Companion diagnostic for early-stage melanoma

Abstract

The disclosure relates to a companion diagnostic immunohistochemical assay for the selection of early-stage melanoma patients at high risk of relapse and / or death for treatment.

Description

[0001] COMPANION DIAGNOSTIC FOR EARLY-STAGE MELANOMA

[0002] RELATED APPLICATION

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63 / 561,877, filed March 6, 2024, which is incorporated by reference herein in its entirety.

[0004] BACKGROUND

[0005] Melanoma is a type of skin cancer that develops in melanocytes, the cells responsible for producing pigment in the skin. It is the deadliest form of skin cancer and has a high mortality rate if not detected and treated early. According to the American Cancer Society, an estimated 106,110 new cases of melanoma were diagnosed in the United States in 2021, and approximately 7,180 people died from the disease. Melanoma can often be cured if it is caught early, but if it spreads to other parts of the body, the prognosis can be poor.

[0006] SUMMARY

[0007] The present disclosure, in some aspects, relates to prognostic tools enabling selection, at the time of diagnosis, of early-stage (e.g., stage I-II) patients with malignant melanoma having a high risk of recurrence and development of distant metastatic disease. This allows for earlier interventions and more personalized care, for example. The technology described herein provides crucial information for clinicians and patients that enables more effective treatment decisions and improved outcomes (e.g., survival rates). Additionally, these prognostic tools can aid in the development of new treatments and help researchers better understand the disease. Thus, aspects of the technology provided herein can be considered a companion diagnostic (CDx) to selectively triage high recurrence risk stage I and stage II melanoma patients for therapies, for example, that combine immune checkpoint inhibitors (e.g., cemiplimab and fianlimab), or combine immune checkpoint inhibitors with neoantigen vaccines (e.g., pembrolizumab and V940).

[0008] Some aspects of the disclosure relate to a method, including (a) selecting a human subject diagnosed with melanoma; (b) selecting the human subject for treatment of the melanoma based on expression levels of two or more biomarkers selected from B-lymphocyte antigen CD20 (CD20), B-cell lymphoma X (Bcl-X), Cyclooxygenase-2 (Cox-2), Bcl-2-associated X protein (Bax), Catenin beta-1 (P-catenin), Phosphatase and tensin homolog (PTEN), and S-mcthyl-5'- thioadenosine phosphorylase (MTAP); and (c) treating the human subject with a combination therapy. In some embodiments, the human subject has stage I or stage II melanoma. In some embodiments, the combination therapy comprises cemiplimab and fianlimab. In other embodiments, the combination therapy comprises pembrolizumab and V940.

[0009] Other aspects of the disclosure relate to a method, including: (a) selecting a human subject diagnosed with melanoma; and (b) selecting or deselecting the human subject for treatment of the melanoma, wherein step (b) includes: (i) preparing one or more section(s) of a tissue obtained from a melanoma lesion in the human subject, wherein cells of the tissue express at least one of the following biomarkers: CD20, Bcl-X, Cox-2, Bax, P-catenin, PTEN, and MTAP; (ii) exposing the one or more section(s) of a tissue to an anti-PTEN antibody, and anti- MTAP antibody, and at least four different antibodies selected from the group consisting of: an anti-CD20 antibody, an anti-Bcl-X antibody, an anti-Cox-2 antibody, an anti-Bax antibody, and an anti-P-catenin antibody, thereby producing one or more immunostained tissue section(s) including immunostained cells; (iii) assigning an expression factor to each of the biomarkers based on two or more characteristics selected from clustering, location, number, type, and staining intensity of the immunostained cells; (iv) calculating a selection score based on the expression factors assigned to the biomarkers in step (iii); and (v) selecting the human subject for treatment of the melanoma when the selection score is higher than a reference score or deselecting the human subject for treatment of the melanoma when the selection score is lower than a reference score.

[0010] Yet other aspects of the disclosure relate to a method, including: (a) selecting a human subject diagnosed with melanoma; and (b) selecting or deselecting the human subject for treatment of the melanoma, wherein step (b) includes: (i) preparing one or more section(s) of a tissue obtained from a melanoma lesion in the human subject, wherein cells of the tissue express at least one of the following biomarkers: CD20, Bcl-X, Cox-2, Bax, P-catenin, PTEN, and MTAP; (ii) exposing the one or more section(s) of a tissue to at least seven different antibodies selected from the group consisting of: an anti-CD20 antibody, an anti-Bcl-X antibody, an anti- Cox-2 antibody, an anti-Bax antibody, an anti-P-catenin antibody, an anti-PTEN antibody, and an anti-MTAP antibody, thereby producing one or more immunostained tissue section(s) including immunostained cells; (iii) assigning an expression factor to each of the biomarkers based on two or more (e.g., three, four or five) characteristics selected from clustering, location, number, type, and staining intensity of the immunostained cells; (iv) calculating a selection score based on the expression factors assigned to the bio markers in step (iii); and (v) selecting the human subject for treatment of the melanoma when the selection score is higher than a reference score or deselecting the human subject for treatment of the melanoma when the selection score is lower than a reference score. In some embodiments, CD20 is assigned an expression factor based on number of CD20+immunostained B cells and clustering of CD20+immunostained B cells. In some embodiments, CD20 is assigned an expression factor of 0 to 3.

[0011] In some embodiments, Bcl-X is assigned an expression factor based on percentage of and staining intensity of Bcl-X+immunostained melanoma cells. In some embodiments, Bcl-X is assigned an expression factor of 0 to 2.

[0012] In some embodiments, Cox-2 is assigned an expression factor based on percentage of and staining intensity of Cox-2+immunostained melanoma cells. In some embodiments, Cox-2 is assigned an expression factor of 0 to 2.

[0013] In some embodiments, Bax is assigned an expression factor based on percentage of and staining intensity of Bax+immunostained melanoma cells. In some embodiments, Bax is assigned an expression factor of 0 to 3.

[0014] In some embodiments, P-catenin is assigned an expression factor based on location and staining intensity of Bax P-catenin+immunostained melanoma cells. In some embodiments, P- catenin is assigned an expression factor of 0 to 3.

[0015] In some embodiments, PTEN is assigned an expression factor based on percentage of and staining intensity of PTEN+immunostained melanoma cells. In some embodiments, PTEN is assigned an expression factor of 0 to 3.

[0016] In some embodiments, MTAP is assigned an expression factor based on percentage of and staining intensity of MTAP+immunostained melanoma cells. In some embodiments, MTAP is assigned an expression factor of 0 to 2.

[0017] In some embodiments, a treatment comprises an immune checkpoint inhibitor therapy.

[0018] In some embodiments, an immune checkpoint inhibitor therapy comprises an antibody selected from anti-PDl antibodies, anti-PD-Ll antibodies, anti-CTLA4 antibodies, and anti- LAG3 antibodies. In some embodiments, an immune checkpoint inhibitor therapy comprises an anti-PDl antibody, for example, selected from pembrolizumab, nivolumab, cemiplimab (Libtayo), toripalimab, tislelizumab, sintilimab, camrelizumab, puzalizumab, and spartalizumab. In some embodiments, an immune checkpoint inhibitor therapy comprises an anti-PD-Ll antibody, for example, selected from atezolizumab, durvalumab, avelumab, sugemalimab, envafolimab, and tislelizumab. In some embodiments, an immune checkpoint inhibitor therapy comprises an anti-CTLA4 antibody, for example, selected from ipilimumab, tremelimumab, quavonlimab, BMS-986218, and ONC-392. In some embodiments, an immune checkpoint inhibitor therapy comprises an anti-LAG3 antibody, for example, selected from fianlimab (REGN3767), relatlimab, favezelimab, ieramilimab (LAG525), TSR-033, MK-4280, and GB- 1107. In some embodiments, a treatment comprises a neoantigen vaccine therapy. In some embodiments, a neoantigen vaccine therapy is a messenger RNA therapy, for example, V940.

[0019] In some embodiments, a treatment is a combination therapy comprising two or more therapies.

[0020] In some embodiments, a combination therapy comprises pembrolizumab and V940.

[0021] In some embodiments, a combination therapy comprises cemiplimab and fianlimab.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 includes New Version 2 expression factor evaluation scheme / algorithm for assigning an expression factor to biomarker CD20 (top) and histological examples with positive expression for CD20 (CD20+).

[0024] FIG. 2 includes New Version 2 expression factor evaluation scheme / algorithm for assigning an expression factor to biomarker Bcl-X (top) and histological examples with positive expression for Bcl-X (Bcl-X+).

[0025] FIG. 3 includes New Version 2 expression factor evaluation scheme / algorithm for assigning an expression factor to biomarker Cox-2 (top) and histological examples with positive expression for Cox-2 (Cox-2+).

[0026] FIG. 4 includes New Version 2 expression factor evaluation scheme / algorithm for assigning an expression factor to biomarker Bax (top) and histological examples with positive expression for Bax (Bax+).

[0027] FIG. 5 includes New Version 2 expression factor evaluation scheme / algorithm for assigning an expression factor to biomarker P-catenin (top) and histological examples with positive expression for P-catenin (P-catenin+).

[0028] FIG. 6 includes New Version 2 expression factor evaluation scheme / algorithm for assigning an expression factor to biomarker PTEN (top) and histological examples with positive expression for PTEN (PTEN+).

[0029] FIG. 7 includes New Version 2 expression factor evaluation scheme / algorithm for assigning an expression factor to biomarker MTAP (top) and histological examples with positive expression for MTAP (MTAP+).

[0030] DETAILED DESCRIPTION

[0031] Current predictive biomarker profiling methods for risk stratification for patients with melanoma (e.g., malignant melanoma) rely on cytoplasmic and nuclear immunoreactivity with a panel of biomarkers. The immunoreactivity is evaluated using a stepwise scoring system, focusing only on melanoma cell staining, independent of the particular biomarker, and independent of the biomarker expression pattern (e.g., a combination of cell location and / or clustering, cell number, and staining intensity). This standardized scoring system (0 to 4+) is based solely on staining intensity in melanoma cells, where no cytoplasmic staining or staining in 0% of cell nuclei is assigned a score of, weak cytoplasmic staining or staining in less than 20% of cell nuclei stained is assigned a score of 1, moderate cytoplasmic staining or staining in 21 to 50% of cell nuclei is assigned a score of 2, strong cytoplasmic staining or staining in 51 to 90% of cell nuclei is assigned a score of 3, and very strong cytoplasmic staining or staining in greater than 90% of cell nuclei is assigned a score of 4.

