the PI score by comparing them with the background population Calculating an integrated score using an integrated model, Normalizing the integrated score by comparing the integrated score with a background population, Determining a risk score for a subject using a final model, the final model including the age of the subject, Comparing the risk score of the subject with a background population to determine whether the risk score of the subject is classified into a risk category, the risk category including a low-risk group, a medium-risk group, or a high-risk group, Providing treatment to the subject based on the risk category of the subject and a method including the same. 38. The method according to arrangement 37, wherein the subject belongs to a high-risk group, and the treatment provided to the subject is RT intensification. 39. The method according to arrangement 37, wherein the subject belongs to a low-risk group, and the treatment provided to the subject is RT de-intensification or omission. 40. The method according to arrangement 37, wherein the subject belongs to a medium-risk group, and the treatment provided to the subject is standard RT. 41. The method according to any one of arrangements 37 to 40, wherein the background population has a matching age. 42. A method for treating breast cancer, determining an immune score (IS) from a tumor sample based on an immunological model composed of genes of an immunological gene set, determining a proliferation index (PI) from a tumor sample based on a tumor-intrinsic model composed of genes of a tumor-intrinsic gene set, integrating the IS and the PI into an integrated model; integrating the integrated model and the age of the subject into a final model to identify whether the subject belongs to a specific risk clinical group, and providing appropriate treatment to the subject based on the specific risk clinical group and a method including the same. 43. A method for treating breast cancer, identifying a set of related genes from an immunological gene set and a tumor-intrinsic gene set, selecting a plurality of related gene sets, generating an IS and a PI from the selected gene sets, integrating the IS and the PI into an integrated model, integrating the integrated model and the age of the subject to generate a final model, identifying, using the final model, whether the subject belongs to a specific risk clinical group, and providing an appropriate treatment to the subject based on the specific risk clinical group comprising, wherein the IS corresponds to the immunological gene set, and the PI corresponds to the tumor-intrinsic gene set. 44. A method for treating breast cancer, determining tumor aggressiveness, wherein the tumor aggressiveness comprises a histological grade of the tumor obtained from at least a portion of a tumor sample provided by the subject, and the histological grade comprises grade I, grade II, and grade III, classifying the sample as low risk if determined to be a grade I tumor, classifying the sample as high risk if determined to be a grade III tumor, determining the tumor aggressiveness of the sample if determined to be a grade II tumor, wherein the tumor aggressiveness further comprises a proliferation index score of the sample, classifying the grade II tumor a) as high risk if the proliferation index score is greater than or equal to the median score of the background population of grade III tumors, or b) as low risk if the proliferation index score is less than the median score of the background population of grade III tumors and classifying it as such, To determine the immunological activity, determining the immune score of the sample, the level of tumor infiltrating lymphocytes (TIL) in the sample, the level of checkpoint molecules in the sample, or any combination thereof, Integrating tumor aggressiveness and immunological activity by training an elastic net having an interaction term between tumor aggressiveness and immunological activity, Determining a treatment plan based on the integration of tumor aggressiveness and immunological activity comprising The proliferation index score is based on the expression of a first group of genes, The immune score is based on the expression of a second group of genes, The checkpoint molecules include programmed cell death protein-1 (PD-1) and programmed death ligand 1 (PD-L1), The immunological activity is i) active when the TIL score is 10% or more and the checkpoint molecule score is 1% or more of lymphocytes that are positively stained for PD-1 or PD-L1; or ii) inactive when the TIL score is less than 10% and the checkpoint molecule score is less than 1% of lymphocytes that are positively stained for both PD-1 and PD-L1, or inactive when the TIL score is 10% or more and the checkpoint molecule score is less than 1% of lymphocytes that are positively stained for both PD-1 and PD-L1 is determined to be Active immunological activity indicates activated immune infiltration, Inactive immunological activity indicates inactive immune infiltration, The first group of genes includes one or more genes listed in Table 6, The second group of genes includes one or more genes listed in Table 4, The treatment plan is c) if the sample is classified as a high-risk grade II tumor and the immunological activity is active, standard radiotherapy, de-escalation of radiotherapy, or omission of radiotherapy; d) if the sample is classified as a high-risk grade II tumor and the immunological activity is inactive, intensification of radiotherapy e) If the sample is classified as a low-risk grade II tumor and the immunological activity is active, intensification of radiotherapy; or f) If the sample is classified as a low-risk grade II tumor and the immunological activity is inactive, de-intensification of radiotherapy, or omission of radiotherapy A method comprising the steps of 45. A method for treating breast cancer, comprising determining tumor aggressiveness, wherein the tumor aggressiveness comprises the histological grade of the tumor obtained from at least a portion of a tumor sample provided by a subject, and the histological grade comprises grade I, grade II, and grade III, classifying the sample as low risk if determined to be a grade I tumor, classifying the sample as high risk if determined to be a grade III tumor, determining the tumor aggressiveness of the sample if determined to be a grade II tumor, wherein the tumor aggressiveness further comprises the proliferation index score of the sample, classifying the grade II tumor as a) high risk if the proliferation index score exceeds a threshold between 60 and 95 percentile compared to a background population of representative grade II tumors, or b) low risk if the proliferation index score is below a threshold below the 60th percentile relative to the background population of representative grade II tumors determining the immunological activity by determining the immune score of the sample, the level of TILs in the sample, the level of checkpoint molecules in the sample, or a combination thereof, integrating tumor aggressiveness and immunological activity by training an elastic net having an interaction term between tumor aggressiveness and immunological activity, determining a treatment plan based on tumor aggressiveness, immunological activity, and the interaction term and comprising wherein the proliferation index score is based on the expression of a first gene group, ​The immune score is based on the expression of a second group of genes, The checkpoint molecules include PD-1 and PD-L1, The immunological activity is i) active when the TIL score is 10% or more and the checkpoint molecule score is 1% or more of lymphocytes with positive staining for PD-1 or PD-L1, or ii) inactive when the TIL score is less than 10% and / or the checkpoint molecule score is less than 1% of lymphocytes that are positively stained for both PD-1 and PD-L1 determined to be Active immunological activity indicates an activated immune infiltration, Inactive immunological activity indicates an inactive immune infiltration, The first group of genes includes one or more genes listed in Table 6, The second group of genes includes one or more genes listed in Table 4, The treatment plan is c) Standard radiotherapy, de-intensification of radiotherapy, or omission of radiotherapy if the sample is classified as a high-risk grade II tumor and the immunological activity is active; d) Intensification of radiotherapy if the sample is classified as a high-risk grade II tumor and the immunological activity is inactive; e) Intensification of radiotherapy if the sample is classified as a low-risk grade II tumor and the immunological activity is active; f) De-intensification of radiotherapy, or omission of radiotherapy if the sample is classified as a low-risk grade II tumor and the immunological activity is inactive; g) Intensification of radiotherapy if the sample is classified as a grade III tumor and the immunological activity is inactive; h) De-intensification of radiotherapy, or omission of radiotherapy if the sample is classified as a grade III tumor and the immunological activity is active; i) Intensification of radiotherapy if the sample is classified as a grade I tumor and the immunological activity is active; or j) If the sample is classified as a grade I tumor and has inactive immunological activity, de-escalation of radiotherapy or omission of radiotherapy A method comprising. 46. A method for treating breast cancer, subtyping the tumor from at least a part of a tumor sample provided by a subject, wherein the subtypes include luminal A, luminal B, HER2 + , and triple-negative / basal; and when the sample is a luminal A tumor or a luminal B tumor, determining the tumor aggressiveness of the sample, wherein the tumor aggressiveness includes a proliferation index score of the tumor sample; and when the sample is subtyped as a luminal A tumor, the sample is a) high risk if the proliferation index score exceeds the threshold of 60-95 percentile compared to the background population of representative luminal A tumors, or b) low risk if the proliferation index score is below the threshold below the 60th percentile for the background population of representative luminal A tumors classified as; and when the sample is subtyped as a luminal B tumor, the sample is c) high risk if the proliferation index score exceeds the threshold of 60-95 percentile compared to the background population of representative luminal B tumors, or d) low risk if the proliferation index score is below the threshold below the 60th percentile for the background population of representative luminal B tumors classified as; and scoring the immune score of the sample, scoring the level of TILs in the sample, scoring the level of checkpoint molecules in the sample, or determining the immunological activity including any combination thereof; and integrating the tumor aggressiveness and the immunological activity by training an elastic net having an interaction term between the tumor aggressiveness and the immunological activity; and Determining a treatment plan based on integrated tumor aggressiveness, immunological activity, and interaction terms comprising scoring of the proliferation index is based on the expression of a first group of genes, scoring of the immune score is based on the expression of a second group of genes, checkpoint molecules include PD-1 and PD-L1, immunological activity is i) active if the TIL score is 10% or more and either of the checkpoint molecule scores is 1% or more, or ii) inactive if the TIL score is less than 10% and the checkpoint molecule score is less than 1% as determined, active immunological activity indicates activated immune infiltration, inactive immunological activity indicates inactive immune infiltration, the first group of genes includes one or more genes listed in Table 6, the second group of genes includes one or more genes listed in Table 4, the treatment plan is e) de-escalation or omission of radiotherapy if the tumor sample is classified as a low-risk luminal A tumor; f) standard radiotherapy, de-escalation of radiotherapy, or omission of radiotherapy if the sample is classified as a high-risk luminal B tumor and the immunological activity is active; or g) intensification of radiotherapy if the tumor sample is classified as a high-risk luminal B tumor and the immunological activity is inactive A method comprising. 