Biomarkers for diagnosis and prognosis of cancer

Abstract

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

Description

[0001] AOS-00725

[0002] BIOMARKERS FOR DIAGNOSIS AND PROGNOSIS OF CANCER

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 689,288, filed on August 30, 2024, the entire content of which is incorporated herein in its entirety by this reference.

[0005] Background of the Invention

[0006] Cancer involves abnormal cell growth with the potential to invade or spread to other parts of the body. Despite decades of cancer research, cancer continues to cause a significant number of deaths (-1,600 deaths per day in the U.S. in 2020) largely due to a lack of means for accurate detection. Accordingly, there is a great need for new biomarkers, e.g., in cancer tissue and / or in blood, to detect cancer and all stages thereof, which would improve the survival of cancer patients.

[0007] Summary of the Inventions

[0008] Gangliosides are glycolipids that comprise a carbohydrate structure and lipid tails that can vary in carbon chain length. The present disclosure is based, at least in part, on the discovery that specific ratios of different gangliosides or lipoforms thereof are useful biomarkers for cancer diagnosis and prognosis. The present disclosure is also based on the discovery that certain gangliosides or lipoforms thereof as well as non-ganglioside biomarkers (e.g., B3GALT4) are useful biomarkers for cancer diagnosis and prognosis.

[0009] Provided herein are methods for cancer diagnosis and prognosis. The surprising and unexpected altered level, activity, and / or ratios of the gangliosides or lipoforms thereof described herein provide novel biomarkers for cancer diagnosis and prognosis.

[0010] Brief Description of Figures

[0011] Fig. lA-Fig. IB show that neuroblastoma stage 2b and late-stage ovarian cancer show elevated levels of GD3. In Fig. 1A, the relative abundance of various individual GD3 species was detected using UHPLC-MS in human serum from healthy controls (n=70), stage 2a (n=10), and stage 2b (n=5) neuroblastoma, early-stage ovarian cancer (n=8), and late-stage ovarian cancer (n=27). The total relative abundance of all detected GD3 species was summed to approximate total GD3 levels (bottom right). In Fig. IB, the relative abundance of various individual GD3 species was detected using UHPLC-MS in human AOS-00725 serum from healthy controls (n=70), stage 2a (n=10) and stage 2b (n=5) neuroblastoma, stage I ovarian cancer (n=5), stage II ovarian cancer (n=3), stage III ovarian cancer (n=21), and stage IV ovarian cancer (n=6). The total relative abundance of all detected GD3 species was summed to approximate total GD3 levels (bottom right).

[0012] Fig. 2A-Fig. 2B show that late-stage ovarian cancer and neuroblastoma stage 2b show elevated levels of GD2. In Fig. 2A, the relative abundance of various individual GD2 species was detected using UHPLC-MS in human serum from healthy controls (n=70), stage 2a (n=10) neuroblastoma, stage 2b (n=5) neuroblastoma, early-stage ovarian cancer (n=8), and late-stage ovarian cancer (n=27). The total relative abundance of all detected GD2 species was summed to approximate total GD2 levels (bottom right). In Fig. 2B, the relative abundance of various individual GD2 species was detected using UHPLC-MS in human serum from healthy controls (n=70), stage 2a (n=10) and stage 2b (n=5) neuroblastoma, stage I ovarian cancer (n=5), stage II ovarian cancer (n=3), stage III ovarian cancer (n=21), and stage IV ovarian cancer (n=6). The total relative abundance of all detected GD2 species was summed to approximate total GD2 levels (bottom right).

[0013] Fig. 3A shows that ovarian cancer (e.g., stage II) and neuroblastoma stage 2a show decreased levels of GDI, neuroblastoma stage 2b shows elevated levels of GDI. The relative abundance of various individual GD2 species was detected using UHPLC-MS in human serum from healthy controls (n=70), stage 2a (n=10) neuroblastoma, stage 2b (n=5) neuroblastoma, early-stage ovarian cancer (n=8), and late-stage ovarian cancer (n=27). The total relative abundance of all detected GD 1 species was summed to approximate total GD 1 levels (bottom right).

[0014] Fig. 3B shows that neuroblastoma stage 2b and certain stages (e.g., stage I and late- stage) of ovarian cancer show elevated levels of GDI. The relative abundance of various individual GDI species was detected using UHPLC-MS in human serum from healthy controls (n=70), stage 2a (n=10) neuroblastoma, stage 2b (n=5) neuroblastoma, stage I ovarian cancer (n=5), stage II ovarian cancer (n=3), stage III ovarian cancer (n=21), and stage IV ovarian cancer (n=6). The total relative abundance of all detected GDI species was summed to approximate total GDI levels (bottom right).

[0015] Fig. 4A-Fig. 4B show that ovarian cancer serum shows altered disialoganglioside profiles compared to healthy serum. Fig. 4A shows a Partial Least Squares Discriminant Analysis (PLSDA) plot comparing serum from all healthy patients (left circle; green) to all AOS-00725 patients with ovarian cancer (right circle; blue). Fig. 4B shows a heat map comparing serum from all healthy patients (red) to all patients with ovarian cancer (green).

[0016] Fig. 5 shows that multiple species of disialo-gangliosides are altered in the serum of ovarian cancer patients compared to healthy controls. Volcano plot displays altered gangliosides, including subclasses GDI, GD2, and GD3.

[0017] Fig. 6A-Fig. 6C show that the ratio of GD2 36: 1 to GDI 36: 1 is increased in all stages of neuroblastoma and ovarian cancer. The relative abundance of GD2 36: 1 and GDI 36: 1 was detected using UHPLC-MS in human serum from healthy controls, neuroblastoma patients, and ovarian cancer patients. Fig. 6A shows the ratio of GD2 36: 1 to GDI 36: 1 in serum from healthy individuals (n=70), all neuroblastoma patients (n=15), and all ovarian cancer patients (n=35). Fig. 6B shows the ratio of GD2 36: 1 to GDI 36: 1 in serum from healthy individuals (n=70), stage 2a neuroblastoma patients (n=10), stage 2b neuroblastoma patients (n=5), early-stage ovarian cancer patients (n=8), and late-stage ovarian cancer patients (n=27). Fig. 6C shows the ratio of GD2 36: 1 to GDI 36: 1 in serum from healthy individuals (n=70), stage 2a neuroblastoma patients (n=10), stage 2b neuroblastoma patients (n=5), and ovarian cancer patients from stage I (n=5), stage II (n=3), stage III (n=21), and stage IV (n=6). ns p > 0.05, * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0018] Fig. 7A-Fig. 7C show that the ratio of GDI 36: 1 to GD2 36: 1 is decreased in all stages of ovarian cancer and neuroblastoma. The relative abundance of GDI 36: 1 and GD2 36: 1 was detected using UHPLC-MS in human serum from healthy controls, neuroblastoma patients, and ovarian cancer patients. Fig. 7A shows the ratio of GDI 36: 1 to GD2 36: 1 in serum from healthy individuals (n=70), all neuroblastoma patients (n=15), and all ovarian cancer patients (n=35). Fig. 7B shows the ratio of GDI 36: 1 to GD2 36: 1 in serum from healthy individuals (n=70), stage 2a neuroblastoma patients (n=10), stage 2b neuroblastoma patients (n=5), early-stage ovarian cancer patients (n=8), and late-stage ovarian cancer patients (n=27). Fig. 7C shows the ratio of GDI 36: 1 to GD2 36: 1 in serum from healthy individuals (n=70), stage 2a neuroblastoma patients (n=10), stage 2b neuroblastoma patients (n=5), and ovarian cancer patients from stage I (n=5), stage II (n=3), stage III (n=21), and stage IV (n=6). ns p > 0.05, * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0019] Fig. 8 shows a synthetic pathway of various gangliosides.

