Mass spectrometry for cancer diagnosis

Abstract

The present disclosure relates to mass spectrometry methods for cancer diagnosis and prognosis.The present invention is based, at least in part, on the discovery that gangliosides (e.g., GD2, GD3, GM2, GD1b, or lipoforms thereof) are useful biomarkers for cancer diagnosis and prognosis, particularly for early detection of cancer.For example, provided herein is a mass spectrometry-based method for detecting and measuring the amount of gangliosides, lipoforms of gangliosides, as well as the lipid length of gangliosides.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 326,974, filed April 4, 2022, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] 2. Background of the Invention Cancer involves abnormal cell growth with the potential to invade or spread to other parts of the body. Despite decades of cancer research, cancer continues to cause a significant number of deaths (nearly 1,600 deaths per day in the United States in 2020), in large part due to the lack of measures for early and / or accurate detection. For example, ovarian cancer is the most lethal gynecologic cancer and the third leading cause of death in women. Serous ovarian cancer is the most aggressive subtype of ovarian cancer and is commonly labeled as the "silent killer" because most patients are diagnosed at advanced stages. Symptoms are vague and easily confused with other conditions. Currently, CA-125 is a blood marker used for both diagnosis and monitoring treatment effectiveness. CA-125 levels are elevated in 80% of epithelial tumors, but most such tumors are at advanced stages. Because its levels are elevated in <50% of stage I ovarian cancers, serum CA-125 has low detection rate and limited specificity for early diagnosis. Thus, there is a great need for new biomarkers, for example in cancer tissue and blood, to detect early stage cancer, which would improve survival of cancer patients. Summary of the Invention [Means for solving the problem]

[0003] Summary of the Invention Gangliosides are glycolipids that contain (i) a carbohydrate structure that specifically defines them by their name, and (ii) a lipid tail that can vary in carbon chain length. The present invention is based, at least in part, on the discovery that gangliosides (e.g., GD2, GD3, GM2, GD1b, or lipoforms thereof) are useful biomarkers for cancer diagnosis and prognosis, particularly for early detection of cancer.

[0004] Provided herein is a method for cancer diagnosis and prognosis. Provided herein is a mass spectrometry-based method for detecting and measuring the amount of gangliosides, ganglioside lipoforms, and ganglioside lipid length. Additionally, provided herein is a method for diagnosing cancer using liquid biopsy (e.g., blood, serum) or solid tissue biopsy (e.g., cancer tissue). The surprising and unexpected prevalence of certain gangliosides, ganglioside lipoforms, and / or ganglioside lipid length leads to the generation of novel biomarkers for cancer diagnosis and prognosis. [Brief description of the drawings]

[0005] [Figure 1A]1A-1B show schematic diagrams of gangliosides. FIG. 1A shows schematic diagrams of GD2 and GD3 gangliosides. Each geometry is a type of sugar. The exposed glycan tree is linked to a ceramide, which is linked to two lipid tails. The sugar heads of normal GM1 and tumor GD3 differ in two sugars, and GD2 and GD3 differ from each other in one sugar. Because GD2 is the biosynthetic product of GD3, GD2 and GD3 can often (but not always) be found on the surface of the same cell. FIG. 1B shows a depiction of how certain lipoforms can assemble differently on the membrane of a cell depending on the homogeneity or heterogeneity of the lipids, resulting in different biological signals or biological events. FIG. 1B also shows a depiction of how sphingosine chains can vary in carbon length and saturation at carbon positions 4-5. The acyl chain can vary more significantly in carbon length, (mono)saturation of the two carbons and hydroxylation at the 2-carbon position. [Figure 1B] Same as above. [Diagram 2] Figure 2 shows LC-MS / MS detection of GD3 in ovarian cancer. All GD3 species are shown combined regardless of lipoform, ANOVA, post hoc Holm Sidak, *p<0.05, **p<0.01. GD3 analytes in early and late stage ovarian cancer (n=13; 4 early stage and 9 late stage) vs. non-cancer (n=2). Ovarian cancer data are absolute quantification (pmol / ml). Glycolipids measured in serum taken at the time of diagnosis. Cancer-free samples have very low total GD3 and cancer samples have significantly higher total GD3. [Figure 3-1]Figures 3A-3S show LC-MS / MS detection of ganglioside lipoforms in ovarian cancer. Figures 3A-3H show lipoforms of GD3. Figure 3I shows lipoforms of GD2. Figures 3J-3P show lipoforms of GD1b. Figure 3Q shows total GD3 detected. Figures 3R and 3S show total GD2 and GD1b detected, respectively. Certain lipoforms show significant increases in cancer samples versus non-cancer samples. For example, Figure 3F demonstrates that GD3 (d18:1 / 23:0) is significantly increased in ovarian cancer samples, thereby providing an excellent marker for cancer diagnosis / prognosis. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above. [Figure 3-5] Same as above. [Diagram 3-6] Same as above. [Diagram 3-7] Same as above. [Diagram 3-8] Same as above. [Diagram 3-9] Same as above. [Figure 3-10] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description of the Invention Gangliosides are a family of more than 40 different sialic acid-containing glycosphingolipids. Each glycan tree is structurally unique and defines each ganglioside by its name. Some gangliosides, such as GM1, are normal and ubiquitous. Other gangliosides, such as GD2 and GD3, are tumor markers (also called tumor marker gangliosides; TMGs). They are expressed at low / absent levels in normal cells and at high levels in cancer. Thus, GD2 and GD3 are pathogenic biomarkers (i.e., biomarkers with essential functions for cancer), and such biomarkers are preferred because cancer does not easily downregulate the expression of the marker.

[0007] GD2 and GD3 regulate membrane fluidity, raft size and function, providing tumors with advantages in growth / metastasis, immune evasion and blockade. The lipid tails are embedded in the outer leaflet of the cell membrane and are variable in length.

[0008] GD2 or GD3 provide a stable, invariant and non-mutated target, whose expression is conserved across mammalian species (the glycan tree is identical), whose expression is uniform and homogeneous in cell lines and primary tumors, and whose density of expression is not downregulated in tumor cells that survive chemotherapy. In addition to being present on the tumor cell surface, GD2 and GD3 can be shed into the extracellular environment.

[0009] However, the examination of GD2 and GD3 expression in tissue or circulation has only been reported in a small number of patient samples. The assays for detecting GD2 or GD3 that can be expressed in tissue or serum are neither quantitative nor standardized; they only produce estimates; and they have led to contradictory conclusions. Because GD2 and GD3 are glycolipids, and the products can be produced by multiple biosynthetic pathways and enzymes, monitoring mutations or mRNA expression is not feasible. Therefore, GD2 and GD3 are still underutilized in cancer diagnosis.

[0010] The present disclosure is based, at least in part, on the discovery that mass spectrometry-based methods provide a powerful means for accurately determining gangliosides associated with cancer, e.g., tumors. Diagnostic methods using novel biomarkers of gangliosides and their lipoforms are further provided herein. Such novel biomarkers show surprising and unexpected prevalence in certain cancer types, e.g., ovarian cancer. Detection of such biomarker(s) provides a highly sensitive and accurate diagnostic method for cancer, especially in the early stages of cancer.

[0011] definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0012] The term "borderline tumor" is recognized in the art and refers to an atypical proliferative tumor with a relatively lower malignant potential. For example, a borderline ovarian tumor is an atypical proliferative tumor with a relatively lower malignant potential, for example, compared to ovarian cancer (see below).

[0013] Lipid length refers to the length of the lipid tail of the ganglioside, which is important for diagnosis. The lipid tail is embedded in the outer leaflet of the cell membrane. The lipids can be variable in length. Notably, such differences occur between gangliosides and when comparing within a single ganglioside. Thus, lipid heterogeneity and its variations are important for the diagnostic methods and methods described herein.

[0014] The term "lipoform" of a ganglioside refers to variants of a ganglioside that differ in their lipid content (see, for example, Kolter (2012) ISRN Biochem 506160).

[0015] The term "minimal residual disease" is recognized in the art and is used to describe a small number of cancer cells in the body during or after cancer treatment when the patient is in remission. The number of remaining cells can be so small that they do not cause any physical signs or symptoms, and in many cases cannot even be detected by traditional methods. This is the main cause of cancer relapse.

[0016] The term "neoadjuvant therapy" refers to treatment given prior to the primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy.

[0017] The term "preventing" is art-recognized and, when used in the context of a condition such as a viral / bacterial infection or a disease such as cancer, is well understood in the art and includes a treatment, e.g., administration of a composition, that reduces the frequency of or delays the onset of symptoms of a medical condition in a subject compared to subjects not receiving the treatment. Thus, preventing cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment compared to an untreated control population by a statistically and / or clinically significant amount, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population.

[0018] The term "remission" is art-recognized and refers to a state in which the signs and symptoms of cancer are reduced.

[0019] As used herein, "subject" refers to any healthy animal, mammal or human, or any animal, mammal or human suffering from cancer. The term "subject" is interchangeable with "patient." The term "non-human animal" includes any vertebrate, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0020] A "therapeutically effective amount" of a compound is that amount which is capable of producing a medically desirable result in a treated patient, e.g., induction of an immune response against gangliosides, reduction in tumor burden, reduction in tumor cell growth, or alleviation of any symptoms associated with cancer, preferably in a human or non-human mammal, at an acceptable benefit:risk ratio.

[0021] The term "treating" includes preventive and / or therapeutic treatment. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration of one or more of the subject compositions to a host. When administered prior to the clinical manifestation of an undesired condition (e.g., a disease or other undesired condition of a host animal), the treatment is preventive (i.e., protects the host from the onset of the undesired condition); whereas, when administered after the manifestation of an undesired condition, the treatment is therapeutic (i.e., intended to reduce, ameliorate or stabilize an existing undesired condition or its side effects).

[0022] Gangliosides Gangliosides are sphingoglycolipids that contain one or more sialic acids. They usually contain a hydrophobic ceramide or sphingoid lipid tail that is anchored to the outer leaflet of the plasma membrane. They also contain an oligosaccharide moiety and are classified according to their carbohydrate structure (ganglio, isoganglio, lacto, etc.). Gangliosides are an important subclass of sphingoglycolipids because they contain a negatively charged sialic acid (N-acetylneuraminic acid or N-glycolylneuraminic acid) linked to a lipooligosaccharide moiety. Gangliosides are named and classified according to the number of sialic acid residues attached to the inner sugar moiety (M for one, D for two, T for three, Q for four) and according to their chromatographic mobility. The numbering of gangliosides (5-x) is based on the number (x) of internal sugar moieties (glucose, galactose or GalNAc) according to the original experimental classification of Svennerholm, so where x is 4, the gangliosides are: GM1, GD1, GT1, x=3 for GM2, GD2, GT2, and x=2 for GM3, GD3 and GT3.

[0023] Heterogeneity is not only found within the glycan portion, but also within the ceramide portion. Heterogeneity can consist of different sphingoid bases of different chain lengths, sphinganine, sphingosine and phytosphingosine, which can be further modified by O-acetylation. In higher animals, C18- and C20-sphingosine are the most abundant sphingoid bases of gangliosides.

[0024] The fatty acids found in the ceramide portion of gangliosides are mostly saturated. α-hydroxylated fatty acids are not found frequently in brain gangliosides, but are abundant, for example, in gangliosides from the intestine, liver or kidney, and in GM4. To designate the lipoforms of gangliosides, nomenclature such as (d18:1 / 18:0)GM3 refers to the 18-carbon sphingosine (d=dihydroxy, 1=1 double bond) and stearoyl residue (18:0) in the ceramide portion. 3 Used for Neu5AcLacCer (see Kolter (2012) ISRN Biochem 506160). The functional consequences of heterogeneity in the lipid components are largely unknown, but the lipid moiety can mask the receptor function of ganglioside glycans through interactions with membrane cholesterol.

[0025] As provided herein, gangliosides are tumor biomarkers. In some embodiments, tumor-associated gangliosides, also referred to as "tumor marker gangliosides" or TMGs, include GD2, GD3, GD1b, GT1b, fucosyl-GM1, GloboH, polysialic acid (PSA), GM2, GM3, sialyl-Lewis, and the like. X , Cialis Lewis Y , Cialis Lewis A , Cialis Lewis B , Lewis Y , any portion thereof and modified versions thereof. In a preferred embodiment, the ganglioside is GD2, GD3, GM2, GD1b or lipoforms thereof.

[0026] Thus, as used herein, the term ganglioside can refer to a ganglioside, a tumor-associated ganglioside, a portion thereof, a lipoform thereof, a glycan variant thereof, or any other isoform / variant thereof.

[0027] Ganglioside Analysis / Detection Ganglioside biomarkers can be analyzed according to the methods described herein and other suitable techniques known in the art. The presence, level or lipid length of gangliosides (e.g., GD1b, GD2, GD3, GM2 or lipoforms thereof) can be detected using methods including, but not limited to, immunodiffusion, immunoelectrophoresis, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemical assay, dot blot assay or slot blot assay. The general techniques to be used in carrying out the various immunoassays described above, alone or in combination with or in place of NMR, MALDI-TOF, LC-MS / MS, gas chromatography and similar methods, as well as other variations of such techniques, such as in situ proximity ligation assay (PLA), fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich ELISA, competitive ELISA, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high diffusion chromatography and others (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn. pp 217-262, 1991, incorporated herein by reference), are known to those of skill in the art.

[0028] Such techniques can also be used to monitor ganglioside levels on cells, in tissues or in blood / plasma.

[0029] In a preferred embodiment, mass spectrometry (e.g., MALDI-TOF, LC-MS / MS, LC-MS, LC-ESI-MS / MS, nanoLC-ESI-MS / MS (also called nLC-ESI-MS / MS or nanobore LC-ESI-MS / MS) or others known in the art) is used to detect the presence, levels or lipid length of gangliosides (e.g., GD1b, GD2, GD3, GM2 or lipoforms thereof) in biological specimens, e.g., liquid biopsies (e.g., blood, saliva, serum, cells / tissues (e.g., cancer cells); see section on samples). Mass spectrometry-based methods are particularly useful in determining the lipid length of gangliosides or heterogeneity of certain lipoforms of gangliosides, which are novel biomarkers of the present disclosure.

[0030] mass spectrometry Liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) is frequently utilized for the sensitive and selective determination of trace-level compounds in biological samples. In particular, electrospray ionization (ESI) can ionize a wide range of compounds, including polar compounds or compounds with large molecular weights, so LC-MS / MS with ESI as the ion source is the most widely used method. By coupling with HPLC, the detected features can be quantified by ultraviolet or MS signal intensity.

[0031] Thus, any mass spectrometry-based method may be applied to the methods of the present disclosure. Exemplary mass spectrometry-based methods include LC-MS-based methods (e.g., LC-MS / MS, LC n -MS n), LC-MS-based methods involving the use of ESI (e.g., LC-ESI-MS, LC-ESI-MS / MS, LC-ESI-CID-MS / MS) and nanobore LC-MS-based methods (e.g., nanobore LC-ESI-MS, nanobore LC-ESI-MS / MS).

[0032] Analytes with suitable hydrophobic structures can be detected with high sensitivity in LC-ESI-MS / MS because (i) hydrophobic ions prefer to be present on the surface of droplets generated by electrospray, and such ions enter the gas phase more easily than ions inside the droplets, resulting in higher signal intensity, (ii) hydrophobic compounds can be sufficiently separated from salts and interfering compounds that have an inhibitory effect on ESI in a reversed-phase column, and (iii) hydrophobic compounds are eluted by a mobile phase with a higher organic solvent content, which is favorable for stable generation of charged droplets by electrospray, thus resulting in higher signal intensity (see, for example, Santa (2013) Drug Discoveries & Therapeutics, 7:9-17, which is incorporated herein by reference in its entirety).

[0033] Gangliosides can be detected using LC-ESI-MS / MS (see, e.g., Fuller et al. (2014) Anal Biochem 458:20-26; Sorensen (2006) Rapid Commun Mass Spectrom 20:3625-33, each of which is incorporated herein by reference in its entirety). Gangliosides and lipoforms can be detected using LC-ESI-MS / MS (see, e.g., Ikeda et al. (2008) J Lipid Res 49:2678-89, each of which is incorporated herein by reference in its entirety). LC-ESI-CID-MS / MS has been widely applied to the detection of gangliosides or glycan sequencing, which shows improved speed and sensitivity.