[0032] The present disclosure provides an unexpected improvement to the standardized biomarker profiling method discussed above. As shown in the Examples and Figures herein, the selection methods provided herein improve sensitivity by about 50% in patients with early-stage melanoma while at the same time not changing the number of patients identified / allocated to the two groups. This is achieved using new algorithms (e.g., a new scoring system), tailored for the specific biomarker assessed, and accounting for a variety of characteristics such as clustering, location, number, type, and staining intensity of immunostained cells. The methods of the disclosure can be used to more accurately select or deselect patients for treatment of melanoma. For example, the treatment of melanoma can include a combination therapy comprising cemiplimab and fianlimab, or a combination therapy comprising pembrolizumab and V940.

[0033] Thus, aspects of the disclosure relate to a method that includes selecting a human subject diagnosed with melanoma. Melanoma is a type of skin cancer that develops from melanocytes, the cells that produce the pigment melanin which gives skin its color. It's considered one of the most serious forms of skin cancer because it tends to spread (metastasize) to other parts of the body more easily than other types. The stages of melanoma are determined based on the thickness of the tumor, involvement of lymph nodes, and the presence of metastasis (spread of cancer to distant organs). Stage 0 (Melanoma in situ) marks the earliest phase of melanoma, where the cancerous cells are confined to the outermost layer of the skin, known as the epidermis. At this stage, the melanoma has not invaded deeper layers of the skin or spread to other parts of the body. Treatment typically involves surgical removal of the affected area of skin to eliminate the cancerous cells, often resulting in a high cure rate.

[0034] Stage I melanoma indicates that the cancer has begun to invade the deeper layers of the skin but remains thin and has not spread to lymph nodes or distant sites. This stage is subdivided into IA and IB, depending on the thickness of the tumor and the presence of ulceration (a break in the skin). Treatment usually involves surgical removal of the tumor along with a margin of normal skin around it to ensure all cancer cells are removed. The prognosis at this stage is generally favorable, but careful monitoring is necessary to detect any recurrence or spread. In some embodiments, a Stage I melanoma patient is treated for melanoma using, for example, a combination therapy comprising cemiplimab and fianlimab, or a combination therapy comprising pembrolizumab and V940.

[0035] Stage II melanoma can be characterized by a thicker tumor that has not yet spread to lymph nodes or distant organs. This stage is further divided into IIA, IIB, and IIC, based on the thickness and ulceration of the tumor. The risk of melanoma spreading or recurring is higher in Stage II than in Stage I. Treatment typically involves more extensive surgical removal of the skin around the tumor, and patients may undergo evaluation of nearby lymph nodes. In some cases, adjuvant therapies may be recommended to reduce the risk of recurrence. In some embodiments, a Stage II melanoma patient is treated for melanoma using, for example, a combination therapy comprising cemiplimab and fianlimab, or a combination therapy comprising pembrolizumab and V940.

[0036] Stage III melanoma signifies that the cancer has spread to nearby lymph nodes, tissues, or satellite tumors close to the primary site but not to distant parts of the body. This stage is more serious and involves various subcategories based on the extent of lymph node involvement and the presence of satellite tumors. Treatment for Stage III melanoma can be more complex and can include surgical removal of the tumor and affected lymph nodes, along with adjuvant treatments such as immunotherapy, targeted therapy, and possibly radiation therapy to reduce the risk of further spread and to manage symptoms.

[0037] Stage IV melanoma is the most advanced stage, indicating that the cancer has spread to distant lymph nodes or organs, such as the lungs, liver, brain, or bones. Treatment at this stage can focus on managing the disease and alleviating symptoms rather than curing the cancer. Options can include systemic treatments such as immunotherapy and targeted therapy, which have shown promise in extending survival and improving quality of life for patients with advanced melanoma. In some cases, surgical removal of metastases or radiation therapy may be used to control symptoms.

[0038] In some embodiments, methods of the disclosure include selecting or deselecting a human subject for treatment of a melanoma. This selection / deselection process can involve several steps, including (i) preparing one or more section(s) of a tissue obtained from a melanoma lesion in a human subject; (ii) exposing the one or more section(s) of a tissue to at least seven different antibodies selected from the group consisting of: an anti-CD20 antibody, an anti-Bcl-X antibody, an anti-Cox-2 antibody, an anti-Bax antibody, an anti-P-catenin antibody, an anti-PTEN antibody, and an anti-MTAP antibody, thereby producing one or more immunostained tissue section(s) comprising immunostained cells, and optionally exposing the one or more section(s) of a tissue to an anti-SlOO antibody; (iii) assigning an expression factor to each of the biomarkers based on number and staining intensity of immunostained cells; (iv) calculating a selection score based on the expression factors assigned to the biomarkers in step (iii); and (v) selecting the human subject for treatment of the melanoma when the selection score is higher than a reference score or deselecting the human subject for treatment of the melanoma when the selection score is lower than a reference score. In some embodiments, the patient is treated for melanoma using, for example, a combination therapy comprising cemiplimab and fianlimab, or a combination therapy comprising pembrolizumab and V940.

[0039] Immunohistochemical Analysis

[0040] Immunohistochemistry (IHC) is a widely used technique to detect and visualize the expression and localization of proteins in tissue samples. The IHC assay involves several basic steps, beginning with tissue preparation. In some embodiments, a method of the disclosure comprises preparing one or more section(s) of a tissue obtained from a melanoma lesion in the human subject. In some embodiments, a method comprises preparing two or more sections of a tissue obtained from a melanoma lesion. For example, a method can comprise preparing three, four, five, six or seven sections of a tissue obtained from a melanoma lesion. In some embodiments, a method comprises preparing no more than two sections of a tissue obtained from a melanoma lesion.

[0041] Tissue samples, in some embodiments, are fixed in formalin and embedded in paraffin (FFPE), then sectioned into thin slices and mounted onto slides (e.g., glass slides). In some embodiments, the tissue sections have a thickness of about 2pm to about 10pm. In some embodiments, the tissue sections have a thickness of about 3pm to about 5pm. For example, the tissue sections may have a thickness of about 2pm to about 5pm, or about 3pm to about 5pm. In some embodiments, the tissue sections have a thickness of about 3pm. In some embodiments, the tissue sections have a thickness of about 4pm. In some embodiments, the tissue sections have a thickness of about 5pm.

[0042] Formalin-fixed paraffin-embedded (FFPE) sections are a widely used form of tissue preparation for immunohistochemical analysis. FFPE is a method of tissue fixation where the tissue is immersed in a formalin solution, which preserves the tissue architecture and prevents degradation of the tissue sample. The fixed tissue is then embedded in paraffin wax and sectioned into thin slices for further analysis.

[0043] Antigen retrieval, in some embodiments, is the next step in IHC, which is often used for FFPE tissue samples to unmask the target antigen and improve the binding of the primary antibody. Non-specific binding sites on the tissue sections are then blocked using a blocking solution, such as serum or BSA, to prevent non-specific binding of the primary antibody. In some embodiments, cells of a tissue express at least one of the following biomarkers: CD20, Bcl-X, Cox-2, Bax, P-catenin, PTEN, and MTAP. In some embodiments, cells of a melanoma express CD20. In some embodiments, cells of a melanoma express Bcl-X. In some embodiments, cells of a melanoma express Cox-2. In some embodiments, cells of a melanoma express Bax. In some embodiments, cells of a melanoma express P-catenin. In some embodiments, cells of a melanoma express PTEN. In some embodiments, cells of a melanoma express MTAP. Thus, in some embodiments, a method of the disclosure includes exposing one or more section(s) of a tissue to at least seven different antibodies, thereby producing one or more immunostained tissue section(s) comprising immunostained cells. The seven different primary antibodies are selected from the group consisting of CD20 antibodies (i.e., anti-CD20 antibodies), Bcl-X antibodies (i.e., anti-Bcl-X antibodies), Cox-2 antibodies (i.e., anti-Cox-2 antibodies), Bax antibodies (i.e., anti-Bax antibodies), P-catenin antibodies (i.e.., anti-P-catenin antibodies), PTEN antibodies (i.e., anti-PTEN antibodies), and MTAP antibodies (i.e., anti- MTAP antibodies).

[0044] CD20 is a biomarker or antigen that is present on the surface of certain types of immune cells, specifically B lymphocytes or B cells. CD20 plays a key role in the normal functioning of B cells, including their development and activation, and it is also used as a target for certain types of cancer therapies. CD20 antibodies for immunohistochemical analysis are used to detect the expression of CD20 in tissue samples. Non-limiting examples include anti-CD20 (L26) from Dako, anti-CD20 (SP148) from Spring Bioscience, anti-CD20 (EP459Y) from Abeam, and anti- CD20 (2H7) from Ventana Medical Systems. These CD20 antibodies have been validated for use in formalin-fixed paraffin-embedded (FFPE) tissue samples and have shown good sensitivity and specificity for the detection of CD20 expression. They are commonly used in research studies as well as in diagnostic settings to aid in the diagnosis and classification of B-cell malignancies.

[0045] Bcl-X (B-cell lymphoma-extra large) is a protein that belongs to the Bcl-2 family of proteins, which regulate apoptosis or programmed cell death. Bcl-X has two isoforms: Bcl-XL (long) and Bcl-XS (short), which have opposing effects on apoptosis. Bcl-XL inhibits apoptosis and promotes cell survival, while Bcl-XS promotes apoptosis and cell death. Bcl-X antibodies for immunohistochemical analysis are used to detect the expression of Bcl-X in tissue samples. Anti-Bcl-X (E63) from Abeam is a rabbit monoclonal antibody that recognizes the N-terminus of Bcl-X. It has been validated for use FFPE tissue samples and has shown good sensitivity and specificity for the detection of Bcl-X expression. Similarly, anti-Bcl-X (44H6) from Cell Signaling Technology, anti-Bcl-X (H-5) from Santa Cruz Biotechnology, and anti-Bcl-X (7D2) from Novus Biologicals are other examples of commercially available Bcl-X antibodies that have been validated for use in FFPE tissue samples and have shown good sensitivity and specificity for the detection of Bcl-X expression. These Bcl-X antibodies are widely used in research studies and diagnostic settings to detect the expression of Bcl-X in tissue samples by immunohistochemical analysis.

