Mass spectrometry method for cancer diagnosis

Abstract

The present disclosure relates to mass spectrometry methods for cancer diagnosis and prognosis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 326,974, filed on April 4, 2022, the entire contents of which are incorporated herein by reference in their entirety. Background Art

[0003] Cancer involves abnormal cell growth, which may invade or spread to other parts of the body. Despite decades of cancer research, cancer still causes a significant number of deaths (about 1,600 deaths per day in the United States in 2020), mainly due to the lack of means for early and / or accurate detection. For example, ovarian cancer is the most deadly gynecological cancer and the third leading cause of death in women. Serous ovarian cancer is the most aggressive subtype of ovarian cancer, and because most patients are diagnosed in the late stage, the serous ovarian cancer is often referred to as the "silent killer". Symptoms are vague and easily confused with other symptoms. Currently, CA-125 is a blood marker for diagnosing and monitoring treatment efficacy. Although CA-125 levels are elevated in 80% of epithelial tumors, most of these tumors are in the late stage. Serum CA-125 has a low detection rate in early diagnosis because its level is elevated in <50% of stage I ovarian cancer and has limited specificity. Therefore, new biomarkers, such as those in cancer tissue and blood, are greatly needed to detect early stages of cancer, which will improve the survival of cancer patients. Summary of the invention

[0004] Gangliosides are glycolipids that include (i) a carbohydrate structure, which is specifically defined by the 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 lipid forms thereof) are useful biomarkers for cancer diagnosis and prognosis, particularly for early detection of cancer.

[0005] Provided herein are methods for cancer diagnosis and prognosis.For example, provided herein are methods based on mass spectrometry for detecting and measuring the amount of gangliosides, the lipid form of gangliosides, and the lipid length of gangliosides.In addition, provided herein are methods for diagnosing cancer using liquid biopsy (e.g., blood, serum) or solid tissue biopsy (e.g., cancerous tissue).The surprising and unexpected prevalence of some gangliosides, the lipid form of gangliosides, and / or the lipid length of gangliosides provides a new generation of biomarkers for cancer diagnosis and prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1A-1BA schematic diagram of gangliosides is shown. Figure 1A Schematic diagrams of GD2 and GD3 gangliosides are shown. Each geometric shape is a type of sugar. The exposed glycan tree is linked to a ceramide, which is linked to two lipid tails. The sugar head of normal GM1 and tumor GD3 differ by 2 sugars, and GD2 and GD3 differ from each other by 1 sugar. Because GD2 is a biosynthetic product of GD3, GD2 and GD3 can often (but not always) be detected on the same cell. Figure 1B Schematic representation showing how certain lipid forms can assemble differently on cell membranes depending on lipid homogeneity or heterogeneity, resulting in different biological signals or biological events. Figure 1B Also shown is a graphic representation of how the sphingosine chain can vary in carbon length and degree of saturation at carbon positions 4-5. Acyl chains can vary more dramatically in carbon length, (mono)saturation at 2 carbons, and hydroxylation at the 2-carbon position.

[0007] Figure 2 LC-MS / MS detection of GD3 in ovarian cancer is shown. All GD3 species were shown to be combined regardless of lipid form, ANOVA, post hoc Holm Sidak, *p<0.05, **p<0.01. GD3 analytes in early and late ovarian cancer (n=13; 4 early and 9 late) versus non-cancer (n=2). Ovarian cancer data are absolute quantification (pmol / ml). Glycolipids were measured in serum collected at diagnosis. Cancer-free samples had very low total GD3, and cancer samples had significantly higher total GD3.

[0008] Figures 3A-3S Shown is LC-MS / MS detection of lipid forms of gangliosides in ovarian cancer. Figure 3A-3H The lipid form of GD3 is shown. Fig. 3I The lipid form of GD2 is shown. Figure 3J-Figure 3P The lipid form of GD1b is shown. Figure 3Q Total GD3 detected is shown. Figure 3R and Figure 3S The total GD2 and GD1b detected are shown separately. Certain lipid forms showed significant increases in cancer samples compared to non-cancer samples. For example, Figure 3F It was shown that GD3 (d18:1 / 23:0) is significantly increased in ovarian cancer samples, thereby providing an excellent marker for cancer diagnosis / prognosis. DETAILED DESCRIPTION

[0009] Gangliosides are a family of glycosphingolipids with >40 different sialic acids. Each polysaccharide tree is unique in structure, and each ganglioside is defined by name. Some gangliosides (such as GM1) are normal and ubiquitous. Other gangliosides (such as GD2 and GD3) are tumor markers (tumor-marker gangliosides; also referred to as TMG). They are low / non-existent in normal cells and expressed at high levels in cancer. Therefore, GD2 and GD3 are etiological biomarkers (i.e., biomarkers with an indispensable function for cancer), which are preferred because cancer is not easy to downregulate the expression of markers.

[0010] GD2 and GD3 regulate membrane fluidity, raft size and function, and provide 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 of variable length.

[0011] GD2 or GD3 provide a stable, invariant and non-mutating target, expression is conserved among mammalian species (the glycan tree is identical), expression is homogenous and uniform in cell lines and primary tumors, and expression density is not downregulated in surviving tumor cells after chemotherapy. In addition to being present on the surface of tumor cells, GD2 and GD3 can be shed into the extracellular environment.

[0012] However, studies on the expression of GD2 and GD3 in tissues or circulation have only been reported in a few patient samples. Assays for detecting GD2 or GD3 that may be expressed in tissues or serum are not quantitative or standardized; only yield estimates are provided; and conflicting conclusions are drawn. Because GD2 and GD3 are glycolipids and the products can be produced by a variety of biosynthetic pathways and enzymes, monitoring mutations or mRNA expression is not feasible. Therefore, GD2 and GD3 remain underutilized in cancer diagnostics.

[0013] The present disclosure is based at least in part on the discovery that mass spectrometry-based methods provide a powerful means to accurately determine gangliosides associated with cancer (e.g., tumors). Further provided herein are diagnostic methods using novel biomarkers of gangliosides and their lipid forms. These novel biomarkers show surprising and unexpected prevalence in certain cancer types (e.g., ovarian cancer). Detection of such biomarkers provides a sensitive and accurate diagnostic method for cancer, especially in the early stages of cancer.

[0014] definition

[0015] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0016] The term "borderline tumor" is recognized in the art and refers to an atypical hyperplastic tumor with relatively low malignant potential. For example, a borderline ovarian tumor is an atypical hyperplastic tumor with relatively low malignant potential compared to, for example, ovarian cancer (see below).

[0017] 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 length of the lipid can be variable. It is worth noting that these differences occur in comparisons between gangliosides and within a single ganglioside. Therefore, lipid heterogeneity and its changes are important for the diagnosis and methods described herein.

[0018] The term "lipid form" of a ganglioside refers to variants of a ganglioside that differ in their lipids (see, e.g., Kolter (2012) ISRN Biochem 506160).

[0019] The term "minimal residual disease" is recognized in the art and is used to describe the small number of cancer cells in the body during or after cancer treatment when a patient is in remission. The number of remaining cells may be so small that they do not cause any physical signs or symptoms, and often cannot even be detected by traditional methods. It is the main reason for cancer recurrence.

[0020] The term "neoadjuvant therapy" refers to treatment given before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy.

[0021] The term "prevention" is art-recognized and well known in the art when used in connection with a condition (e.g., a viral / bacterial infection) or a disease (e.g., cancer), and includes administering a treatment, e.g., a composition that reduces the frequency of symptoms of a medical condition or delays the onset of symptoms of a medical condition in a subject relative to a subject not receiving treatment. Thus, prevention of cancer includes, e.g., reducing the number of detectable cancerous growths in a patient population receiving prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0022] The term "remission" is art-recognized and refers to a condition in which the signs and symptoms of cancer are reduced.

[0023] 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 all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0024] A "therapeutically effective amount" of a compound is an amount capable of producing a medically desirable result in a treated patient, such as inducing an immune response against gangliosides, reducing tumor burden, reducing the growth of tumor cells, or alleviating any symptom associated with cancer, with an acceptable benefit:risk ratio, preferably in a human or non-human mammal.

[0025] The term "treatment" includes preventive and / or therapeutic treatment. The term "preventive or therapeutic" treatment is recognized in the art and includes administering one or more of the subject compositions to the host. If the treatment is administered before the clinical manifestation of an undesirable condition (e.g., a disease or other undesirable state of the host animal), the treatment is preventive (i.e., it protects the host from developing an undesirable condition); however, if the treatment is administered after the manifestation of an undesirable condition, the treatment is therapeutic (i.e., it is intended to reduce, improve or stabilize an existing undesirable condition or its side effects).

[0026] Gangliosides

[0027] Ganglioside is a glycosphingolipid comprising one or more sialic acids. Glycosphingolipids include hydrophobic ceramide or sphingosine lipid tails, which are usually anchored to the outer leaflet of the plasma membrane. The glycosphingolipids also include oligosaccharide moieties and are classified according to the carbohydrate structure (ganglioside, isoganglioside, lactose, etc.). Ganglioside is an important subclass of glycosphingolipids because the gangliosides include negatively charged sialic acids (N-acetylneuraminic acid or N-glycolylneuraminic acid) connected to the lipolytic oligosaccharide moiety. Gangliosides are named and classified according to the number of sialic acid residues connected to the internal sugar moiety (M represents 1; D represents 2; T represents 3; and Q represents 4) and according to their chromatographic mobility. The numbering (5-x) of gangliosides is based on the number (x) of internal sugar moieties (glucose, galactose or GalNAc) classified according to the original experiment of Svennerholm. Thus, if x is 4, the gangliosides are: GM1, GD1, GT1, for GM2, GD2, GT2, x=3, and for GM3, GD3 and GT3, x=2.

[0028] Heterogeneity is found not only within the glycan portion, but also within the ceramide portion. This can consist of different sphingosine bases of different chain lengths, dihydrosphingosine, sphingosine and phytosphingosine, which can be further modified by O-acetylation. In higher animals, C18-sphingosine and C20-sphingosine are the most abundant sphingosine bases in gangliosides.

[0029] The fatty acids found in the ceramide part of gangliosides are mostly saturated. α-Hydroxylated fatty acids are not usually found in brain gangliosides, but are abundant, for example, in gangliosides from the intestine, liver or kidney, and in GM4. To designate the lipid form of gangliosides, names such as (d18:1 / 18:0)GM3 are used for II with 18 carbons of sphingosine (d = dihydroxy, 1 = one double bond) and a stearoyl residue (18:0) in the ceramide part. 3 Neu5AcLacCer (see Kolter (2012) ISRN Biochemistry 506160). The functional consequences of heterogeneity in lipid composition are largely unknown, but the lipid moiety may mask the receptor function of ganglioside glycans through interactions with membrane cholesterol.

[0030] As presented herein, gangliosides are tumor biomarkers. In some embodiments, the tumor-associated ganglioside (also referred to as "tumor marker ganglioside" or TMG) ​​is selected from 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 portion thereof and modified versions thereof. In a preferred embodiment, the ganglioside is GD2, GD3, GM2, GD1b or a lipid form thereof.

[0031] Thus, as used herein, the term ganglioside may refer to gangliosides, tumor-associated gangliosides, portions thereof, lipid forms thereof, glycan variants thereof, or any other isomers / variants thereof.

[0032] Analysis / Detection of Gangliosides

[0033] 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 lipid forms thereof) can be detected using methods including but not limited to immunodiffusion, immunoelectrophoresis, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay or slot blot assay. The general techniques and other variations of the techniques for performing the various immunoassays described above, such as in situ proximity ligation assay (PLA), fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), turbidity 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), ultradiffusion chromatography, etc. (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn., pp. 217-262, 1991, incorporated by reference), are used alone or in combination or alternation with NMR, MALDI-TOF, LC-MS / MS, gas chromatography, and similar methods known to those of ordinary skill in the art.

[0034] Such techniques can also be used to monitor ganglioside levels in cells, tissues, or blood / plasma.

[0035] In a preferred embodiment, mass spectrometry (e.g., MALDI-TOF, LC-MS / MS, LC-MS, LC-ESI-MS / MS, nano-LC-ESI-MS / MS (also known as nLC-ESI-MS / MS or nanopore LC-ESI-MS / MS), or other mass spectrometry methods known in the art) is used to detect the presence, level, or lipid length of gangliosides (e.g., GD1b, GD2, GD3, GM2, or lipid forms thereof) in a biological sample such as a liquid biopsy (e.g., blood, saliva, serum, cells / tissues (e.g., cancer cells); see the section on samples). Mass spectrometry-based methods are particularly useful in determining the lipid length of gangliosides or the heterogeneity of certain lipid forms of gangliosides, which are novel biomarkers of the present disclosure.

[0036] Mass spectrometry

[0037] Liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS) is commonly used to sensitively and selectively determine trace levels of compounds in biological samples. Specifically, the most commonly used method is LC-MS / MS equipped with electrospray ionization (ESI) as an ion source, because ESI can ionize a variety of compounds including polar compounds or large molecular weight compounds. By coupling with HPLC, the detected characteristics can be quantified by UV light or MS signal intensity.

[0038] Therefore, any mass spectrometry-based method can be applied to the methods of the present disclosure. Exemplary mass spectrometry-based methods include, but are not limited to, LC-MS-based methods (e.g., LC-MS / MS, LC n -MS n ), including LC-MS-based methods using ESI (e.g., LC-ESI-MS, LC-ESI-MS / MS, LC-ESI-CID-MS / MS) and nanopore LC-MS-based methods (e.g., nanopore LC-ESI-MS, nanopore LC-ESI-MS / MS).

[0039] In LC-ESI-MS / MS, analytes with appropriate hydrophobic structures can be sensitively detected because (i) hydrophobic ions preferably reside at the surface of droplets generated by electrospray, and these ions are more likely to enter the gas phase and show higher signal intensity than ions inside the droplets. (ii) Hydrophobic compounds can be well separated from salts and interfering compounds that have an inhibitory effect on ESI on a reverse phase column. (iii) Hydrophobic compounds are eluted by a mobile phase with a higher organic solvent content. A higher organic solvent content is suitable for stably generating charged droplets by electrospray, thereby generating a higher signal intensity (see, e.g., Santa (2013) Drug Discoveries & Therapeutics, 7: 9-17, which is incorporated by reference in its entirety).

[0040] 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 by reference in its entirety). Gangliosides and lipid forms can be detected using LC-ESI-MS / MS (see, e.g., Ikeda et al. (2008) J Lipid Res 49:2678-89, which is incorporated by reference in its entirety). LC-ESI-CID-MS / MS has been widely used to detect gangliosides or glycan sequencing, which shows improved speed and sensitivity.

[0041] As noted above, a reversed phase column connected to HPLC or UPLC can be used to effectively separate and quantify hydrophobic compounds, such as gangliosides or their lipid forms. Reversed phase columns or reversed phase HPLC columns are chromatography columns comprising non-polar stationary phases. The sample is placed in a reversed phase column, and then a solvent is added to rinse the sample through the stationary phase. Because the stationary phase in the reversed phase HPLC column is non-polar, the polar components of the sample will first be discharged from the column, followed by the non-polar components. Reversed phase HPLC columns can be filled or capillary, made of glass or metal, and can have many different hydrophobic substances as stationary phases.