[0034] As indicated above, hydrophobic compounds, such as gangliosides or lipoforms thereof, can be efficiently separated and quantified using a reversed-phase column connected to HPLC or UPLC. A reversed-phase column or reversed-phase HPLC column is a chromatographic column that contains a non-polar stationary phase. A sample is placed in the reversed-phase column, and then a solvent is added to run the sample through the stationary phase. Since the stationary phase in a reversed-phase HPLC column is non-polar, the polar components of the sample will run out of the column first, followed by the non-polar components. A reversed-phase HPLC column can be packed or capillary, made of glass or metal, and can have many different hydrophobic materials as the stationary phase.

[0035] Many reversed-phase columns are commercially available.For example, various categories of reversed-phase columns, such as C18 reversed-phase LC column, biphenyl reversed-phase LC column, C4 reversed-phase LC column, C8 reversed-phase column, phenyl reversed-phase LC column, C1 reversed-phase LC column, PFP reversed-phase LC column, C30 reversed-phase LC column, polar embedded reversed-phase LC column, polar end-capped reversed-phase LC column, porous graphitic carbon reversed-phase LC column, phenyl-hexyl reversed-phase LC column and alkyl reversed-phase LC column, are known in the art and are commercially available (Thermo Fisher Scientific, Waltham, MA; Waters Corporation, Milford, MA).

[0036] Analysis of biological samples by MS is challenging due to the limited amount of sample available for analysis, very low concentrations of analytes, and potential interference from the sample matrix. n / MS n The advent of nanobore LC offers a solution to these constraints. nL / min flow rates create much smaller droplets that are more easily desolvated, resulting in higher MS sensitivity. In addition, lower detection limits may be achieved, less sample may be required, and increased resistance to chemical interferences may occur compared to conventional LC flow rates. The interface of the nanobore LC to the MS utilizes an emitter / sprayer with a tip with an internal diameter of approximately 1-30 μm.

[0037] Nanobore liquid chromatography (LC) in conjunction with LC-MS is well known in the art (see, e.g., Valaskovic and Kelleher (2002) Curr Top Med Chem 2:1-12; Siu et al. (2009) J Proteome Res 8:3797-807; Harbourt et al. (2012) Ana Chem 84:98-105, which are incorporated by reference in their entireties), and nanobore columns are readily available commercially (see, e.g., BioBasic™ 18 nanobore HPLC column (Thermo Scientific™ 72105107563), BioBasic™ 85 μm nanobore HPLC column, HPLC column ACE nanobore, and Thermo Scientific® Hypersil GOLD nanobore HPLC column).

[0038] The "level" or "amount" of a biomarker (e.g., one or more gangliosides or lipoforms thereof) in a subject is "significantly" higher or lower than the level of the biomarker in a control (e.g., a normal sample) if the amount of the biomarker is higher or lower, respectively, than the level in the control by an amount that exceeds the standard error of the assay used to assess the amount.

[0039] In some embodiments, the amount is at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, The amount or level of a biomarker in a subject can be considered "significantly" greater or less than the normal and / or control amount if it is greater or less than the normal and / or control amount of the biomarker by 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more, or any range therebetween, such as 5%-100%, etc. Such significant modulation values ​​can apply to any metric described herein, such as ganglioside levels, or changes in ganglioside lipid length heterogeneity, etc.

[0040] The term "lipid length heterogeneity" of at least one ganglioside includes the lipid length level or distribution pattern of at least one ganglioside in a given sample.

[0041] In some embodiments, the lipid length level or distribution pattern of at least one ganglioside in a subject sample is at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 35 ... If the heterogeneity is 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more or any range therebetween, such as 5% to 100%, more or less than that in the normal and / or control sample, there is a change (e.g., increase or decrease) or a significant change in lipid length heterogeneity.

[0042] Similarly, the term "lipid length heterogeneity" of gangliosides includes the distribution pattern of gangliosides with short lipid length versus long lipid length (see Example 6). Gangliosides have two lipid tails: sphingosine and acyl. As used herein, the two lipid tails are not differentiated. Thus, the term "lipid length heterogeneity" as used herein refers to the average length of both lipid tails of a ganglioside. In some embodiments, the amount or level of gangliosides having short lipid lengths (14-24 carbons) in a subject sample is at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, 25 ... There is an alteration or significant alteration in ganglioside lipid length heterogeneity if it is 0%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more, or any range therebetween, such as 5%-100%, more or less than that in normal and / or control samples.

[0043] In some embodiments, the amount or level of gangliosides having long lipid lengths (26-38 carbons) in a subject sample is at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, 2000%, 25 ... There is an alteration or significant alteration in ganglioside lipid length heterogeneity if it is 0%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more, or any range therebetween, such as 5%-100%, more or less than that in normal and / or control samples.

[0044] In some embodiments, the amount or level of gangliosides having short lipid lengths (14-24 carbons) and long lipid lengths (26-38 carbons) in a subject sample is at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 87 In some embodiments, there is an alteration or significant alteration in ganglioside lipid length heterogeneity if the heterogeneity is greater than or equal to 00%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000%, or more, or any range therebetween, such as 5%-100%, more or less than that in the normal and / or control sample.

[0045] Control A control refers to any suitable reference standard, such as a normal patient, a subject, e.g., cultured primary cells / tissues isolated from a normal subject, adjacent normal cells / tissues obtained from the same organ or body part of a patient, a tissue or cell sample isolated from a normal subject, or primary cells / tissues obtained from a depository institution. In other embodiments, a control can include the expression level of gangliosides (e.g., the level of one or more of gangliosides or lipoforms thereof) and / or lipid tail length of a subject, e.g., a normal or healthy subject. In some embodiments, a control can be derived from a diseased subject, e.g., a subject suffering from cancer.

[0046] Control also refers to any reference standard suitable for providing a comparison with the expression product in the test sample.In certain embodiments, control includes obtaining a control sample, in which the level of ganglioside, one or more of its lipoforms, or the lipid length of ganglioside is detected and compared with that from the test sample.Such a control sample can include any suitable sample, including but not limited to a sample from a control cancer patient with known outcome (can be a stored sample or a previous sample measurement); a normal tissue or cell isolated from a subject, for example, a normal patient or a cancer patient, cultured primary cells / tissues isolated from a subject, for example, a normal subject or a cancer patient, adjacent normal cells / tissues obtained from the same organ or body part of a cancer patient, a tissue or cell sample isolated from a normal subject, or primary cells / tissues obtained from a depository institution. In some embodiments, a control can include a reference standard expression product (e.g., ganglioside) level from any suitable source, including, but not limited to, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range in test samples from a group or set of patients with a particular outcome (e.g., 1, 2, 3, 4 year survival, etc.) or undergoing a particular treatment (e.g., standard of care cancer therapy). In some embodiments, a control includes samples drawn or collected longitudinally at different time points to assess changes in the levels of gangliosides, one or more of their lipoforms, or lipid lengths of gangliosides over time. One of skill in the art will understand that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the invention.

[0047] In some embodiments, the amount of ganglioside or its lipoform can be determined in a sample, relative to the amount of another ganglioside or its lipoform in the same sample, or as a ratio.In some embodiments, the control includes expression product level ratio, including but not limited to, determining the ratio of two gangliosides' product levels, ganglioside to total ganglioside lipoforms, or ganglioside short lipid length to long lipid length ratio in a test sample, and comparing it to any suitable ratio of these in a reference standard; determining the product levels of two or more gangliosides or their lipoforms in a test sample, and determining the difference in product levels in any suitable control; and determining the product levels of two or more gangliosides in a test sample, and normalizing these levels to the level of housekeeping gene product in the test sample, and comparing it to any suitable control.In a preferred embodiment, the control includes a control sample of the same series and / or type as the test sample. In other embodiments, the control can include product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer. In some embodiments, a control product level is established, for example, where higher or lower levels of a product compared to a particular percentile are used as a basis for predicting outcome. In other preferred embodiments, a control product level is established using product levels from cancer control patients with known outcomes, and product levels from test samples are compared to the control product levels as a basis for predicting outcome. As demonstrated by the data provided herein, the methods of the present invention are not limited to the use of specific cutoff points in the comparison of product levels in test samples to controls.

[0048] In some embodiments, the predetermined marker amount can be any suitable standard. For example, the predetermined marker amount can be obtained from the same or different human whose patient selection is being assessed. In some embodiments, the predetermined marker amount can be obtained from a previous assessment of the same patient. In such a manner, the progress of the patient's selection can be monitored over time. In addition, if the subject is a human, the control can be obtained from the assessment of another human or humans, for example, a selected group of humans. In such a manner, the degree of selection of the human whose selection is being assessed can be compared to suitable other humans, for example, other humans in a similar situation to the human of interest, such as humans suffering from similar or the same condition(s) and / or humans of the same ethnic group.

[0049] Thus, in preferred embodiments, the control comprises a sample (e.g., serum or tissue) from a normal, healthy person who is cancer-free. In yet other preferred embodiments, the control comprises a sample (e.g., serum or tissue) from a patient being evaluated (e.g., diagnosed or prognosticated). For example, the control sample can comprise (i) a historical sample from the patient, or (ii) a sample obtained from the patient in a longitudinal study, e.g., before or after a therapy (e.g., cancer therapy). The use of such a control allows for comparison of biomarkers present in the same patient over time (e.g., during the progression of cancer).

[0050] Diagnostic Assays The present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample contains gangliosides (or one or more of its lipoforms) and / or whether the levels of gangliosides (or one or more of its lipoforms) are modulated (e.g., upregulated or downregulated), thereby indicating the status of a disorder of interest, such as cancer. In some embodiments, the present invention is useful for classifying a sample (e.g., from a subject) as associated with or at risk for a ganglioside-mediated cancer or subtypes thereof using statistical algorithms and / or empirical data (e.g., the presence, absence, levels, or lipid lengths of gangliosides or their lipoforms).

[0051] An exemplary method for detecting levels of gangliosides, and thus useful for classifying whether a sample is associated with cancer or a clinical subtype thereof or different stages of cancer, involves obtaining a biological sample from a test subject and detecting gangliosides, one or more of their lipoforms, or the lipid tail length of gangliosides in the sample using a mass spectrometry-based method.

[0052] In certain instances, the statistical algorithm is a single learning statistical classifier system.For example, a single learning statistical classifier system can be used to classify a sample as a cancer sample based on prediction or probability value and the presence or level of ganglioside.The use of a single learning statistical classifier system typically classifies a sample as a cancer sample with a sensitivity, specificity, positive predictive value, negative predictive value and / or overall accuracy of at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0053] Other suitable statistical algorithms are well known to those skilled in the art.For example, learning statistical classifier system includes machine learning algorithm techniques that can adapt to complex data sets (e.g., a panel of markers of interest) and make decisions based on such data sets.In some embodiments, a single learning statistical classifier system is used, such as a classification tree (e.g., random forest).In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more learning statistical classifier systems is used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, inductive learning (e.g., decision / classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probabilistic Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NNs), artificial neural networks (ANNs), neuro-fuzzy networks (NFNs), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in known environments such as naive learning, adaptive dynamic learning and time-difference learning, passive learning in unknown environments, active learning in unknown environments, learning action-value functions, applications of reinforcement learning, etc.), and systems using genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss-Newton algorithms, Gaussian mixtures, gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, the methods of the present invention further include sending the sample classification results to a clinician (a non-specialist, e.g., a primary care physician; and / or a specialist, e.g., a histopathologist or oncologist).

[0054] In some embodiments, the method of the present disclosure further provides a diagnosis in the form of a probability that an individual has cancer. For example, an individual can have a probability of having cancer that is about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher. In yet another embodiment, the method of the present invention further provides a prognosis of cancer in an individual. In some instances, the method of classifying a sample as a cancer sample can be further based on the symptoms (e.g., clinical factors) of the individual from whom the sample was obtained. The symptom or group of symptoms can be, for example, lymphocyte count, white blood cell count, erythrocyte sedimentation rate, diarrhea, abdominal pain, abdominal distension, pelvic pain, lower back pain, muscle cramps, fever, anemia, weight loss, anxiety, depression, and combinations thereof. In some instances, the method of classifying a sample as a cancer sample can be further based on genetic mutations and / or cancer predisposition, regardless of symptoms.In some embodiments, diagnosing an individual as having cancer is followed by administering a therapeutically effective amount of a cancer therapy (e.g., a chemotherapeutic agent) to the individual.In some embodiments, diagnosing an individual as having cancer is followed by treating the individual with a cancer therapy.

[0055] In some embodiments, the method further involves obtaining a control biological sample (e.g., a biological sample from a subject without cancer), a biological sample from a subject in remission or prior to the onset of cancer, or a biological sample from a subject undergoing treatment for the onset of cancer.

[0056] In some embodiments, the method comprises analyzing a control sample to detect the presence and / or level of a ganglioside, one or more of its lipoforms, or the lipid length of the ganglioside, such that the presence and / or level of said ganglioside, one or more of its lipoforms, or the lipid length of the ganglioside is detected in a biological sample, and comparing the presence or level of the ganglioside in the control sample with the presence or level of the ganglioside in the test sample.

[0057] Preferred biological samples are serum, blood, saliva, tumor microenvironment, tumor pericellular / tissue or intracellular / tissue, which are isolated by conventional means from subjects.Those skilled in the art will understand that cell or tissue samples may require further processing (e.g., homogenization and / or partial purification of lipid fraction; see Example 1).

[0058] The level and / or heterogeneity of gangliosides, one or more of their lipoforms, or the lipid length of gangliosides determined by the method of the present disclosure correlates with different grades of cancer. Thus, in some embodiments, the method of the present disclosure can be used to determine the grade of cancer based on the level and / or heterogeneity of gangliosides, one or more of their lipoforms, or the lipid length of gangliosides determined as described herein. The grade of cancer describes how abnormal cancer cells and tissues look under a microscope compared to healthy cells. Cancer cells that look and organize most similar to healthy cells and tissues are low-grade tumors. Doctors refer to such cancers as being well-differentiated. Lower grade cancers are typically less invasive and have a better prognosis. The more abnormal the cells look and organize themselves, the higher the grade of the cancer. Cancer cells with high malignancy tend to be more invasive. Such cancer cells are called poorly differentiated or undifferentiated. Some cancers have their own systems for grading tumors. Many other cancers use a standard 1-4 grading scale. Grade 1: The tumor cells and tissue look most similar to healthy cells and tissue. These tumor cells and tissue are called well-differentiated tumors and are considered low-grade. Grade 2: The cells and tissues are slightly abnormal and are called moderately differentiated. These cells and tissues are an intermediate-grade tumor. Grade 3: The cancer cells and tissues look very abnormal. Such cancers are considered poorly differentiated because they no longer have an architectural structure or pattern. Grade 3 tumors are considered high grade. Grade 4: These undifferentiated cancers have the most abnormal-looking cells. They are the highest grade and typically grow and spread faster than lower grade tumors. As used herein, low-grade cancer refers to grade I cancer; high-grade cancer refers to grade 2-4 cancer.

[0059] Similarly, the level and / or heterogeneity of gangliosides, one or more of their lipoforms, or the lipid length of gangliosides determined by the method of the present disclosure correlates with different stages of cancer.Thus, in some embodiments, the composition and method of the present disclosure can be used to determine the grade of cancer based on the level and / or heterogeneity of gangliosides, one or more of their lipoforms, or the lipid length of gangliosides determined as described herein.

[0060] The stage of a cancer describes how large the primary tumor is in the patient's body and how far the cancer has spread. There are several different staging systems, many of which were created for specific types of cancer. Others can be used to describe several types of cancer. One common system that most recognize places cancer on a scale of 0 to IV. Stage 0 refers to abnormal cells that may potentially become cancerous in the future, but have not spread and are not considered to be cancer. This stage is also called "in-situ." Stages I through III relate to cancer that has not spread beyond the primary tumor site or has only spread to nearby tissues. The higher the stage number, the larger the tumor and the more it has spread. Stage IV cancer has spread to distant areas of the body. As used herein, early / low stage cancer refers to stage I cancer; late / high / advanced stage cancer includes stages II through IV cancer.