[0046] Cox-2 (Cyclooxygenase-2) is an enzyme that is involved in the production of prostaglandins, which are substances that play a role in inflammation, pain, and other physiological processes. Cox-2 is expressed at low levels in normal tissues, but is upregulated in response to inflammatory signals, growth factors, and other stimuli. Cox-2 antibodies for immunohistochemical analysis are used to detect the expression of Cox-2 in tissue samples. Anti-COX-2 (SP21) from Abeam, Anti-COX-2 (CX229) from Cell Marque, Anti-COX-2 (NBP1-03711) from Novus Biologicals, and Anti-COX-2 (SP70) from Thermo Fisher Scientific are some non-limiting examples of commercially available COX-2 antibodies for immunohistochemical analysis. These COX-2 antibodies have been validated for use in FFPE tissue samples and have shown good sensitivity and specificity for the detection of COX-2 expression. They are widely used in research studies and diagnostic settings to detect the expression of COX-2 in tissue samples by immunohistochemical analysis.

[0047] Bax is a protein that belongs to the Bcl-2 family of proteins, which regulate apoptosis or programmed cell death. Bax plays a key role in the intrinsic apoptotic pathway, which is triggered by cellular stress signals and leads to the activation of caspases and the dismantling of cellular structures. Bax antibodies for immunohistochemical analysis are used to detect the expression of Bax in tissue samples. Anti-Bax (E63) from Abeam, Anti-Bax (N-20) from Santa Cruz Biotechnology, Anti-Bax (6A7) from BD Biosciences, and Anti-Bax (D2E11) from Cell Signaling Technology are some non-limiting examples of commercially available Bax antibodies for immunohistochemical analysis. These Bax antibodies have been validated for use in formalin-fixed paraffin-embedded (FFPE) tissue samples and have shown good sensitivity and specificity for the detection of Bax expression. They are widely used in research studies and diagnostic settings to detect the expression of Bax in tissue samples by immunohistochemical analysis.

[0048] P-catenin is a protein that plays a key role in cell adhesion and signaling pathways. In the Wnt signaling pathway, P-catenin is activated by binding to Wnt ligands, which leads to its translocation into the nucleus and activation of target genes involved in cell proliferation, differentiation, and survival. P-catenin antibodies for immunohistochemical analysis are used to detect the expression of P-catenin in tissue samples. Anti-P-catenin (E247) from Abeam, Anti-P- catenin (15B8) from Cell Signaling Technology, Anti-P-catenin (E5H1) from Thermo Fisher Scientific, and Anti-P-catenin (610153) from BD Biosciences are some examples of commercially available P-catenin antibodies for immunohistochemical analysis. These P-catenin antibodies have been validated for use in FFPE tissue samples and have shown good sensitivity and specificity for the detection of P-catenin expression.

[0049] PTEN (phosphatase and tensin homolog) is a tumor suppressor gene that regulates cell growth, division, and survival. PTEN is a phosphatase that dephosphorylates phosphatidylinositol-3,4,5-triphosphate (PIP3), a lipid that is produced by the action of phosphoinositide 3-kinase (PI3K) and promotes cell proliferation and survival. PTEN antibodies for immunohistochemical analysis are used to detect the expression of PTEN in tissue samples. Anti-PTEN (138G6) from Cell Signaling Technology, Anti-PTEN (Y184) from Abeam, Anti- PTEN (10P11D12) from Thermo Fisher Scientific, and Anti-PTEN (28H6) from Novus Biologicals are some examples of commercially available PTEN antibodies for immunohistochemical analysis. These PTEN antibodies have been validated for use in FFPE tissue samples and have shown good sensitivity and specificity for the detection of PTEN expression.

[0050] MTAP (methylthioadenosine phosphorylase) is an enzyme that plays a role in the metabolism of sulfur-containing compounds, such as polyamines and methionine. MTAP is frequently deleted or mutated in various types of cancer, including melanoma, and its loss has been associated with tumor progression and resistance to chemotherapy. MTAP antibodies for immunohistochemical analysis are used to detect the expression of MTAP in tissue samples. Anti-MTAP (EPR18502) from Abeam, Anti-MTAP (HPA045141) from Sigma- Aldrich, and Anti-MTAP (MTP-2) from Novus Biologicals are some examples of commercially available MTAP antibodies for immunohistochemical analysis. These MTAP antibodies have been validated for use in FFPE tissue samples and have shown good sensitivity and specificity for the detection of MTAP expression.

[0051] In some embodiments, a method of the disclosure includes exposing one or more section(s) of a tissue to an anti-SlOO antibody. The S-100 protein is expressed in over 95% of malignant melanomas.

[0052] Tissue sections can be incubated with a primary antibody that specifically recognizes the target protein. The primary antibody can be diluted in a buffer solution and incubated with the tissue sections overnight at 4°C or at room temperature for a few hours, for example. In some embodiments, a secondary antibody is used to detect the binding of the primary antibody. The secondary antibody can be conjugated to a detection system, such as an enzyme or a fluorescent dye, and is incubated with the tissue sections for a short period of time.

[0053] The detection system is used to visualize the binding of the secondary antibody to the primary antibody. For example, an enzyme-based detection system may use a chromogen substrate to produce a visible color reaction, while a fluorescent-based detection system may directly visualize the fluorescent signal. In some embodiments, counterstaining with a dye such as hematoxylin is performed to visualize the tissue architecture and aid in the interpretation of the staining pattern.

[0054] The staining pattern of the tissue sections is observed under a microscope, for example, and analyzed to determine the expression and localization of the target protein. The results of the IHC assay are used as provided herein to distinguish between low risk and high-risk patients, and in some embodiments, for the selection of therapy.

[0055] Expression Factor Assignment

[0056] In some embodiments, a method includes assigning an expression factor to each of the biomarkers based on two or more characteristics selected from clustering, location, number (e.g., percentage), type, and staining intensity of the immunostained cells. Immunostained cells includes cells that can be visualized (e.g., chromogenic detection or fluorescence microscopy) following an immuno staining process, for example, with an antibody that binds to a particular biomarker, such as CD20, Bcl-X, Cox-2, Bax, P-catenin, PTEN, or MTAP. Generally, the focus of the evaluation is on the center of the melanoma (e.g., main tumor, proliferative zones).

[0057] Clustering (e.g., in cancer cells) includes the process where cells group together to form clusters. This phenomenon is significant in the context of tumor development, progression, and metastasis. In some embodiments, a cell cluster comprises a particular cell type or mixture of cell types. For example, a cell cluster can include immune cells, such as B cells, melanoma cells, or a combination thereof. Cells in a cell cluster, in some instances, can form a nodular nest, such as a modular next comprising B cells and / or melanoma cells. A nodular nest of cells herein refers to a cluster of greater than 100 cells, for example, a cluster of greater than 100 (e.g., immuno stained) B cells. In some embodiments, assignment of an expression factor is based on the clustering of certain immunostained cell types, for example, CD20+B cells.

[0058] The location of a cell can also be considered when assigning an expression factor to a biomarker. For example, the assignment of an expression factor may be based on whether an immunostained melanoma cell is in an epidermal layer of a tissue (i.e., an epidermal cell) or in a dermal layer of a tissue (i.e., a dermal cell). The epidermis (epidermal layer) is the outset most layer of the skin, while the dermis (dermal layer) is a thicker layer of skin located beneath the epidermis.

[0059] The number or percentage of immunostained cells can also be considered when assigning an expression factor to a biomarker. For example, the assignment of an expression factor may be based on the number of immunostained immune cells, such as B cells, or the number of immunostained melanoma cells. In some embodiments, the assignment of an expression factor is based on a percentage of immunostained cells, such as immunostained melanoma cells.

[0060] Cell type can also factor in the assignment of an expression factor. For example, the presence or absence of a certain number or percentage of immunostained B cells and / or melanoma cells can guide the assignment of an expression factor.

[0061] Staining intensity, for example in immunohistochemistry, is a qualitative measure used to describe the level of antigen expression in the tissue, which can be visualized as the darkness or color density of the stained area. The staining intensity is usually categorized as weak, moderate, or strong, based on visual assessment under a microscope, for example. Weak staining is the lowest level of staining intensity, indicating minimal antigen presence. Weak staining suggests a low level of protein expression or antigen abundance in the tissue. The staining is visible but not bold or vivid. Moderate staining represents a clear, easily observed staining that unmistakably marks the presence of the antigen, without being as intense as strong staining. Moderate staining indicates a significant amount of the protein or antigen in the tissue, suggesting moderate expression levels. Lastly, strong staining is the most intense level of staining, characterized by very dark or highly saturated coloration. This indicates a high level of antigen or protein expression in the tissue. The stained areas are prominently visible and stand out clearly against the background. As is known in the field of immunohistochemistry, the assessment of staining intensity can be subjective and for consistent interpretation, pathologists often refer to control samples or specific criteria to gauge the staining intensity accurately. Exemplary algorithms used to assign an expression factor to an individual biomarker are provided herein (see, e.g., FIGs. 1- 7).

[0062] In some embodiments, the tissue sections are stained with hematoxylin and eosin (H&E), for example, for the overall histological assessment of the tissue.

[0063] In some embodiments, for localization of the tumor areas the tissue sections are stained with anti-SlOO antibody or other marker of melanoma cells.

[0064] In some embodiments, the tissues sections are subjected to antibody diluent staining (omission of the primary antibody, incubation period for chromogen is 5 minutes). In the case of archived tissue, for example, the AEC chromogen can bind to the tissue even without the primary antibody (various factors related to the fixation, embedding and storage of the tissue are suspected). In the case of negative expression in the control tissue and presence of background staining with the AB diluent, the background staining can be subtracted when assessing the expression of the individual markers. In some embodiments, background staining observed with antibody (AB)-diluent samples can be subtracted. CD20 Expression Factor Assignment

[0065] In some embodiments, CD20 is assigned a CD20 expression factor of 0 to 3. See, e.g., FIG. 1. In some embodiments, a tissue section comprising fewer than ten (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1) CD20+B cells is assigned a CD20 expression factor of 0. In some embodiments, a tissue section comprising a cell cluster comprising fewer than five CD20+B cells is assigned a CD20 expression factor of 0. In some embodiments, a tissue section comprising more than ten CD20+B cells is assigned a CD20 expression factor of 1. In some embodiments, a tissue section comprising a cell cluster comprising more than five CD20+B cells, wherein the cell cluster comprises no more than one CD20+B cell nodular nest, is assigned a CD20 expression factor of 1. A “nodular nest” refers to a cluster of at least 100 biomarker positive (e.g., CD20+) cells (e.g., B cells). In some embodiments, a tissue section comprising at least two (e.g., at least 3, 4, or 5) CD20+B cell nodular nests without a ribbon-like connection (e.g., at least two easily distinguished nodular nests) is assigned expression factor 2. In some embodiments, a tissue section comprising a ribbon-like connection of at least three CD20+B cell nodular nests is assigned expression factor 3. In some embodiments, assignment of a CD20 expression factor is based on an evaluation of the extent of inflammatory infiltrate (e.g., H&E staining, antibody diluent stain can be used as a guide) and / or nodular infiltrates that may invade a tumor. In some embodiments, the appearance and arrangement of stained cells (e.g., mostly precursors of B cells) in the inflammatory infiltrate is evaluated. In some embodiments, intensity of staining is not evaluated.