47. A method for treating breast cancer, determining the tumor aggressiveness of a tumor from at least a part of a sample of the tumor provided by a subject, wherein the tumor aggressiveness is such that the tumor a) is high-risk if the proliferation index score of the sample exceeds the threshold of 60-95 percentile compared to a background population of representative tumors, or b) classifying as low-risk if the proliferation index score of the sample is below a threshold that is below the 60th percentile compared to a representative tumor background population, including a proliferation index for classification, determining the immunological activity of the tumor based on the immune score of the sample, integrating the tumor aggressiveness and the immunological activity based on an interaction term between the tumor aggressiveness and the immunological activity, determining a treatment plan based on the tumor aggressiveness, the immunological activity, and the interaction term A method comprising. 48. A method for treating breast cancer, determining the tumor aggressiveness of a tumor from at least a portion of a tumor sample provided by a subject, wherein the tumor aggressiveness classifies the tumor as a) high-risk if the proliferation index score of the sample is greater than or equal to the median score of a representative tumor background population, or b) low-risk if the proliferation index score is less than the median score of a representative tumor background population including a proliferation index score for classification, determining the immunological activity of the tumor based on the immune score of the sample, integrating the tumor aggressiveness and the immunological activity based on an interaction term between the tumor aggressiveness and the immunological activity, determining a treatment plan based on the tumor aggressiveness, the immunological activity, and the interaction term A method comprising. 49. If the tumor is classified as high-risk and the immunological activity is active, the treatment plan includes standard radiotherapy, de-escalation of radiotherapy, or omission of radiotherapy, the method according to arrangement 47 or 48. 50. If the tumor is classified as high-risk and the immunological activity is inactive, the treatment plan includes intensification of radiotherapy, the method according to arrangement 47 or 48. 51. If the tumor is classified as low-risk and the immunological activity is active, the treatment plan includes intensification of radiotherapy, the method according to arrangement 47 or 48. 52. If the tumor is classified as low-risk and the immunological activity is inactive, the treatment plan is the method described in Arrangement 47 or 48, including de-escalation of radiotherapy or omission of radiotherapy. 53. The method according to any one of Arrangements 47 to 52, wherein the integration step includes training an elastic net having an interaction term between tumor aggressiveness and immunological activity. 54. A high proliferation index score indicates an aggressive tumor, If the sample is classified as an aggressive tumor and the immunological activity is inactive, the treatment includes intensification of radiotherapy, according to the method described in any one of Arrangements 47 and 49 to 53. 55. The method according to Arrangement 54, wherein the high proliferation index score includes the proliferation index of a sample that is at least in the 60th percentile compared to a background population of representative tumors. 56. A high proliferation index score indicates an aggressive tumor, If the sample is classified as an aggressive tumor and the immunological activity is inactive, the treatment includes intensification of radiotherapy, according to the method described in any one of Arrangements 48 to 53. 57. The method according to Arrangement 56, wherein the high proliferation index score includes the proliferation index of a sample that is greater than or equal to the median score of a background population of representative tumors. 58. The method according to any one of Arrangements 47 to 57, wherein the scoring of the proliferation index is based on the expression of a first gene group. 59. The method according to Arrangement 58, wherein the first gene group includes one or more genes listed in Table 6. 60. The method according to any one of Arrangements 47 to 59, wherein the scoring of the immune score is based on the expression of a second gene group. 61. The method according to Arrangement 60, wherein the second gene group includes one or more genes listed in Table 4. 62. The method according to any one of Arrangements 47 to 61, wherein determining the immunological activity further includes scoring TILs in the sample. 63. Active immunological activity includes activated tumor infiltration, The activated tumor infiltration includes a TIL score of 10% or more, and the method according to arrangement 62. 64. The method according to any one of arrangements 47 to 60, wherein the step of determining immunological activity further includes measuring the expression of one or more checkpoint molecules. 65. The method according to arrangement 62, wherein the step of determining immunological activity further includes measuring the expression of one or more checkpoint molecules. 66. The method according to arrangement 64 or 65, wherein the checkpoint molecules include PD-1 and PD-L1. 67. Active immunological activity includes activated tumor infiltration, The activated tumor infiltration includes positive staining for the expression of one or more checkpoint molecules, and the method according to arrangement 66. 68. The method according to arrangement 67, wherein the step of determining immunological activity further includes scoring TILs in the sample. 69. The method according to arrangement 68, wherein the activated tumor infiltration further includes a TIL score of 10% or more. 70. The method according to arrangement 62, wherein inactive immunological activity includes a TIL score of less than 10%. 71. The integration step is performed by training an elastic net having an interaction term between tumor aggressiveness and immunological activity, and the method according to any one of arrangements 47 to 70. 72. The method according to any one of arrangements 47 to 71, wherein tumor aggressiveness further includes the histological grade of a tumor sample. 73. The method according to arrangement 72, wherein the histological grade of the tumor sample is determined as a grade II tumor. 74. Further including determining the subtype of the tumor sample before determining tumor aggressiveness, and the subtype includes luminal A, luminal B, HER2 + , and triple-negative / basal, and the method according to any one of arrangements 47 and 49 to 70. 75. The method according to arrangement 74, wherein the subtype is luminal A. 76. The method according to arrangement 74, wherein the subtype is luminal B. 77. A method for treating breast cancer, comprising: determining the histological grade of a tumor from at least a sample of the tumor provided by a subject, wherein the histological grade of the tumor comprises grade I, grade II, or grade III; assigning the tumor aggressiveness of the sample as a) low risk if the sample is determined to be a grade I tumor; b) high risk if the sample is determined to be a grade III tumor; c) low risk if the sample is determined to be a grade II tumor and the proliferation index score of the sample is less than the median score of the background tumor population; d) high risk if the sample is determined to be a grade II tumor and the proliferation index score of the sample is greater than or equal to the median of the background tumor population ; and classifying the immunological activity as active or inactive, the classification comprising e) the immune score of the sample; f) the level of TILs in the sample; g) the level of checkpoint molecules in the sample; or h) any combination of e) - f) ; and integrating the tumor aggressiveness and the immunological activity and determining the benefit of the treatment plan using an interaction term ; and 78. The method according to arrangement 77, wherein when the tumor is classified as high risk and the immunological activity is active, the treatment plan comprises standard radiotherapy, de - escalation of radiotherapy, or omission of radiotherapy. 79. The method according to arrangement 77, wherein when the tumor is classified as high risk and the immunological activity is inactive, the treatment plan comprises intensification of radiotherapy. 80. If the tumor is classified as low-risk and the immunological activity is active, the treatment plan is the method described in Arrangement 77, including intensification of radiotherapy. 81. If the tumor is classified as low-risk and the immunological activity is inactive, the treatment plan is the method described in Arrangement 77, including de-intensification or omission of radiotherapy. 82. The method according to any one of Arrangements 77 to 81, wherein the integration step includes training an elastic net having an interaction term between tumor aggressiveness and immunological activity. 83. A high proliferation index score indicates an aggressive tumor, If the sample is classified as an aggressive tumor and the immunological activity is inactive, the treatment includes intensification of radiotherapy, the method described in any one of Arrangements 77 to 82. 84. The method according to any one of Arrangements 77 to 83, wherein the score of the proliferation index is based on the expression of a first gene group. 85. The method according to Arrangement 84, wherein the first gene group includes one or more genes listed in Table 6. 86. The method according to any one of Arrangements 77 to 85, wherein the immune score is based on the expression of a second gene group. 87. The method according to Arrangement 86, wherein the second gene group includes one or more genes listed in Table 4. 