[0020] Fig. 9 shows the protein expression level of B3GALT4 in various cancers. AOS-00725

[0021] Fig. 10 shows the RNA expression level of B3GALT4 in various cancers. 5.4 FPKM is the calculated median across all female tissue samples, and many cancers show the RNA expression level that is lower than the median.

[0022] Fig. IIA-Fig. 11C show the correlation of the RNA expression level of B3GALT4 and survival rate. Fig. 11A shows that the high expression of B3GALT4 correlates with the decreased 5-year survival rate in ovarian cancer patients. Fig. 11B shows that the high expression of B3GALT4 correlates with the decreased 5-year survival rate in renal cancer. Fig. 11C shows that the high expression of B3GALT4 correlates with the decreased 5-year survival rate in glioma.

[0023] Fig. 12A-Fig. 12C show additional analyses. To evaluate the ability of LCMS- derived tumor-marker gangliosides (TMG) profiles to distinguish ovarian cancer (OC) from non-cancer, LCMS data were generated from serum for 70 non-cancer and 35 OC subjects (n=8 early stage, n=27 late stage), with 31 distinct TMG species measured (n=8 GDI, n=6 GD2, n=17 GD3). Samples were profiled with 3 technical replicates per specimen and averaged for comparative statistical analysis. Evaluation of OC vs. non-cancer groups showed a significant reduction in the level of GD1(36: 1) (adj.P < 0.0076) in OC, as well as a significant increase in multiple GD2 and GD3 species including GD3(41:2), GD3(39: 1), GD2(34: 1), GD2(38: 1), GD2(36: 1), and GD3(35: 1) (adj.P < 0.01 for all comparisons, see Fig. 12A (Volcano plot)). Different strategies for normalizing the LCMS data were further explored, emphasizing sample measures relative to the corresponding level of GD1(36: 1). This approach enhanced the signal between OC and non-cancer subjects, such that there was an improvement in effect size between OC and non-cancer subjects as well as an increase in statistical significance for several quantified species and improved directional trends from non-cancer, to early-stage and finally late-stage cancer (see Fig. 12B (Boxplot)). As an example, after normalization by GD1(36: 1), receiver operating characteristic (ROC) curve analysis found that GD2(36: 1) could distinguish normal from OC subjects with an area under curve (AUC) of 92% (95CI 86-98%), as well as early-stage and late-stage cancers with AUCs of 86% and 94%, respectively (see Fig. 12C (ROC P ).

[0024] Detailed Description of the Invention

[0025] Gangliosides are a family of >40 different sialic acid-containing glycosphingolipids. Each glycan tree is structurally unique and defines each ganglioside subclass by name. AOS-00725

[0026] Certain gangliosides are tumor-markers (Tumor-Marker Gangliosides; also referred to as TMGs). They are low / absent in normal cells, and are expressed at high levels in cancer.

[0027] Gangliosides 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 cell membranes and are variable in length. In addition to being present on the tumor cell surface, gangliosides can be shed into the extracellular environment.

[0028] However, studies of expression of gangliosides in tissues or in circulation have only been reported in a few patient samples. Assays to detect gangliosides that may be expressed in tissue or in serum are not quantitative or standardized; only yield estimations; and resulted in contradictory conclusions. Since gangliosides are glycolipids that can be generated via multiple biosynthetic pathways and enzymes, monitoring mutations or mRNA expression is not feasible. Gangliosides therefore remain underexploited for diagnosis of cancer.

[0029] The present disclosure provides novel biomarkers and their use in cancer diagnosis and prognosis.

[0030] Definitions

[0031] The articles “a” and “an” are used herein to refer to one or to more than one (z.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0032] The term “determining” is art recognized, and includes, without limitation, detecting or identifying.

[0033] The lipid length refers to the length of the lipid tails of a ganglioside. The lipid tails are embedded in the outer leaflet of cell membranes. The lipids can be variable in length.

[0034] The term “lipoform” of a ganglioside refers to a variant of a ganglioside that differs in its lipid (see e.g., Kolter (2012) ISRN Biochem 506160).

[0035] The term “minimal residual disease” is art recognized, and is used to describe a small number of cancer cells in the body during or after cancer treatment, when the patient is in remission. The number of remaining cells may be so small that they do not cause any physical signs or symptoms and often cannot even be detected through traditional methods. It is a major cause of relapse of cancer. AOS-00725

[0036] The term “neoadjuvant therapy” refers to a treatment given before the primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy.

[0037] The term “predictive” includes the use of a biomarker status, e.g., over- or underactivity, emergence, or expression, as well as the growth, remission, recurrence, or resistance of tumors before, during, or after therapy, for determining the likelihood of response of a cancer to a cancer therapy. Such predictive use of the biomarker may be confirmed by, e.g., (1) increased or decreased copy number (e.g., by FISH, FISH plus SKY, single-molecule sequencing, e.g, as described in the art at least at J. Biotechnol., 86:289- 301, or qPCR), overexpression or underexpression of a biomarker nucleic acid (e.g., by ISH, Northern Blot, or qPCR), increased or decreased biomarker protein (e.g., by IHC, mass spectrometry, ELISA), or increased or decreased activity, e.g., in more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed cancers types or cancer samples; (2) its absolute or relatively modulated presence or absence of a biomarker in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g. a human, afflicted with cancer; (3) its absolute or relatively modulated presence or absence of a biomarker in clinical subset of patients with cancer (e.g., those responding to the cancer therapy, or those developing resistance thereto).

[0038] The term “preventing” is art-recognized, and when used in relation to a condition, such as a viral / bacterial infection or a disease such as cancer is well understood in the art, and includes administration of a treatment, e.g., a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the treatment. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0039] The term “remission” is art recognized, and refers to a condition in which the signs and symptoms of the cancer are reduced.

[0040] As used herein, “subject” refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a cancer. The term “subject” is interchangeable AOS-00725 with “patient”. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cat, cow, chickens, amphibians, reptiles, etc.

[0041] The terms “therapeutically-effective amount” and “effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50. Compositions that exhibit large therapeutic indices are preferred. In some embodiments, the LD50 (lethal dosage) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for the agent relative to no administration of the agent. Similarly, the ED50 (z.e., the concentration which achieves a half-maximal inhibition of symptoms) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. Also, Similarly, the IC50 (z.e., the concentration which achieves half-maximal cytotoxic or cytostatic effect on cancer cells) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. In some embodiments, cancer cell growth in an assay can be inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease in a solid malignancy can be achieved.

[0042] The term “treating” is art-recognized and includes administration to the subject of one or more of the compositions of the present disclosure or those known in the art. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal), then the treatment is prophylactic (z. e. , it protects the host against developing the unwanted condition); whereas, if it is administered after AOS-00725 manifestation of the unwanted condition, the treatment is therapeutic (i. e. , it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0043] Gangliosides

[0044] Gangliosides are glycosphingolipids comprising one or more sialic acids. Glycosphingolipids contain a hydrophobic ceramide or sphingoid lipid tail, which is usually anchored to the outer leaflet of the plasma membrane. They also contain an oligosaccharide moiety and are classified according to this carbohydrate structure (ganglio, isoganglio, lacto etc.). Gangliosides are an important subclass of glycosphingolipids, because they contain negatively charged sialic acids (N-acetylneuraminic acid or N-glycolylneuraminic acid) linked to the lipooligosaccharide moiety. Gangliosides are named and classified according to the number of sialic acid residues attached (M for one, D for two, T for 3 and Q for 4) to the inner sugar moiety and according to their chromatographic mobility. The numbering of the gangliosides (5-x) is based on the number (x) of the inner sugar moieties (glucose, galactose or GalNAc) according to the original experimental classification of Svennerhohn. Thus, if x is 4, the gangliosides are: GM1, GDI, GT1, x = 3 for GM2, GD2, GT2, and x = 2 for GM3, GD3, and GT3.