[0066] Bcl-X Expression Factor Assignment

[0067] In some embodiments, Bcl-X is assigned an expression factor of 0 to 2. See, e.g., FIG. 2. In some embodiments, a tissue section in which none of the melanoma cells is Bcl-X+ is assigned expression factor 0. In some embodiments, a tissue section in which fewer than 50% (e.g., fewer than 40%, 30%, 20%, or 10%) of melanoma cells are moderately to strongly Bcl-X+is assigned a Bcl-X expression factor of 1. In some embodiments, a tissue section in which more than 50% (e.g., more than 60%, 70%, 80%, or 90%) of melanoma cells are weakly Bcl-X+is assigned a Bcl-X expression factor of 1. In some embodiments, a tissue section in which at least 50% (e.g., at least 60%, 70%, 80%, or 90%) of melanoma cells are moderately to strongly Bcl- X+is assigned a Bcl-X expression factor of 2.

[0068] Cox-2 Expression Factor Assignment

[0069] In some embodiments, Cox-2 is assigned an expression factor of 0 to 2. See, e.g., FIG. 3. In some embodiments, a tissue section in which none of the melanoma cells is Cox-2+is assigned a Cox-2 expression factor of 0. In some embodiments, a tissue section in which fewer than 50% (e.g., fewer than 40%, 30%, 20%, or 10%) of melanoma cells are moderately to strongly Cox-2+is assigned a Cox-2 expression factor of 1. In some embodiments, a tissue section in which more than 50% (e.g., more than 60%, 70%, 80%, or 90%) of melanoma cells are weakly Cox-2+is assigned a Cox-2 expression factor of 1. In some embodiments, a tissue section in which at least 50% of melanoma cells are moderately to strongly Cox-2+is assigned a Cox-2 expression factor of 2.

[0070] Bax Expression Factor Assignment

[0071] In some embodiments, Bax is assigned an expression factor of 0 to 3. See, e.g., FIG. 4. In some embodiments, a tissue section in which none of the melanoma cells is Bax+is assigned a Bax expression factor of 0. In some embodiments, a tissue section in which fewer than ten melanoma cells are Bax+stained is assigned a Bax expression factor of 0. In some embodiments, a tissue section in which fewer than 50% (e.g., fewer than 40%, 30%, 20%, or 10%)of melanoma cells are Bax+is assigned a Bax expression factor of 1. In some embodiments, a tissue section in which more than 50% (e.g., more than 60%, 70%, 80%, or 90%) of melanoma cells are weakly Bax+is assigned a Bax expression factor of 1. In some embodiments, a tissue section in which at least 50% of melanoma cells are moderately Bax+is assigned a Bax expression factor of 2. In some embodiments, a tissue section in which at least 50% and fewer than 80% of melanoma cells are strongly Bax+is assigned a Bax expression factor of 2. In some embodiments, a tissue section in which at least 80% of melanoma cells are strongly Bax+is assigned a Bax expression factor of 3. In some embodiments, the intensity and strength of Bax expression in melanoma cells is compared with S100 expression, and if the staining pattern is comparable, the Bax biomarker should be assigned an expression factor of 3.

[0072] P-catenin Expression Factor Assignment

[0073] In some embodiments, P-catenin is assigned an expression factor of 0 to 3. See, e.g., FIG. 5. In some embodiments, a tissue section in which none of the melanoma cells is P-catenin+is assigned a P-catenin expression factor of 0. In some embodiments, a tissue section in which only epidermal melanoma cells are P-catenin+is assigned a P-catenin expression factor of 1. In some embodiments, a tissue section in which epidermal and dermal melanoma cells are weakly P- catenin+ is assigned a P-catenin expression factor of 1. In some embodiments, a tissue section in which epidermal and dermal melanoma cells are moderately P-catenin+is assigned a P-catenin expression factor of 2.

[0074] In some embodiments, a tissue section in which epidermal and dermal melanoma cells are strongly P-catenin+is assigned a P-catenin expression factor of 3. PTEN Expression Factor Assignment

[0075] In some embodiments, PTEN is assigned an expression factor of 0 to 3. See, e.g., FIG. 6. In some embodiments, a tissue section in which none of the melanoma cells is PTEN+is assigned a PTEN expression factor of 0. In some embodiments, a tissue section in which fewer than ten melanoma cells are PTEN+is assigned a PTEN expression factor of 0. In some embodiments, a tissue section in which fewer than 50% (e.g., fewer than 40%, 30%, 20%, or 10%) of melanoma cells are PTEN+is assigned a PTEN expression factor of 1. In some embodiments, at least 50% of melanoma cells are weakly PTEN+is assigned a PTEN expression factor of 1. In some embodiments, a tissue section in which at least 50% of melanoma cells are moderately PTEN+is assigned a PTEN expression factor of 2 / In some embodiments, at least 50% and fewer than 80% of melanoma cells are strongly PTEN+is assigned a PTEN expression factor of 2. In some embodiments, a tissue section in which at least 80% of melanoma cells are strongly PTEN+is assigned a PTEN expression factor of 3. In some embodiments, the intensity and strength of PTEN expression in melanoma cells is compared with S100 expression, and if the staining pattern is comparable, the PTEN biomarker should be assigned an expression factor of 3.

[0076] MT P Expression Factor Assignment

[0077] In some embodiments, MTAP is assigned an expression factor of 0 to 2. See, e.g., FIG. 7. In some embodiments, a tissue section in which none of the melanoma cells is MTAP+is assigned an MTAP expression factor of 0. In some embodiments, a tissue section in which fewer than 50% (e.g., fewer than 40%, 30%, 20%, or 10%) of melanoma cells are moderately MTAP+is assigned an MTAP expression factor of 1. In some embodiments, a tissue section in which more than 50% (e.g., more than 60%, 70%, 80%, or 90%) of melanoma cells are weakly MTAP+is assigned an MTAP expression factor of 1. In some embodiments, a tissue section in which more than 50% (e.g., more than 60%, 70%, 80%, or 90%) of melanoma cells are moderately MTAP+or in which more than 30% are strongly MTAP+is assigned expression factor 2.

[0078] Selection Score Calculation

[0079] The Cox proportional hazards model is a widely used statistical technique for the analysis of survival data, allowing researchers to evaluate the effect of several risk factors on survival time simultaneously. It's particularly used in medical research to identify the risk factors associated with outcomes like death, disease recurrence, or recovery. A key component of the Cox model is the regression coefficient (often denoted as [>). Each variable (e.g., expression factor) in the Cox model has an associated coefficient. This coefficient measures the effect of the variable on the hazard rate, which is the rate at which the event of interest (e.g., death) occurs. A positive coefficient (|3 >0) indicates that as the value of the variable increases, the hazard rate (risk of the event occurring) also increases. In practical terms, this means the risk factor is associated with a higher risk of the event happening.

[0080] A negative coefficient (|3 <0) suggests that as the value of the variable increases, the hazard rate decreases. This indicates that the risk factor is associated with a lower risk of the event happening.

[0081] In some embodiments, a method includes calculating a selection score based on the expression factors assigned to the biomarkers. The prognostic value for each of seven biomarkers, for example, can be assayed by univariate Cox proportional hazards regarding overall survival. A risk score can be calculated for each patient (subject) by a linear combination of the univariate Cox regression coefficients and the corresponding expression factors assigned x = {xi,X2, . . . ,XD , where D is the number of markers (e.g., 7) in the signature. The score can be normalized by the number of biomarkers measured. Based on this risk score, patients can be assigned as high risk or low risk.

[0082] In some embodiments, a selection score is calculated using the following formula: / D\ iz, / I y^ a,- I , / / v=i /

[0083] {I , if x / exists

[0084] 0, if jismissing

[0085] , wherein D is the number of biomarkers (e.g., 7), fl is a univariate Cox model coefficient, x is an expression factor determined for one of the seven biomarkers (e.g., CD20, Bcl-X, Cox-2, Bax, P-Catenin, PTEN, or MTAP), and a corrects for negative antibody signal.

[0086] In some embodiments, the coefficient of a univariate Cox model for CD20 is 0.547. In some embodiments, the coefficient of a univariate Cox model for Bcl-X is 0.391. In some embodiments, the coefficient of a univariate Cox model for Cox-2 is 0.297. In some embodiments, the coefficient of a univariate Cox model for Bax is 0.441. In some embodiments, the coefficient of a univariate Cox model for P-Catenin is -0.34. In some embodiments, the coefficient of a univariate Cox model for PTEN is 0.272. In some embodiments, the coefficient of a univariate Cox model for MTAP is -0.621.

[0087] In some embodiments, a reference score is 0.135. Thus, if a selection score is higher than 0.135, the patient is selected for treatment of the melanoma, whereas if a selection score is lower than 0.135, the patient is deselected for treatment of the melanoma. In some embodiments, the patient is selected for treatment using, for example, a combination therapy comprising cemiplimab and fianlimab, or a combination therapy comprising pembrolizumab and V940.

[0088] Therapies

[0089] In some embodiments, a method includes selecting the human subject for treatment of the melanoma when the selection score is higher than a reference score or deselecting the human subject for treatment of the melanoma when the selection score is lower than a reference score.

[0090] Melanoma is a type of skin cancer that can be treated with surgery, radiation therapy, immunotherapy, or chemotherapy. The primary therapy for melanoma is surgery. Surgery is the most effective treatment for early- stage melanoma, when the cancer is still confined to the skin and has not spread to other parts of the body. The goal of surgery is to completely remove the melanoma along with a margin of healthy tissue around it to ensure that all cancer cells are removed. The type of surgery used depends on the location and stage of the melanoma. For small, early-stage melanomas, a wide local excision may be performed, which involves removing the melanoma along with a margin of healthy tissue around it. For larger melanomas or those that have spread to nearby lymph nodes, a more extensive surgery may be required. In addition to surgery, other treatments may be used as adjuvant therapies to reduce the risk of cancer recurrence.