[0061] Similarly, the level and / or heterogeneity of gangliosides, one or more of their lipoforms, or the lipid length of gangliosides determined by the method of the present disclosure correlates with tumor burden. Thus, in some embodiments, the compositions and methods of the present disclosure can be used to determine the tumor burden of a subject based on the lipid length level and / or heterogeneity of at least one ganglioside determined as described herein. Tumor burden (or tumor burden) is defined as the total amount of tumor (cells / mass) distributed in the patient's body, including bone marrow. In Response Evaluation Criteria in Solid Tumors (RECIST) analysis, tumor burden is considered as the sum of the longest diameter of all measurable lesions. Various methods can be used to determine tumor burden in a subject. For example, computed tomography (CT) and magnetic resonance (MR) imaging were used to assess tumor response based on morphological (size, location) criteria, particularly by using RECIST. The RECIST classification describes the size of the lesion and distinguishes between four types of treatment response - stable disease (SD), partial response (PR), complete response (CR) or progressive disease (PD).

[0062] Prognostic Assays The term "prognosis" includes the prediction of the likely course and outcome of cancer, or the likelihood of recovery from disease. In some embodiments, the use of statistical algorithms provides the prognosis of cancer in an individual. For example, the prognosis can be surgery, the development of a clinical subtype of cancer (e.g., solid tumors such as lung cancer, melanoma, and renal cell carcinoma), the development of one or more clinical factors, the development of intestinal cancer, or recovery from disease.

[0063] The assays described herein, such as the preceding diagnostic assays or subsequent assays, can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, immunotherapy, immune checkpoint inhibitor therapy, or other drug candidate) for treating cancer. For example, such methods can be used to determine whether a subject can be effectively treated with one or a combination of agents. Thus, the present disclosure provides a method for determining whether a subject can be effectively treated with one or more agents for treating a cancer for which a test sample has been obtained and gangliosides have been detected.

[0064] Other aspects of the present disclosure include the use of the methods described herein for association and / or stratification analysis, in which the length of gangliosides, one or more of their lipoforms, or lipids of gangliosides in biological samples from individuals with cancer are analyzed, and the information is compared to the length of controls, preferably of similar age and race (e.g., individuals without cancer; controls can also be referred to as "healthy" or "normal" individuals, or can be at an earlier time point in a given time-lapse study). Appropriate selection of patients and controls is critical to the success of association and / or stratification studies. Thus, a pool of individuals with well-characterized phenotypes is highly desirable. Criteria for cancer diagnosis, cancer predisposition screening, prediction of clinical outcomes, cancer prognosis prediction, determination of drug responsiveness (pharmacogenomics), drug toxicity screening, etc. are described herein.

[0065] Different test designs can be used for genetic association and / or stratification studies (Modern Epidemiology, Lippincott Williams & Wilkins (1998), 609-622). Observational studies are most frequently performed in which the patient's response is not interfered with. The first type of observational study identifies a sample from an individual in whom a suspected cause of disease is present and another sample from an individual in whom the suspected cause is not present, and then the frequency of disease occurrence in the two samples is compared. These sampled populations are called cohorts and the study is a prospective study. Other types of observational studies are case-control or retrospective studies. In a typical case-control study, samples are collected from individuals with a phenotype of interest, such as a certain disease manifestation (cases), and individuals without the phenotype (controls) in the population from which conclusions are to be drawn (target population). Possible disease causes are then investigated retrospectively. Because the time and cost of sample collection in case-control studies is considerably less than that in prospective studies, case-control studies are the more commonly used study design in genetic association studies, at least in the discovery and discovery stages.

[0066] After obtaining any relevant phenotypic and / or genotypic information, statistical analysis is performed to determine whether there is any significant correlation between the presence of alleles or genotypes and the phenotypic characteristics of individuals. Preferably, before performing statistical tests for genetic association, data inspection and cleaning is performed first. Epidemiological and clinical data of samples can be summarized by descriptive statistics with tables and graphs, which are well known in the art. Data validation is preferably performed to check data completion, inconsistent entries and outliers. Chi-square test and t-test (Wilcoxon rank sum test if distribution is not normal) can then be used to check for significant differences between cases and controls for discrete and continuous variables, respectively.

[0067] An important decision in the performance of genetic association tests is the determination of the significance level, and when the p-value of the test reaches that level, a significant association can be declared. In exploratory analysis, where positive hits will be followed up in subsequent confirmatory testing, for example, an unadjusted p-value < 0.2 (significance level on the loose side) can be used to hypothesize about the significant association between ganglioside levels and certain phenotypic characteristics of cancer. In order for a level to be considered to have an association with cancer, it is preferred to achieve a p-value < 0.05 (significance level traditionally used in the art). If a hit is followed up in confirmatory analysis with more samples from the same source or different samples from different sources, an adjustment for multiple testing will be performed to avoid an excessive number of hits while maintaining an experimental error rate of 0.05. Different methods exist for adjusting for multiple testing to control different kinds of error rates, but a commonly used, but somewhat conservative, method is the Bonferroni correction to control experimental or family-wise error rates (Multiple comparisons and multiple tests, Westfall et al, SAS Institute (1999)). Permutation tests to control false discovery rates, FDR, can be more powerful (Benjamini and Hochberg, Journal of the Royal Statistical Society, Series B 57, 1289-1300, 1995; Resampling-based Multiple Testing, Westfall and Young, Wiley (1993)). Such methods for controlling multiplicity are preferred when tests are dependent, and control of false discovery rates is sufficient, as opposed to control of experimental error rates.

[0068] Once individual risk factors, whether genetic or non-genetic, are found for disease predisposition, a classification / prediction scheme can be set up to predict the category (e.g., diseased or non-disease) that an individual will belong to depending on its phenotype and / or genotype and other non-genetic risk factors. Logistic regression for discrete traits and linear regression for continuous traits are standard techniques for such tasks (Applied Regression Analysis, Draper and Smith, Wiley (1998)). Furthermore, other techniques can be used to set up classification. Such techniques include, but are not limited to, MART, CART, neural networks and discriminant analysis, which are suitable for use in comparing the performance of different methods (The Elements of Statistical Learning, Hastie, Tibshirani & Friedman, Springer (2002)).

[0069] Exemplary embodiments In certain aspects, methods are provided for detecting the presence, level and / or lipid length of at least one ganglioside and / or at least one lipoform of a ganglioside, comprising detecting said ganglioside and / or at least one lipoform of a ganglioside in a sample using mass spectrometry, optionally wherein the sample is derived from a subject having cancer, a subject suspected of having cancer or a subject free of cancer.

[0070] The mass spectrometry can be any method of this disclosure or any other mass spectrometry method known in the art.

[0071] In some embodiments, the mass spectrometry is selected from LC-MS, LC-MS / MS, LC-ESI-MS / MS, LC-ESI-CID-MS / MS, nanobore LC-ESI-MS and nanobore LC-ESI-MS / MS.

[0072] In certain embodiments, diagnostic and prognostic methods are provided herein.For example, in certain embodiments, a method for diagnosing cancer in a subject comprises: a) determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a subject sample; and b) comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside with the level in a control sample, wherein the level of the subject sample compared with the level in the control sample is A significantly higher level (at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700 and wherein a positive result of 300% or less of the 300% CI, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less is indicative that the subject has cancer.

[0073] In certain embodiments, a method of identifying a subject having cancer comprises the steps of: a) determining a level of at least one ganglioside and / or at least one lipoform of ganglioside in a subject sample; and b) comparing said level of at least one ganglioside and / or at least one lipoform of ganglioside to a level in a control sample, wherein the level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control sample. significantly higher levels (at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1000%, 1000%, 0%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700 %, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less) identify the subject as having cancer.

[0074] In certain embodiments, a method for determining the stage of cancer comprises the steps of: a) determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a subject sample; and b) comparing said level of at least one ganglioside and / or at least one lipoform of ganglioside with a level in a control sample, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, an increase of 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates the subject has an early stage cancer;and / or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1000%, 1000%, %, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1010%, 1020%, 1030%, 1040%, 1050%, 1060%, 1070%, 1080%, 1090%, 1090%, 1095%, 1090%, 1080%, 1095%, 1090%, 1095%, 1090%, 1095%, 1090 20%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820% wherein an increase of 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates that the subject has a later stage cancer.

[0075] In some embodiments, the increase of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample, compared to the level in the control sample, of at least 100% and not more than 200% indicates that the subject has early stage cancer.In some embodiments, the increase of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample, compared to the level in the control sample, of at least 200% indicates that the subject has late stage cancer.

[0076] In certain embodiments, a method for determining the grade of a cancer comprises the steps of: a) determining a level of at least one ganglioside and / or at least one lipoform of a ganglioside in a subject sample; and b) comparing said level of at least one ganglioside and / or at least one lipoform of a ganglioside to a level in a control sample, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290% of the level of at least one ganglioside and / or at least one lipoform of ganglioside in the sample ,300%,310%,320%,330%,340%,350%,360%,370%,380%,390%,400%,410%,420%,430%,440%,450%,460%,470%,480%,490%,500%,510%,520%,530%,540%,550%,560%,570%,580%,590%,600%,610%,620%,630%,640%,650%,660%,670%,680%, an increase of 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates the subject has Grade I cancer;at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, of the level of at least one ganglioside and / or at least one lipoform of a ganglioside in the subject sample compared to the level in the control sample; 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470 %, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, an increase of 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates the subject has Grade II cancer;at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, of the level of at least one ganglioside and / or at least one lipoform of a ganglioside in the subject sample compared to the level in the control sample; 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470 %, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 7 an increase of 80%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates the subject has Grade III cancer;and / or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160% of the level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control sample; 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, and wherein an increase of 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates that the subject has grade IV cancer.

[0077] In some embodiments, the level of at least one ganglioside and / or at least one lipoform of a ganglioside in a subject sample is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150% of the level of at least one ganglioside and / or at least one lipoform of a ganglioside in a subject sample compared to the level in a control sample. ,160%,170%,180%,190%,200%,210%,220%,230%,240%,250%,260%,270%,280%,290%,300%,310%,320%,330%,340%,350%,360%,370%,380%,390%,400%,410%,420%,430%,440%,450%,460%,470%,480%,490%,500%,510%,520%,530%,540%,550%,560%,570%,580%,590%,600%,610%,620%,630%,640%,650%,660%,670%,680%,690%,700%,710%,720%,730%,740%,750%,760%,770%,780%,790%,700%,710%,720%,740%,750%,780%,790%,700%,710%,720%,730%,740%,750%,760%,770%,780%,790%,790%,700%,710%,720%,730%,740%,75 ...00%,710%,720%,730%,740%,750%,790%,790%,700%,700%,700%,700%,7 70%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780 An increase of 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates the subject has a low grade cancer (e.g., Grade I).

[0078] In some embodiments, the level of at least one ganglioside and / or at least one lipoform of a ganglioside in a subject sample is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 0%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 4 80%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, An increase of 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates that the subject has a high-grade cancer (e.g., grade II, III or IV).

[0079] In some embodiments, an increase of at least 100% and no more than 200% in the level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control sample indicates that the subject has a low-grade cancer (e.g., grade I). In some embodiments, an increase of at least 200% in the level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control sample indicates that the subject has a high-grade cancer (e.g., grade II, III or IV).

[0080] In certain embodiments, a method for determining tumor burden of cancer comprises the steps of: a) determining a level of at least one ganglioside and / or at least one lipoform of ganglioside in a subject sample; and b) comparing said level of at least one ganglioside and / or at least one lipoform of ganglioside to a level in a control sample, At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1000%, 1000%, 100 %, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680% , 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less increase indicates that the subject has a low tumor burden;and / or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160% of the level of at least one ganglioside and / or at least one lipoform of a ganglioside in a subject sample compared to the level in a control sample. ,170%,180%,190%,200%,210%,220%,230%,240%,250%,260%,270%,280%,290%,300%,310%,320%,330%,340%,350%,360%,370%,380%,390%,400%,410%,420%,430%,440%,450%,460%,470%,480% %, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 80 wherein an increase of 0%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less is indicative of the subject having a high tumor burden.

[0081] In some embodiments, the increase of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample by at least 100% and not more than 200% compared to the level in the control sample indicates that the subject has low tumor burden.In some embodiments, the increase of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample by at least 200% compared to the level in the control sample indicates that the subject has high tumor burden.

[0082] In certain aspects, provided herein is a method for detecting recurrence of cancer in a subject, the method comprising: a) obtaining or preparing a sample from a subject whose cancer has regressed after undergoing cancer treatment; b) determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample; and c) comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside with the level in a control sample, wherein a significantly higher level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control sample is indicative of recurrence of cancer in the subject.

[0083] In certain aspects, provided herein is a method for detecting minimal residual disease in a subject, the method comprising the steps of: a) obtaining or preparing a sample from a subject in remission; b) determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample; and c) comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside with the level in a control sample, wherein a significantly higher level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control sample indicates that the subject has minimal residual disease.

[0084] In certain aspects, provided herein is a method for stratifying a subject suffering from cancer according to the benefit obtained from a cancer therapy (e.g., immunotherapy), comprising the steps of: a) determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a sample from a subject administered the cancer therapy; b) determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a control; and c) comparing the levels of at least one ganglioside and / or at least one lipoform of ganglioside detected in steps a) and b), wherein a significant change or decrease in the level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control is indicative that the subject suffering from cancer will benefit from the cancer therapy.In certain embodiments, a method for determining whether a subject suffering from cancer is likely to respond, or alternatively, not respond, to a cancer therapy (e.g., immunotherapy) is provided, comprising the steps of: a) determining the level of at least one ganglioside and / or at least one lipoform of a ganglioside in a sample from a subject administered the cancer therapy; b) determining the level of at least one ganglioside and / or at least one lipoform of a ganglioside in a control; and c) determining the level of at least one ganglioside and / or at least one lipoform of a ganglioside detected in steps a) and b). and comparing the levels of lipoforms of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control, wherein a significantly higher level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control is indicative that the subject suffering from cancer will not respond to the cancer therapy; and / or a significant unchanged or decreased level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control is indicative that the subject suffering from cancer will respond to the cancer therapy.

[0085] In certain embodiments, provided herein is a method for predicting a clinical outcome of a subject suffering from cancer, comprising the steps of: a) determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a subject sample; b) determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a control; and c) comparing the levels of at least one ganglioside and / or at least one lipoform of ganglioside determined in steps a) and b), wherein a significantly higher level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the level in the control is indicative of the subject having a poor clinical outcome.

[0086] In certain aspects, provided herein is a method for monitoring the progression of cancer in a subject, comprising: a) detecting the level of at least one ganglioside and / or at least one lipoform of ganglioside in a subject sample at a first time point; b) repeating step a) at a subsequent time point; and c) comparing the levels of at least one ganglioside and / or at least one lipoform of ganglioside detected in steps a) and b) to monitor the progression of cancer in the subject. In some embodiments, a significantly higher level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the control indicates the progression of cancer (e.g., increased tumor burden, increased cancer stage, onset of cancer by at-risk subjects).

[0087] In some embodiments, the method monitors the progression of cancer in a subject who has undergone cancer therapy between a first time point and a subsequent time point. In some embodiments, the subject is at risk of developing cancer.

[0088] In certain embodiments, provided herein is a method of assessing the effectiveness of a cancer therapy in a subject suffering from cancer, comprising the steps of: a) determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a first sample obtained from the subject; b) repeating step a) at least one subsequent time point after administration of the cancer therapy; and c) comparing the levels of at least one ganglioside and / or at least one lipoform of ganglioside detected in steps a) and b), wherein a significantly lower level of at least one ganglioside and / or at least one lipoform of ganglioside in at least one subsequent sample compared to the first sample is indicative of the therapy being effective in treating cancer in the subject.

[0089] In some embodiments, the first and / or at least one subsequent sample is a single sample or a portion of pooled samples obtained from the subject.

[0090] In some embodiments, the cancer therapy is surgery, chemotherapy, a cancer vaccine, a chimeric antigen receptor, radiation therapy, immunotherapy, a modulator of expression of an immune checkpoint inhibitory protein or ligand, or any combination thereof. In some embodiments, the immunotherapy is an immune checkpoint inhibitor therapy. In some embodiments, the cancer therapy is avelumab, durvalumab, atezolizumab, a BRAF / MEK inhibitor, a tyrosine kinase inhibitor, pembrolizumab, nivolumab, ipilimumab, or a combination thereof.

[0091] A number of further embodiments are provided that may be applied to any of the aspects of the invention described herein.

[0092] For example, the diagnostic and prognostic methods described herein can detect and determine the level of at least one ganglioside and / or at least one lipoform of a ganglioside using any method known in the art.