[0042] Many reverse phase columns are commercially available. For example, various types of reverse phase columns (such as C18 reverse phase LC column, biphenyl reverse phase LC column, C4 reverse phase LC column, C8 reverse phase column, phenyl reverse phase LC column, C1 reverse phase LC column, PFP reverse phase LC column, C30 reverse phase LC column, polar embedded reverse phase LC column, polar end-capped reverse phase LC column, porous graphite carbon reverse phase LC column, phenyl-hexyl reverse phase LC column and alkyl reverse phase LC column) are known in the art and are commercially available (Thermo Fisher Scientific, Waltham, MA; Waters Corporation, Milford, MA).

[0043] 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. Nanopore or Nano-LC n / MS nThe advent of nanopore LC provides a solution to these limitations. Nanoliter / minute (nL / min) flow rates produce much smaller droplets that are more easily desolvated and thus improve MS sensitivity. In addition, compared with conventional LC flow rates, detection limits are lower, less sample is required, and tolerance to chemical interferences is increased. Connecting nanopore LC to MS utilizes emitters / nebulizers with tip inner diameters of approximately 1-30 μm.

[0044] Nanopore liquid chromatography (LC) coupled to 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 nanopore columns are readily commercially available (see, e.g., BioBasic TM 18 nanopore HPLC column (Thermo Scientific TM 72105107563), BioBasic TM 8 5μm nanopore HPLC columns, HPLC columns ACE nanopore and Thermo Hypersil GOLD nanopore HPLC column).

[0045] If the amount of a biomarker (e.g., a ganglioside or one or more lipid forms thereof) is higher or lower, respectively, than the level in a control (e.g., a normal sample), and is greater than the standard error of the assay used to assess the amount, then the "level" or "amount" of a biomarker in a subject is "significantly" higher or lower than the level of the biomarker in the control.

[0046] In some embodiments, a biomarker is present if the amount of the biomarker 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%, 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 711%, 712%, 713%, 714%, 715%, 716%, 717%, 718%, 719%, 720%, 721 %, 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%, then the amount or level of a biomarker in a subject can be considered "significantly" above or below a normal and / or control amount. Such significant modulation values ​​can be applied to any metric described herein, such as changes in the level of gangliosides or the heterogeneity of the lipid length of gangliosides, etc.

[0047] The term "heterogeneity of lipid length" of at least one ganglioside includes the level or distribution pattern of lipid length of at least one ganglioside in a given sample.

[0048] In some embodiments, if the level or distribution pattern of lipid length 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%, 201%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 301%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 401%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 511%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 601%, 610%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 701%, 710%, 711%, 720%, 730%, 740%, 750%, %, 0%, 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 or any range therebetween, such as 5%-100%, then there is a change (e.g., an increase or decrease) or a significant change in the heterogeneity of lipid length.

[0049] Similarly, the term "heterogeneity of lipid length" of gangliosides includes the distribution pattern of gangliosides with short lipid lengths versus long lipid lengths (see Example 6). Gangliosides have two lipid tails: sphingosine and acyl. As used herein, the two lipid tails are not distinguished. Therefore, as used herein, the term "heterogeneity of lipid length" refers to the average length of the two lipid tails of gangliosides. In some embodiments, if the amount or level of gangliosides with short lipid length (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%, 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%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 810%, 810%, 810%, 810%, 810%, 810%, 810%, %, 60%, 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 or any range therebetween, such as 5%-100%, then there is a variation or a significant variation in the heterogeneity of the lipid length of the gangliosides.

[0050] In some embodiments, if the amount or level of gangliosides with long lipid length (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%, 201%, 210%, 211%, 220%, 221, 230%, 231, 240%, 251%, 252%, 253%, 254%, 255%, 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%, 279%, 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 300%, %, 60%, 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 or any range therebetween, such as 5%-100%, then there is a variation or a significant variation in the heterogeneity of the lipid length of the gangliosides.

[0051] In some embodiments, if the amount or level of gangliosides with short lipid length (14-24 carbons) and long lipid length (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% higher or lower than the amount or level in a normal and / or control sample, %, 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 or any range therebetween, such as 5%-100%, then there is a variation or a significant variation in the heterogeneity of the lipid length of the gangliosides.

[0052] Comparison

[0053] A control refers to any suitable reference standard, such as a normal patient, a cultured primary cell / tissue isolated from a subject (such as a normal subject), an adjacent normal cell / tissue obtained from the same organ or body location of a patient, a tissue or cell sample isolated from a normal subject, or a primary cell / tissue obtained from a collection. In other embodiments, the control may include the expression level (e.g., the level of ganglioside or one or more of its lipid forms) and / or lipid tail length of a ganglioside of a subject (such as a normal or healthy subject). In some embodiments, the control may be from a diseased subject, such as a subject suffering from cancer.

[0054] Control also refers to any reference standard suitable for comparison with the expression product in the test sample. In some embodiments, the control includes obtaining a control sample, detecting the level of ganglioside, one or more lipid forms thereof, or the lipid length of ganglioside from the control sample, and comparing it with those from the test sample. Such control samples can include any suitable sample, including but not limited to samples from control cancer patients with known results (which can be stored samples or previous sample measurements); normal tissues or cells separated from objects (such as normal patients or cancer patients); cultured primary cells / tissues separated from objects (such as normal subjects or cancer patients); adjacent normal cells / tissues obtained from the same organ or body position of cancer patients; tissues or cell samples separated from normal subjects or primary cells / tissues obtained from a collection center. In some embodiments, the control can include reference standard expression product (e.g., ganglioside) levels from any suitable source, including but not limited to expression product level ranges from normal tissue (or other previously analyzed control samples), expression product level ranges previously determined within a test sample from a group of patients or a group of patients with a particular outcome (e.g., survival for one year, two years, three years, four years, etc.) or receiving a particular treatment (e.g., standard of care cancer therapy). In some embodiments, the control includes samples drawn or collected longitudinally at different times to assess changes in the level of gangliosides, one or more lipid forms thereof, or the lipid length of gangliosides over time. Those skilled in the art will appreciate that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention.

[0055] In some embodiments, the amount of ganglioside or its lipid form in the sample can be determined relative to the amount of another ganglioside or its lipid form in the same sample or as its ratio. In some embodiments, the control includes a ratio conversion of the expression product level, including but not limited to determining the ratio of the product level of two gangliosides, the ratio of the lipid form of gangliosides to total gangliosides, or the ratio of the short lipid length of the ganglioside in the test sample to the long lipid length, and comparing it with any suitable ratio in the reference standard; determining the product level of these two or more gangliosides or their lipid forms in the test sample, and determining the difference in the product level in any suitable control; and determining the product level of these two or more gangliosides in the test sample, standardizing its level to the level of the housekeeping gene product in the test sample, and comparing it with any suitable control. In a preferred embodiment, the control includes a control sample with the same pedigree and / or type as the test sample. In other embodiments, the control may include a product level grouped as a percentile within a group of patient samples (such as all patients with cancer) or based on the percentile of the group of patient samples. In some embodiments, a control product level is established, wherein the higher or lower product level relative to, for example, a specific percentile is used as the basis for predicting the outcome. In other preferred embodiments, the product level from a cancer control patient with a known outcome is used to establish a control product level, and the product level from a test sample is compared with the control product level as the basis for predicting the outcome. As demonstrated by the data provided herein, the method of the present invention is not limited to using a specific cut-off point (cut-point) to compare the product level in a test sample and a control.

[0056] 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 people who are evaluating the patient's selection. In some embodiments, the predetermined marker amount can be obtained from the previous assessment of the same patient. In this way, the progress of the patient's selection can be monitored over time. In addition, if the object is a person, the control can be obtained from the assessment of another person or multiple people (for example, selected groups of people). In this way, the degree of selection of the person whose selection is evaluated can be compared with suitable other people (for example, other people in similar situations with the person concerned, such as suffering from similar or identical symptoms and / or belonging to the same ethnic group).

[0057] Thus, in preferred embodiments, the control comprises a sample (e.g., serum or tissue) from a normal healthy person who does not have cancer. In other preferred embodiments, the control comprises a sample (e.g., serum or tissue) from a patient who is being evaluated (e.g., diagnosis or prognosis). For example, the control sample may include (i) a historical sample of the patient, or (ii) a sample obtained from the patient in a longitudinal study (e.g., before or after therapy (e.g., cancer therapy)). Using such a control, the biomarkers present in the same patient over time (e.g., during cancer progression) can be compared.

[0058] Diagnostic assays

[0059] The present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample includes a ganglioside (or one or more lipid forms thereof) and / or whether the level of a ganglioside (or one or more lipid forms thereof) is modulated (e.g., upregulated or downregulated), thereby indicating the state of a condition of interest (e.g., cancer). In some embodiments, the present invention can be used to classify a sample (e.g., a sample from a subject) as being associated with or at risk for a cancer or a subtype thereof mediated by a ganglioside using statistical algorithms and / or empirical data (e.g., the presence, absence, level, or lipid length of a ganglioside or a lipid form thereof).

[0060] Exemplary methods for detecting the levels of gangliosides and therefore for classifying whether a sample is associated with cancer or a clinical subtype thereof or different stages of cancer involve obtaining a biological sample from a test subject and using a mass spectrometry-based method to detect gangliosides, one or more lipid forms thereof, or the length of the lipid tail of gangliosides in the sample.

[0061] In some cases, 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 a prediction or probability value and the presence or level of gangliosides. The use of a single learning statistical classifier system is usually used to classify a sample as a cancer sample, and its sensitivity, specificity, positive predictive value, negative predictive value and / or overall accuracy are at least or are 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%.

[0062] Other suitable statistical algorithms are well known to those skilled in the art. For example, learning statistical classifier systems include machine learning algorithm techniques that can adapt to complex data sets (e.g., a set 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., a 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 series. Examples of learning statistical classifier systems include, but are not limited to, learning statistical classifier systems using induced learning (e.g., decision / classification trees, such as random forests, classification and regression trees (C&RT), boosted trees, etc.), probabilistic approximately correct (PAC) learning, connection mechanism learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro-fuzzy networks (NFN), network structures, perceptrons (such as multilayer perceptrons), multilayer feedforward 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 temporal difference learning, passive learning in unknown environments, active learning in unknown environments, learning action-value functions, applications of reinforcement learning, etc.), and 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 algorithm, Gauss-Newton algorithm, Gaussian mixtures, gradient descent algorithms, and learning vector quantification (LVQ). In certain embodiments, the methods of the present invention further comprise sending the sample classification results to a clinician (a non-expert, eg, a primary care physician; and / or an expert, eg, a histopathologist or an oncologist).

[0063] In some embodiments, the method of the present disclosure further includes providing a diagnosis in the form of a probability that an individual suffers from cancer. For example, the probability that an individual suffers from cancer can be about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater. In another embodiment, the method of the present invention further provides the prognosis of cancer in an individual. In some cases, the method for classifying a sample as a cancer sample can be further based on the symptoms (e.g., clinical factors) of the individual from which the sample is obtained. Symptoms or symptom groups can be, for example, lymphocyte count, white blood cell count, erythrocyte sedimentation rate, diarrhea, abdominal pain, abdominal distension, pelvic pain, lower back pain, cramps, fever, anemia, weight loss, anxiety, depression and combinations thereof. In some cases, the method for classifying a sample as a cancer sample can be further based on gene mutations and / or cancer susceptibility, and has nothing to do with symptoms. In some embodiments, after the individual is diagnosed with cancer, a therapeutically effective amount of a cancer therapy (eg, a chemotherapeutic agent) is administered to the individual. In some embodiments, after the individual is diagnosed with cancer, the individual is treated with a cancer therapy.

[0064] In some embodiments, the method further involves obtaining a control biological sample (e.g., a biological sample from a subject not having cancer), a biological sample from a subject in remission or prior to developing cancer, or a biological sample from a subject having cancer while undergoing treatment.

[0065] In some embodiments, the method comprises analyzing a control sample to detect gangliosides, one or more lipid forms thereof, or lipid lengths of gangliosides, such that the presence and / or level of the gangliosides, one or more lipid forms thereof, or lipid lengths of gangliosides in the biological sample is detected, and the presence or level of the gangliosides in the control sample is compared to the presence or level of the gangliosides in the test sample.

[0066] Preferred biological samples are serum, blood, saliva, tumor microenvironment, peritumoral cells / tissues or intratumoral cells / tissues separated from a subject by conventional methods. One of ordinary skill in the art will appreciate that cell samples or tissue samples may require further processing (e.g., homogenization and / or partial purification of lipid fractions; see Example 1).

[0067] As determined by the methods of the present disclosure, the levels and / or heterogeneity of gangliosides, one or more lipid forms thereof, or lipid lengths of gangliosides are associated with different grades of cancer. Therefore, in some embodiments, the methods of the present disclosure may be used to determine the grade of cancer based on the levels and / or heterogeneity of gangliosides, one or more lipid forms thereof, or lipid lengths of gangliosides as determined as described herein. The grade of cancer describes the degree of abnormality of cancer cells and tissues under a microscope compared to healthy cells. Cancer cells whose appearance and organization are most like healthy cells and tissues are low-grade tumors. Doctors believe that these cancers are well differentiated. Lower-grade cancers are generally less aggressive and have a better prognosis. The more abnormal the appearance and organization of the cells themselves, the higher the grade of cancer. Cancer cells with high grades tend to be more aggressive. They are referred to as poorly differentiated or undifferentiated. Some cancers have their own systems for grading tumors. Many other cancers use a standard 1-4 grading scale.

[0068] Grade 1: Tumor cells and tissue look most like healthy cells and tissue. These are called well-differentiated tumors and are considered low-grade.

[0069] Grade 2: The cells and tissue are somewhat abnormal and are called moderately differentiated. These are intermediate-grade tumors.

[0070] Grade 3: Cancer cells and tissue look very abnormal. These cancers are considered poorly differentiated because the cancer no longer has an architectural structure or pattern.

[0071] Grade 3 tumors are considered high-grade.

[0072] Grade 4: These undifferentiated cancers have cells that look the most abnormal. These are the highest grade and usually grow and spread faster than lower grade tumors.

[0073] As used herein, low-grade cancer refers to grade 1 cancer; and high-grade cancer refers to grade 2-4 cancer.

[0074] Similarly, the levels and / or heterogeneity of gangliosides, one or more lipid forms thereof, or lipid lengths of gangliosides, as determined by the methods of the present disclosure, are associated with different stages of cancer. Thus, in some embodiments, the compositions and methods of the present disclosure can be used to determine the grade of cancer based on the levels and / or heterogeneity of gangliosides, one or more lipid forms thereof, or lipid lengths of gangliosides determined as described herein.

[0075] The stage of cancer explains how large the primary tumor is and how far the cancer has spread in the patient's body. There are several different grading systems. Many of these grading systems are developed for specific kinds of cancer. Other grading systems can be used to describe several types of cancer. One common system that many people know of places cancer on a scale of 0 to IV.

[0076] Stage 0 refers to abnormal cells that have not spread and are not considered cancer, although they could become cancerous in the future. This stage is also called "in situ."

[0077] Stages I to III are cancers that have not spread beyond the site of the primary tumor or have spread only to nearby tissues. The higher the stage number, the larger the tumor and the more it has spread.

[0078] Stage IV cancer has spread to distant areas of the body.