[0045] Heterogeneity is not only found within the glycan part, but also within the ceramide moiety. This can consist of different sphingoid bases, sphinganine, sphingosine, and phytosphingosine of different chain lengths, which can be further modified by O- acetylation. In higher animals, Cl 8- and C20-sphingosine are the most abundant sphingoid bases of gangliosides.

[0046] The fatty acids found in the ceramide part of gangliosides are mostly saturated, a- Hydroxylated fatty acids are not frequently found in brain gangliosides, but are, for example, abundant in gangliosides from intestine, liver, or kidney, and in GM4. To specify the lipoform of a ganglioside, an exemplary designation such as (dl 8: l / 18:0)GM3 is used for a II3Neu5AcLacCer with a sphingosine (d = dihydroxy, 1 = one double bond) of 18 carbons and a stearoyl residue (18:0) within the ceramide portion (see Kolter (2012) ISRN Biochem 506160, which is incorporated herein by reference in its entirety). The functional consequences of the heterogeneities in the lipid component are largely unknown, but the lipid part can mask the receptor function of ganglioside glycans via interaction with membrane cholesterol. AOS-00725

[0047] As presented herein, certain gangliosides are tumor biomarkers. In some embodiments, a tumor-associated ganglioside, also referred to as a “tumor marker ganglioside” or a TMG, is selected from GD2, GD3, GDlb, GTlb, fucosyl-GMl, GloboH, polysialic acid (PSA), GM2, GM3, sialyl-Lewisx, sialyl-LewisY, sialyl-LewisA, sialyl- LewisB, LewisY, any portion thereof, and modified version thereof. In preferred embodiments, the tumor-associated ganglioside is GD2, GD3, GM2, GDlb, or lipoforms thereof. In some embodiments, the tumor-associated ganglioside is selected from those described herein.

[0048] Accordingly, as used herein, the term ganglioside may refer to a ganglioside, a tumor-associated ganglioside, a portion thereof, a lipoform thereof, a glycan variant thereof, or any other isoform / variant thereof.

[0049] B3GALT4

[0050] B3GALT4 (Beta- 1,3 -Galactosyltransferase 4) is a member of the beta-1, 3- galactosyltransferase (beta3GalT) gene family. This family encodes type II membranebound glycoproteins with diverse enzymatic functions using different donor substrates (UDP-galactose and UDP-N-acetylglucosamine) and different acceptor sugars (N- acetylglucosamine, galactose, N-acetylgalactosamine). The beta3GalT genes are distantly related to the Drosophila Brainiac gene and have the protein coding sequence contained in a single exon. The beta3GalT proteins also contain conserved sequences not found in the beta4GalT or alpha3GalT proteins. The carbohydrate chains synthesized by these enzymes are designated as type 1, whereas beta4GalT enzymes synthesize type 2 carbohydrate chains. The ratio of type l:type 2 chains changes during embryogenesis. By sequence similarity, the beta3GalT genes fall into at least two groups: beta3GalT4 and 4 other beta3GalT genes (beta3GalTl-3, beta3GalT5). This gene is oriented telomere to centromere in close proximity to the ribosomal protein S 18 gene. The functionality of the encoded protein is limited to ganglioseries glycolipid biosynthesis. Diseases associated with B3GALT4 include Gallbladder Papillary Carcinoma and Ehlers-Danlos Syndrome, Spondylodysplastic Type, 2.

[0051] The enzymatic activity of B3GALT4 can be assessed using a galactosyltransferase assays that are known in the art (Deng and Chen (2004) A pH-sensitive assay for galactosyltransferase, Analytical Biochemistry, 330(2):219-226; Hymes and Mullinax (1984) Assay of galactosyltransferase by high-performance liquid chromatography, AOS-00725

[0052] Analytical Biochemistry, 139(l):68-72; Taki et al. (1990) A Simple and Specific Assay of Glycosyltransferase and Glycosidase Activities by an Enzyme-Linked Immunosorbent Assay Method, and Its Application to Assay of Galactosyltransferase Activity in Sera from Patients with Cancer, Journal of Biochemistry, 107(3):493-498; Park and Shin (2007) Carbohydrate Microarrays for Assaying Galactosyltransferase Activity, Organic Letters, 9(9): 1675-1678; each of which is incorporated herein by reference in its entirety). Commercial kits are available for determining the enzymatic activity of B3GALT4, and they include: UDP-Galactosyltransferase Assay Kit (Catalog No. CS 1050; Sigma- Aldrich) and UDP-Galactosyltransferase Assay Kit (Catalog No. Kit-3311; Creative Biomart).

[0053] A person of ordinary skill would readily recognize that the copy number, the expression level, the amount (e.g., mRNA, protein), and / or the presence of at least one mutation (unless a silent mutation) of B3GALT4 affect its activity. At least one mutation in the biomarker may be present within the coding region. Alternatively, the at least one mutation in the biomarker may be present in the non-coding region. A person of ordinary skill would recognize that a mutation in a non-coding region, e.g., enhancer, promoter, or non-coding region that influences the mRNA stability or production, may reduce the activity of the biomarker by interfering with the mRNA production.

[0054] In some embodiments, the activity of B3GALT4 is an enzymatic activity. In some embodiments, the enzymatic activity is a galactosyltransferase activity.

[0055] Antibodies that bind specifically to B3GAL4T are known in the art and are commerically available, and they include: OriGene antibodies Catalog Nos. TA314796, TA373889, TA387999 ; ProSci Catalog No. 15-998 ; Antibodies Online Catalog Nos. ABIN7145371, ABIN6258541, ABIN7076137, ABIN1496798, ABIN522147, ABIN7271197, ABIN7120589, ABIN6281420, ABIN2884868, ABIN7145374 ; Novus Biologicals Catalog Nos. NBP2-15533, NBP2-92226, NBP2-14341 ; Biorbyt antibodies Catalog Nos. orb214816, orb721824 ; Boster antibodies Catalog Nos. A13136, A13136T4 ; and Abeam Catalog Nos. ab 169759, ab249506. Such antibodies can be used to determine the level of the B3GALT4 protein or a fragment thereof.