[0091] Adjuvant therapies are treatments given after the primary treatment to reduce the risk of cancer recurrence. In the case of melanoma, adjuvant therapies may include, for example, immunotherapies, targeted therapies, radiation therapies, and / or regional chemotherapies. In some embodiments, the methods provided herein comprise selecting a melanoma patient for therapy. In other embodiments, the methods provided herein comprise selecting a melanoma patient for therapy and treating the patient with the therapy.

[0092] Neoadjuvant therapies are treatments given before the primary treatment with the intent to reduce the risk of cancer recurrence. In the case of melanoma, neo-adjuvant therapies may include, for example, immunotherapies, and targeted therapies. In some embodiments, the methods provided herein comprise selecting a melanoma patient for therapy. In other embodiments, the methods provided herein comprise selecting a melanoma patient for therapy and treating the patient with the therapy.

[0093] In some embodiments, a treatment comprises a neoantigen vaccine therapy. A neoantigen vaccine therapy is a type of personalized cancer immunotherapy that targets tumor- specific neoantigens — mutated proteins that are unique to a patient’s cancer cells and not found in normal tissues. These vaccines are designed to stimulate the immune system, particularly T cells, to recognize and attack cancer cells displaying these neoantigens. In some embodiments, a neoantigen vaccine therapy is a messenger RNA therapy, for example, V940. V940 (formerly known as mRNA-4157) is an example of an individualized neoantigen vaccine. It is an mRNA- based cancer vaccine designed to enhance the body's immune response against cancer by targeting tumor- specific neoantigens unique to an individual’s cancer.

[0094] In some embodiments, the therapy is an immunotherapy. Immunotherapy works by boosting the body's immune system to fight cancer cells. One type of immunotherapy used in therapy for melanoma is interferon alpha, which is administered through injection. Interferon alpha is thought to stimulate the immune system to attack cancer cells and slow the growth of melanoma. However, it may cause flu-like symptoms such as fever, chills, fatigue, and depression. Another type of immunotherapy drugs are cytotoxic T lymphocyte-associated protein 4 (CTLA-4) immune checkpoint inhibitors and anti-programmed cell death protein 1 (PD-1) monoclonal antibodies, which are taken through injection. Some immunotherapy drugs may cause side effects, most reported being diarrhea, dermatitis and fatigue.

[0095] In some embodiments, a treatment comprises an immune checkpoint inhibitor therapy. Immune checkpoint inhibitor therapy is a type of cancer immunotherapy that blocks immune checkpoint proteins, thereby enhancing T cell activity against cancer cells. These inhibitors help the immune system recognize and attack tumors more effectively by removing inhibitory signals that cancer cells exploit to evade immune destruction. In some embodiments, an immune checkpoint inhibitor therapy comprises an antibody selected from anti-PDl antibodies, anti-PD- L1 antibodies, anti-CTLA4 antibodies, and anti-LAG3 antibodies. In some embodiments, an immune checkpoint inhibitor therapy comprises an anti-PDl antibody, for example, selected from pembrolizumab, nivolumab, cemiplimab (Libtayo), toripalimab, tislelizumab, sintilimab, camrelizumab, puzalizumab, and spartalizumab. In some embodiments, an immune checkpoint inhibitor therapy comprises an anti-PD-Ll antibody, for example, selected from atezolizumab, durvalumab, avelumab, sugemalimab, envafolimab, and tislelizumab. In some embodiments, an immune checkpoint inhibitor therapy comprises an anti-CTLA4 antibody, for example, selected from ipilimumab, tremelimumab, quavonlimab, BMS-986218, and ONC-392. In some embodiments, an immune checkpoint inhibitor therapy comprises an anti-LAG3 antibody, for example, selected from fianlimab (REGN3767), relatlimab, favezelimab, ieramilimab (LAG525), TSR-033, MK-4280, and GB-1107.

[0096] In some embodiments, the therapy is a targeted therapy. Targeted therapy is another type of therapy for melanoma. Targeted therapy drugs specifically attack cancer cells that have a mutation in a particular gene, such as BRAF or MEK. These drugs may be taken orally or through injection. Some targeted therapy drugs may cause side effects, such as fatigue, nausea, diarrhea, and skin rashes. In some embodiments, the therapy is a radiation therapy. Radiation therapy is a type of cancer treatment that uses high-energy radiation to destroy cancer cells. It can be used as a primary treatment for melanoma, or as an adjuvant therapy after surgery to eliminate any remaining cancer cells. Radiation therapy works by damaging the DNA of cancer cells, which causes them to die. The radiation is targeted to the area where the melanoma was located, to minimize damage to healthy cells. The treatment is usually administered through a machine that delivers the radiation beams to the affected area.

[0097] In some embodiments, the therapy is a chemotherapy. Chemotherapy is a type of cancer treatment that uses drugs to kill cancer cells. It is not commonly used as a first-line treatment for melanoma, as other treatments such as surgery, immunotherapy, and targeted therapy have shown to be more effective. However, in some cases, chemotherapy may be used as an adjuvant therapy after surgery to eliminate any remaining cancer cells, or if the melanoma has spread to other parts of the body. Non-limiting examples of chemotherapy drugs for melanoma include dacarbazine and temozolomide.

[0098] In some embodiments, the therapy is a combination therapy. In some embodiments, a treatment is a combination therapy comprising two or more therapies (e.g., two or more immune checkpoint inhibitor therapies, or a combination of a checkpoint inhibitor therapy and another therapy, such as a neoantigen vaccine. For example, a combination therapy can comprise cemiplimab and fianlimab. Cemiplimab (brand name LIBTAYO®) is a PD-1 (programmed death- 1) immune checkpoint inhibitor, and fianlimab is an anti-LAG-3 (lymphocyte-activation gene 3) monoclonal antibody. As another example, a combination therapy can comprise pembrolizumab and V940. Pembrolizumab (brand name KEYTRUDA®) is a monoclonal antibody immune checkpoint inhibitor that targets PD-1 (programmed death- 1), and V940 is a neoantigen mRNA vaccine.

[0099] Some aspects of the disclosure relate to a method, including (a) selecting a human subj ectdiagnosed with melanoma; (b) selecting the human subjectfor treatment of the melanoma based on expression levels of two or more biomarkers selected from B-lymphocyte antigen CD20 (CD20), B-cell lymphoma X (Bcl-X), Cyclooxygenase-2 (Cox-2), Bcl-2-associated X protein (Bax), Catenin beta-1 (P-catenin), Phosphatase and tensin homolog (PTEN), and S- mcthyl-5'-thioadcnosinc phosphorylase (MTAP); and (c) treating the human subjectwith a combination therapy. In some embodiments, the human subjecthas stage I or stage II melanoma. In some embodiments, the combination therapy comprises pembrolizumab and V940. In other embodiments, the combination therapy comprises cemiplimab and fianlimab. Methods

[0100] Aspects of the technology relate to methods of selectively triage, at the time of diagnosis, a high recurrence risk stage I— II patient for adjuvant therapy. Selective treatment of those patients that are more likely to develop distant metastatic disease can lower the burden of unbeatable metastatic melanoma, for example. Thus, in some embodiments, methods herein comprise: diagnosing a patient with stage I or stage II melanoma; identifying the patient as having a high recurrence risk based on expression levels of CD20, Bcl-X, Cox-2, Bax, P-catenin, PTEN, and MTAP; and treating the patient, for example, with a combination of pembrolizumab and V940, or a combination of cemiplimab and fianlimab.

[0101] A “high recurrence risk” refers to the risk a malignant melanoma will develop into distant metastatic disease (e.g., stage IV melanoma, i.e., where cancer cells have spread beyond the original skin tumor to distant organs, lymph nodes, or other parts of the body via the bloodstream or lymphatic system).

[0102] In some embodiments, identification of a high recurrence risk stage I or stage II melanoma patient comprises (i) preparing one or more section(s) of a tissue obtained from a melanoma lesion in the patient, wherein cells of the tissue express at least one of the following biomarkers: CD20, Bcl-X, Cox-2, Bax, P-catenin, PTEN, and MTAP; (ii) exposing the one or more section(s) of a tissue to at least seven different antibodies selected from the group consisting of: an anti-CD20 antibody, an anti-Bcl-X antibody, an anti-Cox-2 antibody, an antiBax antibody, an anti-P-catenin antibody, an anti-PTEN antibody, and an anti-MTAP antibody, thereby producing one or more immunostained tissue section(s) comprising immunostained cells, and optionally exposing the one or more section(s) of a tissue to an anti-SlOO antibody; (iii) assigning an expression factor to each of the biomarkers based on two or more characteristics selected from clustering, location, number, type, and staining intensity of the immunostained cells; (iv) calculating a selection score based on the expression factors assigned to the biomarkers in step (iii); and (v) selecting the patient for treatment of the melanoma when the selection score is higher than a reference score or deselecting the patient for treatment of the melanoma when the selection score is lower than a reference score. In some embodiments, the patient is treated for melanoma using, for example, a combination therapy comprising cemiplimab and fianlimab, or a combination therapy comprising pembrolizumab and V940.

[0103] In some embodiments, CD20 is assigned an expression factor based on number of CD20+immunostained B cells and clustering of CD20+immunostained B cells, Bcl-X is assigned an expression factor based on percentage of and staining intensity of Bcl-X+immunostained melanoma cells, Cox-2 is assigned an expression factor based on percentage of and staining intensity of Cox-2+immunostained melanoma cells, Bax is assigned an expression factor based on percentage of and staining intensity of Bax+immunostained melanoma cells, P-catenin is assigned an expression factor based on location and staining intensity of Bax P-catenin+immunostained melanoma cells, PTEN is assigned an expression factor based on percentage of and staining intensity of PTEN+immunostained melanoma cells, and MTAP is assigned an expression factor based on percentage of and staining intensity of MTAP+immunostained melanoma cells.