[0079] As used herein, early / low stage cancer refers to stage I cancer; and late / high / advanced stage cancer includes stage II to stage IV cancer.

[0080] Similarly, as determined by the method disclosed herein, the level and / or heterogeneity of the lipid length of ganglioside, one or more lipid forms thereof or ganglioside is related to tumor load. Therefore, in some embodiments, the composition and method disclosed herein can be used to determine the tumor load of the object based on the level and / or heterogeneity of the lipid length of at least one ganglioside determined as described herein. Tumor load (or tumor burden) is defined as the total amount of tumor (cell / tumor mass) distributed in the patient's body (including bone marrow). In the solid tumor response evaluation criteria (RECIST) analysis, tumor load is considered to be the sum of the longest diameter of all measurable lesions. Various methods can be used to determine the tumor load of the object. For example, computed tomography (CT) and magnetic resonance (MR) imaging have been used to assess tumor response based on morphological (size, position) criteria, specifically by using RECIST. RECIST classification describes the size of the lesion and distinguishes 4 types of treatment responses-stable disease (SD), partial response (PR), complete response (CR) or progressive disease (PD).

[0081] Prognostic determination

[0082] The term "prognosis" includes a prediction of the likely course and outcome of a cancer or the likelihood of recovery from a disease. In some embodiments, the use of a statistical algorithm provides a prognosis for a cancer in an individual. For example, the prognosis can be the development of a surgical procedure, a clinical subtype of a cancer (e.g., a solid tumor such as lung cancer, melanoma, and renal cell carcinoma), the development of one or more clinical factors, the development of bowel cancer, or recovery from a disease.

[0083] The assays described herein (such as the aforementioned diagnostic assays or the following assays) can be used to determine whether an agent (e.g., an agonist, an antagonist, a peptidomimetic, a polypeptide, a peptide, a nucleic acid, a small molecule, an immunotherapy, an immune checkpoint inhibition therapy, or other drug candidates) can be administered to a subject to treat cancer. For example, such methods can be used to determine whether a subject can be effectively treated with one or a combination of agents. Therefore, the present disclosure provides a method for determining whether a subject can be effectively treated with one or more agents for treating cancer, wherein a test sample is obtained and gangliosides are detected.

[0084] Other aspects of the disclosure include the use of the methods described herein for association and / or stratification analysis, wherein gangliosides, one or more lipid forms thereof, or lipid lengths of gangliosides in biological samples from individuals with cancer are analyzed, and the information is compared with that of controls (e.g., individuals without cancer; controls may also be referred to as "healthy" or "normal" individuals or at earlier time points in a given time-lapse study), preferably controls of similar age and race. Appropriate selection of patients and controls is important for the success of association and / or stratification studies. Therefore, a library of individuals with well-characterized phenotypes is highly desirable. Standards for cancer diagnosis, cancer susceptibility screening, predicting clinical outcomes, cancer prognosis, determining drug responsiveness (pharmacogenomics), drug toxicity screening, etc. are described herein.

[0085] Different research designs can be used for genetic association and / or stratification studies (Modern Epidemiology, Lippincott Williams & Wilkins (1998), 609-622). Observational studies are most frequently conducted, where the patient's response is undisturbed. The first type of observational study determines a group of people's samples with suspected causes and another group of people's samples without suspected causes, and then compares the development frequency of the disease in the two samples. These sampled populations are called groups, 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 (cases) with a target phenotype (such as some manifestations of the disease) and from individuals (controls) without a phenotype, and these individuals are from the population (target population) to draw conclusions. Then, the possible causes of the disease are studied retrospectively. Since the time and cost of collecting samples in case-control studies are significantly less than the time and cost for prospective studies, at least during the exploration and discovery phase, case-control studies are more commonly used research designs in genetic association studies.

[0086] After obtaining all relevant phenotypic and / or genotypic information, statistical analysis is performed to determine whether there is any significant correlation between the existence of allele or genotype and the phenotypic characteristics of individuality. Preferably, before the statistical test for genetic association is performed, data inspection and cleaning are first performed. The epidemiology and clinical data of the sample can be summarized by descriptive statistics using tables and charts known in the art. Preferably, data verification is performed to check data integrity, inconsistent entries and outliers. Chi-squared test (Chi-squared test) and t test (if distribution is abnormal, Wilcoxon rank-sum test (Wilcoxon rank-sum test)) can then be used to check the significant difference between the case and control of discrete variables and continuous variables respectively.

[0087] When performing a genetic association test, an important decision is to determine the significance level, and significant association can be announced when the p value of the test reaches the level. In exploratory analysis, positive hits will be tracked in subsequent confirmatory tests, for example, unadjusted p value <0.2 (significance level on the loose side) can be used to generate the hypothesis that ganglioside levels are significantly associated with certain phenotypic characteristics of cancer. It is preferred to achieve p value <0.05 (the significance level traditionally used in the art) so that the level is considered to be related to cancer. When tracking hits in more samples from the same source or in confirmatory analysis in different samples from different sources, adjustments for multiple tests will be performed to avoid excessive hits, while maintaining the experimental error rate at 0.05. Although there are different methods for adjusting multiple tests to control different types of error rates, a commonly used but conservative method is Bonferroni correction (Bonferroni correction) to control experimental or familial error rates ("Multiple comparisons and multiple tests", Westfall et al., SAS Institute (1999)). Permutation tests for controlling the false discovery rate FDR may be more effective (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 of controlling multiplicity will be preferred when the tests are dependent and it is sufficient to control the false discovery rate without having to control the experimental error rate.

[0088] Once it is found that the risk factors (genetic factors or non-genetic factors) of individual illness, it is possible to establish a classification / prediction scheme, according to its phenotype and / or genotype and other non-genetic risk factors, predict the category (for example, sick or disease-free) that the individual will be in. The logistic regression of discrete traits and the linear regression of continuous traits are standard techniques for such tasks (" Applied Regression Analysis ", Draper and Smith, Wiley Publishing Company (1998)). In addition, other techniques can also be used to establish classification. Such techniques include but are not limited to MART, CART, neural networks and discriminant analysis, which are suitable for comparing the performance of different methods (" The Elements of Statistical Learning ", Hastie, Tibshirani & Friedman, Springer (Springer) (2002)).

[0089] Exemplary Embodiments

[0090] In certain aspects, a method of detecting the presence, level and / or lipid length of at least one ganglioside and / or lipid form of at least one ganglioside, the method comprising detecting the ganglioside and / or lipid form of at least one ganglioside in a sample using mass spectrometry, optionally wherein the sample is from a subject having cancer, a subject suspected of having cancer, or a subject without cancer.

[0091] The mass spectrometry method can be any method of the present disclosure or any other mass spectrometry method known in the art.

[0092] In some embodiments, the mass spectrometry is selected from LC-MS, LC-MS / MS, LC-ESI-MS / MS, LC-ESI-CID-MS / MS, nanopore LC-ESI-MS, and nanopore LC-ESI-MS / MS.

[0093] In certain aspects, diagnostic and prognostic methods are provided herein. For example, in certain aspects, provided herein is a method of diagnosing cancer in a subject, the method comprising: a) determining the level of at least one ganglioside and / or at least one lipid form of a ganglioside in a sample from the subject; and b) comparing the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside with the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control sample, wherein the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the sample from the subject is greater than the level in the control sample. %, 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%, 100%, 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%) indicates that the subject has cancer.

[0094] In certain aspects, provided herein is a method of identifying a subject having cancer, the method comprising: a) determining the level of at least one ganglioside and / or at least one lipid form of a ganglioside in a sample from the subject; and b) comparing the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside to the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control sample, wherein the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the sample from the subject is increased compared to the level in the control sample. %, 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%, %, 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% 940%, 950%, 960%, 970%, 980%, 990%, 1000%) identify the subject as having cancer.

[0095] In certain aspects, provided herein is a method of determining the stage of cancer, the method comprising: a) determining the level of at least one ganglioside and / or at least one lipid form of ganglioside in a sample from a subject; and b) comparing the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside to the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control sample, wherein the level in the sample from the subject is greater than the level in the control sample. The level of the at least one ganglioside is at least, about, or no more than 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%, 541%, 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%, 30%, 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 90%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% indicates that the subject has early stage cancer;and / or wherein the level of the at least one ganglioside in the subject sample is at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 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%, An increase of 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% indicates that the subject has advanced cancer.;

[0096] In some embodiments, an increase of at least 100% and no more than 200% in the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample as compared to the level in the control sample indicates that the subject has early stage cancer. In some embodiments, an increase of at least 200% in the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample as compared to the level in the control sample indicates that the subject has advanced stage cancer.

[0097] In certain aspects, provided herein is a method of determining the grade of cancer, the method comprising: a) determining the level of at least one ganglioside and / or at least one lipid form of ganglioside in a sample from a subject; and b) comparing the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside to the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control sample, wherein the at least one ganglioside in the sample from the subject is increased compared to the level in the control sample. %, 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%, 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% indicates that the subject has grade I cancer;wherein the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample is at least, about or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, %, 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%, 75 an increase of 0%, 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% indicates that the subject has grade II cancer;wherein the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample is at least, about or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, %, 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 an increase of 70%, 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% indicates that the subject has grade III cancer;and / or wherein the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample is at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 70%, 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%, 60%, 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%, 7 An increase of 50%, 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% indicates that the subject has grade IV cancer.;

[0098] In some embodiments, the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample is at least, about, or no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160% as 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%, 0%, 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%, An increase of 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% indicates that the subject has a low grade cancer (e.g., grade I).

[0099] In some embodiments, the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample is at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 80 0%, 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% 0%, 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 0%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% indicates that the subject has a high-grade cancer (e.g., grade II, III or IV).

[0100] In some embodiments, an increase of at least 100% and no more than 200% in the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample as 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 the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample as compared to the level in the control sample indicates that the subject has a high-grade cancer (e.g., grade II, grade III, or grade IV).

[0101] In certain aspects, provided herein is a method of determining tumor burden of a cancer, the method comprising: a) determining the level of at least one ganglioside and / or at least one lipid form of a ganglioside in a sample from a subject; and b) comparing the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside to the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control sample, wherein the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the sample from the subject is increased compared to the level in the control sample. The level of at least one ganglioside and / or the lipid form of the at least one ganglioside is at least, about or no more than 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%, 401%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 601%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 701%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 801%, 810%, 820%, 830%, 840%, 10%, 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% increase indicates that the subject has a low tumor burden;and / or wherein the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample is at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 70%, 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%, 60%, 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%, 7 An increase of 50%, 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% indicates that the subject has a high tumor burden.;

[0102] In some embodiments, an increase of at least 100% and no more than 200% in the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample as compared to the level in the control sample indicates that the subject has a low tumor burden. In some embodiments, an increase of at least 200% in the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample as compared to the level in the control sample indicates that the subject has a high tumor burden.

[0103] In certain aspects, provided herein is a method for detecting recurrence of cancer in a subject, the method comprising: a) obtaining a sample from the subject or providing a sample from the subject, wherein the subject's cancer has regressed after receiving cancer treatment; b) determining the level of at least one ganglioside and / or at least one lipid form of ganglioside in the sample from the subject; and c) comparing the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside with the level of at least one ganglioside and / or the lipid form of at least one ganglioside in a control sample, wherein a significantly higher level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the sample from the subject compared to the level in the control sample indicates recurrence of cancer in the subject.

[0104] In certain aspects, provided herein is a method for detecting minimal residual disease in a subject, the method comprising: a) obtaining a sample from a subject in remission or providing a sample from the subject; b) determining the level of at least one ganglioside and / or at least one lipid form of ganglioside in the sample from the subject; and c) comparing the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside with the level of at least one ganglioside and / or the lipid form of the at least one ganglioside in a control sample, wherein a significantly higher level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the sample from the subject compared to the level in the control sample indicates that the subject has minimal residual disease.

[0105] In certain aspects, provided herein is a method for stratifying subjects having cancer according to the benefit of a cancer therapy (e.g., immunotherapy), the method comprising: a) determining the level of at least one ganglioside and / or at least one lipid form of ganglioside in a sample from a subject to which the cancer therapy is administered; b) determining the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control; and c) comparing the levels of the at least one ganglioside and / or the lipid form of the at least one ganglioside detected in step a) and step b); wherein the absence of a significant change or decrease in the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject's sample as compared to the level in the control indicates that the subject having the cancer will benefit from the cancer therapy. In certain aspects, provided herein is a method for determining whether a subject having cancer will likely respond to, or alternatively will not respond to, a cancer therapy (e.g., an immunotherapy), the method comprising: a) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from a subject to which cancer therapy is administered; b) determining the level of the at least one ganglioside and / or a lipid form of the at least one ganglioside in a control; and c) comparing the levels of the at least one ganglioside and / or a lipid form of the at least one ganglioside detected in step a) and step b); wherein a significantly higher level of the at least one ganglioside and / or a lipid form of the at least one ganglioside in the subject's sample compared to the level in the control indicates that the subject having the cancer will not respond to the cancer therapy; and / or wherein no significant change or decrease in the level of the at least one ganglioside and / or a lipid form of the at least one ganglioside in the subject's sample compared to the level in the control indicates that the subject having the cancer will respond to the cancer therapy.

[0106] In certain aspects, provided herein is a method for predicting a clinical outcome in a subject having cancer, the method comprising: a) determining the level of at least one ganglioside and / or at least one lipid form of ganglioside in a sample from the subject; b) determining the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control; and c) comparing the levels of the at least one ganglioside and / or the lipid form of the at least one ganglioside determined in step a) and step b); wherein a significantly higher level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the sample from the subject compared to the level in the control indicates that the subject has a poor clinical outcome.

[0107] In certain aspects, a method for monitoring the progression of cancer in a subject is provided herein, the method comprising: a) detecting the level of at least one ganglioside and / or the lipid form of at least one ganglioside in a subject sample at a first time point; b) repeating step a) at a subsequent time point; and c) comparing the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside detected in step a) and step b) to monitor the progression of the cancer in the subject. In some embodiments, a significantly higher level of at least one ganglioside and / or the lipid form of at least one ganglioside in a subject sample relative to a control indicates progression of cancer (e.g., increased tumor burden, increased cancer stage, subject at risk of developing cancer).

[0108] In some embodiments, the method monitors progression of cancer in a subject who has received cancer therapy between a first time point and a subsequent time point. In some embodiments, the subject is at risk for developing cancer.

[0109] In certain aspects, provided herein is a method for evaluating the efficacy of a cancer therapy in a subject having cancer, the method comprising: a) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a first sample obtained from the subject; b) repeating step a) during at least one subsequent time point after administration of the cancer therapy; and c) comparing the levels of at least one ganglioside and / or a lipid form of at least one ganglioside detected in step a) and step b), wherein a significantly lower level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in at least one subsequent sample relative to the first sample indicates that the therapy is effective in treating the subject's cancer.

[0110] In some embodiments, the first sample and / or the at least one subsequent sample is a portion of a single sample or a pooled sample obtained from the subject.

[0111] In some embodiments, the cancer therapy is surgery, chemotherapy, cancer vaccine, chimeric antigen receptor, radiotherapy, immunotherapy, immune checkpoint inhibitory protein or ligand expression regulator or any combination thereof. In some embodiments, the immunotherapy is immune checkpoint inhibition therapy. In some embodiments, the cancer therapy is avelumab, durvalumab, atezolizumab, BRAF / MEK inhibitor, tyrosine kinase inhibitor, pembrolizumab, nivolumab, ipilimumab or a combination thereof.