88. The method according to any one of Arrangements 77 to 87, wherein the checkpoint molecules include PD-1 and PD-L1. 89. The immunological activity is f) when it is 10% or more; and g) when it is 1% or more of at least one of the checkpoint molecules is active, the method according to Arrangement 88. 90. The method according to Arrangement 89, wherein the active immunological activity shows activated tumor infiltration. 91. The method according to Arrangement 90, wherein the activated tumor infiltration includes positive staining for the expression of one or more checkpoint molecules. 92. The immunological activity is f) is less than 10%, and / or g) is less than 1% for both checkpoint molecules The method according to arrangement 88, which is inactive in the case. 93. A method for treating breast cancer, comprising: providing a sample of a tumor; undergoing treatment based on an analysis of the sample, wherein the analysis comprises: determining tumor aggressiveness from the sample; determining the immunological activity of the tumor from the sample, and integrating tumor aggressiveness and immunological activity based on an interaction term including; including, the tumor aggressiveness includes a proliferation index score that classifies the tumor as high risk or low risk, the immunological activity is a) the immune score of the sample; b) the level of TIL in the sample; c) the level of checkpoint molecules in the sample; or d) any combination of a) to c) The method comprising. 94. If the tumor e) has a high risk when the proliferation index score of the sample exceeds the threshold of 60-95 percentile compared to a representative tumor background population, or f) has a low risk when the proliferation index score of the sample is below the threshold below 60 percentile compared to a representative tumor background population The method according to arrangement 93, which is classified as. 95. If the tumor g) has a high risk when the proliferation index score of the sample is greater than or equal to the median score of a representative tumor background population, or h) has a low risk when the proliferation index score of the sample is lower than the median score of a representative tumor background population The method according to arrangement 93, which is classified as. 96. If the tumor is classified as high-risk and the immunological activity is active, the treatment plan is the method described in Arrangement 94 or 95, including standard radiotherapy, de-intensification of radiotherapy, or omission of radiotherapy. 97. If the tumor is classified as high-risk and the immunological activity is inactive, the treatment plan is the method described in Arrangement 94 or 95, including intensification of radiotherapy. 98. If the tumor is classified as low-risk and the immunological activity is active, the treatment plan is the method described in Arrangement 94 or 95, including intensification of radiotherapy. 99. If the tumor is classified as low-risk and the immunological activity is inactive, the treatment plan is the method described in Arrangement 94 or 95, including de-intensification of radiotherapy or omission of radiotherapy. 100. A high proliferation index score indicates that the tumor is aggressive, according to the method described in Arrangement 94 or 95. 101. If the tumor is shown to be aggressive, the treatment includes intensification of radiotherapy, according to the method described in Arrangement 100. 102. including activated tumor infiltration with active immunological activity, the activated tumor infiltration includes a TIL score of 10% or more, the inactivated tumor infiltration includes a TIL score of less than 10%, according to any one of Arrangements 94 to 101. 103. The checkpoint molecules include PD-1 and PD-L1, according to any one of Arrangements 94 to 102. 104. The checkpoint molecules include PD-1 and PD-L1, the activated tumor infiltration further includes positive staining for the expression of one or more checkpoint molecules, according to the method described in Arrangement 102. 105. Tumor aggressiveness further includes the histological grade of the tumor sample, according to any one of Arrangements 93 to 104. 106. The histological grades include Grade I, Grade II, and Grade III, according to the method described in Arrangement 105. 107. The tumor analysis further includes determining the subtype of the tumor sample before determining tumor aggressiveness, and the subtype includes luminal A, luminal B, HER2 + , and any one of arrangements 93 to 104 including triple-negative / basal. 108. The method according to arrangement 107, wherein the subtype is luminal A. 109. The method according to arrangement 108, wherein the subtype is luminal B. 110. The method according to any one of arrangements 93 to 109, wherein the proliferation index scoring is based on the expression of a first gene group. 111. The method according to arrangement 110, wherein the first gene group includes one or more genes listed in Table 6. 112. The method according to any one of arrangements 93 to 111, wherein the immune score is based on the expression of a second gene group. 113. The method according to arrangement 112, wherein the second gene group includes one or more genes listed in Table 4. 114. The method according to any one of arrangements 93 to 113, wherein the integration step includes training an elastic net, and the elastic net includes an interaction term between tumor aggressiveness and immunological activity. 115. The method according to arrangement 114, wherein the elastic net further includes age information of the subject who provided the sample. 116. The method according to any one of arrangements 93 to 115, wherein the analysis further includes stratification by premenopausal and postmenopausal states. 117. The premenopausal state includes female subjects under 55 years old, The postmenopausal state includes women 55 years old or older. The method according to arrangement 116. 118. The background population has the same age. The method according to any one of arrangements 44 to 117. 119. A method of treating a subject, determining a proliferation index based on the levels of one or more genes in Table 6, and Determining an immune score based on the expression of one or more genes in Table 4, combining the proliferation index and the immune score, and optionally including the age of the subject as a factor, to determine whether the subject will respond to cancer treatment, and administering cancer treatment if the proliferation index and the immune score, and optionally the age of the subject, indicate that the treatment will be successful A method comprising. 120.a) If the proliferation index score of the sample exceeds the threshold of 60-95 percentile compared to a background population of representative tumors, it is high risk, or b) If the proliferation index score of the sample is below the threshold below the 60th percentile compared to a background population of representative tumors, it is low risk, c) combining the proliferation index and the immune score based on the interaction term between the two, and d) determining cancer treatment based on the proliferation index, the immune score, and the interaction term, the method of arrangement 119. 121. The method according to arrangement 120, wherein the background population has a matching age. 122. A method of treating a subject, including the level of one or more genes in Table 6 as a factor, including the level of one or more genes in Table 4 as a factor, including the age of the subject as a factor, thereby determining whether the subject should receive standard radiotherapy, intensified radiotherapy, de-intensified radiotherapy or omission of radiotherapy A method comprising. 123. The method according to any one of the preceding arrangements, wherein the expression of one or more genes related to IS and / or PI is measured from a sample derived from the subject. 124. The method according to arrangement 123, or any one of the preceding arrangements, wherein the sample comprises a core biopsy or consists essentially of a core biopsy. 125. The method according to arrangement 123, or any one of the preceding arrangements, wherein the sample does not contain TIL. 126. The method according to any one of arrangement 123 or the preceding arrangements, wherein the analysis does not measure the TIL level. 127. The method according to any one of arrangement 123 or the preceding arrangements, wherein the sample consists of a core biopsy. 128. The method according to any one of arrangement 123 or the preceding arrangements, wherein the sample is collected before treatment of the subject for cancer. 129. The method according to any one of arrangement 123 or the preceding arrangements, wherein the sample is collected before therapeutic surgery of the subject for cancer. 130. The method according to any one of the preceding arrangements, wherein age is used as a factor in determining an appropriate treatment or procedure. 131. The method according to arrangement 130, wherein a subject less than 50 years old is regarded as young, and a subject 50 years old or older is regarded as elderly. 132. A method of determining a treatment for cancer treatment, comprising determining an immune score of a core biopsy without determining the level of TIL surrounding the tumor from which the core biopsy was obtained, and using the immune score to determine the amount of radiation therapy to be administered to the subject without including the level of TIL as a factor. 133. The method according to any one of the preceding arrangements, wherein the subject is treated with neoadjuvant immunotherapy. 134. The method according to any one of the preceding arrangements, wherein the subject is 55 years old or older. 135. The method according to any one of arrangements 1 to 133, wherein the subject is over 55 years old. 136. The method according to arrangement 134 or 135, wherein the subject is 65 years old or older. 137. The method according to arrangement 134 or 135, wherein the subject is over 65 years old. 138. The method according to any one of arrangements 1 to 133, wherein the subject is 55 years old or younger. 139. The method according to any one of arrangements 1 to 133, wherein the subject is less than 55 years old. 140. The method according to arrangement 138 or 139, wherein the subject is under 50 years old. 141. The method according to any one of arrangements 1 to 133, wherein the subject is 50 to 65 years old. 142. The method according to any one of the preceding arrangements, wherein the tumor is sampled before neoadjuvant therapy or adjuvant therapy. 143. The method according to any one of the preceding arrangements, wherein the stromal TILS around the tumor are not used and / or not available for analysis, or are used for analysis as part of the method. 144. A method for predicting the effectiveness of cancer treatment, the method comprising analyzing a sample for the presence of one or more of the genes in Table 4 and / or one or more of the genes in Table 6, wherein the variation at the gene level indicates the effectiveness of cancer treatment. 