[0093] In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% of at least one ganglioside and / or at least one lipoform of a ganglioside. ,200%,210%,220%,230%,240%,250%,260%,270%,280%,290%,300%,310%,320%,330%,340%,350%,360%,370%,380%,390%,400%,410%,420%,430%,440%,450%,460%,470%,480%,490%,500%,510%,520%,530%,540%,550%,560%,570%,580%,590%,600%,610%,620%,630%,640%,650%,660%,670%,680%,690%,700%,710%,720%,730%,740%,750%,760%,770%,780%,790%,700%,710%,720%,740%,750%,760%,770%,780%,790%,700%,710%,720%,73 ... 90%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780 %, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or lower higher levels indicate significantly higher levels.

[0094] In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% of at least one ganglioside and / or at least one lipoform of a ganglioside. ,200%,210%,220%,230%,240%,250%,260%,270%,280%,290%,300%,310%,320%,330%,340%,350%,360%,370%,380%,390%,400%,410%,420%,430%,440%,450%,460%,470%,480%,490%,500%,510%,520%,530%,540%,550%,560%,570%,580%,590%,600%,610%,620%,630%,640%,650%,660%,670%,680%,690%,700%,710%,720%,730%,740%,750%,760%,770%,780%,790%,700%,710%,720%,740%,750%,760%,770%,780%,790%,700%,710%,720%,73 ... 90%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780 %, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or lower levels indicate significantly lower levels.

[0095] In some embodiments, the level of at least one ganglioside and / or at least one lipoform of a ganglioside is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500 %, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 8 An increase or decrease of 10%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates no significant change in the level of at least one ganglioside.

[0096] Further provided herein is a diagnostic and prognostic method using the length heterogeneity or homogeneity (e.g., determined using mass spectrometry) of at least one ganglioside lipid.In some embodiments, the significant change in the length heterogeneity of at least one ganglioside lipid in a subject sample compared to a control sample indicates that the subject has cancer.Similarly, in some embodiments, the significant change in the length heterogeneity of at least one ganglioside lipid in a subject sample compared to a control sample identifies the subject as having cancer.

[0097] Thus, in certain embodiments, provided herein is a method for diagnosing cancer in a subject, comprising the steps of: a) determining the lipid length of at least one ganglioside in a subject sample (e.g., using mass spectrometry); and b) comparing the lipid length of said at least one ganglioside with that in a control sample, wherein a significant change in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to the control sample is indicative of the subject having cancer. In certain embodiments, provided herein is a method for determining the stage of cancer, comprising the steps of: a) determining the lipid length of at least one ganglioside in a subject sample using mass spectrometry; and b) comparing the lipid length of said at least one ganglioside with that in a control sample, wherein a significant change in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control sample is indicative of the subject having cancer. At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360% of one sex ,370%,380%,390%,400%,410%,420%,430%,440%,450%,460%,470%,480%,490%,500%,510%,520%,530%,540%,550%,560%,570%,580%,590%,600%,610%,620%,630%,640%,650%,660%,670%,680%,690%,700%,710%,720% , 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less change indicates the subject has an early stage cancer;and / or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1000%, 1050%, %, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 51 0%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 8 wherein a change of 20%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates that the subject has a later stage cancer.

[0098] In some embodiments, at least 100% and no more than 200% change (e.g., increase or decrease) in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control sample indicates that the subject has an early stage of cancer.In some embodiments, at least 200% change (e.g., increase or decrease) in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control sample indicates that the subject has a later stage of cancer.

[0099] In some embodiments, (a) the level of gangliosides having a short lipid length relative to the level of gangliosides having a long lipid length; or (b) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 11% of the level of gangliosides having a long lipid length relative to the level of gangliosides having a short lipid length. 0%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440% ,450%,460%,470%,480%,490%,500%,510%,520%,530%,540%,550%,560%,570%,580%,590%,600%,610%,620%,630%,640%,650%,660%,670%,680%,690%,700%,710%,720%,730%,740%,750%,760%,770%,7 An increase or decrease of 80%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates the subject has early stage or late stage cancer.

[0100] In certain embodiments, a method for determining the grade of cancer comprises the steps of: a) determining the lipid length of at least one ganglioside in a subject sample using mass spectrometry; and b) comparing said lipid length of at least one ganglioside with that in a control sample, wherein the heterogeneity of the lipid length of at least one ganglioside in the subject sample compared to that in the control sample is at least about 10%. ,20%,30%,40%,50%,60%,70%,80%,90%,100%,110%,120%,130%,140%,150%,160%,170%,180%,190%,200%,210%,220%,230%,240%,250%,260%,270%,280%,290%,300%,310%,320%,330%,340%,350%,360%,370%,380%,390%,400%,410%,420%,430%,440%,450%,460%,470%,480%,4 ... 20%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, and wherein a change (e.g., increase or decrease) of 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less is indicative of the subject having grade I, grade II, grade III or grade IV cancer.

[0101] In some embodiments, the heterogeneity in the length of the lipid of at least one ganglioside in a subject sample compared to the length in a control sample is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 97 %, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 86 A change (e.g., increase or decrease) of 0%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates that the subject has a low-grade cancer (e.g., grade I) or a high-grade cancer (e.g., grade II, III or IV).

[0102] In some embodiments, at least 100% and no more than 200% change (e.g., increase or decrease) in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control sample indicates that the subject has a low-grade cancer.In some embodiments, at least 200% change (e.g., increase or decrease) in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control sample indicates that the subject has a high-grade cancer.

[0103] In some embodiments, (a) the level of gangliosides having a short lipid length relative to the level of gangliosides having a long lipid length; or (b) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%. 10%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 10 ... 40%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 7 An increase or decrease of 70%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates the subject has a low-grade or high-grade cancer.

[0104] In certain embodiments, a method of determining tumor burden of cancer comprises the steps of: a) determining the lipid length of at least one ganglioside in a subject sample using mass spectrometry; and b) comparing said lipid length of at least one ganglioside with that in a control sample, wherein the lipid length disparity of at least one ganglioside in the subject sample compared to that in the control sample is detected. of oneness, at least, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1000%, 1010 0%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 7 A change of 20%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates that the subject has a low tumor burden;and / or a decrease in lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in a control sample of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 9 00%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810 wherein a change of 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less is indicative of the subject having a high tumor burden.

[0105] In some embodiments, at least 100% and no more than 200% change (e.g., increase or decrease) in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control sample indicates that the subject has a low tumor burden.In some embodiments, at least 200% change (e.g., increase or decrease) in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control sample indicates that the subject has a high tumor burden.

[0106] In some embodiments, (a) the level of gangliosides having a short lipid length relative to the level of gangliosides having a long lipid length; or (b) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%. 10%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 44 0%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 77 An increase or decrease of 0%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates that the subject has a low or high tumor burden.

[0107] In certain aspects, a method is provided for detecting recurrence of cancer in a subject, the method comprising: a) obtaining or providing a sample from a subject whose cancer has regressed after undergoing cancer treatment; b) determining the lipid length of at least one ganglioside in the subject sample using mass spectrometry; and c) comparing the lipid length of at least one ganglioside with that in a control sample, wherein a significant change in lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control sample is indicative of recurrence of cancer in the subject.

[0108] In certain aspects, provided herein is a method for detecting minimal residual disease in a subject, the method comprising: a) obtaining or providing a sample from a subject in remission; b) determining the lipid length of at least one ganglioside in the subject sample using mass spectrometry; and c) comparing the lipid length of at least one ganglioside with that in a control sample, wherein a significant change in lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control sample is indicative of the subject having minimal residual disease.

[0109] In certain aspects, provided herein is a method of stratifying a subject suffering from cancer according to the benefit obtained from a cancer therapy (e.g., immunotherapy), comprising the steps of: a) determining using mass spectrometry the lipid length of at least one ganglioside in a sample from a subject administered the cancer therapy; b) determining the lipid length of at least one ganglioside in a control; and c) comparing the lipid length of at least one ganglioside detected in steps a) and b), wherein no significant change in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to that in the control is indicative that the subject suffering from cancer will benefit from the cancer therapy.

[0110] In certain aspects, provided herein is a method for determining whether a subject suffering from cancer is likely to respond to a cancer therapy, the method comprising the steps of: a) determining using mass spectrometry the lipid length of at least one ganglioside in a sample from a subject administered the cancer therapy; b) determining the lipid length of at least one ganglioside in a control; and c) comparing the lipid length of at least one ganglioside detected in steps a) and b), wherein a significant change in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to the length in the control is indicative of the subject suffering from cancer not responding to the cancer therapy; and / or no significant change in the lipid length heterogeneity of at least one ganglioside in the subject sample compared to the length in the control is indicative of the subject suffering from cancer responding to the cancer therapy.

[0111] In certain aspects, provided herein is a method for predicting clinical outcome of a subject suffering from cancer, comprising the steps of: a) determining the lipid length of at least one ganglioside in a subject sample using mass spectrometry; b) determining the lipid length of at least one ganglioside in a control; and c) comparing the lipid length of at least one ganglioside determined in steps a) and b), wherein a significant change in lipid length heterogeneity of at least one ganglioside in the subject sample compared to the control sample is indicative of the subject having a poor clinical outcome.

[0112] In certain aspects, provided herein is a method for monitoring the progression of cancer in a subject, comprising: a) using mass spectrometry to detect at least one ganglioside lipid length in a subject sample at a first time point; b) repeating step a) at a subsequent time point; and c) comparing the length heterogeneity of at least one ganglioside lipid detected in steps a) and b) to monitor the progression of cancer in a subject, where the subject is at risk of developing cancer. In some embodiments, a significant change in the length heterogeneity of at least one ganglioside lipid in a subject sample compared to a control indicates the progression of cancer (e.g., increased tumor burden, increased cancer stage, cancer development by at-risk subjects).

[0113] In some embodiments, between the first and subsequent time points, the subject is undergoing cancer therapy.

[0114] In certain aspects, provided herein is a method of assessing the effectiveness of a cancer therapy in a subject suffering from cancer, comprising the steps of: a) determining the lipid length of at least one ganglioside in a first sample obtained from the subject using mass spectrometry; b) repeating step a) at least one subsequent time point after administration of the cancer therapy; and c) comparing the levels of at least one ganglioside detected in steps a) and b), wherein a significant change in lipid length heterogeneity of the at least one ganglioside in the second sample compared to the first sample is indicative of the therapy being effective in treating cancer in the subject.

[0115] In some embodiments, the first and / or at least one subsequent sample is a single sample or a portion of pooled samples obtained from the subject.

[0116] The methods of the present disclosure (e.g., the diagnostic and prognostic methods described herein) can detect and determine the lipid length heterogeneity of at least one ganglioside using any method known in the art. In a preferred embodiment, the lipid length heterogeneity of at least one ganglioside is determined by mass spectrometry (e.g., LC-MS, LC-MS / MS, LC-ESI-MS / MS, LC-ESI-CID-MS / MS, nanobore LC-ESI-MS, nanobore LC-ESI-MS / MS or any other mass spectrometry method disclosed herein or known in the art).

[0117] In some embodiments, the lipid length heterogeneity or homogeneity of at least one ganglioside is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 0%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 5 00%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, A change of 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates a significant change in heterogeneity or homogeneity.

[0118] In some embodiments, (a) the level of gangliosides having a short lipid length relative to the level of gangliosides having a long lipid length; or (b) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%. 10%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 10 ... 40%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 7 An increase or decrease of 70%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less indicates a significant change in lipid length heterogeneity or uniformity.

[0119] In some embodiments, the lipid length heterogeneity or homogeneity of at least one ganglioside is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, %, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500% ,510%,520%,530%,540%,550%,560%,570%,580%,590%,600%,610%,620%,630%,640%,650%,660%,670%,680%,690%,700%,710%,720%,730%,740%,750%,760%,770%,780%,790% , 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less change indicates no significant change in heterogeneity or homogeneity.

[0120] In some embodiments, (a) the level of gangliosides having a short lipid length relative to the level of gangliosides having a long lipid length; or (b) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 11% of the level of gangliosides having a long lipid length relative to the level of gangliosides having a short lipid length. 0%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440 %, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770% , 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less increase or decrease indicates no significant change in lipid length heterogeneity or uniformity.

[0121] Further provided are numerous embodiments that can be applied to any aspect of the invention described herein.For example, in some embodiments, the cancer therapy is surgery, chemotherapy, cancer vaccine, chimeric antigen receptor, radiation therapy, immunotherapy, modulator of expression of immune checkpoint inhibitory protein or ligand, or any combination thereof.In some embodiments, the immunotherapy is immune checkpoint inhibitor therapy.In some embodiments, the cancer therapy is avelumab, durvalumab, atezolizumab, BRAF / MEK inhibitor, tyrosine kinase inhibitor, pembrolizumab, nivolumab, ipilimumab, or combination thereof.

[0122] In some embodiments, the at least one ganglioside and / or at least one lipoform of a ganglioside is a tumor-associated ganglioside and / or a lipoform of a tumor-associated ganglioside. In some embodiments, the tumor-associated ganglioside is GD2, GD3, GD1b, GT1b, Fucosyl-GM1, GloboH, polysialic acid (PSA), GM2, GM3, sialyl-Lewis. X , Cialis Lewis Y , Cialis Lewis A , Cialis Lewis B , Lewis Y , any portion thereof, any lipoform or isoform thereof, and any combination of two or more thereof.

[0123] In a preferred embodiment, the at least one ganglioside and / or at least one lipoform of a ganglioside comprises GM2, GD3, GD2, GD1b, any lipoform thereof, or any combination thereof.

[0124] In some embodiments, the at least one ganglioside and / or at least one lipoform of a ganglioside comprises a combination of two or more selected from GM2, GD3, GD2, GD1b, and any lipoform thereof.

[0125] In some embodiments, at least one ganglioside and / or at least one lipoform of a ganglioside comprises GD3(d18:1 / 16:0), GD3(d18:1 / 23:0), GD3(d18:1 / 24:1), GD2(d18:1 / 16:0), GD1b(d18:1 / 16:0), GD1b(d18:1 / 24:1), GD1b(d18:1 / 18:1), or any combination of two or more thereof.

[0126] In some embodiments, at least one ganglioside and / or at least one lipoform of a ganglioside is selected from GD3(d18:1 / 16:0), GD3(d18:1 / 23:0), GD3(d18:1 / 24:1), GD2(d18:1 / 16:0), GD1b(d18:1 / 16:0), GD1b(d18:1 / 24:1), GD1b(d18:1 / 18:1), and any combination of two or more thereof.

[0127] In some embodiments, at least one ganglioside and / or at least one lipoform of a ganglioside comprises GD3(d18:1 / 23:0) and / or GD3(d18:1 / 24:1).

[0128] In some embodiments, at least one ganglioside and / or at least one lipoform of a ganglioside is GD3(d18:1 / 23:0) and / or GD3(d18:1 / 24:1).

[0129] In some embodiments, at least one ganglioside and / or at least one lipoform of a ganglioside comprises GD3(d18:1 / 23:0).

[0130] In some embodiments, at least one ganglioside and / or at least one lipoform of a ganglioside is GD3(d18:1 / 23:0).

[0131] In some embodiments, at least one ganglioside and / or at least one lipoform of a ganglioside comprises acyl chains 24:1.

[0132] In some embodiments, the acyl chain 24:1 is present in GD3 and / or GD1b.

[0133] In some embodiments, the cancer is selected from the group consisting of neuroblastoma, lymphoma, leukemia, melanoma, glioma, small cell lung cancer, breast cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, Ewing's sarcoma, desmoplastic round cell tumor, rhabdomyosarcoma, retinoblastoma, non-small cell lung cancer, renal cell carcinoma, Wilms' tumor, prostate cancer, gastric cancer, endometrial cancer, pancreatic cancer, and colon cancer.

[0134] In some embodiments, the cancer is selected from the group consisting of neuroblastoma, lymphoma, leukemia, melanoma, glioma, small cell lung cancer, breast cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, Ewing's sarcoma, desmoplastic round cell tumor, rhabdomyosarcoma, and retinoblastoma.

[0135] In some embodiments, the cancer is ovarian cancer, melanoma, renal cancer, or lung cancer.

[0136] In a preferred embodiment, the cancer is ovarian cancer.

[0137] In some embodiments, the cancer is a borderline tumor.