[0112] A number of embodiments are further provided which may be applied to any aspect of the invention described herein.

[0113] For example, the diagnostic and prognostic methods described herein can use any method known in the art to detect and determine the level of at least one ganglioside and / or the lipid form of at least one ganglioside.

[0114] In some embodiments, at least one ganglioside and / or the lipid form of at least one ganglioside is at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 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%, 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%, 7 60%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% higher levels indicate significantly higher levels.

[0115] In some embodiments, at least one ganglioside and / or the lipid form of at least one ganglioside is at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 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%, 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%, 7 60%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% lower levels indicate significantly lower levels.

[0116] In some embodiments, the level of at least one ganglioside and / or the lipid form of at least one ganglioside is at least, about, or no more than 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%, %, 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 or decrease of 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% indicates that the level of at least one ganglioside has not changed significantly.

[0117] Further provided herein are diagnostic and prognostic methods that use the heterogeneity or homogeneity of lipid length of at least one ganglioside (e.g., as determined using mass spectrometry). In some embodiments, a significant change in the heterogeneity of lipid length of at least one ganglioside in a subject sample compared to a control sample indicates that the subject has cancer. Similarly, in some embodiments, a significant change in the heterogeneity of lipid length of at least one ganglioside in a subject sample compared to a control sample identifies the subject as having cancer.

[0118] Thus, in certain aspects, provided herein is a method for diagnosing cancer in a subject, the method comprising: a) determining the lipid length of at least one ganglioside in a sample from the subject (e.g., using mass spectrometry); and b) comparing the lipid length of the at least one ganglioside to the lipid length of at least one ganglioside in a control sample, wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the sample from the subject compared to the control sample indicates that the subject has cancer. In certain aspects, provided herein is a method for determining the stage of cancer, the method comprising: a) determining the lipid length of at least one ganglioside in a sample from the subject using mass spectrometry; and b) comparing the lipid length of the at least one ganglioside to the lipid length of at least one ganglioside in a control sample, wherein the heterogeneity of the lipid length of the at least one ganglioside in the sample from the subject compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample. at least, about, or no more than 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%, 0%, 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% change indicates that the subject has early stage cancer;and / or the heterogeneity of the lipid length of the at least one ganglioside in the subject sample is at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 70%, 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%, 60%, 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%, 7 A change of 50%, 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% indicates that the subject has advanced cancer.;

[0119] In some embodiments, a change of at least 100% and no more than 200% in the heterogeneity of lipid length of at least one ganglioside in a subject sample compared to the heterogeneity of lipid length of at least one ganglioside in a control sample (e.g., an increase or decrease) indicates that the subject has early-stage cancer. In some embodiments, a change of at least 200% in the heterogeneity of lipid length of at least one ganglioside in a subject sample compared to the heterogeneity of lipid length of at least one ganglioside in a control sample (e.g., an increase or decrease) indicates that the subject has advanced cancer.

[0120] In some embodiments, (a) the level of gangliosides with a short lipid length relative to the level of gangliosides with a long lipid length; or (b) the level of gangliosides with a long lipid length relative to the level of gangliosides with a short lipid length is increased or decreased by at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 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% 0%, 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%, and 1000% indicate that the subject has early stage cancer or advanced stage cancer.

[0121] In certain aspects, provided herein is a method of determining the grade of cancer, the method comprising: a) determining the lipid length of at least one ganglioside in a sample from a subject using mass spectrometry; and b) comparing the lipid length of the at least one ganglioside to the lipid length of at least one ganglioside in a control sample, wherein the heterogeneity of the lipid length of the at least one ganglioside in the sample from the subject is at least 100% compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample. , about or no more than 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%, 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%, A change (e.g., an increase or decrease) of 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% indicates that the subject has grade I, II, III or IV cancer.

[0122] In some embodiments, the heterogeneity in lipid length of at least one ganglioside in the subject sample is at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 800%, 800%, 0%, 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% change (e.g., increase or decrease) 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).

[0123] In some embodiments, a change of at least 100% and no more than 200% in the heterogeneity of lipid length of at least one ganglioside in a subject sample compared to the heterogeneity of lipid length of at least one ganglioside in a control sample (e.g., an increase or decrease) indicates that the subject has a low-grade cancer. In some embodiments, a change of at least 200% in the heterogeneity of lipid length of at least one ganglioside in a subject sample compared to the heterogeneity of lipid length of at least one ganglioside in a control sample (e.g., an increase or decrease) indicates that the subject has a high-grade cancer.

[0124] In some embodiments, (a) the level of gangliosides with a short lipid length relative to the level of gangliosides with a long lipid length; or (b) the level of gangliosides with a long lipid length relative to the level of gangliosides with a short lipid length is increased or decreased by at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 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% 0%, 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% indicate that the subject has low-grade cancer or high-grade cancer.

[0125] In certain aspects, provided herein is a method of determining tumor burden of a cancer, the method comprising: a) determining the lipid length of at least one ganglioside in a sample from a subject using mass spectrometry; and b) comparing the lipid length of the at least one ganglioside to the lipid length of at least one ganglioside in a control sample, wherein the lipid length of the at least one ganglioside in the sample from the subject is greater than the heterogeneity of the lipid length of the at least one ganglioside in the control sample. The heterogeneity of the degree of at least, about or no more than 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% change indicates that the subject has a low tumor burden;and / or the heterogeneity of the lipid length of the at least one ganglioside in the subject sample is at least, about, or no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160% greater than the heterogeneity of the lipid length of the at least one ganglioside 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%, 7 A change of 50%, 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% indicates that the subject has a high tumor burden.;

[0126] In some embodiments, a change of at least 100% and no more than 200% in the heterogeneity of lipid length of at least one ganglioside in a subject sample compared to the heterogeneity of lipid length of at least one ganglioside in a control sample (e.g., an increase or decrease) indicates that the subject has a low tumor burden. In some embodiments, a change of at least 200% in the heterogeneity of lipid length of at least one ganglioside in a subject sample compared to the heterogeneity of lipid length of at least one ganglioside in a control sample (e.g., an increase or decrease) indicates that the subject has a high tumor burden.

[0127] In some embodiments, (a) the level of gangliosides with a short lipid length relative to the level of gangliosides with a long lipid length; or (b) the level of gangliosides with a long lipid length relative to the level of gangliosides with a short lipid length is increased or decreased by at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 30%, 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% indicates that the subject has a low tumor burden or a high tumor burden.

[0128] In certain aspects, a method of detecting recurrence of cancer in a subject is provided, the method comprising: a) obtaining a sample from the subject or providing a sample from the subject, wherein the cancer in the subject has regressed after receiving cancer treatment; b) determining the lipid length of at least one ganglioside in the sample from the subject using mass spectrometry; and c) comparing the lipid length of the at least one ganglioside with the lipid length of at least one ganglioside in a control sample, wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the sample from the subject compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates recurrence of cancer in the subject.

[0129] In certain aspects, provided herein is a method for detecting minimal residual disease in a subject, the method comprising: a) obtaining a sample from a subject in remission or providing a sample from the subject; b) determining the lipid length of at least one ganglioside in the subject's sample using mass spectrometry; and c) comparing the lipid length of the at least one ganglioside with the lipid length of at least one ganglioside in a control sample, wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the subject's sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates that the subject has minimal residual disease.

[0130] In certain aspects, provided herein is a method for stratifying subjects having cancer according to the benefit of a cancer therapy (e.g., immunotherapy), the method comprising: a) determining the lipid length of at least one ganglioside in a sample from a subject administered the cancer therapy using mass spectrometry; b) determining the lipid length of the at least one ganglioside in a control; and c) comparing the lipid length of the at least one ganglioside detected in step a) and step b); wherein the absence of a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the subject sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control indicates that the subject having the cancer will benefit from the cancer therapy.

[0131] In certain aspects, provided herein is a method of determining whether a subject having cancer will likely respond to a cancer therapy, the method comprising:

[0132] a) determining the lipid length of at least one ganglioside in a sample from a subject to which a cancer therapy is administered using mass spectrometry; b) determining the lipid length of the at least one ganglioside in a control; and c) comparing the lipid length of the at least one ganglioside detected in step a) and step b); wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the subject's sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control indicates that the subject suffering from the cancer will not respond to the cancer therapy; and / or wherein no significant change in the heterogeneity of the lipid length of the at least one ganglioside in the subject's sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control indicates that the subject suffering from the cancer will respond to the cancer therapy.

[0133] In certain aspects, provided herein is a method for predicting a clinical outcome in a subject having cancer, the method comprising: a) determining the lipid length of at least one ganglioside in a sample from the subject using mass spectrometry; b) determining the lipid length of the at least one ganglioside in a control; and c) comparing the lipid length of the at least one ganglioside determined in step a) and step b); wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the sample from the subject compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates that the subject has a poor clinical outcome.

[0134] In certain aspects, provided herein is a method for monitoring the progression of cancer in a subject, the method comprising: a) detecting the lipid length of at least one ganglioside in a subject sample using mass spectrometry at a first time point; b) repeating step a) at a subsequent time point; and c) comparing the heterogeneity of the lipid length of the at least one ganglioside detected in step a) and step b) to monitor the progression of the cancer in the subject, optionally wherein the subject is at risk of developing cancer. In some embodiments, a significant change in the heterogeneity of the lipid length of at least one ganglioside in a subject sample relative to a control indicates progression of cancer (e.g., increased tumor burden, increased cancer stage, subject at risk of developing cancer).

[0135] In some embodiments, between the first time point and the subsequent time point, the subject has received cancer therapy.

[0136] In certain aspects, provided herein is a method for evaluating the efficacy of a cancer therapy in a subject having cancer, the method comprising: 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) during at least one subsequent time point after administration of the cancer therapy; and c) comparing the levels of the at least one ganglioside detected in step a) and step b), wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the second sample relative to the first sample indicates that the therapy is effective in treating the cancer in the subject.

[0137] In some embodiments, the first sample and / or the at least one subsequent sample is a portion of a single sample or a pooled sample obtained from the subject.

[0138] The methods of the present disclosure (e.g., the diagnostic and prognostic methods described herein) can detect and determine the heterogeneity of the lipid length of at least one ganglioside using any method known in the art. In a preferred embodiment, the heterogeneity of the lipid length 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, nanopore LC-ESI-MS, nanopore LC-ESI-MS / MS, or any other mass spectrometry disclosed herein or mass spectrometry known in the art).

[0139] In some embodiments, the heterogeneity or homogeneity of lipid length of at least one ganglioside is at least, about, or no more than 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%, 80 20%, 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%, Changes of 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000% indicate significant changes in heterogeneity or homogeneity.

[0140] In some embodiments, (a) the level of gangliosides with a short lipid length relative to the level of gangliosides with a long lipid length; or (b) the level of gangliosides with a long lipid length relative to the level of gangliosides with a short lipid length is increased or decreased by at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 0%, 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%, indicating a significant change in the heterogeneity or homogeneity of lipid length.

[0141] In some embodiments, the heterogeneity or homogeneity of lipid length of at least one ganglioside is at least, about, or no more than 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%, 0%, 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 The changes in the percentage of patients with AIDS were 7, 80%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, and 1000%, indicating that there was no significant change in heterogeneity or homogeneity.

[0142] In some embodiments, (a) the level of gangliosides with a short lipid length relative to the level of gangliosides with a long lipid length; or (b) the level of gangliosides with a long lipid length relative to the level of gangliosides with a short lipid length is increased or decreased by at least, about, or no more than 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%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 0%, 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% 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%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, indicating that there was no significant change in the heterogeneity or homogeneity of lipid length.

[0143] Further provided are many 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, radiotherapy, immunotherapy, immune checkpoint inhibitory protein or ligand expression modulator or any combination thereof. In some embodiments, the immunotherapy is immune checkpoint inhibition therapy. In some embodiments, the cancer therapy is avelumab, durvalumab, atezolizumab, BRAF / MEK inhibitor, tyrosine kinase inhibitor, pembrolizumab, nivolumab, ipilimumab or a combination thereof.

[0144] In some embodiments, the at least one ganglioside and / or the lipid form of the at least one ganglioside is a tumor-associated ganglioside and / or a lipid form of a tumor-associated ganglioside. In some embodiments, the tumor-associated ganglioside is selected from 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 portion thereof, any lipid form or isomer thereof, and any combination of two or more thereof.

[0145] In a preferred embodiment, the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises GM2, GD3, GD2, GD1b, any lipid form thereof, or any combination thereof.

[0146] In some embodiments, the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises a combination of two or more selected from GM2, GD3, GD2, GD1b, and any lipid form thereof.

[0147] In some embodiments, the at least one ganglioside and / or the lipid form of the at least one 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.

[0148] In some embodiments, the at least one ganglioside and / or the lipid form of the at least one 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.

[0149] In some embodiments, the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises GD3 (d18:1 / 23:0) and / or GD3 (d18:1 / 24:1).

[0150] In some embodiments, the at least one ganglioside and / or the lipid form of the at least one ganglioside is GD3 (d18:1 / 23:0) and / or GD3 (d18:1 / 24:1).

[0151] In some embodiments, the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises GD3 (d18:1 / 23:0).

[0152] In some embodiments, the at least one ganglioside and / or the lipid form of the at least one ganglioside is GD3 (d18:1 / 23:0).

[0153] In some embodiments, the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises an acyl chain 24:1.

[0154] In some embodiments, the acyl chain 24:1 is present in GD3 and / or GD1b.

[0155] 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.

[0156] 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.

[0157] In some embodiments, the cancer is ovarian cancer, melanoma, renal cancer, or lung cancer.

[0158] In a preferred embodiment, the cancer is ovarian cancer.

[0159] In some embodiments, the cancer is a borderline tumor.

[0160] In some embodiments, the sample comprises cells, serum, blood, peritumoral tissue and / or intratumoral tissue (e.g., biopsy) obtained from a subject. In a preferred embodiment, the sample comprises a liquid biopsy (comprising a liquid). Thus, in some embodiments, the sample comprises serum or blood.

[0161] In some embodiments, the significantly higher level of at least one ganglioside and / or lipid form of at least one ganglioside comprises at least a 20% increase in the level of at least one ganglioside and / or lipid form of at least one ganglioside.

[0162] In some embodiments, the significantly lower level of at least one ganglioside and / or lipid form of at least one ganglioside comprises at least a 20% decrease in the level of at least one ganglioside and / or lipid form of at least one ganglioside.

[0163] In preferred embodiments, the significantly higher or lower level of at least one ganglioside and / or at least one lipid form of a ganglioside is a level that is at least or about 50% higher or lower than the level of a control (e.g., a non-cancerous sample). In other embodiments, the significantly higher or lower level of at least one ganglioside and / or at least one lipid form of a ganglioside is a level that is at least or about 25% higher or lower than the level of a previous reading of a subject in a longitudinal study.

[0164] In some embodiments, a significant change in the heterogeneity of lipid length of at least one ganglioside comprises a change (eg, an increase or decrease) of at least 20% in a subject sample relative to a control sample.

[0165] In some embodiments, the control sample is a sample from a cancer-free subject.