145. The method according to arrangement 144, further comprising administering an appropriate therapy to the subject based on the analysis of one or more genes in Table 4 and / or Table 6. 146. A method for treating breast cancer, determining the histological grade of the tumor from at least a sample of the tumor provided by the subject, the histological grade of the tumor including grade I, grade II, or grade III, determining the tumor aggressiveness of the sample a) low risk if the sample is determined to be a grade I tumor; b) low risk if the sample is determined to be a grade II tumor and the proliferation index score of the sample is less than the median of the background population of high-risk tumors; c) high risk if the sample is determined to be a grade II tumor and the proliferation index score of the sample is greater than or equal to the median score of the background population of high-risk tumors; or d) the sample is i) a grade III tumor, ii) the proliferation index score of the sample is greater than or equal to the median score of the background population of high-risk tumors, iii) ER negative, iv) HER2 positive, v) high Ki67 level, vi) high mutation load, or vii) any combination of i) - vi) assigned as high risk when determined as such, classifying the immunological activity, including the level of TILs in the sample; integrating tumor aggressiveness and immunological activity using interaction terms to determine a treatment plan and a method comprising the same. 147. The method according to arrangement 146, wherein the Ki67 level is 10% or more. 148. The method according to arrangement 146, wherein the Ki67 level is 20% or more. 149. The method according to arrangement 146, wherein the Ki67 level is 30% or more. 150. The method according to any one of arrangements 146 - 149, wherein the mutation load is 5 mutations or more per genomic megabase (mutations / MB). 151. The method according to any one of arrangements 146 - 149, wherein the mutation load is 7 mutations / MB or more. 152. The method according to any one of arrangements 146 - 149, wherein the mutation load is 10 mutations / MB or more. 153. A method for diagnosing breast cancer, determining the histological grade of a tumor from at least a sample of the tumor provided by a subject, wherein the histological grade of the tumor includes grade I, grade II, or grade III, the tumor aggressiveness of the sample a) low risk when the sample is determined to be a grade I tumor; b) low risk when the sample is determined to be a grade II tumor and the proliferation index score of the sample is less than the median of the background population of high - risk tumors; c) If the sample is determined to be a Grade II tumor and the proliferation index score of the sample is greater than or equal to the median score of the background population of high-risk tumors, then it is high-risk; or d) If the sample is i) a Grade III tumor, ii) the proliferation index score of the sample is greater than or equal to the median score of the background population of high-risk tumors, iii) ER-negative, iv) HER2-positive, v) the level of Ki67 is high, vi) the mutation load is high, or vii) any combination of i) - vi), then it is determined to be high-risk and assigned as such, classify the immunological activity, including the level of TILs in the sample, integrate tumor aggressiveness and immunological activity using interaction terms A method comprising the above steps. 154. The method according to arrangement 153, wherein the level of Ki67 is 10% or more. 155. The method according to arrangement 153, wherein the level of Ki67 is 20% or more. 156. The method according to arrangement 153, wherein the level of Ki67 is 30% or more. 157. The method according to any one of arrangements 153 - 156, wherein the mutation load is 5 mutations or more per megabase of the genome (mutations / MB). 158. The method according to any one of arrangements 153 - 156, wherein the mutation load is 7 mutations / MB or more. 159. The method according to any one of arrangements 153 - 156, wherein the mutation load is 10 mutations / MB or more. 160. The method according to any one of arrangements 153 - 159, wherein the classification of immunological activity further includes the level of checkpoint molecules in the sample. 161. The method according to arrangement 160, wherein the checkpoint molecules include PD-1 and PD-L1. 162. A method for treating breast cancer, comprising: determining the histological grade of a tumor from at least a sample of the tumor provided by a subject, wherein the histological grade of the tumor comprises Grade I, Grade II, or Grade III; assigning tumor aggressiveness to the sample; classifying immunological activity, including the level of TILs in the sample; integrating tumor aggressiveness and immunological activity using an interaction term and determining a treatment plan; and a method comprising the steps above. 163. Assigning high-risk tumor aggressiveness to a sample is determined by the sample being i) a Grade III tumor; ii) the proliferation index score of the sample being greater than or equal to the median score of the tumor background population; iii) ER negative; iv) HER2 positive; v) a high level of Ki67; vi) a high mutation burden, or vii) any combination of i) to vi); and the method according to arrangement 162. 164. The method according to arrangement 163, wherein the level of Ki67 is 10% or more. 165. The method according to arrangement 163, wherein the level of Ki67 is 20% or more. 166. The method according to arrangement 163, wherein the level of Ki67 is 30% or more. 167. The method according to any one of arrangements 163 to 166, wherein the mutation burden is 5 mutations / MB or more. 168. The method according to any one of arrangements 163 to 166, wherein the mutation burden is 7 mutations / MB or more. 169. The method according to any one of arrangements 163 to 166, wherein the mutation burden is 10 mutations / MB or more. 170. Assigning a high risk of tumor aggressiveness includes determining the sample as a grade II tumor and the proliferation index score of the sample being greater than or equal to the median score of the tumor background population, the method according to arrangement 162. 171. The method according to any one of arrangements 146 to 170, wherein the tumor aggressiveness is high risk. 172. The level of TIL in the sample is in the range of 50% to 90%, The treatment plan includes omission of radiotherapy, the method according to arrangement 171. 173. The level of TIL in the sample is in the range of 10% to 49%, The treatment plan includes omission of boost, the method according to arrangement 171. 174. The classification of immunological activity further includes the level of checkpoint molecules in the sample, the method according to any one of arrangements 146 to 170. 175. The method according to arrangement 174, wherein the checkpoint molecules include PD-1 and PD-L1. 176. The level of PD-1 is 1% or more, the method according to arrangement 175. 177. The level of PD-L1 is 1% or more, the method according to arrangement 176. 178. The level of TIL is 10% or more, the method according to arrangement 177. 179. The treatment plan includes omission of RT, the method according to arrangement 178. 180. The level of PD-1 is less than 1%, the method according to arrangement 175. 181. The level of PD-L1 is less than 1%, the method according to arrangement 180. 182. The level of TIL is 10% or more, the method according to arrangement 181. 183. The treatment plan includes omission of RT boost, the method according to arrangement 182. 184. The subject is 55 years old or older, the method according to any one of arrangements 146 to 183. 185. The method according to any one of arrangements 146 to 183, wherein the subject is over 55 years old. 186. The method according to any one of arrangements 146 to 183, wherein the subject is 65 years old or older. 187. The method according to any one of arrangements 146 to 183, wherein the subject is over 65 years old. 188. The method according to any one of arrangements 146 to 183, wherein the subject is 55 years old or younger. 189. The method according to any one of arrangements 146 to 183, wherein the subject is under 55 years old. 190. The method according to any one of arrangements 146 to 183, wherein the subject is 50 to 65 years old. 191. The method according to any one of arrangements 146 to 183, wherein the subject is under 50 years old. 192. The method according to any one of arrangements 146 to 191, wherein the background group has a matching age. 193. The method according to any one of arrangements 146 to 192, wherein the sample includes a core biopsy. 194. A method for treating breast cancer, comprising: determining the tumor-intrinsic aggressiveness of a sample provided by a subject, wherein the tumor-intrinsic aggressiveness includes the proliferation index of the sample, the histological grade of the sample, the Ki67 level of the sample, the HER2 expression of the sample, the ER status of the sample, or a combination thereof; determining the immune infiltration of the sample, wherein the immune infiltration includes the immune score of the sample, the level of TIL in the sample, the level of PD-1 in the sample, the level of PD-L1 in the sample, or a combination thereof; providing treatment based on the tumor-intrinsic aggressiveness and the immune infiltration; and wherein the subject is 65 years old or older. 195. The tumor-intrinsic risk is whether the proliferation index exceeds the 60th percentile compared to the background population of representative patients and tumors; the histological grade is 3; Whether the histology is 2 and the proliferation index is equal to or greater than the median of the background population of tumors with a grade of 3; Whether HER2 expression is amplified; Whether the ER status of the sample is negative; or That combination If so, the method described in arrangement 194, which is high. 196. Immune infiltration is Whether the immune score exceeds the 60th percentile threshold compared to the background population of representative tumors; Whether the level of TIL in the sample is greater than 10%; Whether the level of PD-1 in the sample is greater than 1%; Whether the level of PD-L1 in the sample is greater than 1%, or That combination If so, the method described in arrangement 194 or 195, which is activated. 197. The tumor intrinsic risk is high and immune activation is inactive, The method described in arrangement 196, where the treatment includes RT boost. 198. The tumor intrinsic risk is high and immune activation is inactive, the subject has no comorbidities, The method described in arrangement 196, where the treatment includes intensification of chemotherapy. 199. The method described in arrangement 198, where the treatment further includes omission of immunotherapy. 200. The sample is ER positive, The method described in arrangement 198 or 199, where the treatment further includes endocrine therapy. 201. The tumor intrinsic risk is high and immune activation is inactive, the subject has no comorbidities, The method described in arrangement 196, where the treatment includes omission of immunotherapy. 