[0056] The nucleic acid copy number (e.g., DNA, mRNA, cDNA) of B3GALT4 can be determined using various techniques described herein or those known in the art (e.g., FISH, Northern, PCR, qPCR, RT-PCT, RT-qPCR) using the nucleic acid sequence provided herein (e.g., SEQ ID NO: 1) or those known in the art. AOS-00725

[0057] B3GALT4 human (GenBank: Y15061. 1) mRNA or cDNA sequence (SEQ ID NO: 1) 1 1

[0058] B3GT4_HUMAN Beta- 1,3 -galactosyltransferase 4 amino acid sequence (SEQ ID NO: 2)

[0059] 1 MQLRLFRRLL LAALLLVIVW TLFGPSGLGE ELLSLSLASL LPAPASPGPP

[0060] 51 LALPRLLI PN QEACSGPGAP PFLLILVCTA PENLNQRNAI RASWGGLREA

[0061] 101 RGLRVQTLFL LGEPNAQHPV WGSQGSDLAS ESAAQGDILQ AAFQDSYRNL

[0062] 151 TLKTLSGLNW AEKHCPMARY VLKTDDDVYV NVPELVSELV LRGGRWGQWE

[0063] 201 RSTEPQREAE QEGGQVLHSE EVPLLYLGRV HWRVNPSRTP GGRHRVSEEQ

[0064] 251 WPHTWGPFPP YASGTGYVLS ASAVQLI LKV ASRAPLLPLE DVFVGVSARR

[0065] 301 GGLAPTQCVK LAGATHYPLD RCCYGKFLLT SHRLDPWKMQ EAWKLVGGSD

[0066] 351 GERTAPFCSW FQGVLGILRC RAIAWLQS

[0067] There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

[0068] GENETIC CODE

[0069] Alanine (Ala, A) GCA, GCC, GCG, GCT

[0070] Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT

[0071] Asparagine (Asn, N) AAC, AAT

[0072] Aspartic acid (Asp, D) GAC, GAT AOS-00725

[0073] Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAG Glutamine (Gin, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGT Histidine (His, H) CAC, CAT Isoleucine (He, I) ATA, ATC, ATT Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAG Methionine (Met, M) ATG Phenylalanine (Phe, Fj TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT Threonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAC, TAT Valine (Vai, V) GTA, GTC, GTG, GTT Termination signal (end) TAA, TAG, TGA

[0074] An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0075] In view of the foregoing, the nucleotide sequence of a DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) can be used to derive the polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given AOS-00725 amino acid sequence). Thus, description and / or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and / or disclosure of a polypeptide amino acid sequence herein should be considered to also include description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0076] Finally, nucleic acid and amino acid sequence information for nucleic acid and polypeptide molecules useful in the present invention are well-known in the art and readily available on publicly available databases, such as the National Center for Biotechnology Information (NCBI).

[0077] Analyzing / Detecting Biomarkers

[0078] A. Detecting gangliosides or lipoforms thereof

[0079] The biomarkers of the present disclosure can be analyzed according to the methods described herein and other suitable techniques known in the art. The presence or the level of a biomarker (e.g., gangliosides or lipoforms thereof or non-ganglioside markers) can be detected using methods including, without limitation, immunodiffusion, immunoelectrophoresis, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, Western blot, or a slot blot assay. General techniques to be used in performing the various immunoassays noted above and other variations of the techniques, such as fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich ELISA, competitive ELISA, ELISpot (Enzyme -Linked ImmunoSpot), agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference) alone or in combination or alternatively with NMR, MALDL TOF, LC-MS / MS, gas chromatography and similar methods that are known to those of ordinary skill in the art.

[0080] Such techniques can also be used to monitor the biomarker levels on a cell, in tissues, or in blood / plasma. AOS-00725

[0081] In preferred embodiments, a mass spectrometry (e.g., MALDI-TOF, LC-MS / MS, LC-MS, LC-ESI-MS / MS, nano-LC-ESI-MS / MS (also called nLC-ESI-MS / MS or nanobore LC-ESI-MS / MS), or others known in the art) is used to detect the presence or the level of a biomarker (e.g., gangliosides or lipoforms thereof or non-ganglioside markers) in a biological specimen, such as liquid biopsy (e.g., blood, saliva, serum, cell / tissue (e.g., cancer cell); see the section on Samples). The mass spectrometry-based method is particularly useful in determining the presence or level of lipoforms of gangliosides.

[0082] Mass Spectrometry

[0083] Liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) has been frequently utilized for the sensitive and selective determination of the trace level compounds in biological samples. In particular, LC-MS / MS equipped with electrospray ionization (ESI) as the ion source is most often used method, since ESI can ionize wide range of the compounds including the polar compounds or large molecular weight compounds. Through the coupling with HPLC, the detected characteristics can be quantified by ultraviolet light or MS signal intensity.

[0084] Accordingly, any mass spectrometry -based methods can be applied to the methods of the present disclosure. Exemplary mass spectrometry -based methods include but not limited to LC-MS-based methods (e.g., LC-MS / MS, LCn-MSn), LC-MS-based methods comprising the use of ESI (e.g., LC-ESLMS, LC-ESI-MS / MS, LC-ESI-CID-MS / MS), and nanobore LC-MS-based methods (e.g., nanobore LC-ESLMS, nanobore LC-ESI-MS / MS).

[0085] In LC-ESI-MS / MS, the analytes having the appropriate hydrophobic structures can be sensitively detected, since (i) the hydrophobic ions prefer to reside at the droplet surface generated by electrospray and these ions enter the gas phase more readily than those in the droplet interior and show the higher signal intensities, (ii) The hydrophobic compounds can be well separated on the reversed phase column from salts and interfering compounds possessing suppression effects on ESI. (iii) The hydrophobic compounds are eluted by the mobile phase with the higher organic solvent content. The higher organic solvent content is suitable for the stable generation of charged droplets by electrospray and thus gives the higher signal intensities (see e.g., Santa (2013) Drug Discoveries & Therapeutics, 7:9-17, which is incorporated by reference in its entirety).

[0086] Gangliosides can detected using LC-ESI-MS / MS (see e.g., Fuller et al. (IQ Anal Biochem 458:20-26; Sorensen (2006) Rapid Commun Mass Spectrom 20:3625-33; each of which is incorporated by reference in their entirety). Gangliosides and lipforms can be AOS-00725 detected using LC-ESI-MS / MS (see, e.g., Ikeda et a / . (2008) J Lipid Res 49:2678-89), which is incorporated by reference in its entirety). LC-ESI-CID-MS / MS has been widely applied to detect gangliosides or glycan sequencing, which shows improved speed and sensitivity.

[0087] As indicated above, hydrophobic compounds, e.g., gangliosides or their lipoforms, can be efficiently separated and quantified using a reverse phase column connected to HPLC or UPLC. A reverse phase column, or reversed-phase HPLC columns, are chromatography columns that contain a non-polar stationary phase. A sample is placed into a reverse phase column and then solvent is added to flush the sample through the stationary phase. Because the stationary phase in a reversed-phase HPLC column is non-polar, the polar components of the sample will drain from the column first, followed by the non-polar components. Reversed-phase HPLC columns can be packed or capillary, made of glass or metal, and can have many different hydrophobic substances as the stationary phase.

[0088] A number of reverse phase columns are available commercially. For example, various categories of reverse phase columns, such as C18 Reversed Phase LC Columns, Biphenyl Reversed Phase LC Columns, C4 Reversed Phase LC Columns, C8 Reversed Phase Columns, Phenyl Reversed Phase LC Columns, Cl Reversed Phase LC Columns, PFP Reversed Phase LC Columns, C30 Reversed Phase LC Columns, Polar Embedded Reversed Phase LC Columns, Polar Endcapped Reversed Phase LC Columns, Porous Graphitic Carbon Reversed Phase LC Columns, PhenyLHexyl Reversed Phase LC Columns, and Alkyl Reversed Phase LC Columns are known in the art and commercially available (Thermo Fisher Scientific, Waltham, MA; Waters Corporation, Milford, MA).

[0089] Analysis of biological samples by MS is challenging due to the limited amount of sample available for analysis, the very low concentration of analyte, and the potential for interference from sample matrix. The advent of nanobore or nano LCn / MSnoffers a solution to these limitations. The nL / min flow rate creates much smaller droplets that are more readily desolvated and result in higher MS sensitivity. In addition, lower detection limits are achieved, less sample is required, and there can be an increased tolerance to chemical interferences compared to conventional LC flow rates. Interfacing nanobore LC to MS utilizes emitters / sprayers that have tips with inner diameters of -1-30 pm.