[0104] In some embodiments, CD20 is assigned an expression factor of 0 to 3, Bcl-X is assigned an expression factor of 0 to 2, Cox-2 is assigned an expression factor of 0 to 2, Bax is assigned an expression factor of 0 to 3, P-catenin is assigned an expression factor of 0 to 3, PTEN is assigned an expression factor of 0 to 3, and MTAP is assigned an expression factor of 0 to 2. In some embodiments, an expression factor is assigned to CD20 as follows: a tissue section comprising fewer than ten CD20+B cells or a cell cluster comprising fewer than five CD20+B cells is assigned a CD20 expression factor of 0; a tissue section comprising more than ten CD20+B cells or a cell cluster comprising more than five CD20+B cells, wherein the cell cluster comprises no more than one CD20+B cell nodular nest, is assigned a CD20 expression factor of 1; a tissue section comprising at least two CD20+B cell nodular nests without a ribbon-like connection is assigned a CD20 expression factor of 2; and a tissue section comprising a ribbonlike connection of at least three CD20+B cell nodular nests is assigned a CD20 expression factor of 3.

[0105] In some embodiments, an expression factor is assigned to Bcl-X in step (iii) as follows: a tissue section in which none of the cells is Bcl-X+is assigned a Bcl-X expression factor of 0, a tissue section in which fewer than 50% of melanoma cells are moderately to strongly Bcl-X+or in which more than 50% of melanoma cells are weakly Bcl-X+, is assigned a Bcl-X expression factor of 1, and a tissue section in which at least 50% of melanoma cells are moderately to strongly Bcl-X+is assigned a Bcl-X expression factor of 2.

[0106] In some embodiments, an expression factor is assigned to Cox-2 in step (iii) as follows: a tissue section in which none of the cells is Cox-2+is assigned a Cox-2 expression factor of 0, a tissue section in which fewer than 50% of melanoma cells are moderately to strongly Cox-2+, or in which more than 50% of melanoma cells are weakly Cox-2+, is assigned a Cox-2 expression factor of 1, and a tissue section in which at least 50% of melanoma cells are moderately to strongly Cox-2+is assigned a Cox-2 expression factor of 2.

[0107] In some embodiments, an expression factor is assigned to Bax in step (iii) as follows: a tissue section in which none of the cells is Bax+or in which fewer than ten melanoma cells are weakly Bax+stained is assigned a Bax expression factor of 0, a tissue section in which fewer than 50% of melanoma cells are Bax+or in which more than 50% of melanoma cells are weakly Bax+is assigned a Bax expression factor of 1, a tissue section in which at least 50% of melanoma cells are moderately Bax+or in which at least 50% and fewer than 80% of melanoma cells are strongly Bax+is assigned a Bax expression factor of 2, and a tissue section in which at least 80% of melanoma cells are strongly Bax+is assigned a Bax expression factor of 3.

[0108] In some embodiments, an expression factor is assigned to P-catenin in step (iii) as follows: a tissue section in which none of the cells is P-catenin+is assigned a P-catenin expression factor of 0, a tissue section in which only epidermal melanoma cells are P-catenin+or in which epidermal and dermal melanoma cells are weakly P-catenin+is assigned a P-catenin expression factor of 1, a tissue section in which epidermal and dermal melanoma cells are moderately P-catenin+is assigned a P-catenin expression factor of 2, and a tissue section in which epidermal and dermal melanoma cells are strongly P-catenin+is assigned a P-catenin expression factor of 3.

[0109] In some embodiments, an expression factor is assigned to PTEN in step (iii) as follows: a tissue section in which none of the cells is PTEN+or in which fewer than ten melanoma cells are faintly PTEN+is assigned a PTEN expression factor of 0, a tissue section in which fewer than 50% of melanoma cells are PTEN+or at least 50% of melanoma cells are weakly PTEN+is assigned a PTEN expression factor of 1, a tissue section in which at least 50% of melanoma cells are moderately PTEN+or at least 50% and fewer than 80% of melanoma cells are strongly PTEN+is assigned a PTEN expression factor of 2, and a tissue section in which at least 80% of melanoma cells are strongly PTEN+is assigned a PTEN expression factor of 3.

[0110] In some embodiments, an expression factor is assigned to MTAP in step (iii) as follows: a tissue section in which none of the cells is MTAP+is assigned an MTAP expression factor of 0, a tissue section in which fewer than 50% of melanoma cells are moderately MTAP+or in which more than 50% of melanoma cells are weakly MTAP+is assigned an MTAP expression factor of 1, and a tissue section in which more than 50% of melanoma cells are moderately or strongly MTAP+is assigned an MTAP expression factor of 2.

[0111] In some embodiments, a selection score is calculated using the following formula:

[0112] , wherein D is 7, / > is a univariate Cox model coefficient, x is an expression factor assigned for one of the biomarkers, and a corrects for negative antibody signal. In some embodiments, (a) the coefficients of a univariate Cox model are 0.547 for CD20, 0.391 for Bcl-X, 0.297 for Cox-2, 0.441 for Bax, -0.34 for P-Catenin, 0.272 for PTEN, and - 0.621 for MTAP.; and / or (b) wherein the reference score is 0.135

[0113] In some embodiments, a method further comprises exposing the one or more section(s) of a tissue to hematoxylin and eosin (H&E) staining.

[0114] In some embodiments, no more than two sections of tissue are prepared in step (b).

[0115] In some embodiments, the tissue is from a primary melanoma tumor tissue.

[0116] In some embodiments, the patient is diagnosed with stage IB melanoma.

[0117] In some embodiments, the patient is diagnosed with stage IIA melanoma.

[0118] In some embodiments, the patient is treated if the selection score is greater than the reference score.

[0119] In some embodiments, the treatment is selected from the group consisting of: surgery, chemotherapy, radiation therapy, immunotherapy, and targeted drug therapy.

[0120] Additional Embodiments - NEW VERSION 1 ALGORITHM

[0121] The present disclosure also relates to the embodiments described in the following numbered paragraphs:

[0122] Paragraph 1. A method, comprising: (a) selecting a human subject diagnosed with melanoma; and (b) selecting or deselecting the human subject for treatment of the melanoma, wherein step (b) comprises: (i) preparing one or more section(s) of a tissue obtained from a melanoma lesion in the human subject, wherein cells of the tissue express at least one of the following biomarkers: CD20, Bcl-X, Cox-2, Bax, P-catenin, PTEN, and MTAP; (ii) exposing the one or more section(s) of a tissue to at least seven different antibodies selected from the group consisting of: an anti-CD20 antibody, an anti-Bcl-X antibody, an anti-Cox-2 antibody, an antiBax antibody, an anti-P-catenin antibody, an anti-PTEN antibody, and an anti-MTAP antibody, thereby producing one or more immunostained tissue section(s) comprising immunostained cells, and optionally exposing the one or more section(s) of a tissue to an anti-SlOO antibody; (iii) assigning an expression factor to each of the biomarkers based on two or more characteristics selected from clustering, location, number, type, and staining intensity of the immunostained cells; (iv) calculating a selection score based on the expression factors assigned to the biomarkers in step (iii); and (v) selecting the human subject for treatment of the melanoma when the selection score is higher than a reference score or deselecting the human subject for treatment of the melanoma when the selection score is lower than a reference score.

[0123] Paragraph 2. The method of Paragraph 1, wherein in step (iii): CD20 is assigned an expression factor based on number of CD20+ immunostained B cells and clustering of CD20+ immunostained B cells; Bcl-X is assigned an expression factor based on percentage of and staining intensity of Bcl-X+ immunostained melanoma cells; Cox-2 is assigned an expression factor based on percentage of and staining intensity of Cox-2+ immunostained melanoma cells; Bax is assigned an expression factor based on percentage of and staining intensity of Bax+ immunostained melanoma cells; P-catenin is assigned an expression factor based on location and staining intensity of Bax P-catenin+ immunostained melanoma cells; PTEN is assigned an expression factor based on percentage of and staining intensity of PTEN+ immunostained melanoma cells; and / or MTAP is assigned an expression factor based on percentage of and staining intensity of MTAP+ immunostained melanoma cells.

[0124] Paragraph 3. The method of Paragraph 1 or 2, wherein in step (iii) CD20 is assigned an expression factor of 0 to 3, Bcl-X is assigned an expression factor of 0 to 2, Cox-2 is assigned an expression factor of 0 to 2, Bax is assigned an expression factor of 0 to 3, P-catenin is assigned an expression factor of 0 to 3, PTEN is assigned an expression factor of 0 to 3, and MTAP is assigned an expression factor of 0 to 2.

[0125] Paragraph 4. The method of any one of the preceding Paragraphs, wherein an expression factor is assigned to CD20 in step (iii) as follows: a tissue section comprising fewer than three CD20+ B cells is assigned a CD20 expression factor of 0; a tissue section comprising more than two CD20+ B cells is assigned a CD20 expression factor of 1; a tissue section comprising more than two CD20+ B cells that cluster is assigned a CD20 expression factor of 2; and a tissue section comprising more than two CD20+ B cells that form a nodular nest is assigned a CD20 expression factor of 3.

[0126] Paragraph 5. The method of any one of the preceding Paragraphs, wherein an expression factor is assigned to Bcl-X in step (iii) as follows: a tissue section in which fewer than 2 cells are Bcl-X+ melanoma cells is assigned a Bcl-X expression factor of 0; a tissue section in which more than one cell is a Bcl-X+ melanoma cell is assigned a Bcl-X expression factor of 1; and a tissue section in which a cluster of melanoma cells is strongly Bcl-X+ or in which more than 25% of cells are moderately Bcl-X+ melanoma cells is assigned a Bcl-X expression factor of 2.

[0127] Paragraph 6. The method of any one of the preceding Paragraphs, wherein an expression factor is assigned to Cox-2 in step (iii) as follows: a tissue section in which fewer than two cells are Cox-2+ melanoma cells is assigned a Cox-2 expression factor of 0; a tissue section in which more than 1 cell is a Cox-2+ melanoma cell is assigned a Cox-2 expression factor of 1; and a tissue section in which more than 25% of cells are moderately Cox-2+ melanoma cells is assigned a Cox-2 expression factor of 2.

[0128] Paragraph 7. The method of any one of the preceding Paragraphs, wherein an expression factor is assigned to Bax in step (iii) as follows: a tissue section in which fewer than two cells are Bax+ melanoma cells is assigned a Bax expression factor of 0; a tissue section in which more than one cell is Bax+ melanoma cell is assigned a Bax expression factor of 1; a tissue section in which more than 25% of cells are moderately Bax+ melanoma cells is assigned a Bax expression factor of 2; and a tissue section in which more than 50% of cells are strongly Bax+ melanoma cells is assigned a Bax expression factor of 3.