[0166] In other embodiments, the control sample is a sample from a subject having cancer.

[0167] In some embodiments, the control sample is a sample from the subject (eg, the first sample collected from the subject in a longitudinal collection to assess changes in the level of the at least one ganglioside over time).

[0168] In a preferred embodiment, the diagnosis and / or prognosis methods described herein further include treating the subject with a cancer therapy disclosed herein or a cancer therapy known in the art. For example, a subject determined to be in need of treatment based on the results of the diagnosis and / or prognosis methods disclosed herein can be treated with cancer therapy. In some embodiments, the diagnosis and / or prognosis methods further include recommending, prescribing and / or administering a cancer therapy disclosed herein (e.g., immune checkpoint inhibition therapy) or a cancer therapy known in the art to the subject.

[0169] In some embodiments, the subject has cancer. In some such embodiments, the subject is asymptomatic.

[0170] In some embodiments, the subject is a mammal (eg, a human, a pet (eg, a dog, a cat), livestock).

[0171] In some embodiments, the subject is a cancer animal model, dog, cat, or human.

[0172] In a preferred embodiment, the subject is a human.

[0173] Treating patients and monitoring outcomes during clinical trials

[0174] The methods of the present disclosure may be followed by treatment of the subject whose sample is tested in the methods. For example, a subject diagnosed with cancer using the methods described herein may be treated with a cancer therapy (e.g., standard of care or a cancer therapy described herein or a cancer therapy known in the art).

[0175] Monitoring the effect of a medicament (e.g., a compound, drug or small molecule described herein, immunotherapy, cancer therapy, or a medicament known in the art) on the level of gangliosides and / or the lipid form of at least one ganglioside can be applied not only to basic drug screening, but also to clinical trials. For example, the effectiveness of reducing the level of gangliosides by a medicament determined by a screening assay as described herein can be monitored in a clinical trial of a subject, and the effectiveness can be detected by a mass spectrometry-based method. 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 specific cell, tissue, or system.

[0176] In a preferred embodiment, the present disclosure provides a method for monitoring the effectiveness of treating a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, immunotherapy, immune checkpoint inhibition therapy or other drug candidate), the method comprising the following steps: (i) obtaining a pre-administration sample from the subject before administering the agent; (ii) detecting the level of at least one ganglioside and / or the lipid form of at least one ganglioside in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of at least one ganglioside and / or the lipid form of at least one ganglioside in the post-administration sample; (v) comparing the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the pre-administration sample with the at least one ganglioside and / or the lipid form of the at least one ganglioside in the one or more post-administration samples; and (vi) altering the administration of the agent to the subject accordingly. For example, it may be desirable to increase the administration of the medicament so that the level of at least one ganglioside and / or the lipid form of at least one ganglioside is reduced to a level lower than the detected level, i.e., increase the effectiveness of the medicament. According to such embodiments, the lipid form of at least one ganglioside and / or at least one ganglioside can be used as an indicator of the effectiveness of the medicament, even in the absence of an observable phenotypic response. Similarly, the lipid form of at least one ganglioside and / or at least one ganglioside analysis (such as by a mass spectrometry-based method) can also be used to select patients who will receive cancer therapy (e.g., immunotherapy, immune checkpoint inhibition therapy).

[0177] sample

[0178] Biological samples can be collected from a variety of sources from the subject, including body fluid samples, cell samples or tissue samples. Body fluid refers to liquids excreted or secreted from the body and liquids that are not usually excreted or secreted (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, spleen and cerumen, Cowper's fluid or pre-ejaculatory fluid, chyle, chyme, feces, female ejaculation, interstitial fluid, intracellular fluid, lymphatic fluid, menstruation, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomitus). In some embodiments, the subject and / or control sample is selected from the group consisting of: cells, cell lines, whole blood, serum, plasma, cheek scrapings, saliva, cerebrospinal fluid and bone marrow. In some embodiments, the sample can include living cells / tissues, fresh frozen cells, fresh tissues, biopsies, fixed cells / tissues, cells / tissues embedded in a medium (such as paraffin), histological slides, or any combination thereof. In some embodiments, the sample can include living cells / tissues, fresh frozen cells, fresh tissues, and / or biopsies.

[0179] Samples can be collected repeatedly from an individual over a longitudinal period of time (eg, once or more over a period of days, weeks, months, year, year, etc.).

[0180] Sample preparation and separation can involve any procedure, depending on the type of sample collected and / or the analysis of the biomarker measurements. By way of example only, such procedures include concentration, dilution, pH adjustment, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids. In some embodiments, certain cell types are purified based on at least one marker present on the cell surface.

[0181] A sample may include an immobilized molecule. A molecule is "immobilized" or "attached" to a substrate if it is covalently or non-covalently associated with the substrate, and the substrate can be washed with a fluid (e.g., standard citrate water, pH 7.4) without a significant portion of the molecule dissociating from the substrate.

[0182] As described herein, in some embodiments, the level of at least one ganglioside and / or the lipid form of at least one ganglioside measurement value in a sample from an object is compared with a control biological sample (e.g., a biological sample from an object not suffering from cancer), a control biological sample from an object in remission or before suffering from cancer, or a control biological sample from an object during treatment for cancer. In some embodiments, the control biological sample is from an object before treatment with a certain therapy. In some embodiments, where the object 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 object sample during such treatment. For example, the object sample after the third round of therapy can be compared with the control object sample after the first round of therapy.

[0183] In some embodiments, the level of at least one ganglioside and / or the lipid form of at least one ganglioside measurement value in the sample from the object is compared with a predetermined control (standard) sample. The sample from the object is usually from a diseased tissue, such as a cancer cell or tissue. The control sample can be from the same object or a different object. The control sample is usually a normal, non-diseased sample. However, in some embodiments, as used for disease staging or for evaluating therapeutic efficacy, the control sample can be from a diseased tissue. The control sample can be a combination of samples from several different objects. In some embodiments, the biomarker amount and / or activity measurement value from the object is compared with a predetermined level. The predetermined level is usually obtained from a normal sample.

[0184] As described herein, "predetermined" biomarker amount measurement value can be a biomarker amount measurement value for (only by way of example) evaluating an object that can be selected for treatment, evaluating a response to a cancer therapy and / or evaluating a combination of anticancer therapies. Predetermined biomarker amounts and / or activity measurements can be determined in a patient population with or without cancer. Predetermined biomarker amount measurement values ​​can be a single number that is equally applicable to each patient, or predetermined biomarker amount measurement values ​​can vary according to a specific patient subgroup. The age, weight, height and other factors of the object may affect the predetermined biomarker amount measurement value of an individual. In addition, predetermined biomarker amounts can be determined separately for each object. In some embodiments, the amount determined and / or compared in the methods described herein is based on absolute measurements.

[0185] In some embodiments, the amount determined and / or compared in the methods described herein is based on relative measurements, such as ratios (e.g., biomarker levels before and after treatment, such biomarker measurements relative to spiked or artificial controls, such biomarker measurements relative to the expression of housekeeping genes, etc.). For example, relative analysis can be based on the ratio of biomarker measurements before treatment compared to biomarker measurements after treatment. Pre-treatment biomarker measurements can be performed at any time before anticancer therapy is started. Post-treatment biomarker measurements can be performed at any time after anticancer therapy is started. In some embodiments, post-treatment biomarker measurements are performed 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or longer after anticancer therapy is started, and even longer to continue monitoring indefinitely. Treatment can include one or more anticancer therapies, such as immune checkpoint inhibitors.

[0186] The predetermined biomarker amount measurement value can be any suitable standard. For example, the predetermined biomarker amount measurement value can be obtained from the same or different people who are evaluating the patient's selection. In some embodiments, the predetermined biomarker amount measurement value can be obtained from the previous assessment of the same patient. In this way, the progress of the patient's selection can be monitored over time. In addition, if the object is a person, the control can be obtained from the assessment of another person or multiple people (for example, selected groups of people). In this way, the degree of selection of the person whose selection is evaluated can be compared with suitable other people (for example, other people in similar situations with the person concerned, such as people suffering from similar or identical symptoms and / or belonging to the same ethnic group).

[0187] In some embodiments of the present disclosure, the change in the biomarker amount measurement relative to the predetermined level is about 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1.0 times, 1.5 times, 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times or 5.0 times or greater or any range therebetween, including end values. When the measurement is based on relative changes, such as based on the ratio of the biomarker measurement before treatment to the biomarker measurement after treatment, such cutoff values ​​are equally applicable.

[0188] cancer

[0189] Cancer, tumor or hyperproliferative disorder refers to the presence of cells with typical characteristics of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain unique morphological features. Cancer cells are usually in the form of tumors, but such cells can exist alone in an animal or can be non-tumorigenic cancer cells, such as leukemia cells. Cancer includes, but is not limited to, B-cell cancers, such as multiple myeloma, Waldenstrom's macroglobulinemia ( macroglobulinemia), heavy chain disease (e.g., alpha chain disease, gamma chain disease, and mu chain disease), benign monoclonal gammopathy and immune cell 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 cancer or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, blood tissue cancer, etc. Other non-limiting examples of cancer types suitable for the methods encompassed by the invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer (SCLC), bladder cancer, epithelial carcinoma, glioma, astrocytoma In some embodiments, the cancer is epithelial in nature and includes, but is 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 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 various other ways, including but not limited to serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0190] 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. Notably, 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 predict borderline tumors, including but not limited to borderline ovarian tumors.

[0191] Borderline tumors

[0192] Borderline tumors are a heterogeneous group of lesions defined histologically as a proliferation of atypical epithelium without stromal invasion.

[0193] Similarly, borderline tumors of the ovary (also called tumors of low malignant potential) are a heterogeneous group of lesions defined histologically as atypical epithelial proliferations without stromal invasion (Seidman et al. (2002) Blaustein's pathology of the female genital tract, p. 791; Seidman et al. (2003) Hematol Oncol Clin North Am, 17:909). These tumors behave differently from low-grade ovarian cancers, and these tumors are considered distinct clinical entities.

[0194] Borderline epithelial ovarian tumors are non-invasive tumors with occasional intraperitoneal spread. This group of tumors exhibits behavior intermediate between benign cystadenomas and invasive carcinomas. These tumors are referred to by different names including: borderline, atypical proliferation, and tumor of low malignant potential. Borderline tumor is the name currently most widely used by pathologists, gynecologists, and oncologists and has been adopted into the World Health Organization (WHO) classification.

[0195] Borderline tumors account for 14-15% of all primary ovarian tumors.

[0196] Borderline tumors occur in a variety of histologies, such as in epithelial ovarian cancer. Most cases are serous or mucinous. In some cases, endometrioid, clear cell, or transitional cell (Brenner) borderline tumors are found.

[0197] Cancer Therapy

[0198] The method disclosed herein (e.g., diagnosis and / or prognosis method) can be followed by cancer therapy described herein or cancer therapy known in the art (e.g., standard care treatment for cancer well known to those skilled in the art, chemotherapeutic agents, hormones, anti-angiogenesis, radiolabeled compounds) or by surgery, cryotherapy, immunotherapy, cancer vaccines, immune cell engineering (e.g., CAR-T) and / or radiotherapy to treat patients. The aforementioned treatment methods can be administered in combination with other forms of cancer therapy, administered continuously with the cancer therapy, before or after the cancer therapy. For example, immunotherapy can be administered together with a therapeutically effective dose of a chemotherapeutic agent, for example, immunotherapy can be administered in combination with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The Physician's Desk Reference (PDR) discloses the dosage of chemotherapeutic agents that have been used in the treatment of various cancers. The dosage regimen and dosage of these above-mentioned chemotherapeutic drugs that are effective in treatment will depend on the specific cancer being treated, the extent of the disease, and other factors familiar to physicians in the art, and can be determined by the physician.

[0199] Immunotherapy is a targeted therapy, which may 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 without harming normal cells, making them potentially useful in cancer therapy. The replication of oncolytic viruses not only promotes tumor cell destruction, but also produces dose amplification at the tumor site. The oncolytic virus can also serve as a carrier of anti-cancer genes, thereby allowing them to be specifically delivered to the tumor site. Immunotherapy may involve passive immunity for short-term protection of the host, which is achieved by administering preformed antibodies for cancer antigens or disease antigens (e.g., administering monoclonal antibodies for tumor antigens, optionally connected to chemotherapeutic agents or toxins). For example, it is known that anti-VEGF is effective in treating renal cell carcinoma. Immunotherapy can also focus on epitopes recognized by cytotoxic lymphocytes using cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc. can be used to selectively regulate biomolecules related to the initiation, progression and / or pathology of tumors or cancers.

[0200] Immunotherapy also encompasses immune checkpoint modulators, which are a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by downregulating 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, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3 and A2aR (see, e.g., WO 2012 / 177624). The inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling, thereby raising the immune response, thereby more effectively treating cancer. In some embodiments, a cancer vaccine is administered in combination with one or more inhibitors of immune checkpoints (immune checkpoint inhibition therapy), such as PD1, PD-L1, and / or CD47 inhibitors.

[0201] Immunotherapy based on adoptive cells can be combined with the therapy of the present invention. Well-known immunotherapy forms based on adoptive cells, including but not limited to irradiated autologous or allogeneic tumor cells, tumor lysates or apoptotic tumor cells, immunotherapy based on antigen presenting cells, immunotherapy based on dendritic cells, adoptive T cell transfer, adoptive CAR T cell therapy, autologous immune enhancement therapy (AIET), cancer vaccines and / or antigen presenting cells. Such cell-based immunotherapy can be further modified to express one or more gene products to further regulate immune response, such as expressing cytokines, such as GM-CSF, and / or expressing tumor-associated antigens (TAA) antigens, such as Mage-1, gp-100, etc.

[0202] The term "chimeric antigen receptor" or "CAR" refers to an engineered T cell receptor (TCR) with desired antigen specificity. T lymphocytes recognize specific antigens through the interaction of T cell receptors (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or class II molecules. For initial activation and clonal expansion, naive T cells rely on professional antigen presenting cells (APCs) that provide additional co-stimulatory signals. In the absence of co-stimulation, TCR activation may lead to unresponsiveness and clonal anergy. In order to bypass immunity, different methods for deriving cytotoxic effector cells with transplanted recognition specificity have been developed. CARs composed of binding domains of antibodies derived from natural ligands or cell surface components of TCR-related CD3 complexes have been constructed. After antigen binding, such chimeric antigen receptors are connected to endogenous signal transduction pathways in effector cells and produce activation signals similar to activation signals initiated by TCR complexes. Since the first reports of chimeric antigen receptors, the concept has been steadily refined and the molecular design of chimeric receptors has been optimized and routinely uses any number of well-known binding domains, such as scFV and another protein binding fragment described herein.

[0203] In other embodiments, immunotherapy includes non-cell-based immunotherapy. In some embodiments, a composition comprising an antigen with or without a vaccine enhancing adjuvant is used. Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, recombinant antigens including fusion proteins, etc. In some embodiments, immunomodulatory cytokines such as interferon, G-CSF, imiquimod, TNFα, etc., and their regulators (e.g., blocking antibodies or more effective or more lasting forms) are used. In some embodiments, immunomodulatory interleukins such as IL-2, IL-6, IL-7, IL-12, IL-17, IL-23, etc. and their regulators (e.g., blocking antibodies or more effective or more lasting forms) are used. In some embodiments, immunomodulatory chemokines such as CCL3, CCL26 and CXCL7, etc., and their regulators (e.g., blocking antibodies or more effective or more lasting forms) are used. In some embodiments, immunomodulatory molecules such as STAT3 signaling regulators, NFκB signaling regulators, and immune checkpoint regulators are used for targeted immunosuppression.