[0090] Nanobore Liquid Chromatography (LC) coupled with LC-MS is well known in the art (see, e.g., Valaskovic and Kelleher (2002) Curr Top Med Chem 2: 1-12; Siu et al. (2009) J Proteome Res 8:3797-807; Harbourt et al. (2012) Ana Chem 84:98-105, which are AOS-00725 incorporated by reference in their entirety), and nanobore columns are readily available commercially (see, e.g., BioBasic™ 18 Nanobore HPLC column (Thermo Scientific™ 72105107563), BioBasic™ 8 5 pm Nanobore HPLC Columns, HPLC Column ACE Nanobore, and Thermo Scientific® Hypersil GOLD Nanobore HPLC Columns).

[0091] In some embodiments, the “level” or “amount” of a biomarker (e.g., a ganglioside or one or more of its lipoforms) in a subject is “significantly” higher or lower than the control, if the amount of the biomarker is greater or less, respectively, than the level in a control by an amount greater than the standard error, standard error of the mean, or the like, of the assay employed to assess amount.

[0092] In some embodiments, the amount or level of a biomarker in a subject is “significantly” higher or lower than the normal and / or control amount if the amount is at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000%, or more, or any range in between, such as 5%-100%, higher or lower, respectively, than the normal and / or control amount of the biomarker. Such significant modulation values can be applied to any metric described herein, such as the level of a ganglioside or lipoform thereof, a ratio of gangliosides or lipoforms thereof, and a non-ganglioside biomarker (e.g., the level or copy number of B3GALT4).

[0093] B. Methods for detecing a copy number / mutation of a non-ganglioside biomarker nucleic acid

[0094] Methods of evaluating the copy number of a nucleic acid biomarker (also referred to as the level of the nucleic acid biomarker) are well-known to those of skill in the art. The presence or absence of chromosomal gain or loss can be evaluated simply by a determination of copy number of the regions or markers described herein.

[0095] In some embodiments, a biological sample is tested for the presence of copy number changes in genomic loci containing the genomic marker. A copy number of less than 1 of B3GALT4 is predictive of poorer outcome of cancer.

[0096] Methods of evaluating the copy number of a biomarker locus include, but are not limited to, hybridization-based assays. Hybridization-based assays include, but are not limited to, traditional “direct probe” methods, such as Southern blots, in situ hybridization AOS-00725

[0097] (e.g., FISH and FISH plus SKY) methods, and “comparative probe” methods, such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array -based approaches.

[0098] In some embodiments, evaluating the biomarker gene copy number in a sample involves a Southern Blot. In a Southern Blot, the genomic DNA (typically fragmented and separated on an electrophoretic gel) is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal genomic DNA (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid. Alternatively, a Northern blot may be utilized for evaluating the copy number of encoding nucleic acid in a sample. In a Northern blot, mRNA is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal RNA (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid. Alternatively, other methods well-known in the art to detect RNA can be used, such that higher or lower expression relative to an appropriate control (e.g., a non-amplified portion of the same or related cell tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid.

[0099] An alternative means for determining genomic copy number is in situ hybridization (e.g., Angerer (1987) Meth. Enzymol 152: 649). Generally, in situ hybridization comprises the following steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments. The reagent used in each of these steps and the conditions for use vary depending on the particular application. In a typical in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., AOS-00725 cells) are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained. The probes are typically labeled, e.g., with radioisotopes or fluorescent reporters. In one embodiment, probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. Probes generally range in length from about 200 bases to about 1000 bases. In some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, human genomic DNA, or Cot-I DNA is used to block non-specific hybridization.

[0100] An alternative means for determining genomic copy number is comparative genomic hybridization. In general, genomic DNA is isolated from normal reference cells, as well as from test cells e.g., tumor cells) and amplified, if necessary. The two nucleic acids are differentially labeled and then hybridized in situ to metaphase chromosomes of a reference cell. The repetitive sequences in both the reference and test DNAs are either removed or their hybridization capacity is reduced by some means, for example by prehybridization with appropriate blocking nucleic acids and / or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization. The bound, labeled DNA sequences are then rendered in a visualizable form, if necessary. Chromosomal regions in the test cells which are at increased or decreased copy number can be identified by detecting regions where the ratio of signal from the two DNAs is altered. For example, those regions that have decreased in copy number in the test cells will show relatively lower signal from the test DNA than the reference compared to other regions of the genome. Regions that have been increased in copy number in the test cells will show relatively higher signal from the test DNA. Where there are chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels will be detected and the ratio will provide a measure of the copy number. In another embodiment of CGH, array CGH (aCGH), the immobilized chromosome element is replaced with a collection of solid support bound target nucleic acids on an array, allowing for a large or complete percentage of the genome to be represented in the collection of solid support bound targets. Target nucleic acids may comprise cDNAs, genomic DNAs, oligonucleotides e.g., to detect single nucleotide polymorphisms) and the like. Array -based CGH may also be performed with single-color labeling (as opposed to labeling the control and the possible tumor sample with two different dyes and mixing them prior to hybridization, which will yield a ratio due to competitive hybridization of probes on the arrays). In single color AOS-00725

[0101] CGH, the control is labeled and hybridized to one array and absolute signals are read, and the possible tumor sample is labeled and hybridized to a second array (with identical content) and absolute signals are read. Copy number difference is calculated based on absolute signals from the two arrays. Methods of preparing immobilized chromosomes or arrays and performing comparative genomic hybridization are well-known in the art (see, e.g., U.S. Pat. Nos: 6,335,167; 6,197,501; 5,830,645; and 5,665,549 and Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc I). In another embodiment, the hybridization protocol of Pinkel et al. (1998) Nature Genetics 20: 207-211, or of Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992) is used.

[0102] In some embodiments, amplification-based assays can be used to measure copy number. In such amplification-based assays, the nucleic acid sequences act as a template in an amplification reaction (e.g, Polymerase Chain Reaction (PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls, e.g. healthy tissue, provides a measure of the copy number.

[0103] Methods of “quantitative” amplification are well-known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications , Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis is described in Ginzonger et al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR may also be used in the methods encompassed by the present invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and SYBR green.

[0104] Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence AOS-00725 replication (Guatelli et al. (1990) Proc. Nat. Acad. Set. USA 87: 1874), dot PCR, and linker adapter PCR, etc.

[0105] Loss of heterozygosity (LOH) and major copy proportion (MCP) mapping (Wang, Z.C. et al. (2004) Cancer Res 64(1):64-71; Seymour, A. B. et al. (1994) Cancer Res 54, 2761-4; Hahn, S. A. et al. (1995) Cancer Res 55, 4670-5; Kimura, M. et al. (1996) Genes Chromosomes Cancer 17, 88-93; Li et al., (2008) A / BC Bioinform. 9, 204-219) may also be used to identify regions of amplification or deletion.

[0106] A person of ordinary skill would understand that a mutation (e.g., substitution, deletion, addition, inversion, etc.) that compromises the function or activity of a nonganglioside biomarker (e.g., B3GALT4) can also be determined using the techniques described herein (e.g., PCR using specific primers, pPCR, sequencing, etc) or those known in the art.

[0107] C. Methods for detecting the expression level of a non-ganglioside biomarker nucleic acid Biomarker expression may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cellsurface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0108] In preferred embodiments, activity of a particular gene is characterized by a measure of gene transcript (e.g. mRNA), by a measure of the quantify of translated protein, or by a measure of gene product activity. Marker expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.

[0109] In another embodiment, detecting or determining expression levels of a biomarker and functionally similar homologs thereof, including a fragment or genetic alteration thereof (e.g., in regulatory or promoter regions thereof) comprises detecting or determining RNA levels for the marker of interest. In one embodiment, one or more cells from the AOS-00725 subject to be tested are obtained and RNA is isolated from the cells. In a preferred embodiment, a sample of breast tissue cells is obtained from the subject.