[0138] In some embodiments, the sample comprises cells, serum, blood, peritumoral tissue and / or intratumoral tissue obtained from a subject (e.g., a biopsy). In preferred embodiments, the sample comprises a liquid biopsy (comprises a liquid). Thus, in some embodiments, the sample comprises serum or blood.

[0139] In some embodiments, a significantly higher level of at least one ganglioside and / or at least one lipoform of a ganglioside comprises at least a 20 percent increase in the level of at least one ganglioside and / or at least one lipoform of a ganglioside.

[0140] In some embodiments, a significantly lower level of at least one ganglioside and / or at least one lipoform of a ganglioside comprises at least a 20 percent decrease in the level of at least one ganglioside and / or at least one lipoform of a ganglioside.

[0141] In a preferred embodiment, the significantly higher or lower level of at least one ganglioside and / or at least one lipoform of ganglioside is at least or about 50% higher or lower than the level of a control (e.g., non-cancerous sample).In another embodiment, the significantly higher or lower level of at least one ganglioside and / or at least one lipoform of ganglioside is at least or about 25% higher or lower than the level of the subject's previous reading in a longitudinal study.

[0142] In some embodiments, a significant change in lipid length heterogeneity of at least one ganglioside comprises at least a 20 percent change (eg, an increase or decrease) in a subject sample compared to a control sample.

[0143] In some embodiments, the control sample is a sample from a subject without cancer.

[0144] In other embodiments, the control sample is a sample from a subject with cancer.

[0145] In some embodiments, the control sample is a sample from a subject (e.g., the first sample collected from a subject in a longitudinal collection to assess changes in the level of at least one ganglioside over time).

[0146] In a preferred embodiment, the diagnostic and / or prognostic method described herein further comprises treating the subject with a cancer therapy of the present disclosure or a cancer therapy known in the art. For example, a subject determined to need treatment based on the results of the diagnostic and / or prognostic method of the present disclosure can be treated with a cancer therapy. In some embodiments, the diagnostic and / or prognostic method further comprises recommending, prescribing and / or administering to the subject a cancer therapy of the present disclosure (e.g., immune checkpoint inhibitor therapy) or a cancer therapy known in the art.

[0147] In some embodiments, the subject has cancer. In some such embodiments, the subject is asymptomatic.

[0148] In some embodiments, the subject is a mammal (eg, a human, a pet (eg, a dog, a cat), a farm animal).

[0149] In some embodiments, the subject is an animal model of cancer, a dog, a cat, or a human.

[0150] In a preferred embodiment, the subject is a human.

[0151] Patient Treatment & Clinical Trial Outcome Monitoring Following the method of the present disclosure, the subject whose sample is examined in the method can be treated.For example, the subject diagnosed with cancer using the method described herein can be treated with cancer therapy (e.g., standard therapy or cancer therapy described herein or known in the art).

[0152] Monitoring the effect of an agent (e.g., a compound, drug or small molecule, immunotherapy, cancer therapy described herein or known in the art) on the level of gangliosides and / or at least one lipoform of gangliosides can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by the screening assay described herein to reduce the level of gangliosides can be monitored in subject clinical trials and can be detectable by mass spectrometry-based methods. In such clinical trials, the level of gangliosides and / or symptoms or other markers of cancer can be used as a "readout" or marker of the phenotype of a particular cell, tissue or system.

[0153] In a preferred embodiment, the disclosure provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, immunotherapy, immune checkpoint inhibitor therapy or other drug candidate), comprising: (i) obtaining a pre-administration sample from the subject prior to administration of the agent; (ii) detecting the level of at least one ganglioside and / or at least one lipoform of a ganglioside in the pre-administration sample; and (iii) obtaining one or more post-administration samples from the subject. (iv) detecting the level of at least one ganglioside and / or at least one lipoform of ganglioside in the post-administration sample; (v) comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside in the pre-administration sample with the level of at least one ganglioside and / or at least one lipoform of ganglioside in the post-administration sample(s); and (vi) modifying the administration of the agent to the subject accordingly. For example, increasing the administration of the agent may be desirable to reduce the level of at least one ganglioside and / or at least one lipoform of ganglioside to a level lower than that detected, i.e., to increase the effectiveness of the agent. In such an embodiment, the at least one ganglioside and / or at least one lipoform of ganglioside can be used as an indicator of the effectiveness of the agent even in the absence of an observable phenotypic response. Similarly, analysis of at least one ganglioside and / or at least one lipoform of a ganglioside, such as by mass spectrometry-based methods, can also be used to select patients to receive cancer therapy (e.g., immunotherapy, immune checkpoint inhibitor therapy).

[0154] sample Biological samples can be collected from various sources from a subject, including bodily fluid samples, cell samples, or tissue samples. Bodily fluid refers to fluids that are excreted or secreted from the body, as well as fluids that are not normally excreted or secreted (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, earwax and ear wax, Cowper's or bulbourethral fluid, chyle, chyme, feces, female ejaculate, interstitial fluid, intracellular fluid, lymph, menstruation, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In some embodiments, the subject and / or control sample is selected from the group consisting of cells, cell lines, whole blood, serum, plasma, buccal scraping, saliva, cerebrospinal fluid, and bone marrow. In some embodiments, the sample may contain living cells / tissues, fresh frozen cells, fresh tissue, biopsies, fixed cells / tissues, cells / tissues embedded in media such as paraffin, histological slides, or any combination thereof. In some embodiments, the sample may contain living cells / tissues, fresh frozen cells, fresh tissue, and / or biopsies.

[0155] Samples can be collected from an individual repeatedly over a longitudinal period of time (eg, on the order of days, weeks, months, annually, once every two years or more, etc.).

[0156] Sample preparation and separation can involve any of a number of procedures depending on the type of sample collected and / or the analysis of the biomarker measurement(s). Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of the sample, enrichment of sample proteins, lipid extraction and purification. In some embodiments, a particular cell type is purified based on at least one marker present on the cell surface.

[0157] A sample can include immobilized molecules. A molecule is "immobilized" or "anchored" to a substrate when it is covalently or non-covalently associated with the substrate such that the substrate can be rinsed with a fluid (e.g., normal saline citrate, pH 7.4) without a significant proportion of the molecule dissociating from the substrate.

[0158] As described herein, in some embodiments, the level of at least one ganglioside and / or at least one lipoform measure(s) of ganglioside in a sample from a subject is compared to a control biological sample (e.g., a biological sample from a subject without cancer), a control biological sample from a subject in remission or before the onset of cancer, or a control biological sample from a subject undergoing treatment for the onset of cancer. In some embodiments, the control biological sample is from a subject prior to treatment with a particular therapy. In some embodiments, when a subject is treated with multiple rounds of one or more therapies, the control biological sample can be from an earlier or later time point relative to the subject sample during such treatment. For example, a subject sample after the third round of therapy can be compared to a control subject sample after the first round of therapy.

[0159] In some embodiments, the level of at least one ganglioside and / or at least one lipoform of ganglioside measurement(s) in a sample from a subject is compared to a predetermined control (standard) sample. The sample from a subject is typically from a diseased tissue, such as a cancer cell or tissue. The control sample can be from the same subject or from a different subject. The control sample is typically a normal, non-diseased sample. However, in some embodiments, the control sample can be from a diseased tissue, such as for staging a disease or evaluating the effectiveness of a treatment. The control sample can be a combination of samples from several different subjects. In some embodiments, the biomarker amount and / or activity measurement(s) from a subject is compared to a predetermined level. This predetermined level is typically obtained from a normal sample.

[0160] As described herein, the "predetermined" biomarker quantity measurement(s) may be, by way of example only, a biomarker quantity measurement(s) used to evaluate a subject who may be selected for treatment, to evaluate response to a cancer therapy, and / or to evaluate response to a combination of anti-cancer therapies. The predetermined biomarker quantity and / or activity measurement(s) may be determined in a population of patients with or without cancer. The predetermined biomarker quantity measurement(s) may be a single number, equally applicable to all patients, or the predetermined biomarker quantity measurement(s) may vary according to specific subpopulations of patients. The subject's age, weight, height, and other factors may affect the predetermined biomarker quantity measurement(s) of an individual. Furthermore, the predetermined biomarker quantity may be determined individually for each subject. In some embodiments, the amounts determined and / or compared in the methods described herein are based on absolute measurements.

[0161] In some embodiments, the amounts determined and / or compared in the methods described herein are based on relative measurements such as ratios (e.g., biomarker levels before treatment vs. after treatment, e.g., biomarker measurements compared to spiked or artificial controls, e.g., biomarker measurements compared to housekeeping gene expression, etc.). For example, the relative analysis can be based on the ratio of pre-treatment biomarker measurements compared to post-treatment biomarker measurements. Pre-treatment biomarker measurements can occur at any time prior to initiation of anti-cancer therapy. Post-treatment biomarker measurements can occur at any time after initiation of anti-cancer therapy. In some embodiments, post-treatment biomarker measurements occur 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more after initiation of anti-cancer therapy, and even longer times toward indefiniteness for continued monitoring. Treatment can include one or more anti-cancer therapies, e.g., immune checkpoint inhibitors.

[0162] The predetermined biomarker quantity measurement(s) can be any suitable standard. For example, the predetermined biomarker quantity measurement(s) can be obtained from the same or different human whose patient selection is being assessed. In some embodiments, the predetermined biomarker quantity measurement(s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the patient selection can be monitored over time. In addition, if the subject is a human, a control can be obtained from the assessment of another human or humans, e.g., a selected group of humans. In such a manner, the degree of selection of the human whose selection is being assessed can be compared to suitable other humans, e.g., other humans in a similar situation to the human of interest, such as humans suffering from a similar or the same condition(s) and / or humans of the same ethnic group.

[0163] In some embodiments of the present disclosure, the change in biomarker amount measurement(s) from a pre-determined level is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 fold or greater, or any range inclusive therebetween. Such cutoff values ​​apply equally when measurements are based on relative changes, such as based on the ratio of pre-treatment biomarker measurements compared to post-treatment biomarker measurements.

[0164] cancer Cancer, tumor or hyperproliferative disorder refers to the presence of cells that possess characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, although such cells can exist alone in an animal or can be non-tumorigenic cancer cells, such as leukemia cells. Cancers include, but are not limited to, B cell cancers, e.g., multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and others. Other non-limiting examples of types of cancer applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, Brain tumors, testicular cancer, lung cancer, small cell lung cancer (SCLC), bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's hypergammaglobulinemia, and heavy chain disease.In some embodiments, the cancer is epithelial in nature, including but not limited to bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In yet other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers can be characterized in a variety of other ways, including but not limited to serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0165] The compositions and methods of the present invention can be used to detect various cancers, including ovarian cancer, small cell lung cancer (SCLC) or melanoma.In particular, the compositions and methods of the present invention are particularly useful in detecting borderline tumors.Therefore, the present invention can be used to diagnose or prognose borderline tumors, including but not limited to borderline ovarian tumors.

[0166] Borderline tumors Borderline tumors are a heterogeneous group of lesions histologically defined by atypical epithelial proliferation without stromal invasion.

[0167] Similarly, borderline tumors of the ovary (also called low-grade tumors) are a heterogeneous group of lesions histologically defined by atypical epithelial proliferation without stromal invasion (Seidman et al. (2002) Blaustein's pathology of the female genital tract, page 791;Seidman et al. (2003) Hematol Oncol Clin North Am, 17:909). The behavior of such tumors is distinct from low-grade ovarian carcinoma, and they are considered a separate clinical entity.

[0168] Borderline ovarian epithelial neoplasms are non-invasive neoplasms that sometimes have intraperitoneal spread. This group of neoplasms exhibits intermediate behavior between benign cystadenomas and invasive carcinomas. It has been referred to by different terms, including borderline, atypical proliferative, and low-grade tumors. Borderline neoplasm is the nomenclature currently most widely used by pathologists, gynecologists, and oncologists, and has been adopted into the World Health Organization (WHO) classification.

[0169] Borderline tumors account for 14 to 15 percent of all primary ovarian neoplasms.

[0170] Borderline tumors occur in a variety of histologies, such as epithelial ovarian cancer. The majority of cases are serous or mucinous. In some cases, endometrioid, clear cell, or transitional cell (Brenner) borderline tumors are found.

[0171] Cancer Treatment Following the methods of the present disclosure (e.g., diagnostic and / or prognostic methods), the patient can be treated with the cancer therapy described herein or known in the art, such as standard of care treatments for cancer known to those skilled in the art, chemotherapeutic agents, hormones, antiangiogenic agents, radiolabeled compounds, or by surgery, cryotherapy, immunotherapy, cancer vaccines, immune cell engineering (e.g., CAR-T), and / or radiation therapy. The preceding treatment methods can be administered in conjunction with other forms of cancer therapy, either consecutively with, prior to, or subsequent to said cancer therapy. For example, immunotherapy can be administered with a therapeutically effective dose of a chemotherapeutic agent, e.g., immunotherapy can be administered in conjunction with chemotherapy to enhance the activity and effectiveness of the chemotherapeutic agent. The Physicians' Desk Reference (PDR) discloses the dosages of chemotherapeutic agents that have been used in the treatment of various cancers. The dosage regimens and dosages of these aforementioned chemotherapeutic agents that are therapeutically effective depend on the particular cancer being treated, the extent of the disease, and other factors familiar to the skilled physician, and can be determined by the physician.

[0172] Immunotherapy is a targeted therapy that can include, for example, the use of cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are viruses that can infect and lyse cancer cells while leaving normal cells unharmed, making them potentially useful in cancer therapy. Oncolytic virus replication facilitates tumor cell destruction and also results in dose amplification at the tumor site. It can also act as a vector for anti-cancer genes, allowing them to be delivered specifically to the tumor site. Immunotherapy can involve passive immunity for short-term protection of the host, achieved by administration of preformed antibodies made against cancer or disease antigens (e.g., administration of monoclonal antibodies against tumor antigens, optionally linked to chemotherapeutic agents or toxins). For example, anti-VEGF is known to be effective in the treatment of renal cell carcinoma. Immunotherapy can also focus on the use of cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, and the like, can be used to selectively modulate biomolecules implicated in tumor or cancer initiation, progression and / or pathology.

[0173] Immunotherapies also include immune checkpoint modulators. Immune checkpoints are groups of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by down-modulating or inhibiting anti-tumor immune responses. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3 and A2aR (see, for example, WO2012 / 177624). Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling, thereby upregulating the immune response to more effectively treat cancer. In some embodiments, the cancer vaccine is administered in combination with one or more inhibitors of immune checkpoints (immune checkpoint blockade therapy), such as PD1, PD-L1 and / or CD47 inhibitors.

[0174] Adoptive cell-based immunotherapy can be combined with the therapeutic method of the present invention. Well-known adoptive cell-based immunotherapy modalities include, but are not limited to, irradiated autologous or allogeneic tumor cells, tumor lysate or apoptotic tumor cells, antigen-presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell transfer, adoptive CAR T cell therapy, autologous immune enhancement therapy (AIET), cancer vaccine, and / or antigen-presenting cells. Such cell-based immunotherapy can be further modified to express one or more gene products for further modulating immune response, such as expression of cytokines such as GM-CSF, and / or to express tumor-associated antigen (TAA) antigens such as Mage-1, gp-100, etc.

[0175] The term "chimeric antigen receptor" or "CAR" refers to an engineered T cell receptor (TCR) with a desired antigen specificity. T lymphocytes recognize specific antigens through the interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, naive T cells depend on professional antigen presenting cells (APCs) that provide additional costimulatory signals. TCR activation in the absence of costimulation can result in unresponsiveness and clonal anergy. To circumvent immunization, different approaches have been developed for the derivation of cytotoxic effector cells with grafted recognition specificity. CARs have been constructed that consist of binding domains derived from natural ligands or antibodies specific for cell surface components of the TCR-associated CD3 complex. After antigen binding, such chimeric antigen receptors couple to endogenous signaling pathways in the effector cells and generate activation signals similar to those initiated by the TCR complex. Since the first report of chimeric antigen receptors, the concept has been steadily refined and the molecular design of chimeric receptors has been optimized and now routinely uses any number of well-known binding domains, such as the scFVs and other protein-binding fragments described herein.