[0204] In yet other embodiments, immunomodulatory drugs such as immune cell suppressive drugs, glucocorticoids, cytostatics, immunophilins and their modulators (e.g., rapamycin, calcineurin inhibitors, tacrolimus, cyclosporine, pimecrolimus, abetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (Cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, acetic acid fludrocortisone, deoxycorticosterone acetate (doca), aldosterone, non-glucocorticoid steroids, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid analogs, methotrexate, antithymocyte globulin, antilymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, opioid, IMPDH inhibitors, mycophenolic acid, myriocin, fingolimod, NF-xB inhibitors, raloxifene, drotrecogin alfa alfa), denosumab, NF-xB signaling cascade inhibitors, disulfiram, olmesartan, dithiocarbamates, proteasome inhibitors, bortezomib, MG132, Pro1, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, nonsteroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroepoxymethylquinomycin (DHMEQ), I3C (indole-3-methanol) / DIM (diindolylmethane) (13C / DIM), Bay11-7082, luteolin, cell permeable peptide SN-50, IKBa.-super inhibitor overexpression, NFKB decoy oligodeoxynucleotide (ODN) or any derivative or analog thereof. In some other embodiments, immunomodulatory antibodies or proteins are used. For example, antibodies that bind to CD40, Toll-like receptor (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; revulizumab; anti-CD20 antibodies; afutuzumab ab); ocrelizumab; ofatumumab; pascolizumab; rituximab; anti-CD23 antibody; lumiliximab; anti-CD40 antibody; teneliximab; toralizumab; anti-CD40L antibody; ruplizumab; anti-CD62L antibody; aselizumab; anti-CD80 antibody; galiximab; anti-CD147 antibody ; gavilimomab; B-lymphocyte stimulator (BLyS) inhibitory 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; o odulimomab; anti-CD25 antibody; basiliximab; daclizumab; inolimomab; anti-CD5 antibody; zolimomab; anti-CD2 antibody; siplizumab; nerelimomab; faralimomab; atlizumab; torolimumab; cedelizumab; dorlimomab aritox; dorlixizumab;fontolizumab; gantenerumab; gomiliximab; lebrilizumab; maslimomab; morolimumab; pexelizumab; reslizumab; rovelizumab; talizumab; telimomab aritox; vapaliximab; vepalimomab; aflibercept; alefacept; rilonacept; IL-1 receptor antagonist; anakinra; anti-IL-5 antibody; mepolizumab; IgE inhibitor; omalizumab; talizumab; IL12 inhibitor; IL23 inhibitor; ustekinumab, etc. ;

[0205] Nutritional supplements that enhance immune responses (eg, vitamin A, vitamin E, vitamin C, etc.) are well 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.

[0206] Similarly, various agents or combinations thereof can be used to treat cancer. For example, chemotherapy, radiotherapy, epigenetic modifiers (e.g., histone deacetylase (HDAC) modifiers, methylation modifiers, phosphorylation modifiers, etc.), targeted therapies, etc. are well known in the art.

[0207] In some embodiments, chemotherapy is used. Chemotherapy includes administering a chemotherapeutic agent. Such chemotherapeutic agents can be, but are not limited to, chemotherapeutic agents selected from the group consisting of platinum compounds, cytotoxic antibiotics, antimetabolites, antimitotic agents, 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, docetaxel; 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 antimitotic agents: halichondrin, colchicine, and root toxin. Compositions comprising 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, PARP (e.g., PARP-1 and / or PARP-2) inhibitors are used, and such inhibitors are well known in the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-1001 (Inotek Pharmaceuticals, Inc.); PARP inhibitors include 1,2-diaminobenzamide (Trevogen Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001; Pacher et al., 2002b); 3-aminobenzamide (Trevigen); 4-amino-1,8-naphthalimide (Trevigen); 6(5H)-phenanthridinone (Trevogen); benzamide (U.S. Reissue Patent 36,397); and NU1025 (Bowman et al.). The mechanism of action is generally related to the ability of PARP inhibitors to bind to PARP and reduce its activity. PARP catalyzes the conversion of β-nicotinamide adenine dinucleotide (NAD+) to nicotinamide and poly-ADP-ribose (PAR).Both poly(ADP-ribose) and PARP are involved in the regulation of transcription, cell proliferation, genome stability and carcinogenesis (Bouchard VJ et al. Experimental Hematology, Vol. 31, No. 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 477, No. 1, June 2, 2001, pp. 97-110(14)). Poly (ADP-ribose) polymerase 1 (PARP1) is a key molecule in 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). Knockout of SSB repair by inhibiting PARP1 function induces DNA double-strand breaks (DSBs), triggering 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 illustrative and not intended to be limiting.

[0208] In other embodiments, radiotherapy is used. The radiation used in radiotherapy can be ionizing radiation. Radiotherapy can also be gamma rays, X-rays or proton beams. Examples of radiotherapy include, but are not limited to, external beam radiotherapy, interstitial implantation of radioisotopes (I-125, palladium, iridium), radioisotopes such as strontium-89, chest radiotherapy, intraperitoneal P-32 radiotherapy and / or whole abdomen and pelvic radiotherapy. For a general overview of radiotherapy, see Hellman, Chapter 16: "Principles of Cancer Management: Radiation Therapy", 6th edition, 2001, edited by DeVita et al., JB Lippincott Company, Philadelphia. Radiotherapy can be administered as external beam radiation or teleradiotherapy, in which radiation is guided from a remote source. Radiotherapy can also be administered as internal therapy or brachytherapy, in which a radioactive source is placed in the body close to cancer cells or tumor masses. Also included is the use of photodynamic therapy, including the administration of photosensitizers such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and 2BA-2-DMHA.

[0209] In other embodiments, hormone therapy is used. Hormonal therapeutic treatment can include, for example, hormone agonists, hormone antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprorelin acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progesterone), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antiprogestins (e.g., mifepristone, onapristone) or antiandrogens (e.g., cyproterone acetate).

[0210] In other embodiments, photodynamic therapy (also known as PDT, photoradiation therapy, phototherapy, or photochemotherapy) is used to treat some types of cancer. The photodynamic therapy is based on the discovery that certain chemicals called photosensitizers can kill single-cell organisms when the organisms are exposed to certain types of light.

[0211] In yet other embodiments, laser therapy is used to destroy cancer cells using high-intensity light. This technology is often used to relieve symptoms of cancer, such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. The technology can also be used to treat cancer by shrinking or destroying tumors.

[0212] Clinical efficacy / response to therapy

[0213] Clinical efficacy can be measured by any method known in the art. For example, the response to therapy relates to any response of cancer (e.g., tumor) to therapy, preferably to changes in tumor mass and / or volume after the start of neoadjuvant or adjuvant chemotherapy. Tumor response can be assessed in neoadjuvant or adjuvant situations, where the size of the tumor after systemic intervention can be compared with the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation, and the cell structure of the tumor can be estimated histologically and compared with the cell structure of the tumor biopsy performed before starting treatment. The response can also be assessed by caliper measurement or tumor pathology examination after biopsy or surgical resection. Responses can be recorded quantitatively, such as percent change in tumor volume or cellularity, or using semiquantitative scoring systems, such as residual cancer burden (Symmans et al., J. Clin. Oncol. (2007) 25:4414-4422) or Miller-Payne score (Ogston et al. (2003) Breast (Edinburgh, Scotland) 12:320-327), qualitatively, such as "pathological complete response" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinically stable disease" (cSD), "clinically progressive disease" (cPD) or other qualitative criteria. Assessment of tumor response can be performed early after the start of neoadjuvant or adjuvant therapy, such as hours, days, weeks, or preferably months later. Typical endpoints for response assessment are at the termination of neoadjuvant chemotherapy or surgical resection of residual tumor cells and / or tumor bed.

[0214] In some embodiments, the clinical efficacy of the therapeutic treatment 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) at a time point of at least 6 months after the end of therapy, the number of patients in partial remission (PR), and the number of patients with stable disease (SD). The abbreviation of this formula is CBR=CR+PR+SD for more than 6 months. In some embodiments, the CBR of a specific anti-immune checkpoint treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more.

[0215] Additional criteria for assessing responses to cancer therapy are related to "survival", which includes all of the following: survival before death, also known as overall survival (wherein the death may be etiologically independent or tumor-related); "recurrence-free survival" (wherein the term recurrence shall include local recurrence and distant recurrence); metastasis-free survival; disease-free survival (wherein the term disease shall include cancer and diseases related thereto). The length of the survival can be calculated by reference to a defined starting point (e.g., time of diagnosis or start of treatment) and an 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 time period, and the probability of tumor recurrence.

[0216] For example, in order to determine an appropriate threshold, a specific anticancer treatment regimen can be applied to a subject population, and the result can be associated with a biomarker measurement value determined before any cancer therapy is applied. The result value can be a pathological response to a therapy given in a neoadjuvant setting. Alternatively, the result measurement of the subject after a cancer therapy for which the biomarker measurement value is known can be monitored over a period of time, such as total survival and disease-free survival. In certain embodiments, the same dose of anticancer agent is applied to each subject. In a related embodiment, the dose applied is a standard dose of an anticancer agent known in the art. The time period for monitoring the subject can vary. For example, the subject 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 biomarker measurement threshold value associated with the result of the cancer therapy can be determined using methods such as the method described in the embodiment section.

[0217] Example

[0218] Example 1: Materials and Methods

[0219] patient

[0220] Patient samples (TMA and liquid biopsy) were obtained from the LDI / JHG biobank under ethical approval. A retrospective study of clinical history was used to evaluate the correlation between TMG and CA-125 levels, cancer disease stage and survival, and menopausal stage. A total of 176 patients (n=9 normal; n=16 borderline; n=151 ovarian) were studied.

[0221] Extraction and Isolation of Gangliosides from Tissue

[0222] Total lipids in homogenized tissue (e.g., tumor) (500 mg) were extracted with 9 ml of chloroform-methanol (2:1; v / v) and then with 7.6 ml of chloroform-methanol-water (1:2:0.8; v / v / v). The extract was dried under a gentle nitrogen stream, redissolved with 7.5 ml of chloroform-methanol-water (30:60:8; v / v / v), and applied to a DEAE-Sephadex A-25 column (6×45 mm, acetate form) (Amersham Biosciences AB, Sweden). After neutral lipids were washed from the column with 15 ml of chloroform-methanol-water (30:60:8; v / v / v), the acidic glycolipid mixture containing gangliosides was eluted with 7.5 ml of chloroform-methanol-0.8 M sodium acetate (30:60:8; v / v / v).

[0223] Extraction and Isolation of Gangliosides from Liquid Biopsies

[0224] Monophasic lipid extraction of polar gangliosides with a solvent volume ratio of 0.85:2:1:0.75 MeOH:EtOH:CHCl3:H2O. The method has been adapted from Svennerholm and Fredman (1980) Biochim Biophys Acta 617:97-109; and Lydic et al. (2014) Journal of Lipid Research 55:1797-809, both of which are incorporated by reference in their entirety.

[0225] Liquid chromatography / electrospray ionization-MS

[0226] Liquid chromatography (LC) separation was achieved by using an octadecylsilane (ODS) reverse phase LC (RPLC) column (150×0.3 mm inner diameter) (LC PACKINGS; Amsterdam, The Netherlands) at 45°C. The mobile phase was prepared from methanol (A), isopropanol (B) and water (containing 200 mM ammonium formate) (C), and the composition was prepared by mixing these solvents. The gradient consisted of holding solvent (A / B / C 55:25:20) (D) for 5 minutes, then linearly converting solvent D to solvent (A / B 60:40) (E) for 50 minutes and holding solvent E for 35 minutes. The mobile phase was pumped at a flow rate of 7.5 microliters / minute for gradient elution. Typically, 3 μl of sample solution was applied.

[0227] Electrospray ionization-tandem mass spectrometry analysis

[0228] Electrospray ionization (ESI)-MS analysis was performed using a 4000Q TRAP quadrupole-linear ion trap hybrid MS (Applied Biosystems, Foster City, CA) with an UltiMate 3000 nano / cap / micro LC system (Dionex Corporation, Sunnyvale, CA) in combination with a HTS PAL autosampler (CTC Analytics AG, Zwingen, Switzerland).

[0229] The scanning range of the instrument was set to m / z 200 to 2,300 at a scanning speed of 1,000 atomic mass units / second. The well fill time was 10 milliseconds in the positive ion mode and 20 milliseconds in the negative ion mode. Other MS scanning conditions were operated using 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 the positive ion mode and an ion spray voltage of -4,500 V, a DP of -120 V, and a CID of -5 V in the negative ion mode. Tandem mass spectrometry (MS / MS) analysis of ceramide molecular species derived from in-source decay of gangliosides was performed under each of the following CID conditions: 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).

[0230] Multiple reaction monitoring (MRM) analysis

[0231] MRM analysis was performed using the same instrument system used for LC / ESI-MS analysis. The DP was set to -120 V, and the CID conditions for each ganglioside were optimized as follows: GM1, GM2, and GM3 (-95 V), GD1, GD2, and GD3 (-55 V), GT1, GT2, GT3, and GQ1 (-60 V). Precursor and product ion pairs for MRM analysis were selected by these MS / MS spectra. The following MRM m / z transitions were monitored: parent ion to sialic acid ion (SA - ) or sialic acid-containing sugar chains of gangliosides. For the analysis of regioisomeric gangliosides, specific fragments originating from the terminal regions of the sugar chains in these isomers were selected and operated under the same conditions as for the MRM analysis of each ganglioside.

[0232] Example 2: LC-MS Assay in Liquid Biopsy - Ganglioside Levels

[0233] Liquid biopsies from 6 cancer patients (2 melanoma patients, 2 renal cancer patients, 2 small cell lung cancer patients (SCLC_newly diagnosed untreated) and 3 non-cancer controls (2 donors studied individually and 1 pooled plasma from 30 donors) were analyzed using nLC-ESI-MS / MS (LC-MS) (Tables 5 and 6). The LC-MS method was developed to simultaneously investigate all tumor gangliosides (e.g., cancer ganglioside matrix, including 20 tumor gangliosides out of at least 5,000 analytes).

[0234] Cancer patients show elevated tumor gangliosides, which are specific to each cancer. Melanoma samples showed significant increases in GM2, GD3, and GD1b levels compared to non-cancer samples, as assessed by LC-MS; renal cancer samples showed significant increases in GD3 and GD2 levels; and lung cancer samples showed significant increases in GD3 and GM2 levels. In a study of ovarian cancer samples specifically evaluating GD3, 12 / 13 ovarian cancer samples showed a significant increase in GD3 compared to 2 non-cancer control samples. Control non-cancer samples showed very low or below the detection threshold level (Table 1). Internal standards include cholesterol (not shown) and phosphatidylcholine (not shown) present in all samples at high levels. Phosphatidylserine (PS), that is, a non-specific positive marker for general stress (cancer, inflammation, diabetes, infection, sepsis, apoptosis), is higher in all cancer samples than in control non-cancer samples.