[0129] Paragraph 8. The method of any one of the preceding Paragraphs, wherein an expression factor is assigned to P-catenin in step (iii) as follows: a tissue section in which fewer than two cells are P-catenin+ melanoma cells is assigned a P-catenin expression factor of 0; a tissue section in which more than 1 cell is a P-catenin+ melanoma cell is assigned a P-catenin expression factor of 1; a tissue section in which more than 25% of dermal cells are moderately P- catenin+ melanoma cells is assigned a P-catenin expression factor of 2; and a tissue section in which more than 50% of dermal cells are strongly P-catenin+ melanoma cells is assigned a P- catenin expression factor of 3.

[0130] Paragraph 9. The method of any one of the preceding Paragraphs, wherein an expression factor is assigned to PTEN in step (iii) as follows: a tissue section in which fewer than 2 of the cells are PTEN+ melanoma cells is assigned a PTEN expression factor of 0; a tissue section in which more than 1 cell is a PTEN+ melanoma cell is assigned a PTEN expression factor of 1; a tissue section in which clusters of melanoma cells are moderately PTEN+ or in which more than 25% of the cells are moderately PTEN+ melanoma cells is assigned a PTEN expression factor of 2; and a tissue section in which at least 50% of the cells are strongly PTEN+ melanoma cells is assigned a PTEN expression factor of 3.

[0131] Paragraph 10. The method of any one of the preceding Paragraphs, wherein an expression factor is assigned to MTAP in step (iii) as follows: a tissue section in which fewer than 2 of the cells are MTAP+ melanoma cells is assigned an MTAP expression factor of 0; a tissue section in which more than 1 cell is an MTAP+ melanoma cell is assigned an MTAP expression factor of 1; and a tissue section in which more than 25% of cells are moderately or strongly MTAP+ melanoma cells is assigned an MTAP expression factor of 2.

[0132] Paragraph 11. The method of any one of the preceding Paragraphs, wherein the selection score is calculated using the following formula: DRAWING, wherein D is 7, P is a univariate Cox model coefficient, x is an expression factor assigned for one of the biomarkers, and a corrects for negative antibody signal.

[0133] Paragraph 12. The method of Paragraph 11, wherein the coefficients of a univariate Cox model are 0.547 for CD20, 0.391 for Bcl-X, 0.297 for Cox-2, 0.441 for Bax, -0.34 for P-Catenin, 0.272 for PTEN, and -0.621 for MTAP.

[0134] Paragraph 13. The method of Paragraph 12, wherein the reference score is 0.135. Paragraph 14. The method of any one of the preceding Paragraphs, wherein no more than two sections of tissue are prepared in step (b).

[0135] Paragraph 15. The method of any one of the preceding Paragraphs, wherein the tissue is from a primary melanoma tumor tissue.

[0136] Paragraph 16. The method of any one of the preceding Paragraphs, wherein the human subject has been diagnosed with early-stage melanoma.

[0137] Paragraph 17. The method of Paragraph 16, wherein the human subject has been diagnosed with stage IB melanoma.

[0138] Paragraph 18. The method of Paragraph 16, wherein the human subject has been diagnosed with stage IIA melanoma.

[0139] Paragraph 19. The method of any one of the preceding Paragraphs, further comprising treating the human subject if the selection score is greater than the reference score.

[0140] Paragraph 20. The method of Paragraph 19, wherein the treatment is selected from the group consisting of: surgery, chemotherapy, radiation therapy, immunotherapy, and targeted drug therapy.

[0141] EXAMPLES

[0142] Evaluation Scheme - NEW VERSION 2 ALGORITHM (FIGURES 1-7)

[0143] The slides were evaluated by trained staff (e. g. biologists, dermato-histopathologist, etc.) according to the NEW VERSION 2 ALGORITHM shown in FIGs. 1-7 and described elsewhere herein. The scans were evaluated using the ndp.view 2 or NZconnect software provided by Hamamatsu Photonics. The monitor was calibrated to ensure a uniform setting of the colors.

[0144] To exclude mechanical instability and ensure sufficient drying of the stained tissue section, the evaluation on the microscope or the scanning procedure was carried out after 72 hours. To exclude incorrect evaluation due to the fading of the stain, evaluation on the microscope or the scanning procedure was performed within 14 days.

[0145] If the sections are folded after completion of the staining process, if they have air bubbles or if only part of the original tissue material is present, it was considered whether the evaluation could be carried out without any doubt. If in doubt, the staining was repeated up to two times per marker.

[0146] Positive and negative controls were included for all marker stains that are processed (one positive and one negative control per run). Before evaluating the expression of the markers, these controls were examined and confirmed. The following additional stains were processed for each marker (if sufficient tissue is available):

[0147] H&E stain: Standard staining for the overall histological assessment of the tissue. S100 stain: For localization of the tumor areas (in case of negative expression of S100, the tissue cannot be assessed; the S100 stain should then be repeated if necessary).

[0148] Antibody diluent staining (omission of the primary antibody, incubation period for chromogen is 5 minutes): in the case of archived tissue, the AEC chromogen may bind to the tissue even without the primary antibody (various factors related to the fixation, embedding and storage of the tissue are suspected). In case of negative expression in the control tissue and presence of background staining with the AB diluent, the background staining may have to be subtracted when assessing the expression of the individual markers.

[0149] Criteria for rating were the percentage of stained melanoma cells, the intensity (weak, moderate, strong) of the staining, and the location / arrangement of the stained cells. This applies to all markers except CD20. The rating of CD20 was based on the arrangement of the stained CD20 positive cells from the B-cell lineage (B lymphocyte precursors up to plasmablasts) in the inflammatory infiltrate peripheral or inside the tumor.

[0150] Calculation

[0151] The selection score (risk score) was calculated using the following formula, to determine risk class (high risk (HR) v. low risk (LR)): wherein D is 7,

[0152] P is a univariate Cox model coefficient, x is an expression factor assigned for one of the biomarkers, and a corrects for negative antibody signal.

[0153] The coefficients of the univariate Cox model were 0.547 for CD20, 0.391 for Bcl-X, 0.297 for Cox-2, 0.441 for Bax, -0.34 for P-Catenin, 0.272 for PTEN, and -0.621 for MTAP.

[0154] The reference score was 0.135

[0155] If one biomarker (except PTEN or MTAP) could not be evaluated due to incomplete tissue section, folding or air bubbles on the tissue even after repeating the staining twice, the risk score minus the corresponding factor weighting could still be reliably determined; if two or more biomarkers were missing, the evaluation of the tissue block was discarded. The STANDARD SCORING ALGORITHM was validated within the frame described by Meyer et al, PLoS ONE 2012; 7(6): e38222.

[0156] The NEW VERSION 1 ALGORITHM was validated within the frame of three independent clinical studies, one prospective study (Meyer et al. European Journal of Cancer 2023; 182: 77- 86) and two archival cohorts (Reschke et al. Cancers 2021; 13: 2902; and University of Homburg).

[0157] Meyer et al. (2023) prospectively enrolled all patients who presented with cutaneous melanoma at University of Regensburg between 2010 and mid-2012. Tissue blocks of patients were tested with NEW VERSION 1 ALGORITHM. Follow-up was collected until 2020. Endpoints were RFS (relapse-free survival) and MSS (melanoma- specific survival).

[0158] Two further archival cohorts were evaluated from University of Leipzig (Reschke et al, 2021) and University of Homburg. These studies enrolled patients diagnosed between 2005-2016. Tissue blocks of patients were tested with NEW VERSION 1 ALGORITHM. Endpoints were RFS (relapse-free survival) and MSS (melanoma- specific survival).

[0159] The NEW VERSION 2 ALGORITHM was validated within the frame below. Tissue blocks of patients were tested with NEW VERSION 2 ALGORITHM. Endpoints were RFS (relapse-free survival) and MSS (melanoma- specific survival).

[0160] Background: For patients (pts) with resected cutaneous melanoma (CM), adjuvant anti-PDl is approved for AJCC v8 stage IIB-IV and BRAFi / MEKi for stage III-IV disease. Nonetheless, there remains a subgroup of pts within stages IB / IIA, which is at high risk of relapse and death, accounts for a considerable share of overall CM mortality, and lacks access to such therapy. Thus, adjuvant clinical trials in this “early”-stage setting may be merited. However, this subgroup is not detectable via AJCC staging alone. A prognostic 7-marker IHC assay (7-IHC) assay that was developed, and analytically and prospectively clinically validated to identify stage IB / IIA pts at high risk of relapse and death, is useful in selecting pts for adjuvant trials. Here, we report results from the multicenter study of that assay.

[0161] Methods: This study enrolled consecutive pts diagnosed with stage IB / IIA CM from 2000- 16, with available formalin-fixed paraffin-embedded primary melanoma. Specimens were analyzed by 7-IHC and pts classified as either high-risk or low-risk. Kaplan-Meier survival analysis and multivariate Cox regression analysis (MVA) against Breslow thickness, ulceration, age, and sex were performed.

[0162] Results: This study included 382 pts; 247 (65%) pts in stage IB, 135 (35%) in stage IIA, all

[0163] 5 sentinel node-negative. Median Breslow thickness was 1.6 mm, median age 60Y. 212 pts (55%) were classified as 7-IHC high-risk, 170 pts (45%) as 7-IHC low-risk. Median follow-up for recurrence-free survival (RFS) was 90 mos, for melanoma- specific survival (MSS) 98 mos. Comparing 7-IHC high-risk vs low-risk pts, 5Y RFS was 79% vs 100%, 10Y RFS, 72% vs 99%, 5Y MSS, 92% vs 100%, and 10Y MSS 82% vs 100% (Table; logrank p<0.001). In MVA, 7-IHC

[0164] 10 risk class (hazard ratio [HR] 22.3; 95% CI 7.0-70.9), Breslow thickness (1.5; 1.1-1.9), and age (1.61; 1.0-2.5) were significant independent prognosticators of RFS. 7-IHC HR for MSS could not be computed due to 7-IHC's 100% sensitivity. Prognostic performance variables (95% CI) for RFS were: sensitivity 98% (91 %— 100%), positive predictive value 26% (20%-33%), negative predictive value 99% (97%- 100%). Conclusions: 7-IHC risk categorization identified

[0165] 15 98% of relapses and 100% of CM-related deaths in stage IB / IIA pts. In MVA, 7-IHC risk class was the strongest independent prognosticator of survival endpoints. 7-IHC high-risk pts have a relapse rate comparable to that of later-stage pts for whom adjuvant therapy is approved. This study provides further evidence supporting use of 7-IHC to identify early-stage pts at high risk of relapse and death who may benefit from adjuvant therapy trials. This study enrolled patients

[0166] 20 diagnosed between 2005-2016.