[0176] In other embodiments, the immunotherapy includes non-cell-based immunotherapy. In some embodiments, compositions comprising antigens with or without vaccine-enhancing adjuvants are used. Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, recombinant antigens including fusion proteins, and others. In some embodiments, immunomodulatory cytokines, such as interferons, G-CSF, imiquimod, TNF alpha, and others, and their modulators (e.g., blocking antibodies or more potent or longer-lasting forms), are used. In some embodiments, immunomodulatory interleukins, such as IL-2, IL-6, IL-7, IL-12, IL-17, IL-23, and others, and their modulators (e.g., blocking antibodies or more potent or longer-lasting forms), are used. In some embodiments, immunomodulatory chemokines, such as CCL3, CCL26, and CXCL7, and others, and their modulators (e.g., blocking antibodies or more potent or longer-lasting forms), are used. In some embodiments, immunomodulatory molecules that target immunosuppression are used, such as STAT3 signaling modulators, NFkappaB signaling modulators, and immune checkpoint modulators.

[0177] In yet other embodiments, immunomodulatory agents, such as immune cell proliferation inhibitors, glucocorticoids, cell proliferation inhibitors, immunophilins and their modulators (e.g., rapamycin, calcineurin inhibitors, tacrolimus, cyclosporin (cyclosporin), pimecrolimus, avetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hepatitis C, rheumatoid arthritis ... Drocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (doca), aldosterone, non-glucocorticoid steroids, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid analogues, methotrexate, antithymocyte globulin riboflavin, antilymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, opioids, IMPDH inhibitors, mycophenolic acid, myriocin, fingolimod, NF-xB inhibitors, raloxifene, drotrecogin alfa, denosumab, NF-xB signaling cascade inhibitors, disulfiram, olmesartan, dithiocarbamate, proteasome inhibitors, bortezomib, MG132 , Prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, nonsteroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethylepoxyquinomycin (DHMEQ), I3C (indole-3-carbinol) / DIM (di-indole methane) (13C / DIM), Bay11-7082, luteolin, cell-penetrating peptide SN-50, IKBa.-super repressor overexpression, NFKB decoy oligodeoxynucleotide (ODN), or any derivative or analogue thereof is used. In yet other embodiments, immunomodulatory antibodies or proteins are used. For example, antibodies that bind to CD40, Toll-like receptors (TLR), OX40, GITR, CD27 or 4-1BB, T cell bispecific antibodies, anti-IL-2 receptor antibodies, anti-CD3 antibodies, OKT3 (muromonab), otelixizumab, teplizumab, visilizumab, anti-CD4 antibodies, clenoliximab, keliximab, zanolimumab, anti-CD11a antibodies, efalizumab, anti-CD18 antibodies, erlizumab, rovelizumab, and the like. izumab), anti-CD20 antibody, afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, anti-CD23 antibody, rumiliximab, anti-CD40 antibody, teneliximab, toralizumab, anti-CD40L antibody, ruplizumab, anti-CD62L antibody, acelizumab, anti-CD80 antibody, galiximab Mab, anti-CD147 antibody, gavilimomab, B-lymphocyte stimulator (BLyS) inhibitor antibody, belimumab, CTLA4-Ig fusion protein, abatacept, belatacept, anti-CTLA4 antibody, ipilimumab, tremelimumab, anti-eotaxin 1 antibody, bertilimumab, anti-a4-integrin antibody, natalizumab, anti-IL-6R antibody, tocilizumab, anti-LFA-1 antibody, odulimomab ulimomab, anti-CD25 antibody, basiliximab, daclizumab, inolimomab, anti-CD5 antibody, zolimomab, anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atolimumab, cedelizumab, dorlimomabaritox, dorlixizumab, fontolizumab, gantenerumab, gomiliximab, lebrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, lovelizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab, aflibercept, alefacept, rilonacept, IL-1 receptor antagonists, anakinra, anti-IL-5 antibodies, mepolizumab, IgE inhibitors, omalizumab, talizumab, IL12 inhibitors, IL23 inhibitors, ustekinumab and others.

[0178] Nutritional supplements that enhance immune response, such as vitamin A, vitamin E, vitamin C, and others, are known in the art (see, e.g., U.S. Pat. Nos. 4,981,844 and 5,230,902 and PCT Publication No. WO 2004 / 004483) and can be used in the methods described herein.

[0179] Similarly, various agents or combinations thereof can be used to treat cancer, such as chemotherapy, radiation, epigenetic modifiers (e.g., histone deacetylase (HDAC) modifiers, methylation modifiers, phosphorylation modifiers, etc.), targeted therapies, etc., as are well known in the art.

[0180] In some embodiments, chemotherapy is used. Chemotherapy includes the administration of a chemotherapeutic agent. Such chemotherapeutic agent can be, but is not limited to, a chemotherapeutic agent selected from the following compound groups: platinum compounds, cytotoxic antibiotics, antimetabolites, mitotic inhibitors, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogs, plant alkaloids and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol and mitomycin; antifolates: methotrexate, mycophenolic acid and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphidicolin glycinate and pyrazoloimidazole; and mitotic inhibitors: halichondrin, colchicine and rhizoxin. Compositions containing one or more chemotherapeutic agents (e.g., FLAG, CHOP) can also be used. FLAG includes fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP includes cyclophosphamide, vincristine, doxorubicin, and prednisone.In another embodiment, a PARP (e.g., PARP-1 and / or PARP-2) inhibitor is used, such inhibitors being well known in the art (e.g., olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001; Pacher et al., 2002b); 3-aminobenzamide (Trevigen); 4-amino-1,8-naphthalimide; (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (US Pat. Re. 36,397); and NU1025 (Bowman et al.)). The mechanism of action is generally related to the ability of the PARP inhibitor to bind to and reduce the activity of PARP. PARP catalyzes the conversion of beta-nicotinamide adenine dinucleotide (NAD+) to nicotinamide and poly-ADP-ribose (PAR). Both poly(ADP-ribose) and PARP have been implicated in the regulation of transcription, cell proliferation, genomic stability and carcinogenesis (Bouchard VJ et.al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9);Herceg Z.; Wang Z.-Q. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 Jun. 2001, pp. 97-110(14)).Poly(ADP-ribose) polymerase 1 (PARP1) is a critical molecule for the repair of DNA single-strand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame JC, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang ZQ, et al. (1997) Genes Dev 11:2347-2358). Knocking out SSB repair by inhibiting PARP1 function induces DNA double-strand breaks (DSBs) that can induce synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant HE, et al. (2005) Nature 434:913-917; Farmer H, et al. (2005) Nature 434:917-921). The above examples of chemotherapeutic agents are for illustrative purposes and are not intended to be limiting.

[0181] In other embodiments, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or protons. Examples of radiation therapy include, but are not limited to, external beam radiation therapy, interstitial implantation of radioisotopes (I-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or total abdominal and pelvic radiation therapy. For a general review of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., JB Lippencott Company, Philadelphia. Radiation therapy can be administered as external beam radiation or teletherapy, whereby radiation is directed from a remote source. Radiation treatment can also be administered as internal therapy or brachytherapy, whereby a radioactive source is placed in the body near the cancer cells or tumor mass. Also included is the use of photodynamic therapy, which includes the administration of photosensitizers such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanines, photosensitizer Pc4, demethoxy-hypocrellin A; and 2BA-2-DMHA.

[0182] In other embodiments, hormone therapy is used.Hormonal therapeutic treatment can include, for example, hormone agonist, hormone antagonist (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonist), hormone biosynthesis and processing inhibitor, and steroid (e.g., dexamethasone, retinoid, deltoid, betamethasone, cortisol, cortisone, prednisone, dihydrotestosterone, glucocorticoid, mineralocorticoid, estrogen, testosterone, progestin), vitamin A derivative (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analog; antigestagen (e.g., mifepristone, onapristone) or antiandrogen (e.g., cyproterone acetate).

[0183] In other embodiments, photodynamic therapy (also called PDT, photoradiotherapy, phototherapy or photochemotherapy) is used to treat some types of cancer. It is based on the discovery that certain chemicals, known as photosensitizers, can kill single-celled organisms when they are exposed to certain types of light.

[0184] In yet other embodiments, laser therapy is used to utilize high intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer, such as bleeding or blockages, especially when other treatments cannot cure the cancer. It can also be used to treat cancer by shrinking or destroying tumors.

[0185] Clinical efficacy / response to treatment Clinical efficacy can be measured by any method known in the art.For example, response to therapy refers to the response of any cancer, e.g., tumor, to therapy, preferably the change in tumor mass and / or volume after the start of neoadjuvant or adjuvant chemotherapy.Tumor response can be assessed in the neoadjuvant or adjuvant setting, whereby the size of tumor after systemic intervention, measured by CT, PET, mammogram, ultrasound or palpation, can be compared with the initial size and dimensions, and the cellularity of tumor can be estimated histologically and compared with the cellularity of tumor biopsy taken before the start of treatment.Response can also be assessed by caliper measurement or pathological examination of tumor after biopsy or surgical resection. Responses can be recorded in a quantitative manner, such as percentage change in tumor volume or cellularity, or using semi-quantitative scoring systems, such as residual cancer burden (Symmans et al., J. Clin. Oncol. (2007) 25:4414-4422), or in a qualitative manner, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria, such as the Miller-Payne score (Ogston et al., (2003) Breast (Edinburgh, Scotland) 12:320-327). Assessment of tumor response can be performed early, for example, hours, days, weeks, or preferably months, after the initiation of neoadjuvant or adjuvant therapy. Typical endpoints for response assessment are following completion of neoadjuvant chemotherapy or following surgical removal of residual tumor cells and / or tumor bed.

[0186] In some embodiments, the clinical efficacy of the therapeutic treatments described herein can be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the percentage of patients in complete remission (CR), the number of patients in partial remission (PR), and the number of patients with stable disease (SD) at least 6 months after the end of the treatment. A shorthand for this formula is CBR=CR+PR+SD over 6 months. In some embodiments, the CBR of a particular anti-immune checkpoint treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more.

[0187] Additional criteria for evaluating response to cancer therapy relate to "survival", including all of the following: survival until death, also known as overall survival (death may be unrelated to associated cause or tumor); "recurrence-free survival" (the term recurrence includes both localized and distant recurrence); metastasis-free survival; disease-free survival (the term disease includes cancer and its associated diseases). The length of survival can be calculated by reference to a defined starting point (e.g., time of diagnosis or treatment initiation) and end point (e.g., death, recurrence or metastasis). In addition, the criteria for efficacy of treatment can be expanded to include the probability of survival, the probability of metastasis within a given period, and the probability of tumor recurrence.

[0188] For example, to determine an appropriate threshold, a particular anti-cancer treatment regimen can be administered to a population of subjects, and the outcome can be correlated with a biomarker measurement determined prior to administration of any cancer therapy. The outcome measurement can be a pathological response to a therapy given in a neoadjuvant setting. Alternatively, outcome measures such as overall survival and disease-free survival can be monitored over a period of time for subjects following a cancer therapy for which the value of the biomarker measurement is known. In certain embodiments, the same dose of anti-cancer drug is administered to each subject. In related embodiments, the dose administered is a standard dose known in the art for anti-cancer drugs. The period of time that subjects are monitored can vary. For example, subjects can be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. The threshold value of the biomarker measurement that correlates with the outcome of the cancer therapy can be determined using methods such as those described in the Examples section. EXAMPLES

[0189] Example 1 material and method patient Patient samples (TMA and liquid biopsies) were obtained from the LDI / JHG biobank under ethical approval. A retrospective study of clinical history was used to assess the correlation between TMG and CA-125 levels, cancer disease stage and survival, and menopausal stage. A total of 176 patients were studied (n=9 normal; n=16 borderline; n=151 ovarian).

[0190] Extraction and isolation of gangliosides from tissues Total lipids from homogenized tissue (e.g., tumor) (500 mg) are extracted with 9 ml chloroform-methanol (2:1; v / v) and then with 7.6 ml chloroform-methanol-water (1:2:0.8; v / v / v). The extract is dried under a gentle stream of nitrogen, redissolved in 7.5 ml chloroform-methanol-water (30:60:8; v / v / v) and applied to a DEAE-Sephadex A-25 column (6 x 45 mm, acetate form) (Amersham Biosciences AB, Sweden). After washing the neutral lipids from the column with 15 ml chloroform-methanol-water (30:60:8; v / v / v), the acidic glycolipid mixture containing gangliosides is eluted with 7.5 ml chloroform-methanol-0.8 M sodium acetate (30:60:8; v / v / v).

[0191] Extraction and isolation of gangliosides from liquid biopsies Monophasic lipid extraction of polar gangliosides with a solvent volume ratio of 0.85:2:1:0.75 MeOH:EtOH:CHCl3:H2O. Methods were adapted from Svennerholm and Fredman (1980) Biochim Biophys Acta 617:97-109; and Lydic et al. (2014) J Lipid Res 55:1797-809, both of which are incorporated by reference in their entirety.

[0192] Liquid chromatography / electrospray ionization-MS Liquid chromatographic (LC) separation is achieved by using an octadecylsilane (ODS) reversed-phase LC (RPLC) column (150 x 0.3 mm internal diameter) (LC PACKINGS; Amsterdam, The Netherlands) at 45 °C. The mobile phase is prepared by methanol (A), isopropanol (B) and water (containing 200 mM ammonium formate) (C), and the composition is produced by mixing these solvents. The gradient consists of holding solvent (A / B / C 55:25:20) (D) for 5 min, followed by a linear conversion of solvent D to solvent (A / B 60:40) (E) for 50 min, and holding solvent E for 35 min. The mobile phase is pumped at a flow rate of 7.5 μl / min for gradient elution. Typically, 3 μl of sample solution is applied.

[0193] Electrospray ionization-tandem mass spectrometry analysis Electrospray ionization (ESI)-MS analysis is performed by using a 4000Q TRAP quadrupole-linear ion trap hybrid MS (Applied Biosystems, Foster City, CA) equipped with an UltiMate 3000 nano / cap / micro LC system (Dionex Corporation, Sunnyvale, CA) coupled with an HTS PAL autosampler (CTC Analytics AG, Zwingen, Switzerland).

[0194] The instrument scan range is set to m / z 200-2,300 with a scan speed of 1,000 amu / s. The trap fill time is 10 ms in positive ion mode and 20 ms in negative ion mode. Other MS scan conditions are operated with an ion spray voltage of 5,500 V, a declustering potential (DP) of 80 V, and a collision-induced dissociation (CID) of 10 V in positive ion mode, and with an ion spray voltage of -4,500 V, a DP of -120 V, and a CID of -5 V in negative ion mode. Tandem mass spectrometry (MS / MS) analysis of ceramide species derived from in-source decay of gangliosides is performed under each CID condition: d16:1-18:0 / d18:1-16:0 (60 V), d18:1-18:0 and d20:1-18:0 (85 V) and d20:1-20:0 / d18:1-22:0 (75 V).

[0195] Multiple reaction monitoring (MRM) analysis MRM analysis is performed with the same instrument system used for LC / ESI-MS analysis. The DP is set at -120V, and the CID conditions for each ganglioside are optimized as follows: GM1, GM2 and GM3 (-95V), GD1, GD2 and GD3 (-55V), GT1, GT2, GT3 and GQ1 (-60V). Precursor and product ion pairs for MRM analysis are selected by these MS / MS spectra. The following MRM m / z transitions are monitored: parent ion to sialic acid ion (SA - ) or sialic acid-containing glycans for gangliosides. For the analysis of positional isomeric gangliosides, specific fragments derived from the terminal regions of the glycans in these isomers are selected and run under the same conditions of MRM analysis for each ganglioside.

[0196] Example 2 LC-MS assay in liquid biopsies - Ganglioside levels Using nLC-ESI-MS / MS (LC-MS), liquid biopsies from six cancer patients (two melanoma, two renal cancer, two small cell lung cancer (SCLC_newly diagnosed treatment naive) and three non-cancer controls (two donors tested individually and one plasma pooled from 30 donors) were analyzed (Tables 5 and 6). An LC-MS method was developed to test all tumor gangliosides at once (e.g., a cancer gangliosome matrix containing 20 tumor gangliosides within at least 5,000 analytes).