202. The tumor intrinsic risk is high and immune activation is inactive, the subject has no comorbidities, the sample is ER positive, The method described in arrangement 196, where the treatment includes endocrine therapy. 203. The tumor-intrinsic risk is not high, the immune infiltration is not activated, and the subject does not have a co-existing disease, The method according to arrangement 196, wherein the treatment includes omission of RT. 204. The method according to arrangement 203, wherein the treatment further includes omission or de-escalation of chemotherapy. 205. The method according to arrangement 204, wherein the treatment further includes endocrine therapy. 206. The tumor-intrinsic risk is not high, the immune infiltration is not activated, and the subject does not have a co-existing disease, The method according to arrangement 196, wherein the treatment includes omission or de-escalation of chemotherapy. 207. The tumor-intrinsic risk is not high, the immune infiltration is not activated, and the subject does not have a co-existing disease, The method according to arrangement 196, wherein the treatment includes endocrine therapy. 208. The tumor-intrinsic risk is not high, the immune infiltration is activated, and the subject does not have a co-existing disease, The method according to arrangement 196, wherein the treatment includes RT. 209. The method according to arrangement 208, wherein the treatment further includes escalation of chemotherapy. 210. The method according to arrangement 209, wherein the treatment further includes endocrine therapy. 211. The tumor-intrinsic risk is not high, the immune infiltration is activated, and the subject does not have a co-existing disease, The method according to arrangement 196, wherein the treatment includes escalation of chemotherapy. 212. The tumor-intrinsic risk is not high, the immune infiltration is activated, and the subject does not have a co-existing disease, The method according to arrangement 196, wherein the treatment includes endocrine therapy. 213. The method according to any one of arrangements 194 to 196, wherein the subject has a co-existing disease. 214. The method according to arrangement 213, wherein the treatment includes omission of chemotherapy and de-escalation of RT. The method according to any one of arrangements 198 to 214, wherein the coexisting disease comprises coronary artery disease, heart failure, chronic obstructive pulmonary disease, previous stroke (ischemic or hemorrhagic), uncontrolled hypertension, diabetes, osteoporosis, one or more other cancers, or a combination thereof. 216. A method for treating breast cancer, comprising: constructing an integrated model from a sample provided by a subject and generating an integrated score, wherein the integrated model includes a proliferation index and an immune score; constructing a final model based on the integrated model and the age of the subject and generating a final score; providing treatment based on the integrated / final score including the integrated score or the final score; and wherein the subject is 65 years of age or older. 217. The method according to arrangement 216, wherein the integrated / final score is high if the integrated score or the final score exceeds the 60th percentile of a representative background population of patients and tumors. 218. The method according to arrangement 217, wherein the background population has a matching age. 219. The method according to arrangement 217, wherein the background population has the same age as the subject. 220. The method according to any one of arrangements 216 to 219, further comprising determining the immune infiltration of the sample, wherein the immune infiltration includes the immune score of the sample. 221. The method according to arrangement 220, wherein the immune infiltration further includes the level of TIL in the sample, the level of PD-1 in the sample, the level of PD-L1 in the sample, or a combination thereof. 222. The immune infiltration is when the immune score exceeds the 60th percentile threshold compared to a representative tumor background population; when the level of TIL in the sample is greater than 10%; when the level of PD-1 in the sample is greater than 1%; when the level of PD-L1 in the sample is greater than 1%, or The method according to arrangement 221, which is activated when in that combination. 223. The method according to arrangement 220, wherein the immune infiltration is activated when the immune score exceeds the 60th percentile threshold compared to a representative patient and tumor background population. 224. The method according to arrangement 223, wherein the background population has the same age. 225. The method according to arrangement 223, wherein the background population has the same age as the subject. 226. The integrated / final score is high, The method according to any one of arrangements 216 - 225, wherein the treatment includes RT boost. 227. The integrated / final score is not high, the immune infiltration is active, The method according to any one of arrangements 222 - 225, wherein the treatment includes omission of chemotherapy or de-escalation of chemotherapy. 228. The method according to arrangement 227, wherein the treatment further includes immunotherapy. 229. The sample is ER positive, The method according to arrangement 228, wherein the treatment further includes endocrine therapy. 230. The integrated / final score is high, the immune infiltration is activated, The method according to any one of arrangements 222 - 225, wherein the treatment includes immunotherapy. 231. The integrated / final score is not high, the immune infiltration is not activated, The method according to any one of arrangements 222 - 225, wherein the treatment includes RT omission. 232. The method according to arrangement 231, wherein the treatment further includes omission or de-escalation of chemotherapy. 233. The method according to arrangement 232, wherein the treatment further includes endocrine therapy. 234. The integrated / final score is not high, the immune infiltration is activated, The method according to any one of arrangements 222 - 225, wherein the treatment includes standard RT. The method according to arrangement 234, wherein the treatment further comprises escalation of chemotherapy. 236. The integrated / final score is not high, the immune infiltration is activated, The method according to any one of arrangements 222 to 225, wherein the treatment comprises escalation of chemotherapy. 237. The method according to any one of arrangements 234 to 236, wherein the treatment further comprises endocrine therapy. 238. A method for treating breast cancer, Determining the tumor-intrinsic aggressiveness of a sample provided by a subject, wherein the tumor-intrinsic aggressiveness includes the growth index of the sample, the histological grade of the sample, the Ki67 level of the sample, the HER2 expression of the sample, the ER status of the sample, or a combination thereof, Determining the immune infiltration of the sample, wherein the immune infiltration includes the immune score of the sample, the level of TIL of the sample, the level of PD-1 of the sample, the level of PD-L1 of the sample, or a combination thereof, Providing treatment based on tumor-intrinsic aggressiveness and immune infiltration comprising The method, wherein the subject is less than 55 years old. 239. A method for treating breast cancer, Constructing an integrated model from a sample provided by a subject and generating an integrated score, wherein the integrated model includes the growth index and the immune score, Constructing a final model based on the integrated model and the age of the subject and generating a final score, Providing treatment based on the integrated / final score including the integrated score or the final score comprising The method, wherein the subject is less than 55 years old. 240. The method according to arrangement 239, wherein the integrated / final score is less than the 5th percentile of a representative background population of the patient and the tumor. 241. The method according to arrangement 240, wherein the treatment comprises RT de-escalation. 242. The method according to arrangement 239, wherein the integrated / final score is greater than the 50th percentile of a representative background population of patients and tumors. 243. The method according to arrangement 242, wherein the treatment includes RT escalation as compared to standard RT. 244. The method according to arrangement 243, wherein the RT escalation includes an RT boost. 245. The method according to arrangement 239, wherein the integrated / final score is greater than the 95th percentile of a representative background population of patients and tumors. 246. The method according to arrangement 245, wherein the treatment includes surgical escalation. 247. The method according to arrangement 246, wherein the surgical escalation includes mastectomy. 248. The method according to arrangement 246, wherein the surgical escalation further includes a wider resection margin. 249. The method according to arrangement 247 or 248, wherein the treatment further includes escalation of systemic therapy. 250. The method according to arrangement 245, wherein the treatment includes escalation of systemic therapy. 251. The method according to any one of arrangements 240 to 250, wherein the background population has a matching age. 252. The method according to any one of arrangements 240 to 250, wherein the background population has an age matching the subject. 253. A method for treating breast cancer, comprising: constructing an integrated model from a sample provided by a subject and generating an integrated score, wherein the integrated model includes a proliferation index and an immune score; constructing a final model based on the integrated model and the age of the subject and generating a final score; providing treatment based on an integrated / final score including the integrated score or the final score; and wherein the subject is 55 years of age or older. 254. The method according to arrangement 253, wherein the integrated / final score exceeds the 70th percentile of a representative background population of patients and tumors. 255. The method according to arrangement 254, wherein the treatment includes RT escalation. 256. The method according to arrangement 255, wherein the RT escalation includes RT boost. 257. The method according to arrangement 253, wherein the integrated / final score exceeds the 85th percentile of a representative background population of patients and tumors. 258. The method according to arrangement 257, wherein the treatment includes mastectomy. 259. The method according to arrangement 257, wherein the treatment includes escalated systemic therapy. 260. The method according to any one of arrangements 253 to 259, wherein the background population has the same age. 261. The method according to any one of arrangements 253 to 259, wherein the background population has the same age as the subject. 262. The method according to any one of the preceding arrangements regarding treatment, wherein the subject is administered or receives the indicated or disclosed treatment or therapy.