[0235] Table 1: LC-MS-MS detection of gangliosides as a diagnostic for cancer.

[0236]

[0237] Data are presented as relative quantification. TMG in the serum of cancer patients has discrete lengths of its lipid chains that vary from cancer to cancer. The length can range from 16 to 44 carbons, so this feature was analyzed in detail.

[0238] The left graph (melanoma, kidney cancer, lung cancer, and non-cancer) is 1 patient per case with relative quantification. The limit of quantitative detection (LOQD) is about 1-20 units. The right graph (ovarian cancer and non-cancer) is absolute quantification (pmol / ml). Glycolipids measured in serum collected at diagnosis, the average of 13 ovarian cancer samples (4 early and 9 late) was compared with the average of 2 non-cancer samples.

[0239] Figure 2 Further shown are GD3 analytes in early and advanced ovarian cancer versus non-cancer (*p<0.05 and **p<0.01).

[0240] In both data sets (Table 1 and Figure 2 ), tumor gangliosides in serum differ quantitatively in terms of total quantification, depending on whether non-cancer samples or cancer samples (where TMG is elevated in cancer) are evaluated. FIG. 3 also shows that TMG also differs in terms of its lipid chain length, which differs qualitatively and quantitatively depending on whether non-cancer samples or cancer samples are evaluated.

[0241] Example 3: LC-MS Assays in Liquid Biopsies - Lipid Tail Length

[0242] In addition to demonstrating by LC-MS that tumor ganglioside levels are elevated in cancer patients, this paper provides the surprising and unexpected finding that tumor gangliosides have lipid tail heterogeneity patterns that are specific to each cancer.

[0243] In melanoma, GM2 with a short chain form (lipid tail) predominates and increases 10-fold relative to normal, while the long chain form increases from undetectable in normal to 90 units in cancer. For GD3, the short chain form increases 40-fold relative to normal. For GD1, the short chain form increases from undetectable in normal to 70 units. GD3 and GD2 of renal cancer increase significantly (particularly the short lipid chain form). GD3 of lung cancer increases significantly or changes (particularly the long lipid chain form) and has a change of GM2 to the short lipid chain form (Table 2). The markers of all control samples are very low or below the threshold, but normal gangliosides such as GM1 are detected.

[0244] As shown herein, the present disclosure uses LC-MS to detect and quantify the lipid variable length of gangliosides, and establishes a correlation between lipid tail length and cancer diagnosis. In addition, as further shown herein, the LC-MS method detects other ganglioside tumor markers such as GM2 and GD1, which are currently not quantified in ELISA. Detection of specific markers for many tumor gangliosides (e.g., cancer ganglionic stroma) is valuable for expanded applications and detection / diagnosis / prognosis of tumors.

[0245] Table 2: LC-MS-MS detection of gangliosides as diagnostics for human cancer.

[0246]

[0247] Serum glycolipids and lipids were studied at diagnosis. Only relevant data compared to normal controls are shown in relative units. For simplicity, lipid tails are short (14-24 carbons) or long (26-38 carbons). Examples shown include melanoma with extensive metastatic disease; renal cancer and non-small cell lung cancer. Patients were compared to average values ​​of non-cancer donors. Data examples from approximately 5,000 analytes (significant changes from normal are shown in bold). Other gangliosides remain unchanged or are undetectable. The currently estimated limit of quantifiable detection (LOQD) is approximately 40 units or 0.010-0.24pmol / mL.

[0248] Example 4: LC-MS assay in liquid biopsy identified GD3 as a molecule with acyl chains of 16-24 carbons in length. An important biomarker for ovarian cancer

[0249] To address the need to quantify ganglioside levels and furthermore to identify the molecular identity of potential biomarkers, a reversed phase liquid chromatography-mass spectrometry method 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, 5 volumes of cold methanol were added, centrifuged to precipitate proteins and undesirable molecules, and a single phase supernatant including gangliosides was collected.

[0250] The total level of GD3 is assessed, and the lipid species of GD3 are assessed. The data show that (a) the total concentration of GD3 (in nM) increases in cancer, and (b) the increased GD3 is a species with a short acyl-lipid chain with a length range of C16-C24. Short acyl chains are defined in the title of Table 2 (see above). These data show that when lipid species increase in cancer, lipid species can be identified, and a measurement method can be used to identify cancer. In the present embodiment, the GD3 species with short acyl chains has been identified as increasing in ovarian cancer.

[0251] Table 3: LC-MS / MS detection of gangliosides as diagnostics for human cancer.

[0252]

[0253] Total GD3 was increased in ovarian cancer (OVCA) serum compared to serum collected from patients with benign gynecological tumors or from normal healthy individuals (n=5 for each, averaged). The acyl chains of GD3 increased in OVCA had a range of C16-C24 carbon lengths (defined and classified as "short" lipids herein) and were shown to be increased 2.4-fold in cancer (normalized to normal).

[0254] Example 5: LC-ESI-MS / MS assay in liquid biopsy identifies certain lipid forms, such as GD3 (d18:1 / 23:0), as an important biomarker for ovarian cancer

[0255] To address the need to quantify ganglioside levels and identify the molecular identity of potential biomarkers, a reversed-phase nanopore liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS / MS) method was used for the disialogangliosides GD1b, GD2, and GD3. Levels of all GD3, GD1, and GD1b species (with one exception) were comparable between all groups.

[0256] Notably, GD3 (d18:1 / 23:0) levels were below the limit of detection in healthy controls but were robustly detected at high abundance in the sera of both ovarian cancer patients and patients with borderline tumors.

[0257] Table 4: LC-ESI-MS / MS detection and quantification of disialoganglioside (GD).

[0258] 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 measuring each 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 analyte, and IDA-EPI scans were collected in a linear ion trap, allowing complete structural characterization of each ganglioside species. For statistical purposes, when the control values ​​of GD3 (d18:1 / 23:0) were lower than the LLOD values, they were assigned to values ​​2 times lower than the LLOD.

[0259]

[0260]

[0261] Example 6: Pan-cancer screening based on LC / MS liquid biopsy test for tumor ganglioside quantification

[0262] A major challenge in cancer research is the early detection of tumors when treatment therapies are more likely to be successful. For some cancers, it may take decades for a tumor to progress to advanced disease, whereas in others, it may take only months. 1,2 Early detection offers tremendous opportunity to intervene before a cancer progresses to advanced disease or metastasizes to distant sites or acquires additional mutations or heterogeneity, making 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, and have limited sensitivity and specificity. This short list highlights the lack of effective early screening tests for all other types of cancer. 3

[0263] Recent technologies focus on genomics (using next-generation sequencing for circulating tumor DNA) and proteomics, enabling liquid biopsy blood tests that detect signals from multiple cancers. This is called multicancer early detection (MCED), and some have promising initial screening results, such as CancerSEEK, PanSeer, and Galleri. 4–7

[0264] However, the diagnostic performance of these tests is suboptimal, especially at early-stage cancers. For example, the overall sensitivity of the Galleri test across 12 cancer types and stages is 51.5% (39% stage I to 92% stage IV). 7 These tests have not been shown to improve clinical outcomes. 8 Modeled performance of MCED tests in representative populations suggests that if combined with usual care, they could significantly reduce overall cancer mortality by enabling stage-based metastatic diagnosis. 9 A key hurdle for these emerging liquid biopsy-based diagnostic MCED technologies is that these technologies must have very high specificity to minimize overtreatment while having sufficiently high sensitivity to improve outcomes. 8

[0265] Liquid biopsy assays based on liquid chromatography-mass spectrometry (LC-MS) technology are provided herein. The assay relates to a novel liquid biopsy method that quantifies a previously undeveloped family of biomarkers called tumor marker glycolipids (TMG) from blood samples. TMG has almost exclusively persistent and homogeneous expression in cancer, expression is not downregulated, and has etiological effects in cancer that make it an ideal target for use as a biomarker. Although genomics and proteomics have been used as tools in precision medicine; it is foreseen that the glycomics of TMG will become the next frontier for diagnosis and screening.

[0266] Tumor marker glycolipid (TMG)

[0267] All cells are covered with a dense coat of glycans, which are carbohydrate chains of variable length and structure, which can be bound to proteins or lipids. 10–12 Glycolipids are lipids with covalently attached carbohydrate chains. Glycolipids can be components of cell membranes, where the lipids are located 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 basic roles of glycolipids are to maintain cell membrane stability and to facilitate cell recognition, intercellular communication, tissue formation11,14,15 and immune responses. 15 A subgroup of glycolipids are gangliosides, which have a carbohydrate head tree including at least one sialic acid, which is linked to a sphingosine ceramide tail consisting of 2 lipids embedded in the membrane.

[0268] Gangliosides are a family of >40 different sialic acid-containing glycosphingolipids. Each glycan head is structurally unique and defines each ganglioside by its name. Some gangliosides are ubiquitous and present in normal cells, while others are tumor markers that are low / absent in normal cells and expressed at high levels in embryonic tissues and cancers. 16–22

[0269] The expression of gangliosides in certain species has been described to change during cell proliferation, differentiation, and tumor formation. 17 Aberrant and elevated expression of gangliosides has also been observed in different types of cancer cells. A subgroup of gangliosides, termed TMG, includes a family of approximately 20 different gangliosides that are preferentially or almost exclusively present at high densities on the cell surface of certain cancers, such as cancer ganglionic matrix (CGM). 11

[0270] As the term “tumor marker” implies, TMG expression is closely associated with malignancy. The expression of tumor gangliosides provides a survival advantage to tumors, as some TMGs are known to provide tumors with immune evasion or immunosuppression, growth factor-independent growth, and improved blood supply, all of which promote tumor growth, metastasis, and survival. 17 In addition, gangliosides are actively released from tumor cell membranes, thereby systemically impairing antitumor immunity. 17

[0271] Table 5: Tumor Marker Ganglioside Targets. Adapted from Bartish et al.11 Selected cancers where there was evidence of TMG expression in >50% of all patients in the indicated malignancy, exemplifying the prevalence of TMG. Cells not entered reflect a prevalence of ≤50% or were omitted from literature deemed unreliable because very few biopsies were phenotyped. The range listed is from approximately 95% (neuroblastoma) to 80% (melanoma), to approximately 50% (head and neck) of patients. When expressed in patients, TMG is uniformly present in tumor nodules and cells.

[0272]

[0273]

[0274] Furthermore, TMG is present in extracellular fluids and can also be detected in serum, as it can be shed from tumor membranes81–89. However, surprisingly, although TMG in tissue or serum appears to be an ideal and high-value etiological biomarker, TMG has not yet been fully utilized in the diagnosis of any cancer.

[0275] Liquid chromatography-mass spectrometry (LC-MS)

[0276] The LC-MS method allows the analysis of multiple ganglioside analytes, so all tumor marker gangliosides can be evaluated, including GD2, GD3, GD1b, GT1b, fucosyl-GM1, GloboH, polysialic acid, GM2, GM3, sialyl-LewisX, sialyl-LewisY, sialyl-LewisA, sialyl-LewisB and LewisY. In addition, LC-MS allows the physical properties of tumor gangliosides to be examined, such as ganglioside lipid length and saturation state heterogeneity. These features of the lipid portion of gangliosides can produce hundreds of permutations (for example, carbon chain lengths vary from 14-40, and unsaturation states range from 0 to 2). These features have never been elucidated in detail, let alone used for diagnosis. Early proof of concept data showed that one could identify unique features of lipid length and saturation that were specific and limited to specific cancers and could be used as another diagnostic layer.

[0277] result

[0278] Liquid biopsies from 6 cancer patients (2 melanoma patients, 2 renal cancer patients, 2 small cell lung cancer patients (newly diagnosed, treatment-naive SCLC patients) and 3 non-cancer controls (2 donors studied individually and 1 pooled plasma from 30 donors) were analyzed using nLC-ESI-MS / MS (LC-MS). The LC-MS method was developed to simultaneously investigate all tumor gangliosides (e.g., cancer ganglioside matrix, including 20 tumor gangliosides with at least 5,000 analytes).

[0279] Cancer patients have elevated TMG and lipid tail heterogeneity patterns, which seem to be specific to each cancer. GM2 and GD3 and GD1 of melanoma significantly increase. For GM2, the short-chain form increased 10 times relative to normal, and the long-chain form increased from 90 units in cancer to less than normal detection. For GD3, the short-chain form increased 40 times relative to normal. For GD1, the short-chain form increased from less than normal detection to 70 units. GD3 and GD2 of renal carcinoma significantly increase (particularly short lipid chain form). The GD3 of lung cancer significantly increases or changes (particularly long lipid chain form) and has the change of GM2 to short lipid chain form. The marking of all control samples is very low or below the threshold, but normal gangliosides such as GM1 are detected. Internal standard (not shown) includes cholesterol and phosphatidylcholine present in all samples at high levels. Phosphatidylserine (PS) is a non-specific positive marker for general stress (cancer, inflammation, diabetes, infection, sepsis, apoptosis).

[0280] Melanoma, renal cancer, lung cancer and non-cancer were 1 patient each with relative quantification (Table 1). The quantifiable limit of detection (LOQD) was approximately 1-20 units.

[0281] In another LC-MS study, 12 / 13 OC serum samples had significantly increased GD3 compared to 2 non-cancer serum samples. Controls were very low or below threshold ( Figure 2 ).

[0282] These studies demonstrate the potential of LC-MS for quantification of GD2 / GD3. The findings demonstrate up-regulated expression of GD3 during both stages and suggest its utility in early screening of ovarian cancer.

[0283] In both data sets, tumor gangliosides in serum differed quantitatively in terms of total quantification, depending on whether non-cancer or cancer samples (where TMG is elevated in cancer) were assessed.

[0284] GD3 (d18:1 / 23:0)-an important biomarker for ovarian cancer.

[0285] To address the need to identify the molecular identity of lipid forms, a reversed-phase nanopore liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS / MS) approach was used for GD2 and GD3. Interestingly, 70% of GD3 in the serum of OC patients had only one type of relatively short lipid chain (Table 4).

[0286] This signature can be used to distinguish one cancer from another. Additional bioinformatics analysis showed that GD3 ( Figure 2) have discrete and highly restricted lipid chain lengths, while other gangliosides are highly heterogeneous combinations of chain lengths. This emphasizes the importance of LC-MS analysis in asymptomatic screening. Notably, among more than 500 possible lipid form permutations, the GD3 (d18:1 / 23:0) isomer was the most represented, accounting for 70% of the total GD3 mass in 12 of the 13 OC patients (127 / 181; p=0.0005) (Table 4).

[0287] The results provided herein show exemplary data for a pan-cancer liquid biopsy test for multiple indications for detecting TMG isolated from patient serum. By exploiting the characteristics of lipid tail heterogeneity, tumor origin can be identified. This is a pioneering new cancer screening that uses advances in lipidomics science to detect specific cancer signals (changes in tumor marker gangliosides). Compared to existing technologies, the new method will lay the foundation for a new cancer screening paradigm with potentially higher specificity and clinically useful sensitivity.

[0288] These data further establish a pan-cancer early detection screening test. Liquid biopsy technology provides cancer screening at a population level, with great potential to help people get effective treatment earlier and substantially reduce overall cancer mortality.