[0197] Cancer patients showed elevated tumor gangliosides, which were specific to each cancer. As assessed by LC-MS and compared to non-cancer samples, melanoma samples showed significantly increased levels of GM2, GD3, and GD1b; renal cancer samples showed significantly increased levels of GD3 and GD2; lung cancer samples showed significantly increased levels of GD3 and GM2. In a test of ovarian cancer samples made specifically to evaluate GD3, 12 / 13 ovarian cancer samples demonstrated a significant increase in GD3 compared to the two non-cancer control samples. The control non-cancer samples showed very low or below the detection threshold (Table 1). Internal standards included cholesterol (not shown) and phosphatidylcholine (not shown), which were present at high levels in all samples. Phosphatidylserine (PS), a non-specific positive marker of general stress (cancer, inflammation, diabetes, infection, sepsis, apoptosis), was elevated in all cancer samples over the control non-cancer samples. [Table 1]

[0198] Data are presented by relative quantification. TMG in the serum of cancer patients has lipid chains of distinct lengths that differ among cancers. The length can vary from 16 to 44 carbons, and this feature was analyzed in detail.

[0199] Left panels (melanoma, renal cancer, lung cancer and non-cancer) are one patient per case with relative quantification. Quantifiable limits of detection (LOQD) were approximately 1-20 units. Right panels (ovarian cancer and non-cancer) are absolute quantification (pmol / ml). Glycolipids were measured in serum taken at the time of diagnosis and the average of 13 ovarian cancer samples (4 early stage and 9 late stage) was compared to the average of 2 non-cancer samples.

[0200] FIG. 2 further shows GD3 analytes in early and late stage ovarian cancer vs. non-cancer (*p<0.05 and **p<0.01).

[0201] In both data sets (Table 1 and Figure 2), tumor gangliosides in serum are quantitatively different in terms of total quantification depending on whether non-cancer or cancer samples are evaluated (TMG is elevated in cancer). Figure 3 also shows that TMG is also different in terms of its lipid chain length, which is qualitatively and quantitatively different depending on whether non-cancer or cancer samples are evaluated.

[0202] Example 3 LC-MS Assay for Liquid Biopsies - Lipid Tail Length In addition to the demonstration by LC-MS that cancer patients have elevated levels of tumor gangliosides, provided herein is the surprising and unexpected finding that tumor gangliosides have lipid tail heterogeneity patterns that are specific to each cancer.

[0203] In melanoma, GM2 with short chain forms (lipid tails) was predominant and increased 10-fold above normal, while the long chain forms increased from undetectable in normal to 90 units in cancer. For GD3, the short chain forms increased 40-fold above normal. For GD1, the short chain forms increased from undetectable in normal to 70 units. Renal cancer had a significant increase in GD3 and GD2 (especially the short lipid chain forms). Lung cancer had a significant increase or shift in GD3 (especially the long lipid chain forms) and a shift in GM2 to the short lipid chain forms (Table 2). All control samples had very low or below threshold signatures, but normal gangliosides such as GM1 were detected.

[0204] As demonstrated herein, using LC-MS, the present disclosure detects and quantifies the variable length of ganglioside lipids and establishes the relationship between cancer diagnosis and lipid tail length.In addition, as further demonstrated herein, the LC-MS method detects other ganglioside tumor markers, such as GM2 and GD1, which cannot currently be quantified in ELISA.Detection of specific signatures of many tumor gangliosides (e.g., cancer gangliosomal matrix) is valuable for extended application and tumor detection / diagnosis / prognosis prediction. [Table 2]

[0205] Serum glycolipids and lipids were tested at the time of diagnosis. Only relevant data compared to normal controls are shown in relative units. For simplicity, lipid tails are categorized as short (14-24 carbons) or long (26-38 carbons). Examples shown include melanoma with widespread metastatic disease; renal cancer and non-small cell lung cancer. Patients were compared to the average of non-cancer donors. Example data from nearly 5,000 analytes (significant changes versus normal shown in bold). Other gangliosides remained unchanged or undetectable. The current estimated quantifiable limit of detection (LOQD) is approximately 40 units or 0.010-0.24 pmol / mL.

[0206] Example 4 LC-MS assays on liquid biopsies identified GD3, with acyl chains between 16 and 24 carbons long, as a key biomarker for ovarian cancer To address the need to quantify ganglioside levels and further identify the molecular identity of potential biomarkers, reversed-phase liquid chromatography-mass spectrometry was applied to gangliosides extracted from donors with cancer or normal healthy individuals. Gangliosides from serum samples were purified using standard methods. In this case, we used the addition of 5 volumes of cold methanol, centrifugation to precipitate proteins and unwanted molecules, and collection of the monophasic supernatant containing the gangliosides.

[0207] The total level of GD3 was assessed and lipid species of GD3 were evaluated. The data indicate that (a) the total concentration of GD3 (in nM) is increased in cancer and (b) the increased GD3 is a species with short acyl-lipid chains ranging in length from C16 to C24. The short acyl chains are as defined in the caption of Table 2 (see above). These data indicate that lipid species can be identified as increased in cancer and that the measurement method can be used to identify cancer. In this example, GD3 species with short acyl chains were identified as increased in ovarian cancer. [Table 3]

[0208] Total GD3 in serum is increased in ovarian cancer (OVCA) compared to serum collected from patients with benign gynecologic tumors or from normal healthy individuals (n=5 each, average). The acyl chains of GD3 increased in OVCA range in length from C16 to C24 carbons (defined and categorized herein as "short" lipids), showing a 2.4-fold increase in cancer (normalized to normal).

[0209] Example 5 LC-ESI-MS / MS assays on liquid biopsies have identified certain lipoforms, such as GD3 (d18:1 / 23:0), as important biomarkers for ovarian cancer. To address the need to quantify ganglioside levels and identify the molecular identity of potential biomarkers, a reversed-phase nanobore liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS / MS) method was performed for disialogangliosides GD1b, GD2, and GD3. With one exception, the levels of all GD3, GD1, and GD1b species were comparable between all groups.

[0210] Notably, GD3 (d18:1 / 23:0) levels were below the lower limit of detection in healthy controls, but were robustly detected at high abundance in the sera of both ovarian cancer and borderline tumor patients. [Table 4] Serum was collected at the time of diagnosis. Quantification of GD1b, GD2 and GD3 molecular lipid species was performed using calibration curves and regression equations obtained from the respective measurements of GD1b, GD2 and GD3 lipid species in standard solutions. Molecular identity was confirmed by LC-selected reaction monitoring (SRM)-information dependent acquisition (IDA)-enhanced product ion (EPI) experiments, in which SRM was used as a survey scan to identify the target analytes and IDA-EPI scans were acquired in a linear ion trap, allowing complete structural characterization of each ganglioside species. For statistical purposes, if the control value for GD3 (d18:1 / 23:0) fell below the LLOD value, it was assigned a value of half the LLOD.

[0211] Example 6 LC / MS liquid biopsy assay for pan-cancer screening based on quantification of tumor gangliosides A major challenge in cancer research is early detection of tumors, where treatment therapies are more likely to be successful. For certain cancers, it may take decades for a tumor to progress to later stage disease, whereas in others, it takes only a few months. 1,2 Early detection offers a golden opportunity to intervene before the cancer progresses to later stage disease, metastasizes to distant sites, or acquires additional mutations or heterogeneity that would make the cancer more difficult or impossible to treat. However, currently available routine cancer screening tests detect only five cancer types: breast, lung, colorectal, cervical, and prostate, again with limited sensitivity and specificity. This short list highlights the lack of effective early screening tests for all other cancer types. 3

[0212] Recent technologies focused on genomics (using next-generation sequencing for circulating tumor DNA) and proteomics have enabled liquid biopsy blood tests that detect signals from multiple cancers. This has been termed Multiple Cancer Early Detection (MCED), some of which have promising preliminary screening outcomes, such as CancerSEEK, PanSeer, and Galleri. 4-7

[0213] However, the diagnostic performance of these tests is suboptimal, especially for early stage cancers. As an example, the overall sensitivity of Galleri across 12 cancer types and stages was 51.5% (39% stage I to 92% stage IV). 7 These tests have not yet been shown to improve clinical outcomes. 8 Modeled performance of the MCED test in representative populations suggests that it could substantially reduce overall cancer mortality by stage-shifting diagnostics when added to usual care. 9 A key barrier for these emerging liquid biopsy-based diagnostic MCED technologies is that they must have very high specificity to minimize overtreatment, with sufficiently high sensitivity to improve outcomes. 8

[0214] A liquid biopsy assay based on liquid chromatography-mass spectrometry (LC-MS) technology is presented herein. The assay involves a novel liquid biopsy method of quantification of a previously unexplored family of biomarkers called tumor marker glycolipids (TMGs) from blood samples. TMGs have persistent and uniform expression almost exclusively in cancers, their expression is not downregulated, and they have a pathogenic role in cancer, making them an ideal target to be explored as a biomarker. Although genomics and proteomics have been used as tools in precision medicine; applicants foresee the glycomics of TMGs as the next frontier to be used in diagnosis and screening.

[0215] Tumor marker glycolipid (TMG) All cells are covered with a dense coat of glycans, which are carbohydrate chains of variable length and structure that can be attached to proteins or lipids. 10-12 Glycolipids are lipids to which carbohydrate chains are covalently attached. Glycolipids can be components of cell membranes, where the lipid is in the membrane and the carbohydrate chains are exposed to the outside. Glycolipids are found on the surface of all eukaryotic cell membranes. 10-13 The essential role of glycolipids is to maintain cell membrane stability and facilitate cell recognition, cell-cell communication, tissue formation, 11,14,15 and immune responses. 15 A subset of glycolipids are gangliosides, which have carbohydrate head trees containing at least one sialic acid, and the carbohydrate is attached to a sphingosine ceramide containing two lipid tails embedded in the membrane.

[0216] Gangliosides are a family of >40 different sialic acid-containing glycosphingolipids. Each glycan head tree is structurally unique and defines each ganglioside by its name. Some gangliosides are ubiquitous and present in normal cells, whereas others are low / absent in normal cells and expressed at high levels in embryonic tissues and cancers, being tumor markers. 16-22

[0217] Altered expression of certain species of gangliosides has been described to occur during cell proliferation, differentiation, and carcinogenesis. 17 Aberrant and elevated expression of gangliosides has also been observed in different types of cancer cells. A subset of gangliosides, termed TMG, comprises a family of about 20 different gangliosides that are present preferentially or almost exclusively and at high density on the cell surface of certain cancers (e.g., cancer gangliosome matrix (CGM)). 11

[0218] As the term “tumor marker” suggests, TMG expression is closely associated with malignant lesions. Expression of tumor gangliosides provides a survival advantage to tumors, as some TMGs are known to provide immune evasion or suppression, growth factor-independent growth, and better blood supply to tumors, all of which promote tumor growth, metastasis, and survival. 17 Furthermore, gangliosides are actively released from the membrane of tumor cells, systemically impairing antitumor immunity. 17 [Table 5] Moreover, TMG can be shed from tumor membranes,81-89 so that it is present in extracellular fluids and can be detected in serum as well. Surprisingly, however, TMG has been underutilized for the diagnosis of any cancer, although it seems to be an ideal and high-value pathogenic biomarker in tissue or serum.

[0219] Liquid chromatography-mass spectrometry (LC-MS) The LC-MS method allows for the analysis of multiple ganglioside analytes, and therefore all tumor marker gangliosides can be evaluated, including GD2, GD3, GD1b, GT1b, Fucosyl-GM1, GloboH, Polysialic acid, GM2, GM3, Sialyl-Lewis X, Sialyl-Lewis Y, Sialyl-Lewis A, Sialyl-Lewis B and Lewis Y. In addition, LC-MS allows for the examination of physical properties of tumor gangliosides, such as the heterogeneity of the lipid length and saturation status of the gangliosides. These features of the lipid moiety of the gangliosides can generate hundreds of permutations (e.g., carbon chain lengths varying from 14 to 40, and unsaturation status ranging from 0 to 2). These features have not been elucidated in detail, and even less have been exploited in diagnostics. Our initial proof-of-concept data indicates that we can identify unique signatures of lipid length and saturation that are specific and restricted to specific cancers and can be exploited as another diagnostic layer.

[0220] result Using nLC-ESI-MS / MS (LC-MS), we analyzed liquid biopsies from six cancer patients (two melanoma, two renal cancer, two small cell lung cancer (SCLC newly diagnosed treatment naive) and three non-cancer controls (two donors tested individually and one plasma pooled from 30 donors). An LC-MS method was developed to test all tumor gangliosides at once (e.g., cancer gangliosomal matrix containing 20 tumor gangliosides with at least 5,000 analytes).

[0221] Cancer patients had elevated TMG and lipid tail heterogeneity patterns that appeared to be specific to each cancer. Melanoma had significant increases in GM2 and GD3 and GD1. For GM2, the short chain form was increased 10-fold above normal, and the long chain form was increased from undetectable in normal to 90 units in cancer. For GD3, the short chain form was increased 40-fold above normal. For GD1, the short chain form was increased from undetectable in normal to 70 units. Renal cancer had significant increases in GD3 and GD2 (especially the short lipid chain forms). Lung cancer had significant increases or shifts in GD3 (especially the long lipid chain forms) and a shift in GM2 to short lipid chain forms. All control samples had very low or below threshold signatures, but normal gangliosides such as GM1 were detected. Internal standards (not shown) included cholesterol and phosphatidylcholine, which were present at high levels in all samples. Phosphatidylserine (PS) is a non-specific positive marker of common stresses (cancer, inflammation, diabetes, infection, sepsis, apoptosis).

[0222] Melanoma, renal cancer, lung cancer and non-cancer were quantified with one patient per case by relative quantification (Table 1). Quantifiable limits of detection (LOQD) were approximately 1-20 units.

[0223] In another LC-MS study, 12 / 13 OC serum samples had a significant increase in GD3 compared to 2 non-cancer serum samples, with the controls either very low or below the threshold (Figure 2).

[0224] These studies demonstrate the potential of LC-MS to quantify GD2 / GD3. Applicants' findings demonstrate an upregulation in the expression of GD3 at both stages, suggesting its utility in early screening of ovarian cancer.

[0225] In both data sets, tumor gangliosides in serum are quantitatively different in terms of total quantification depending on whether non-cancer or cancer samples are evaluated (TMG is elevated in cancer).

[0226] GD3(d18:1 / 23:0) - An important biomarker for ovarian cancer To address the need to identify the molecular identity of lipoforms, we used a reversed-phase nanobore liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS / MS) method for GD2 and GD3. Interestingly, 70% of GD3 in serum from OC patients has only one type of relatively short lipid chain (Table 4).

[0227] This feature can be exploited to distinguish one cancer from another. Further bioinformatics analysis suggests that GD3 in the serum of 12 of 13 OC patients (Figure 2) has a distinct and highly restricted lipid chain length, while other gangliosides are a highly heterogeneous combination of chain lengths. This highlights the importance of LC-MS analysis in asymptomatic screening. Of note, among the more than 500 possible lipoform permutations, the GD3 (d18:1 / 23:0) isoform is the most represented, constituting 70% of the total GD3 mass in 12 of 13 OC patients (127 / 181; p=0.0005) (Table 4).

[0228] The results presented herein describe exemplary data on a pan-cancer liquid biopsy test for multiple indications for the detection of TMG isolated from patient serum. By utilizing the signature of lipid tail heterogeneity, the tumor of origin can be identified. This is an innovative new type of cancer screening that utilizes advances in lipidomics science to detect specific cancer signals (variants in tumor marker gangliosides). Applicants' new approach will set the stage for a new cancer screening paradigm with potentially higher specificity and clinically useful sensitivity compared to existing technologies.

[0229] These data further establish a pan-cancer early detection screening test. Applicants' liquid biopsy technology provides cancer screening at a population level, with enormous potential to help people access earlier effective treatment and substantially reduce overall cancer mortality.

[0230] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0231] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. In the case of conflict, the present application, including any definitions herein, shall control.

[0232] Any polynucleotide and amino acid sequences that reference an accession number that correlates to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov, are also incorporated by reference herein in their entirety.

[0233] Equal Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A method for detecting the presence, level, and / or length of at least one ganglioside and / or at least one lipoform of a ganglioside, comprising the step of using mass spectrometry to detect the ganglioside and / or at least one lipoform of a ganglioside in a sample, wherein, optionally, (a) the sample is derived from a subject having cancer, a subject suspected of having cancer, or a subject without cancer, and / or (b) the mass spectrometry is selected from LC-ESI-MS / MS, LC-ESI-CID-MS / MS, nanobore LC-ESI-MS, and nanobore LC-ESI-MS / MS.

2. A method for obtaining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a target sample as an indicator that the subject has cancer or cancer recurrence, a) A step of determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in the sample of interest using the mass spectrometry described in claim 1, b) A step of comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside with a control, A significantly higher level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the control indicates cancer or cancer recurrence in the subject, and A method that includes this.