Example

[0257] Example 1 The influence of local immune infiltration in tumor progression is closely related to tumor-intrinsic factors. Genomically unstable tumors with a high tumor mutation burden benefit more from immune infiltration. The aim is to investigate whether clinically high-risk patients who could be candidates for de-escalation of radiotherapy (RT) can be identified by integrating immunological factors and tumor-intrinsic factors.

[0258] For the SweBCG91RT trial, 1,178 stage I-IIA breast cancer patients participated, were randomly assigned to a group with or without the combination of breast-conserving surgery and adjuvant RT after surgery, and were followed up for a median of 15.2 years. A total of 8% received systemic therapy. Using a publicly available cohort, two models were trained, each designed to capture immune activity and immune-modulatory tumor-intrinsic properties. Subsequently, an analysis was conducted to determine whether high-risk tumors with a favorable prognosis could be identified without RT boost by combining these two variables.

[0259] The immunological model (immune score) and the tumor-intrinsic model (proliferation index) showed a strong interaction (p = 0.01). Aggressive tumors with low immunological activity (i.e., high scores on the tumor-intrinsic model called the proliferation index) had the highest risk of ipsilateral breast tumor recurrence (IBTR). By integrating the immunological model and the tumor-intrinsic model, patients who were irradiated with grade III tumors under 70 years old, and patients under 60 years old regardless of histological grade, were identified, and despite no RT boost and a low frequency of systemic therapy, their 10-year IBTR risk was less than 10%.