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[0379] Incorporated by Reference

[0380] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0381] Also incorporated by reference in their entirety are any polynucleotide and amino acid sequences referenced by accession numbers associated with entries in public databases, such as those maintained by The Institute for Genomic Research (TIGR) at tigr.org on the World Wide Web and / or the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov on the World Wide Web.

[0382] Equivalent solutions

[0383] 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. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for detecting the presence, level and / or lipid length of at least one ganglioside and / or at least one lipid form of a ganglioside, the method comprising detecting the ganglioside and / or the lipid form of the at least one ganglioside in a sample using mass spectrometry, optionally wherein the sample is from a subject having cancer, a subject suspected of having cancer, or a subject without cancer.

2. The method of claim 1, wherein the mass spectrometry is selected from the group consisting of LC-ESI-MS / MS, LC-ESI-CID-MS / MS, nanopore LC-ESI-MS, and nanopore LC-ESI-MS / MS.

3. A method of diagnosing cancer in a subject, the method comprising: a) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from a subject using mass spectrometry according to claim 1 or 2; and b) comparing said level of said at least one ganglioside and / or said at least one lipid form of a ganglioside with the level of at least one ganglioside and / or at least one lipid form of a ganglioside in a control sample, Wherein a significantly higher level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample as compared to the control sample indicates that the subject has cancer.

4. A method for determining the grade of cancer, the method comprising: a) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from a subject using mass spectrometry according to claim 1 or 2; and b) comparing said level of said at least one ganglioside and / or said at least one lipid form of a ganglioside with the level of at least one ganglioside and / or at least one lipid form of a ganglioside in a control sample, wherein an increase of at least 100% and not more than 200% in said level of said at least one ganglioside and / or said lipid form of said at least one ganglioside in said subject sample as compared to said level in said control sample indicates that said subject has a low-grade cancer; and / or wherein an increase of at least 200% in said level of said at least one ganglioside and / or said lipid form of said at least one ganglioside in said subject sample as compared to said level in said control sample indicates that said subject has an advanced cancer.

5. A method for determining the tumor burden of a cancer, the method comprising: a) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from a subject using mass spectrometry according to claim 1 or 2; and b) comparing said level of said at least one ganglioside and / or said at least one lipid form of a ganglioside with the level of at least one ganglioside and / or at least one lipid form of a ganglioside in a control sample, wherein an increase of at least 100% and not more than 200% in said level of said at least one ganglioside and / or said lipid form of said at least one ganglioside in said subject sample as compared to said level in said control sample indicates that said subject has a low tumor burden; and / or wherein an increase of at least 200% in said level of said at least one ganglioside and / or said lipid form of said at least one ganglioside in said subject sample compared to said level in said control sample indicates that said subject has a high tumor burden.

6. A method for detecting recurrence of cancer in a subject, the method comprising: a) obtaining a sample from a subject whose cancer has regressed after receiving cancer treatment or providing a sample from said subject; b) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from the subject using mass spectrometry according to claim 1 or 2; and c) comparing said level of said at least one ganglioside and / or said at least one lipid form of a ganglioside with the level of at least one ganglioside and / or at least one lipid form of a ganglioside in a control sample, wherein a significantly higher level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample compared to the level in the control sample indicates a recurrence of cancer in the subject.

7. A method for detecting minimal residual disease in a subject, the method comprising: a) obtaining a sample from a subject in remission or providing a sample from said subject; b) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from the subject using mass spectrometry according to claim 1 or 2; and c) comparing said level of said at least one ganglioside and / or said at least one lipid form of a ganglioside with the level of at least one ganglioside and / or at least one lipid form of a ganglioside in a control sample, wherein a significantly higher level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample as compared to the level in the control sample indicates that the subject has minimal residual disease.

8. A method of stratifying subjects suffering from cancer according to benefit from cancer therapy, the method comprising: a) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from a subject administered cancer therapy using mass spectrometry according to claim 1 or 2; b) determining the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control; and c) comparing said levels of said at least one ganglioside and / or said at least one lipid form of ganglioside detected in step a) and step b); Wherein said level of said at least one ganglioside and / or a lipid form of said at least one ganglioside in said subject sample as compared to said level in said control is not significantly changed or is decreased indicates that said subject having said cancer will benefit from said cancer therapy.

9. A method of determining whether a subject having cancer will likely respond to a cancer therapy, the method comprising: a) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from a subject administered cancer therapy using mass spectrometry according to claim 1 or 2; b) determining the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control; and c) comparing said levels of said at least one ganglioside and / or said at least one lipid form of ganglioside detected in step a) and step b); wherein a significantly higher level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the subject sample as compared to the level in the control indicates that the subject having the cancer will not respond to the cancer therapy; and / or Wherein said level of said at least one ganglioside and / or a lipid form of said at least one ganglioside in said subject sample as compared to said level in said control is not significantly changed or is decreased indicates that said subject having said cancer will respond to said cancer therapy.

10. A method for predicting clinical outcome in a subject suffering from cancer, the method comprising: a) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from a subject using mass spectrometry according to claim 1 or 2; b) determining the level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in a control; and c) comparing said levels of said at least one ganglioside and / or of said at least one lipid form of ganglioside determined in step a) and step b); wherein a significantly higher level of the at least one ganglioside and / or the lipid form of the at least one ganglioside in the sample from the subject compared to the level in the control indicates that the subject has a poor clinical outcome.

11. A method of monitoring the progression of cancer in a subject, the method comprising: a) detecting the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a sample from a subject at a first time point using mass spectrometry according to claim 1 or 2; b) repeating step a) at a subsequent time point; and c) comparing said levels of said at least one ganglioside and / or lipid form of said at least one ganglioside detected in step a) and step b) to monitor the progression of cancer in said subject, optionally wherein said subject is at risk of developing cancer.

12. The method of claim 11, wherein between the first time point and the subsequent time point, the subject has received cancer therapy.

13. A method of evaluating the efficacy of a cancer therapy in a subject suffering from cancer, the method comprising: a) determining the level of at least one ganglioside and / or a lipid form of at least one ganglioside in a first sample obtained from a subject using mass spectrometry according to claim 1 or 2; b) repeating step a) at at least one subsequent time point after administration of said cancer therapy; and c) comparing the levels of said at least one ganglioside and / or the lipid form of said at least one ganglioside detected in step a) and step b), wherein a significantly lower level of the at least one ganglioside and / or a lipid form of the at least one ganglioside in at least one subsequent sample relative to the first sample indicates that the therapy is effective in treating cancer in the subject.

14. The method according to any one of claims 11 to 13, wherein the first sample and / or the at least one subsequent sample is part of a single sample or a pooled sample obtained from the subject.

15. A method of diagnosing cancer in a subject, the method comprising: a) determining the lipid length of at least one ganglioside in a sample of a subject using mass spectrometry according to claim 1 or 2; as well as b) comparing said lipid length of said at least one ganglioside to the lipid length of at least one ganglioside in a control sample, wherein a significant change in the lipid length heterogeneity of the at least one ganglioside in the subject sample compared to the control sample indicates that the subject has cancer.

16. A method of determining the grade of cancer, the method comprising: a) determining the lipid length of at least one ganglioside in a sample of a subject using mass spectrometry according to claim 1 or 2; as well as b) comparing said lipid length of said at least one ganglioside to the lipid length of at least one ganglioside in a control sample, wherein a change (e.g., an increase or decrease) of at least 100% and no more than 200% in the heterogeneity of the lipid length of the at least one ganglioside in the subject sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates that the subject has a low-grade cancer; and / or wherein a change (e.g., an increase or decrease) of at least 200% in the heterogeneity of the lipid length of the at least one ganglioside in the subject sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates that the subject has an advanced cancer.

17. A method of determining tumor burden of cancer, the method comprising: a) determining the lipid length of at least one ganglioside in a sample of a subject using mass spectrometry according to claim 1 or 2; as well as b) comparing said lipid length of said at least one ganglioside to the lipid length of at least one ganglioside in a control sample, wherein a change (e.g., an increase or decrease) of at least 100% and no more than 200% in the heterogeneity of the lipid length of the at least one ganglioside in the subject sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates that the subject has a low tumor burden; and / or wherein at least a 200% change (e.g., an increase or decrease) in the heterogeneity of the lipid length of the at least one ganglioside in the subject sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates that the subject has a high tumor burden.

18. A method for detecting recurrence of cancer in a subject, the method comprising: a) obtaining a sample from a subject whose cancer has regressed after receiving cancer treatment or providing a sample from said subject; b) determining the lipid length of at least one ganglioside in the subject sample using mass spectrometry according to claim 1 or 2; and c) comparing said lipid length of said at least one ganglioside to the lipid length of at least one ganglioside in a control sample, wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the subject sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates recurrence of cancer in the subject.

19. A method for detecting minimal residual disease in a subject, the method comprising: a) obtaining a sample from a subject in remission or providing a sample from said subject; b) determining the lipid length of at least one ganglioside in the subject sample using mass spectrometry according to claim 1 or 2; and c) comparing said lipid length of said at least one ganglioside to the lipid length of at least one ganglioside in a control sample, wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the subject sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates that the subject has minimal residual disease.

20. A method of stratifying subjects having cancer according to benefit from a cancer therapy (e.g., immunotherapy), the method comprising: a) determining the lipid length of at least one ganglioside in a sample from a subject administered cancer therapy using mass spectrometry according to claim 1 or 2; b) determining the lipid length of the at least one ganglioside in the control; and c) comparing the lipid length of the at least one ganglioside detected in step a) and step b); Wherein the heterogeneity of the lipid length of the at least one ganglioside in the subject sample is not significantly changed compared to the heterogeneity of the lipid length of the at least one ganglioside in the control indicates that the subject having the cancer will benefit from the cancer therapy.

21. A method of determining whether a subject having cancer will likely respond to a cancer therapy (e.g., immunotherapy), the method comprising: a) determining the lipid length of at least one ganglioside in a sample from a subject administered cancer therapy using mass spectrometry according to claim 1 or 2; b) determining the lipid length of the at least one ganglioside in the control; and c) comparing the lipid length of the at least one ganglioside detected in step a) and step b); wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the subject sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control indicates that the subject having the cancer will not respond to the cancer therapy; and / or Wherein the heterogeneity of the lipid length of the at least one ganglioside in the subject sample is not significantly changed compared to the heterogeneity of the lipid length of the at least one ganglioside in the control indicates that the subject having the cancer will respond to the cancer therapy.

22. A method for predicting clinical outcome in a subject suffering from cancer, the method comprising: a) determining the lipid length of at least one ganglioside in a sample of a subject using mass spectrometry according to claim 1 or 2; b) determining the lipid length of the at least one ganglioside in the control; and c) comparing the lipid lengths of the at least one ganglioside determined in step a) and step b); wherein a significant change in the heterogeneity of the lipid length of the at least one ganglioside in the subject sample compared to the heterogeneity of the lipid length of the at least one ganglioside in the control sample indicates that the subject has a poor clinical outcome.

23. A method of monitoring the progression of cancer in a subject, the method comprising: a) detecting the lipid length of at least one ganglioside in a sample of a subject at a first time point using mass spectrometry according to claim 1 or 2; b) repeating step a) at a subsequent time point; and c) comparing the heterogeneity of said lipid length of said at least one ganglioside detected in step a) and step b) to monitor the progression of cancer in said subject, optionally wherein said subject is at risk of developing cancer.

24. The method of claim 23, wherein between the first time point and the subsequent time point, the subject has received cancer therapy.

25. A method of evaluating the efficacy of a cancer therapy in a subject suffering from cancer, the method comprising: a) determining the lipid length of at least one ganglioside in a first sample obtained from the subject using mass spectrometry according to claim 1 or 2; b) repeating step a) at at least one subsequent time point after administration of said cancer therapy; and wherein a significant change in the lipid length heterogeneity of the at least one ganglioside in the second sample relative to the first sample indicates that the therapy is effective in treating cancer in the subject.

26. The method of any one of claims 23 to 25, wherein the first sample and / or at least one subsequent sample is part of a single sample or a pooled sample obtained from the subject.

27. The method of any one of claims 3 to 26, further comprising treating the subject with a cancer therapy (eg, recommending, prescribing, and / or administering a cancer therapy to the subject).

28. The method of any one of claims 8, 9, 12 to 14, 20, 21, and 24 to 27, wherein 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.

29. The method of claim 28, wherein the immunotherapy is immune checkpoint inhibition therapy.

30. The method of any one of claims 8, 9, 12-14, 20, 21, and 24-29, wherein the cancer therapy is avelumab, durvalumab, atezolizumab, a BRAF / MEK inhibitor, pembrolizumab, nivolumab, ipilimumab, or a combination thereof.

31. The method according to any one of claims 1 to 30, wherein the at least one ganglioside and / or the lipid form of the at least one ganglioside is a tumor-associated ganglioside and / or a lipid form of a tumor-associated ganglioside.

32. The method according to any one of claims 1 to 31, wherein the gangliosides include 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 portion thereof, any lipid form thereof, or any combination thereof.

33. The method of any one of claims 1 to 32, wherein the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises GM2, GD3, GD2, GDlb, any lipid form thereof, or any combination thereof.

34. The method of any one of claims 1 to 34, wherein the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises a combination of two or more selected from GM2, GD3, GD2, GD1b, and any lipid form thereof.

35. The method of any one of claims 1 to 34, wherein the at least one ganglioside and / or the lipid form of the at least one 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.

36. The method according to any one of claims 1 to 35, wherein the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises GD3 (d18:1 / 23:0) and / or GD3 (d18:1 / 24:1).

37. The method according to any one of claims 1 to 36, wherein the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises GD3 (d18:1 / 23:0).

38. The method according to any one of claims 1 to 36, wherein the at least one ganglioside and / or the lipid form of the at least one ganglioside comprises an acyl chain 24:

1.

39. The method of claim 38, wherein the acyl chain 24:1 is present in GD3 and / or GD1b.

40. The method of any one of claims 1 to 39, wherein 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, 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.

41. The method of any one of claims 1 to 40, wherein the cancer or tumor is ovarian cancer, melanoma, renal cancer, or lung cancer, optionally wherein the cancer or tumor is ovarian cancer.

42. The method of any one of claims 1 to 41, wherein the cancer is a borderline tumor.

43. The method of any one of claims 1 to 42, wherein the sample comprises cells, serum, blood, peritumoral tissue and / or intratumoral tissue obtained from the subject.

44. The method of any one of claims 1 to 43, wherein the sample comprises serum or blood.

45. The method of any one of claims 3 to 44, wherein the significantly higher level of the at least one ganglioside comprises an increase of at least 20% of the level of the at least one ganglioside.

46. ​​The method of any one of claims 3 to 45, wherein the significantly lower level of the at least one ganglioside comprises at least a 20% reduction in the level of the at least one ganglioside.

47. The method of any one of claims 3 to 46, wherein the control sample is a sample from a cancer-free subject.

48. The method of any one of claims 3 to 47, wherein the control sample is a sample from the subject (e.g., the first sample collected from the subject in a longitudinal collection to assess changes in the level of the at least one ganglioside over time).

49. The method of any one of claims 3 to 48, wherein the subject is a mammal (eg, a subject suffering from cancer, eg, an asymptomatic subject).

50. The method of any one of claims 3 to 49, wherein the subject is a cancer animal model, a dog, a cat, or a human.

51. The method of any one of claims 3 to 50, wherein the subject is a human.

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