3. A method for obtaining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a target sample as an indicator of cancer grade or tumor burden of cancer, a) A step of determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in the sample of interest using the mass spectrometry described in claim 1, b) A step of comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside with a control, An increase of at least 100% and / or not more than 200% in the levels of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the control indicates that the subject has low-grade cancer or a low tumor burden, and / or A step in which, compared to a control, an increase of at least 200% in the level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample indicates that the subject is a high-grade cancer or has a high tumor burden. A method that includes this.

4. A method for obtaining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a target sample as an indicator of minimal residual disease in the target, a) A step of determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a sample derived from a subject in remission, using the mass spectrometry described in claim 1; b) A step of comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside with a control, A significantly higher level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample compared to the control indicates that the subject has minimal residual disease. A method that includes this.

5. A method for obtaining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a target sample, stratified according to the benefits obtained from cancer treatment, as an indicator for subjects suffering from cancer, a) A step of determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in the sample derived from a subject administered the cancer treatment, using the mass spectrometry described in claim 1, b) A step of comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside with a control, The fact that, compared to the control, the levels of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample do not change or decrease significantly indicates that the subject with cancer will benefit from the cancer treatment, step and A method that includes this.

6. A method for obtaining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a target sample as an indicator for determining whether a subject with cancer is likely to respond to cancer treatment, a) A step of determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a sample derived from a subject administered a cancer treatment using the mass spectrometry described in claim 1, b) A step of comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside with a control, Compared to the control, a significantly higher level of the at least one ganglioside and / or at least one lipoform of ganglioside in the control sample indicates that the subject with cancer is not responding to the cancer treatment, and / or The step is that, compared to the control, the level of at least one ganglioside and / or at least one lipoform of ganglioside in the target sample does not change or decreases significantly, indicating that the subject with cancer responds to the cancer treatment. A method that includes this.

7. A method for obtaining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a target sample as an indicator of clinical outcome for a subject with cancer, a) A step of determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in the sample of interest using the mass spectrometry described in claim 1, b) A step of comparing the level of at least one ganglioside and / or at least one lipoform of ganglioside with a control, Compared to the control, a significantly higher level of at least one ganglioside and / or at least one lipoform of ganglioside in the subject sample indicates that the subject has a poor clinical outcome, and A method that includes this.

8. A method for obtaining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a target sample as an indicator of cancer progression in the target, a) A step of detecting the level of at least one ganglioside and / or at least one lipoform of a ganglioside in the target sample at a first time point using the mass spectrometry described in claim 1, b) A step in which step a) is repeated at a later point in time, c) A step of monitoring the progression of the cancer in the subject by comparing the levels of the at least one ganglioside and / or at least one lipoform of a ganglioside detected in steps a) and b), If necessary, the steps include: (i) the subject is at risk of developing cancer; (ii) between the first time point and the subsequent time point, the subject is undergoing cancer treatment; and / or (iii) the first and / or at least one subsequent sample is a single sample or a portion of a pooled sample obtained from the subject. A method that includes this.

9. A method for obtaining the level of at least one ganglioside and / or at least one lipoform of ganglioside in a first sample obtained from a subject, as an indicator of the effectiveness of a cancer treatment in a subject suffering from cancer, a) A step of determining the level of at least one ganglioside and / or at least one lipoform of ganglioside in the first sample obtained from a subject using the mass spectrometry described in claim 1, b) A step of repeating step a) at at least one subsequent time point after administration of the cancer treatment, c) A step of comparing the levels of at least one ganglioside and / or at least one lipoform of ganglioside detected in steps a) and b), A step in which, compared to the first sample, a significantly lower level of the at least one ganglioside and / or at least one lipoform of ganglioside in the at least one subsequent sample indicates that the treatment is effective in treating cancer in the subject, If necessary, the first and / or at least one subsequent sample is a single sample or a portion of a pooled sample obtained from the subject, step and A method that includes this.

10. A method for obtaining heterogeneity in the length of lipids of at least one ganglioside in a target sample as an indicator of cancer or cancer recurrence in the target, a) A step of determining the length of the lipid of at least one ganglioside in the target sample using the mass spectrometry described in claim 1, b) A step of comparing the length of the lipid of at least one ganglioside with a control, A significant change in the heterogeneity of the lipid length of at least one ganglioside in the target sample compared to the control indicates cancer or cancer recurrence in the target, and A method that includes this.

11. A method for obtaining the heterogeneity of the lipid length of at least one ganglioside in a target sample as an indicator of cancer grade or tumor burden of cancer, a) A step of determining the length of the lipid of at least one ganglioside in the target sample using the mass spectrometry described in claim 1, b) A step of comparing the length of the lipid of at least one ganglioside with a control, A change of at least 100% and no more than 200% (e.g., increase or decrease) in the heterogeneity of the lipid length of at least one ganglioside in the subject sample compared to the control indicates that the subject is a low-grade cancer or has a low tumor burden, and / or A change of at least 200% (e.g., increase or decrease) in the heterogeneity of the length of the lipids of at least one ganglioside in the subject sample compared to the control indicates that the subject is a high-grade cancer or has a high tumor burden. A method that includes this.

12. A method for obtaining non-uniformity of the lipid length of at least one ganglioside in a target sample as an indicator of minimal residual disease in the target, a) A step of determining the length of the lipid of at least one ganglioside in a sample derived from a subject in remission using the mass spectrometry described in claim 1, b) A step of comparing the length of the lipid of at least one ganglioside with a control, A significant change in the heterogeneity of the lipid length of at least one ganglioside in the target sample compared to the control indicates that the target has minimal residual disease. A method that includes this.

13. A method for obtaining an index of cancer-suffering subjects, stratified according to the benefits obtained from cancer treatment (e.g., immunotherapy), of heterogeneity in the length of lipids of at least one ganglioside in a sample derived from a subject administered a cancer treatment, a) A step of determining the length of the lipid of at least one ganglioside in the sample derived from a subject administered the cancer treatment, using the mass spectrometry described in claim 1, b) A step of comparing the length of the lipid of at least one ganglioside with a control, The absence of a significant change in the heterogeneity of the lipid lengths of at least one ganglioside in the target sample compared to the control indicates that the subject suffering from cancer will benefit from the cancer treatment, step and A method that includes this.

14. A method for obtaining heterogeneity in the length of at least one ganglioside lipid in a sample derived from a subject administered a cancer treatment, as an indicator for determining whether a subject with cancer is likely to respond to a cancer treatment (e.g., immunotherapy), a) A step of determining the length of the lipids of at least one ganglioside in a sample derived from a subject administered a cancer treatment using the mass spectrometry described in claim 1, b) A step of comparing the length of the lipid of at least one ganglioside with a control, Compared to the control, a significant change in the heterogeneity of the lipid lengths of at least one ganglioside in the subject sample indicates that the subject with cancer is not responding to the cancer treatment, and / or The absence of a significant change in the heterogeneity of the lipid lengths of at least one ganglioside in the target sample compared to the control indicates that the subject suffering from cancer responds to the cancer treatment, step and A method that includes this.

15. A method for obtaining heterogeneity of the lipid length of at least one ganglioside in a target sample as an indicator of clinical outcome for a subject with cancer, a) A step of determining the length of the lipid of at least one ganglioside in the target sample using the mass spectrometry described in claim 1, b) A step of comparing the length of the lipid of at least one ganglioside with a control, A significant change in the heterogeneity of the lipid length of at least one ganglioside in the subject sample compared to the control indicates that the subject has a poor clinical outcome. A method that includes this.

16. A method for obtaining non-uniformity of the lipid length of at least one ganglioside in a target sample as an indicator of cancer progression in the target, a) A step of detecting the length of the lipid of at least one ganglioside in the target sample at a first time point using the mass spectrometry described in claim 1, b) A step in which step a) is repeated at a later point in time, c) A step of monitoring the progression of the cancer in the subject by comparing the heterogeneity of the lipid lengths of the at least one ganglioside detected in steps a) and b), If necessary, the steps include: (i) the subject is at risk of developing cancer; (ii) between the first time point and the subsequent time point, the subject is undergoing cancer treatment; and / or (iii) the first and / or at least one subsequent sample is a single sample or a portion of a pooled sample obtained from the subject. A method that includes this.

17. A method for obtaining heterogeneity in the length of at least one ganglioside lipid in a first sample and a second sample as an indicator of the effectiveness of a cancer treatment method in a patient suffering from cancer, a) A step of determining the length of the lipid of at least one ganglioside in the first sample obtained from the subject using the mass spectrometry described in claim 1, b) A step of repeating step a) at at least one subsequent point in time after the administration of the cancer treatment, A significant change in the heterogeneity of the lipid length of at least one ganglioside in the second sample compared to the first sample indicates that the treatment method is effective in treating cancer in the subject. If necessary, the first and / or at least one subsequent sample is a single sample or a portion of a pooled sample obtained from the subject, step and A method that includes this.

18. The method according to any one of claims 2 to 17, wherein (i) the subject is treated with a cancer treatment, or (ii) a cancer treatment is recommended, prescribed and / or administered to the subject.

19. (a) The cancer treatment method is surgery, chemotherapy, cancer vaccine, chimeric antigen receptor, radiotherapy, immunotherapy, an expression modulator of an immune checkpoint inhibitor protein or ligand, or any combination thereof. (b) The cancer treatment is an immunotherapy which is an immune checkpoint inhibitor therapy, and / or (c) The cancer treatment is avelumab, durvalumab, atezolizumab, BRAF / MEK inhibitors, pembrolizumab, nivolumab, ipilimumab, or a combination thereof. The method according to any one of claims 5, 6, 8, 9, 13, 14, 16, and 17.

20. (a) The at least one ganglioside and / or at least one lipoform of a ganglioside is a tumor-associated ganglioside and / or lipoform of a tumor-associated ganglioside (b) The ganglioside comprises GD2, GD3, GD1b, GT1b, fucosyl-GM1, GloboH, polysialic acid (PSA), GM2, GM3, sialyl-Lewis X, sialyl-Lewis Y, sialyl-Lewis A, sialyl-Lewis B, Lewis Y, any part thereof, any lipoform thereof, or any combination thereof. (c) The at least one ganglioside and / or at least one lipoform of ganglioside comprises GM2, GD3, GD2, GD1b, any of the lipoforms thereof, or any combination thereof. (d) The at least one ganglioside and / or at least one lipoform of a ganglioside comprises two or more combinations selected from GM2, GD3, GD2, GD1b and any of the lipoforms, (e) The at least one ganglioside and / or the at least one lipoform of the ganglioside comprises GD3(d18:1 / 16:0), GD3(d18:1 / 23:0), GD3(d18:1 / 24:1), GD2(d18:1 / 16:0), GD1b(d18:1 / 16:0), GD1b(d18:1 / 24:1), GD1b(d18:1 / 18:1), GD1b(d18:1 / 13:1), GD3(d18:1 / 18:0), GD3(d18:1 / 20:1), GD3(d18:1 / 18:1), GD1b(d18:1 / 17:1), GD1b(d18:1 / 24:0), or any combination of two or more of these. (f) At least one lipoform of ganglioside comprises GD3(d18:1 / 23:0) and / or GD3(d18:1 / 24:1), (g) The at least one ganglioside and / or the at least one lipoform of the ganglioside comprises GD3 (d18:1 / 23:0), (h) The at least one ganglioside and / or the at least one lipoform of the ganglioside comprises an acyl chain 24:1, and optionally the acyl chain 24:1 is present in GD3 and / or GD1b. (i) The cancer or tumor is selected from the group consisting of neuroblastoma, lymphoma, leukemia, melanoma, glioma, small cell lung cancer, breast cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, Ewing's sarcoma, fibroplastic round cell tumor, rhabdomyosarcoma, retinoblastoma, non-small cell lung cancer, renal cell carcinoma, Wilms' tumor, prostate cancer, gastric cancer, endometrial cancer, pancreatic cancer, serous ovarian cancer, cervical cancer, gynecological cancer, and colon cancer. (j) The cancer or tumor is ovarian cancer, melanoma, kidney cancer or lung cancer, and if necessary, the cancer or tumor is ovarian cancer. (k) The cancer is a borderline tumor. (l) The sample includes cells, serum, blood, peritumoral tissue and / or intratumoral tissue obtained from the subject, (m) The sample contains serum or blood, (n) The significantly higher level of at least one ganglioside includes an increase of at least 20 percent in the level of at least one ganglioside, (o) The significantly lower level of at least one ganglioside includes a reduction of at least 20 percent in the level of at least one ganglioside, (p) The control is the level of at least one ganglioside and / or at least one lipoform of ganglioside in a sample derived from a cancer-free subject. (q) The control is the level of at least one ganglioside and / or at least one lipoform of ganglioside in a sample derived from the subject (for example, a first sample collected from the subject in a long-term collection to evaluate the change over time in the level of at least one ganglioside), (r) The comparison is a reference standard, (s) The subject is a mammal (for example, a subject suffering from cancer, for example, an asymptomatic subject), (t) The subject is an animal model of cancer, a dog, a cat, or a human. (u) The subject is a human being. (v) The level of the at least one ganglioside and / or the lipoform of the ganglioside, or the length of the lipid of the at least one ganglioside, is detected according to the method of claim 1. (w) The mass spectrometry is selected from LC-ESI-MS / MS, LC-ESI-CID-MS / MS, nanobore LC-ESI-MS, and nanobore LC-ESI-MS / MS. (x) The method is an in vitro method or an ex vivo method. (y) The change or significant change in the heterogeneity of the lipid length of the at least one ganglioside includes a change in gangliosides having a shorter lipid length (14 to 24 carbons) (e.g., a higher or lower amount or level), which, if necessary, is at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, compared to the control. 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more, higher or lower, (z) The change or significant change in the heterogeneity of the lipid length of the at least one ganglioside includes a change in gangliosides having a longer lipid length (26 to 38 carbon atoms) (e.g., a higher or lower amount or level), which, if necessary, is at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% compared to the control. 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more, higher or lower, (aa) The change or significant change in the heterogeneity of the lipid length of the at least one ganglioside includes a change in gangliosides having short lipid lengths (14 to 24 carbons) and long lipid lengths (26 to 38 carbons) (e.g., a higher or lower amount or level), and, if necessary, at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 6% compared to the control. 5%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000% or more, higher or lower, (ab) The significantly high or low levels of the at least one ganglioside and / or at least one lipoform of ganglioside are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160% compared to the control. %, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 4 70%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770% , including changes of 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% or less (e.g., higher or lower amounts or levels), (ac) The method further includes obtaining or providing a sample derived from the subject, (ad) The method further comprises determining the level of the at least one ganglioside and / or at least one lipoform of ganglioside in a control sample. (ae) The method further comprises determining the length of the lipid of at least one ganglioside in a control sample, (af) The at least one ganglioside and / or at least one lipoform of a ganglioside comprises d18:1, (ag) The at least one ganglioside and / or at least one lipoform of a ganglioside comprises a C16-C24 acyl chain, (ah) The at least one ganglioside and / or at least one lipoform of a ganglioside is selected from any one or more gangliosides listed in Tables 2 to 5. (ai) The heterogeneity of the lipid length of the at least one ganglioside includes the at least one ganglioside listed in Table 2. (aj) The cancer is melanoma, and the heterogeneity of the length of the lipids of at least one ganglioside includes a higher level of short lipids compared to the long lipids for GM2, GD3, GD2 and / or GD1, and optionally the short lipids contain 14 to 24 carbon atoms and / or the long lipids contain 26 to 38 carbon atoms. (ak) The cancer is renal cancer, and the heterogeneity of the length of the lipids of at least one ganglioside includes (i) a higher level of short lipids compared to the long lipids for GD3 and / or GD2, and / or (ii) a higher level of long lipids compared to the short lipids for GM2, wherein the short lipids optionally contain 14 to 24 carbon atoms and / or the long lipids contain 26 to 38 carbon atoms, and / or (al) The cancer is lung cancer, and the heterogeneity of the length of the lipids of the at least one ganglioside includes (i) a higher level of short lipids compared to the long lipids for GD2, and / or (ii) a higher level of long lipids compared to the short lipids for GM2 and / or GD3, wherein the short lipids contain 14 to 24 carbon atoms and / or the long lipids contain 26 to 38 carbon atoms, The method according to any one of claims 1 to 17.

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