[0110] In one embodiment, RNA is obtained from a single cell. For example, a cell can be isolated from a tissue sample by laser capture microdissection (LCM). Using this technique, a cell can be isolated from a tissue section, including a stained tissue section, thereby assuring that the desired cell is isolated (see, e.g, Bonner et al. (1997) Science 278: 1481; Emmert-Buck et al. (1996) Science 274:998; Fend et al. (1999) Am. J. Path. 154: 61 and Murakami et al. (2000) Kidney Int. 58: 1346). For example, Murakami et al., supra, describe isolation of a cell from a previously immunostained tissue section.

[0111] It is also be possible to obtain cells from a subject and culture the cells in vitro, such as to obtain a larger population of cells from which RNA can be extracted. Methods for establishing cultures of non-transformed cells, i.e., primary cell cultures, are known in the art.

[0112] When isolating RNA from tissue samples or cells from individuals, it may be important to prevent any further changes in gene expression after the tissue or cells has been removed from the subject. Changes in expression levels are known to change rapidly following perturbations, e.g., heat shock or activation with lipopolysaccharide (LPS) or other reagents. In addition, the RNA in the tissue and cells may quickly become degraded. Accordingly, in a preferred embodiment, the tissue or cells obtained from a subject is snap frozen as soon as possible.

[0113] RNA can be extracted from the tissue sample by a variety of methods, e.g. , the guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al., 1979, Biochemistry 18:5294-5299). RNA from single cells can be obtained as described in methods for preparing cDNA libraries from single cells, such as those described in Dulac, C. (1998) Curr. Top. Dev. Biol. 36, 245 and Jena et al. (1996) J. Immunol. Methods 190: 199. Care to avoid RNA degradation must be taken, e.g., by inclusion of RNAsin.

[0114] The RNA sample can then be enriched in particular species. In some embodiments, poly(A)+ RNA is isolated from the RNA sample. In general, such purification takes advantage of the poly -A tails on mRNA. In particular and as noted above, poly-T oligonucleotides may be immobilized within on a solid support to serve as affinity ligands for mRNA. Kits for this purpose are commercially available, e.g., the MessageMaker kit (Life Technologies, Grand Island, NY). AOS-00725

[0115] In preferred embodiments, the RNA population is enriched in marker sequences. Enrichment can be undertaken, e.g., by primer-specific cDNA synthesis, or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription (see, e.g. , Wang et al. (1989) roc. Natl. Acad. Sci. U.S.A. 86: 9717; Dulac et al. , supra, and Jena et al. , supra).

[0116] The population of RNA, enriched or not in particular species or sequences, can further be amplified. As defined herein, an “amplification process” is designed to strengthen, increase, or augment a molecule within the RNA. For example, where RNA is mRNA, an amplification process such as RT-PCR can be utilized to amplify the mRNA, such that a signal is detectable or detection is enhanced. Such an amplification process is beneficial particularly when the biological, tissue, or tumor sample is of a small size or volume.

[0117] Various amplification and detection methods can be used. For example, it is within the scope encompassed by the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall et al., PCR Methods and Applications 4: 80-84 (1994). Real time PCR may also be used.

[0118] Other known amplification methods which can be utilized herein include but are not limited to the so-called “NASBA” or “3SR” technique described in PNAS USA 87: 1874- 1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta amplification as described in published European Patent Application (EP A) No. 4544610; strand displacement amplification (as described in G. T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European Patent Application No. 684315; target mediated amplification, as described by PCT Publication WO9322461; PCR; ligase chain reaction (LCR) (see, e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)); self-sustained sequence replication (SSR) (see, e.g., Guatelli et al. , Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)); and transcription amplification (see, e.g., Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)).

[0119] Many techniques are known in the state of the art for determining absolute and relative levels of gene expression, commonly used techniques suitable for use in the present invention include Northern analysis, RNase protection assays (RPA), microarrays and PCR- based techniques, such as quantitative PCR and differential display PCR. For example, AOS-00725

[0120] Northern blotting involves running a preparation of RNA on a denaturing agarose gel, and transferring it to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed and analyzed by autoradiography.

[0121] In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. The samples may be stained with hematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion. Non-radioactive labels such as digoxigenin may also be used.

[0122] Alternatively, mRNA expression can be detected on a DNA array, chip or a microarray. Labeled nucleic acids of a test sample obtained from a subject may be hybridized to a solid surface comprising biomarker DNA. Positive hybridization signal is obtained with the sample containing biomarker transcripts. Methods of preparing DNA arrays and their use are well-known in the art (see, e.g., U.S. Pat. Nos: 6,618,6796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S. 20030157485 and Schena et al. (1995) Science 20, 467-470; Gerhold et al. (1999) Trends In Biochem. Sci. 24, 168-173; and Lennon et al. (2000) Drug Discovery Today 5, 59-65, which are herein incorporated by reference in their entirety). Serial Analysis of Gene Expression (SAGE) can also be performed (See for example U.S. Patent Application 20030215858).

[0123] To monitor mRNA levels, for example, mRNA is extracted from the biological sample to be tested, reverse transcribed, and fluorescently-labeled cDNA probes are generated. The microarrays capable of hybridizing to marker cDNA are then probed with the labeled cDNA probes, the slides scanned and fluorescence intensity measured. This intensity correlates with the hybridization intensity and expression levels.

[0124] Types of probes that can be used in the methods described herein include cDNA, riboprobes, synthetic oligonucleotides and genomic probes. The type of probe used will generally be dictated by the particular situation, such as riboprobes for in situ hybridization, and cDNA for Northern blotting, for example. In one embodiment, the probe is directed to nucleotide regions unique to the RNA. The probes may be as short as is required to differentially recognize marker mRNA transcripts, and may be as short as, for example, 15 bases; however, probes of at least 17, 18, 19 or 20 or more bases can be used. In one embodiment, the primers and probes hybridize specifically under stringent conditions to a AOS-00725

[0125] DNA fragment having the nucleotide sequence corresponding to the marker. As herein used, the term “stringent conditions” means hybridization will occur only if there is at least 95% identity in nucleotide sequences. In another embodiment, hybridization under “stringent conditions” occurs when there is at least 97% identity between the sequences.

[0126] The form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example,32P and35S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases.

[0127] In one embodiment, the biological sample contains polypeptide molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.

[0128] In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting marker polypeptide, mRNA, genomic DNA, or fragments thereof, such that the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in the biological sample, and comparing the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, in the control sample with the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof in the test sample.

[0129] An mRNA level can also be determined using Next Generation sequencing, including RNA-seq, which provides quantitative assessment of the mRNA level.

[0130] D. Methods for detecting a non-ganglioside biomarker protein expression

[0131] The activity or level of a biomarker protein can be detected and / or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well-known to those of skill in the art. Aberrant levels of polypeptide expression of the polypeptides encoded by a biomarker nucleic acid and functionally similar homologs thereof, including a fragment or genetic alteration thereof (e.g., in regulatory or promoter regions thereof) are associated with the likelihood of response of a condition that would benefit from an increased immune response to inhibitors of PTPN2. Any method known in the art for detecting polypeptides can be used. Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELIS As, including e.g., conventional ELISA, sandwish ELISA, competitive ELISA, ELISpot (Enzyme-Linked AOS-00725

[0132] ImmunoSpot)), immunofluorescent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference). Preferred are binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.

[0133] For example, ELISA and RIA procedures may be conducted such that a desired biomarker protein standard is labeled (with a radioisotope such as125I or35S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabeled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, the biomarker protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-biomarker protein antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.