[0167] Table 1. Comparison of New Version 1 Algorithm and Standard Scoring Algorithm

[0168] Table 2. Comparison of New Version 2 Algorithm and Standard Scoring Algorithm

[0169] Table 3. Comparison of New Version 1 Algorithm and New Version 2 Algorithm

[0170] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0171] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0172] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0173] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0174] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0175] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

What is claimed is:CLAIMS1. A method, comprising:(a) selecting a human subject diagnosed with melanoma; and(b) selecting or deselecting the human subject for treatment of the melanoma, wherein step (b) comprises:(i) preparing one or more section(s) of a tissue obtained from a melanoma lesion in the human subject, wherein cells of the tissue express at least one of the following biomarkers: CD20, Bcl-X, Cox-2, Bax, P-catenin, PTEN, and MTAP;(ii) exposing the one or more section(s) of a tissue to at least seven different antibodies selected from the group consisting of: an anti-CD20 antibody, an anti-Bcl-X antibody, an anti-Cox-2 antibody, an anti-Bax antibody, an anti-P-catenin antibody, an anti-PTEN antibody, and an anti-MTAP antibody, thereby producing one or more immunostained tissue section(s) comprising immunostained cells, and optionally exposing the one or more section(s) of a tissue to an anti-SlOO antibody;(iii) assigning an expression factor to each of the biomarkers based on two or more characteristics selected from clustering, location, number, type, and staining intensity of the immunostained cells;(iv) calculating a selection score based on the expression factors assigned to the biomarkers in step (iii); and(v) selecting the human subject for treatment of the melanoma when the selection score is higher than a reference score or deselecting the human subject for treatment of the melanoma when the selection score is lower than a reference score.

2. The method of claim 1, wherein in step (iii):CD20 is assigned an expression factor based on number of CD20+immunostained B cells and clustering of CD20+immunostained B cells;Bcl-X is assigned an expression factor based on percentage of and staining intensity of Bcl-X+immunostained melanoma cells;Cox-2 is assigned an expression factor based on percentage of and staining intensity of Cox-2+immunostained melanoma cells;Bax is assigned an expression factor based on percentage of and staining intensity of Bax+immunostained melanoma cells;P-catenin is assigned an expression factor based on location and staining intensity of Bax P-catenin+immunostained melanoma cells;PTEN is assigned an expression factor based on percentage of and staining intensity of PTEN+immunostained melanoma cells; and / orMTAP is assigned an expression factor based on percentage of and staining intensity of MTAP+immunostained melanoma cells.

3. The method of claim 1 or 2, wherein in step (iii) CD20 is assigned an expression factor of 0 to 3, Bcl-X is assigned an expression factor of 0 to 2, Cox-2 is assigned an expression factor of 0 to 2, Bax is assigned an expression factor of 0 to 3, P-catenin is assigned an expression factor of 0 to 3, PTEN is assigned an expression factor of 0 to 3, and MTAP is assigned an expression factor of 0 to 2.

4. The method of any one of the preceding claims, wherein an expression factor is assigned to CD20 in step (iii) as follows: a tissue section comprising fewer than ten CD20+B cells or a cell cluster comprising fewer than five CD20+B cells is assigned a CD20 expression factor of 0; a tissue section comprising more than ten CD20+B cells or a cell cluster comprising more than five CD20+B cells, wherein the cell cluster comprises no more than one CD20+B cell nodular nest, is assigned a CD20 expression factor of 1; a tissue section comprising at least two CD20+B cell nodular nests without a ribbon-like connection is assigned a CD20 expression factor of 2; and a tissue section comprising a ribbon-like connection of at least three CD20+B cell nodular nests is assigned a CD20 expression factor of 3.

5. The method of any one of the preceding claims, wherein an expression factor is assigned to Bcl-X in step (iii) as follows: a tissue section in which none of the cells is Bcl-X+is assigned a Bcl-X expression factor of O; a tissue section in which fewer than 50% of melanoma cells are moderately to strongly Bcl-X+or in which more than 50% of melanoma cells are weakly Bcl-X+, is assigned a Bcl-X expression factor of 1; and a tissue section in which at least 50% of melanoma cells are moderately to strongly Bcl- X+is assigned a Bcl-X expression factor of 2.

6. The method of any one of the preceding claims, wherein an expression factor is assigned to Cox-2 in step (iii) as follows:a tissue section in which none of the cells is Cox-2+is assigned a Cox-2 expression factor of O; a tissue section in which fewer than 50% of melanoma cells are moderately to strongly Cox-2+, or in which more than 50% of melanoma cells are weakly Cox-2+, is assigned a Cox-2 expression factor of 1; and a tissue section in which at least 50% of melanoma cells are moderately to strongly Cox- 2+is assigned a Cox-2 expression factor of 2.

7. The method of any one of the preceding claims, wherein an expression factor is assigned to Bax in step (iii) as follows: a tissue section in which none of the cells is Bax+or in which fewer than ten melanoma cells are weakly Bax+stained is assigned a Bax expression factor of 0; a tissue section in which fewer than 50% of melanoma cells are Bax+or in which more than 50% of melanoma cells are weakly Bax+is assigned a Bax expression factor of 1; a tissue section in which at least 50% of melanoma cells are moderately Bax+or in which at least 50% and fewer than 80% of melanoma cells are strongly Bax+is assigned a Bax expression factor of 2; and a tissue section in which at least 80% of melanoma cells are strongly Bax+is assigned a Bax expression factor of 3.

8. The method of any one of the preceding claims, wherein an expression factor is assigned to P-catenin in step (iii) as follows: a tissue section in which none of the cells is P-catenin+is assigned a P-catenin expression factor of 0; a tissue section in which only epidermal melanoma cells are P-catenin+or in which epidermal and dermal melanoma cells are weakly P-catenin+is assigned a P-catenin expression factor of 1 ; a tissue section in which epidermal and dermal melanoma cells are moderately P-catenin+is assigned a P-catenin expression factor of 2; and a tissue section in which epidermal and dermal melanoma cells are strongly P-catenin+is assigned a P-catenin expression factor of 3.

9. The method of any one of the preceding claims, wherein an expression factor is assigned to PTEN in step (iii) as follows:a tissue section in which none of the cells is PTEN+or in which fewer than ten melanoma cells are faintly PTEN+is assigned a PTEN expression factor of 0; a tissue section in which fewer than 50% of melanoma cells are PTEN+or at least 50% of melanoma cells are weakly PTEN+is assigned a PTEN expression factor of 1; a tissue section in which at least 50% of melanoma cells are moderately PTEN+or at least 50% and fewer than 80% of melanoma cells are strongly PTEN+is assigned a PTEN expression factor of 2; and a tissue section in which at least 80% of melanoma cells are strongly PTEN+is assigned a PTEN expression factor of 3.

10. The method of any one of the preceding claims, wherein an expression factor is assigned to MTAP in step (iii) as follows: a tissue section in which none of the cells is MTAP+is assigned an MTAP expression factor of 0; a tissue section in which fewer than 50% of melanoma cells are moderately MTAP+or in which more than 50% of melanoma cells are weakly MTAP+is assigned an MTAP expression factor of 1 ; and a tissue section in which more than 50% of melanoma cells are moderately or strongly MTAP+is assigned an MTAP expression factor of 2.

11. The method of any one of the preceding claims, wherein the selection score is calculated using the following formula:wherein D is 7, / > is a univariate Cox model coefficient, x is an expression factor assigned for one of the biomarkers, and a corrects for negative antibody signal.

12. The method of claim 11, wherein (a) the coefficients of a univariate Cox model are 0.547 for CD20, 0.391 for Bcl-X, 0.297 for Cox-2, 0.441 for Bax, -0.34 for P-Catenin, 0.272 for PTEN, and -0.621 for MTAP.; and / or (b) wherein the reference score is 0.13513. The method of any one of the preceding claims, further comprising exposing the one or more section(s) of a tissue to hematoxylin and eosin (H&E) staining.

14. The method of any one of the preceding claims, wherein no more than two sections of tissue are prepared in step (b).

15. The method of any one of the preceding claims, wherein the tissue is from a primary melanoma tumor tissue.

16. The method of any one of the preceding claims, wherein the human subject has been diagnosed with early-stage melanoma.

17. The method of claim 16, wherein the human subject has been diagnosed with stage IB melanoma.

18. The method of claim 16, wherein the human subject has been diagnosed with stage IIA melanoma.

19. The method of any one of the preceding claims, further comprising treating the human subject if the selection score is greater than the reference score.

20. The method of claim 19, wherein the treatment is selected from the group consisting of: surgery, chemotherapy, radiation therapy, immunotherapy, and targeted drug therapy.

21. The method of claim 20, wherein the treatment comprises an immune checkpoint inhibitor therapy.

22. The method of claim 21, wherein the immune checkpoint inhibitor therapy comprises an antibody selected from anti-PDl antibodies, anti-PD-Ll antibodies, anti-CTLA4 antibodies, and anti-LAG3 antibodies.

23. The method of claim 22, wherein the immune checkpoint inhibitor therapy comprises an anti-PDl antibody, optionally selected from pembrolizumab, nivolumab, cemiplimab (Libtayo), toripalimab, tislelizumab, sintilimab, camrelizumab, puzalizumab, and spartalizumab.

24. The method of claim 22 or 23, wherein the immune checkpoint inhibitor therapy comprises an anti-PD-Ll antibody, optionally selected from atezolizumab, durvalumab, avelumab, sugemalimab, envafolimab, and tislelizumab.

25. The method of any of claim 22-24, wherein the immune checkpoint inhibitor therapy comprises an anti-CTLA4 antibody, optionally selected from ipilimumab, tremelimumab, quavonlimab, BMS-986218, and ONC-392.

26. The method of any of claim 22-25, wherein the immune checkpoint inhibitor therapy comprises an anti-LAG3 antibody, optionally selected from fianlimab (REGN3767), relatlimab, favezelimab, ieramilimab (LAG525), TSR-033, MK-4280, and GB-1107.

27. The method of any of claims 20-25, wherein the treatment comprises a neoantigen vaccine therapy.

28. The method of claim 26, wherein the neoantigen vaccine therapy is a messenger RNA therapy, optionally V940.

29. The method of any of claims 19-28, wherein the treatment is a combination therapy comprising two or more therapies.

30. The method of claim 29, wherein the combination therapy comprises pembrolizumab and V940.

31. The method of claim 29, wherein the combination therapy comprises cemiplimab and fianlimab.

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