[0260] Model training Training cohort The publicly available breast cancer dataset was downloaded using the R package MetaGxBreast [1] (Table 3). For the TCGA cohort, updated follow-up data were obtained using the R package curatedTCGAData [2]. Cohorts with available outcome data (n = 21) were selected as the training cohort. The Pam50 subtype was estimated using the genefu package [3]. The endpoints used were selected in the following order based on availability: 1. distant metastasis. 2. any recurrence. 3. overall survival. Age was included as a covariate for overall survival. The follow-up period was 10 years for distant metastasis and any recurrence, and 15 years for overall survival.

[0261] The rank-based method showed robustness in analysis across different array platforms. To minimize batch effects, the rank-based single-sample gene set enrichment analysis (ssGSEA) method was selected. HGNChelper was used to harmonize gene names between different training cohorts. Without being bound by theory, gene sets from msigdb were used as features because there is a hypothesis that biological processes rather than individual genes facilitate prognosis and treatment prediction. Different from individual genes that are involved in several different biological pathways and may be expressed in various cell types, gene sets are more specific to a given underlying biological process, which was important because accurate assessment of immune infiltration was required to test this hypothesis. Finally, since the enrichment score is calculated based on genes available in each gene set, the analysis did not need to be limited to genes profiled in all 21 training cohorts. In other words, the enrichment score was calculated for each included gene set using the ssGSEA method. Gene sets and coefficients are shown in Table 4 and / or Table 6. Next, the enrichment score for each gene set is centered so that the mean is 0 and the standard deviation is 1. Each enrichment score is multiplied by its respective coefficient, and the values are summed to obtain the immune score or the proliferation index score. The integrated score is obtained by centering the mean of the immune score and the proliferation index to 0 and the standard deviation to 1. Next, the integrated score is obtained using the formula in Table 8.

Table 3-1

Table 3-2

[0262] Immune score In a non-limiting example, since less aggressive subtypes show a more heterogeneous immunological prognostic signal, immune models were based on basal and HER2 +Training was performed within tumors (n = 2230). ssGSEA scores were obtained using the msigdb and GSVA packages for gene sets from the C7 category (immunological signature gene sets) and all additional gene sets, including any keyword of "LYMPHOCYTE|T_CELL|PD1|PD-1|PDL1|PD-L1|LAG3|CHECKPOINT_RECEPTOR|B_CELL|PERFORIN|GRANZYME|NK_CELL|CD8|CYTOTOXIC" (cell type signature gene sets) (N = 5661 gene sets, including over 20,000 genes in total).

[0263] The characteristics of the gene sets were analyzed in a Cox regression model, and the p-values were saved as two-sided (each p-value multiplied by the sign of each coefficient), and then converted to one-sided p-values using the metap package [4]. Next, meta-analysis of the 21 p-values for each feature was performed using the weighted sum Z (Stouffer’s) method [5]. The square root of the number of observations in each cohort was used as the weight. The top 50 gene sets were selected, and Spearman correlations were calculated for each possible pairwise combination of the 50 gene sets within each cohort. The mean value of each pairwise correlation was generated using all 22 cohorts. To reduce the multicollinearity of the variables, the gene set with the lowest p-value among all gene sets where the mean value of the pairwise correlation exceeded 0.7 or was less than -0.7 was removed as showing a strong association. This resulted in the 22 remaining gene sets shown in Table 4, which were then scaled and centered within each cohort and the cohorts were integrated. An elastic net model was fitted using the caret package [6] (Figure 1). The resulting model was named the immune score (Figure 1). In some embodiments, this model is based on an immunological model, which includes genes from immunological gene sets, some of which are shown in Table 4. Regarding correlations, in some embodiments, different thresholds may be selected to perform collinearity filtering (i.e., removal of highly correlated gene sets). In some embodiments, the top number of gene sets selected may be the same as that of the general model described above: in some embodiments, the top 5 gene sets are selected; in some embodiments, the top 200 gene sets are selected; or, in some embodiments, any number from the top 5 gene sets to the top 200 gene sets is selected (e.g., the top 10 gene sets, the top 15 gene sets,... the top 100 gene sets,... etc.).

[0264] Since this model is trained for prognosis, it measures the quantity and quality of the local immune response. However, since direct measurements of immune infiltration were not used in developing the model, its relevance was verified by comparing it to the immunohistochemical evaluation of stromal tumor-infiltrating lymphocytes (TIL) on whole tissue sections scored as described above [7], which is hereby incorporated by reference in its entirety. Additionally, xCell [8] and ESTIMATE [9], deconvolution methods developed to quantify immune infiltration from gene expression data, were used as controls for correlation with TIL. The results of the present inventors show that the immune score has a stronger Spearman correlation with TIL than ESTIMATE and xCell (rho 0.42, p<0.001 vs rho 0.33, p<0.001, rho 0.27, p<0.001, respectively). In other words, the immunological model (immune score) disclosed herein functions better than the already established methods xCell and ESTIMATE. This is also evident from the fact that the correlation between the immune score and TIL is stronger than the correlation between ESTIMATE / xCell and TIL.

Table 4-1

Table 4-2

Table 4-3

Table 5

[0265] Calculation of the risk score In this non-limiting example for calculating a risk score, Table 4 here serves as an example of how the risk score is calculated from the values provided in the table. Multiply the average value of one or more genes for each gene set by their respective coefficients. Then sum the product (average value of the gene × coefficient) to obtain the risk score. The intercept is a constant and does not change. To show an example of the first two gene sets, AGR2 and AQP3 are used from the first gene set, and ADA and CCNB2 are used from the second gene set. As an example, the measured values of gene expression are as follows: AGR2: 0.5; AQP3: 1; ADA: 0.25; CCNB2: 0.35. Based on these gene expression measurements, the average of the genes in the first gene set (AGR2, AQP3) is 0.75 ((0.5 + 1) / 2). The average of the genes in the second gene set (ADA, CCNB2) is 0.3. The values provided by these two gene sets for risk score calculation are 0.75 × 0.00777260484210833 and 0.3 × -0.0101018153391127. Sum this value with the values of other gene sets (calculated in the same way) and the intercept to calculate the final risk score. To determine an appropriate threshold, whether the risk score exceeds / falls below a certain threshold is determined, for example, by one of the following methods: 1) Using the percentile as a cut-off and comparing it with a representative background population (for example, a value exceeding the 75th percentile is considered high risk); and 2) When the gene is measured by a method that generates an absolute value (for example, qPCR normalized / standardized against a housekeeping gene), an absolute threshold is used, and that value does not necessarily need to be compared with the background population.

[0266] When selecting only one gene from each gene set, multiply each gene by its respective coefficient. When selecting multiple genes from each gene set, multiply the average value of the genes corresponding to a particular gene set by their respective coefficients. In order to determine high / low scores without associating with percentiles from the background population, any method that can measure and normalize gene expression at the single sample level can be used. This can be achieved by the qPCR method, where the absolute transcript number is measured and normalized against a housekeeping gene. To achieve this, qPCR analysis needs to be performed on a sufficiently large cohort (once the final gene set to be included in future studies is identified), and absolute thresholds need to be set based on the distribution of values. These thresholds can be applied to a new cohort at the single sample level (using exactly the same method) without the need to associate the values with the background population. Alternatively, absolute risk scores can also be provided using microarray and single channel array normalization (SCAN) methods. When the Affymetrix microarray platform is used for a new population and the SCAN normalization method is used, theoretically an absolute threshold is set and decisions regarding appropriate treatment methods can be derived without associating the values with the background population. When using the Final model, age is included as a factor. However, this is not the optimal method in the clinical setting, and methods such as qPCR using housekeeping genes are more appropriate than microarray and SCAN normalization methods.