[0134] The above techniques may be conducted essentially as a “one-step” or “two-step” assay. A “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. A “two-step” assay involves washing before contacting, the mixture with labeled antibody. Other conventional methods may also be employed as suitable.

[0135] In one embodiment, a method for measuring biomarker protein levels comprises the steps of: contacting a biological specimen with an antibody or variant (e.g. , fragment) thereof which selectively binds the biomarker protein, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of the biomarker protein.

[0136] Enzymatic and radiolabeling of biomarker protein and / or the antibodies may be effected by conventional means. Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of AOS-00725 the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.

[0137] It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient.

[0138] It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support.

[0139] Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.

[0140] Other techniques may be used to detect biomarker protein according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-biomarker protein antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including125I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.

[0141] Immunohistochemistry may be used to detect expression of biomarker protein, e.g., in a biopsy sample. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabeling. The assay is scored visually, using microscopy. AOS-00725

[0142] Anti-biomarker protein antibodies, including antibodies and intrabodies, may also be used for imaging purposes, for example, to detect the presence of biomarker protein in cells and tissues of a subject. Suitable labels include radioisotopes, iodine (1251,121I), carbon (14C), sulphur (35S), tritium (3H), indium (112In), and technetium (99mTc), fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0143] For in vivo imaging purposes, antibodies are not detectable, as such, from outside the body, and so must be labeled, or otherwise modified, to permit detection. Markers for this purpose may be any that do not substantially interfere with the antibody binding, but which allow external detection. Suitable markers may include those that may be detected by X-radiography, NMR or MRI. For X-radiographic techniques, suitable markers include any radioisotope that emits detectable radiation but that is not overtly harmful to the subject, such as barium or cesium, for example. Suitable markers for NMR and MRI generally include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by suitable labeling of nutrients for the relevant hybridoma, for example.

[0144] The size of the subject, and the imaging system used, will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of technetium-99. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain biomarker protein. The labeled antibody or antibody fragment can then be detected using known techniques.

[0145] Antibodies that may be used to detect biomarker protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the biomarker protein to be detected. An antibody may have a Ka of at most about 10'6M, 10'7M, 10'8M, 10'9M, 10'10M, 10-11M, 10"12M. The phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. An antibody may bind preferentially to the biomarker protein relative to other proteins, such as related proteins.

[0146] Antibodies are commercially available or may be prepared according to methods known in the art. AOS-00725

[0147] Antibodies and derivatives thereof that may be used encompass polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR-grafted), veneered or single-chain antibodies as well as functional fragments, i. e. , biomarker protein binding fragments, of antibodies. For example, antibody fragments capable of binding to a biomarker protein or portions thereof, including, but not limited to, Fv, Fab, Fab' and F(ab') 2 fragments can be used. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab') 2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain.

[0148] Synthetic and engineered antibodies are described in, e.g., Cabilly et al., U.S. Pat. No. 4,816,567 Cabilly et al., European Patent No. 0,125,023 Bl; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 Bl; Neuberger, M. S. et al., WO 86 / 01533; Neuberger, M. S. et al., European Patent No. 0,194,276 Bl; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen et al., European Patent No. 0451216 Bl; and Padlan, E. A. et al., EP 0519596 Al. See also, Newman, R. et al., BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988)) regarding single-chain antibodies. Antibodies produced from a library, e.g., phage display library, may also be used.

[0149] In some embodiments, agents that specifically bind to a biomarker protein other than antibodies are used, such as peptides. Peptides that specifically bind to a biomarker protein can be identified by any means known in the art. For example, specific peptide binders of a biomarker protein can be screened for using peptide phage display libraries.

[0150] E. Methods for Detection of Biomarker Structural Alterations

[0151] The following illustrative methods can be used to identify the presence of a structural alteration in a biomarker nucleic acid and / or biomarker polypeptide molecule in order to, for example, identify a non-ganglioside biomarker (e.g., B3GALT4). AOS-00725

[0152] In certain embodiments, detection of the alteration involves the use of a probe / primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in a biomarker nucleic acid such as a biomarker gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a biomarker gene under conditions such that hybridization and amplification of the biomarker gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and / or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

[0153] Alternative amplification methods include: self-sustained sequence replication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh, D. Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[0154] In alternative embodiments, mutations in a biomarker nucleic acid from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0155] In other embodiments, genetic mutations in biomarker nucleic acid can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density AOS-00725 arrays containing hundreds or thousands of oligonucleotide probes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J. et al. (1996) Nat. Med. 2:753-759). For example, biomarker genetic mutations can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al. (1996) supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential, overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene. Such biomarker genetic mutations can be identified in a variety of contexts, including, for example, germline and somatic mutations.

[0156] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence a biomarker gene and detect mutations by comparing the sequence of the sample biomarker with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560 or Sanger (1977) Proc. Natl. Acad Set. USA 74:5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve (1995) Biotechniques 19:448-53), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94 / 16101; Cohen et al. (1996) Adv. Chromatogr. 36: 127- 162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38: 147-159).

[0157] Other methods for detecting mutations in a biomarker gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA / RNA or RNA / DNA heteroduplexes (Myers et al. (1985) Science 230: 1242). In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type biomarker sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA / DNA duplexes can be treated with RNase and DNA / DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions. In other AOS-00725 embodiments, either DNA / DNA or RNA / DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397 and Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

[0158] In some embodiments, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in biomarker cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coll cleaves A at G / A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G / T mismatches (Hsu et al. (1994) Carcinogenesis 15: 1657-1662). According to an exemplary embodiment, a probe based on a biomarker sequence, e.g., a wild-type biomarker treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like (e.g., U.S. Pat. No. 5,459,039.)

[0159] In other embodiments, alterations in electrophoretic mobility can be used to identify mutations in biomarker genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766; see also Cotton (1993) Mutat. Res. 285: 125-144 and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73- 79). Single-stranded DNA fragments of sample and control biomarker nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In preferred embodiments, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

[0160] In yet other embodiments, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE AOS-00725 is used as the method of analysis, DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high- melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265: 12753).

[0161] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324: 163; Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

[0162] Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6: 1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88: 189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[0163] Control

[0164] A control refers to any suitable reference standard, such as a normal patient, cultured primary cells / tissues isolated from a subject such as a normal subject, adjacent normal cells / tissues obtained from the same organ or body location of the patient, a tissue or cell sample isolated from a normal subject, or a primary cells / tissues obtained from a AOS-00725 depository. In some embodiments, the control may comprise an expression level of a ganglioside biomarker (e.g., the level of a ganglioside or one or more of its lipoforms, a ratio of gangliosides or lipoforms), or the presence of at least one mutation, the level, and / or activity of a non-ganglioside biomarker of a subject, such as a normal or healthy subject. In some embodiments, the control may be from a diseased subject, e.g., a subject afflicted with a cancer, e.g., a subject whose cancer stage or tumor burden is known.