[0267] Proliferation index In this non-limiting example, gene sets from the categories of H (hallmark), C2 (curated gene set), and C6 (oncogenic signature gene set) were selected as potential features of the tumor-intrinsic model (n = 6529 gene sets, containing over 9000 genes in total). To identify biological processes that act as effect modifiers of active immune infiltration, a Cox regression analysis including all tumors (n = 7008) was performed using the interaction term between each gene set and the immune score, which is an immunological signature created. A meta-analysis of the interaction p-values of all gene sets was conducted to select the top 50 ranked gene sets and exclude processes with high correlation (rho > 0.7 or < -0.7) in the same way as the immune score. A total of three gene sets were included in the resulting model (Table 6). Without being bound by theory, biological processes related to tumor aggressiveness are thought to modify the prognostic impact of local immune infiltration. As a result, to separate the prognostic signal and the immune regulatory signal of the aggressive tumor-intrinsic pathway, the model was trained within the tumors in the lowest one-third of the immune scores (n = 2312) where the prognostic signal is not attenuated by immune infiltration. This model showed that, without being bound by theory, processes related to proliferation, which are hypothesized to correlate with genomic instability, were clearly dominant. Therefore, the resulting model is called the Proliferative Index as shown in Figure 1. In some embodiments, the model is based on a tumor-intrinsic model, and the tumor-intrinsic model contains genes from tumor-intrinsic gene sets, some of the gene sets and their related genes are shown in Table 6.

Table 6

Table 7

[0268] Integrated Model To answer the clinical question of whether the integration of tumor-intrinsic and immunological factors enables the down-grading of high-risk tumors, an integrated model was created that weights these dimensions together. This was done by scaling, centering, and calculating the immune score and proliferation index for each training cohort. Next, the cohorts were overlaid and a new model was trained. The variables included were the immune score, proliferation index, and an interaction term given by the formula: (immune score + proliferation index)^2 (Table 8). The integrated model was used to stratify patients under 70 years of age with grade III tumors, or patients under 60 years of age based on the median. This subgroup was selected because RT boost might be recommended based on today's guidelines. Without being bound by theory, the hypothesis is raised that this could identify a high-risk clinical group in which RT boost might be omitted.

Table 8

[0269] Validation cohort The publicly available Servant cohort (n = 341)

[10] and Sjostrom cohort (n = 172)

[11] , with detailed annotations, included radiotherapy patients for whom local recurrence was tracked (Table 9). These cohorts were used to evaluate the meaning of the immune score with respect to the local recurrence risk based on the tumor proliferation index in irradiated patients.

Table 9

[0270] The SweBCG91RT cohort. To understand how genomic instability of tumors affects the benefit of RT in relation to an active immune infiltrate, the randomized SweBCG91RT cohort was used. Briefly, 1178 patients with node-negative (N0) stage I or IIA breast cancer were randomly assigned between 1991 and 1997 to receive either breast-conserving surgery combined with whole-breast RT or not, and were followed for a median of 15.2 years (Figure 8, Table 1). GeneChip Human Exon 1.0 ST Arrays (Thermo Fisher Scientific, South San Francisco, CA) were used to obtain gene expression data (GEO GSE119295). Overall, 7% of the patients received endocrine therapy, 1% received chemotherapy, and 0.4% received both endocrine and chemotherapy. Analyses were performed on untreated tumor samples. Invasive cancer was histologically confirmed by a certified pathologist. No significant differences were observed in clinical variables except for tumor size and histological grade. Excluded patients had slightly smaller tumors (median size: 11 mm vs 12 mm) and lower histological grades compared to included patients (Table 18). Gene expression data are registered in the Gene Expression Omnibus under accession number GSE119295. Due to ethical review board regulations and laws regarding patient privacy, all clinical information is not publicly available.

[0271] This study and follow-up were approved by the regional ethical review board (approval numbers 2010 / 127 and 2015 / 548) and were conducted in accordance with the Declaration of Helsinki.

[0272] Statistical methods The time to ipsilateral breast tumor recurrence (IBTR) as the first event within 10 years from the diagnosis date was defined as the primary endpoint. Other recurrences and deaths were considered competing risks for IBTR. Multivariable regression within the framework of flexible parametric survival analysis (Stata macro stpm2

[12] ) was used to estimate the Royston-Parmer model and hazard ratio (HR) and predict the 10-year cumulative incidence of IBTR. Time was measured as the period from the randomization date (SweBCG91RT cohort) or diagnosis date (Servant and Sjostrom cohorts) until an IBTR event, competing event, or death, or until the end of the last follow-up. The analysis was conducted with up to 10 years of follow-up. Continuous values of the immune score, proliferation index, and integrated score were used in interaction analyses

[16] . The regression model included the time-dependent effects of covariates that did not satisfy the proportional hazards assumption. Interactions were tested by comparing models with and without interaction terms using the likelihood ratio test. Covariates included in the SweBCG91RT cohort were age, ER status, histological grade, and tumor diameter. Variables for the immune score and proliferation index were normalized and rescaled to have a mean of 0 and a standard deviation of 1. The predicted cumulative incidence by percentile of the immune score and proliferation index obtained from the regression model was plotted together with 95% confidence intervals. A p-value < 0.05 was considered statistically significant. The full model is shown in Table 19. Numerical values of the cumulative incidence were generated based on a multivariable Cox model of subhazard for different endpoints according to the method of Fine and Gray

[14] . The P-value of the subhazard ratio between comparison groups was denoted as P CIF in the plot and calculated using the weighted log-rank test as described by Geskus

[15] (using the Stata stcrprep command). R version 4.1.2 and Stata / MP 17.0 (for Mac) were used for statistical analysis.

[0273] Results Correlation. The immune score was correlated with TIL (rho 0.42, p < 0.001) (Figure 15). The Y-axis is standardized so that the mean is 0 and the standard deviation is 1. ESTIMATE and xCell showed correlations with TIL of 0.33 (p < 0.001) and 0.27 (p < 0.001), respectively. Furthermore, the immune score was correlated with histological grade (rho 0.25, p < 0.001) and inversely correlated with ER status (rho -0.26, p < 0.001) and age (rho -0.076, p = 0.038) (Table 10). As shown in Figure 15, the immune score showed a stronger correlation with tumor-infiltrating lymphocytes than other methods developed to quantify the degree of immune infiltration. Also, the proliferation index has been shown to correlate with Ki67 in the SweBCG91RT cohort (Figure 16). Ki67 levels were evaluated by a certified pathologist on the TMA.

[0274] The immune score and the proliferation index were correlated (rho 0.23, p < 0.001) and were concentrated in tumors with generally unfavorable features. The immune score was correlated with TIL (rho 0.36, p < 0.001) and histological grade (rho 0.25, p < 0.001). It was inversely correlated with ER status (rho -0.26, p < 0.001) and age (rho -0.076, p = 0.038) (Table 10). The proliferation index showed sim...

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