[0165] A control also refers to any reference standard suitable to provide a comparison to the expression products in the test sample. In certain embodiments, the control comprises obtaining a control sample from which the level of a ganglioside, one or more of its lipoforms, a ratio of gangliosides or lipoforms, or the presence of at least one mutation, the level, and / or activity of a non-ganglioside biomarker is detected and compared to the same from the test sample. Such a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells / tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells / tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells / tissues obtained from a depository. In some embodiments, the control may comprise a reference standard expression product (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms, or a non-ganglioside biomarker) level from any suitable source, including but not limited to an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.; cancer with a known stage of cancer or a known amount of tumor burden) or receiving a certain treatment (for example, standard of care cancer therapy). In some embodiments, the control comprises samples drawn or collected longitudinally at different times, to evaluate a change in the level of a ganglioside, one or more of its lipoforms, a ratio of gangliosides or lipoforms, or the presence of at least one mutation, the level, and / or activity a non-ganglioside biomarker over time. It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention. AOS-00725

[0166] In some embodiments, the amount of a ganglioside or a lipoform thereof may be determined within a sample relative to, or as a ratio of, the amount of another ganglioside or a lipoform thereof in the same sample. In some embodiments, the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of product levels of two gangliosides, a ratio of a lipoform of a ganglioside vs. total ganglioside, or the ratio of the nucleic acid / protein / activity level of a non-ganglioside biomarker vs. total nucleic acid / protein / activity level or that of a house-hold gene nucleic acid / protein / activity level in the test sample and comparing it to any suitable ratio of the same in a reference standard; determining product levels of the two or more gangliosides or lipoforms thereof in the test sample and determining a difference in product levels in any suitable control; and determining product levels of the two or more gangliosides in the test sample, normalizing their level to the level of housekeeping gene products in the test sample, and comparing to any suitable control. In preferred embodiments, the control comprises a control sample which is of the same lineage and / or type as the test sample. In other embodiments, the control may comprise product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer. In some embodiments, a control product level is established wherein higher or lower levels of product relative to, for instance, a particular percentile, are used as the basis for predicting outcome. In other preferred embodiments, a control product level is established using product levels from cancer control patients with a known outcome, and the product levels from the test sample are compared to the control product level as the basis for predicting outcome. As demonstrated by the data provided herein, the methods of the present invention are not limited to use of a specific cut-point in comparing the level of product in the test sample to the control.

[0167] In some embodiments, a pre-determined marker amount can be any suitable standard. For example, the pre-determined marker amount can be obtained from the same or a different human for whom a patient selection is being assessed. In some embodiments, the pre-determined marker amount can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time. In addition, the control can be obtained from an assessment of another human or multiple humans, e.g., selected groups of humans, if the subject is a human. In such a manner, the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g., other humans who are in a similar situation to AOS-00725 the human of interest, such as those suffering from similar or the same condition(s) and / or of the same ethnic group.

[0168] Accordingly, in some embodiments, a control comprises a sample (e.g., serum or tissues) from a normal healthy person without cancer. In yet other embodiments, a control comprises a sample (e.g., serum or tissues) from a patient who is being evaluated (e.g., diagnosis or prognosis). For example, the control sample may comprise (i) a historical sample of the patient, or (ii) the sample obtained from the patient in longitudinal studies, e.g., pre-therapy or post-therapy (e.g., cancer therapy). The use of such control allows comparison of a biomarker present in the same patient over time (e.g., during the progression of cancer).

[0169] Uses and Methods of the Invention

[0170] In certain aspects, provided herein are diganostic methods, prognostic methods, treatment methods, or any combination thereof.

[0171] 1. Predictive Medicine

[0172] The present invention pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect encompassed by the present invention relates to diagnostic assays for determining the amount and / or activity level of a biomarker described herein in the context of a biological sample (e.g., blood, serum, cells, or tissue) to thereby determine whether an individual afflicted with a cancer. Such assays can be used for prognostic or predictive purpose alone, or can be coupled with a therapeutic intervention to thereby prophylactically treat an individual prior to the onset or after recurrence of a disorder characterized by or associated with a certain level of the biomarkers of the present disclosure (a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or the presence of at least one mutation, the level, and / or activity a non-ganglioside biomarker (e.g., B3GALT4)). The skilled artisan will appreciate that any method can use one or more (e.g., combinations) of biomarkers described herein, such as those in the tables, figures, examples, and otherwise described in the specification.

[0173] Another aspect encompassed by the present disclosure pertains to monitoring the influence of agents (e.g., drugs, compounds, and small nucleic acid-based molecules) on the AOS-00725 level or activity of a biomarker described herein. These and other agents are described in further detail in the following sections.

[0174] The skilled artisan will also appreciated that, in certain embodiments, the methods encompassed by the present invention implement a computer program and computer system. For example, a computer program can be used to perform the algorithms described herein. A computer system can also store and manipulate data generated by the methods encompassed by the present invention which comprises a plurality of biomarker signal changes / profiles which can be used by a computer system in implementing the methods of this invention. In certain embodiments, a computer system receives biomarker expression data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate controls) to determine the state of informative biomarkers from cancerous or pre-cancerous tissue. In other embodiments, a computer system (i) compares the determined expression biomarker level to a threshold value; and (ii) outputs an indication of whether said biomarker level is significantly modulated (e.g., above or below) the threshold value, or a phenotype based on said indication.

[0175] In certain embodiments, such computer systems are also considered part encompassed by the present invention. Numerous types of computer systems can be used to implement the analytic methods of this invention according to knowledge possessed by a skilled artisan in the bioinformatics and / or computer arts. Several software components can be loaded into memory during operation of such a computer system. The software components can comprise both software components that are standard in the art and components that are special to the present invention (e.g., dCHIP software described in Lin et al. (2004) Bioinformatics 20, 1233-1240; radial basis machine learning algorithms (RBM) known in the art).

[0176] The methods encompassed by the present invention can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms. Such packages include, e.g., Matlab from Mathworks (Natick, Mass.), Mathematica from Wolfram Research (Champaign, Ill.) or S-Plus from MathSoft (Seattle, Wash.).

[0177] In certain embodiments, the computer comprises a database for storage of biomarker data. Such stored profiles can be accessed and used to perform comparisons of interest at a later point in time. For example, biomarker expression profiles of a sample AOS-00725 derived from the non-cancerous tissue of a subject and / or profiles generated from population-based distributions of informative loci of interest in relevant populations of the same species can be stored and later compared to that of a sample derived from the cancerous tissue of the subject or tissue suspected of being cancerous of the subject.

[0178] In addition to the exemplary program structures and computer systems described herein, other, alternative program structures and computer systems will be readily apparent to the skilled artisan. Such alternative systems, which do not depart from the above described computer system and programs structures either in spirit or in scope, are therefore intended to be comprehended within the accompanying claims.

[0179] 2, Diagnostic Assays

[0180] The present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample comprises a ganglioside (or one or more of its lipoforms) and / or whether the levels of a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or the presence of at least one mutation, the level, and / or activity a non-ganglioside biomarker (e.g., B3GALT4) are modulated (e.g. , upregulated or downregulated), thereby indicative of the state of a disorder of interest, such as cancer. In some embodiments, the present invention is useful for classifying a sample (e.g., from a subject) as associated with or at risk for cancer or a subtype thereof, mediated by a ganglioside using a statistical algorithm and / or empirical data (e.g., the presence, absence, level, or the lipid length of a ganglioside or its lipoforms).

[0181] An exemplary method for detecting the level of a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or the presence of at least one mutation, the level, and / or activity a non-ganglioside biomarker (e.g., B3GALT4), and thus useful for classifying whether a sample is associated with a cancer or a clinical subtype thereof or different stages of a cancer involves obtaining a biological sample from a test subject and detecting a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or the presence of at least one mutation, the level, and / or activity a non-ganglioside biomarker (e.g., B3GALT4) of the ganglioside in the sample using any one or more methods described herein or those known in the art.

[0182] In certain instances, the statistical algorithm is a single learning statistical classifier system. For example, a single learning statistical classifier system can be used to classify a sample as a cancer sample based upon a prediction or probability value and the presence or level of ganglioside. The use of a single learning statistical classifier system typically AOS-00725 classifies the sample as a cancer sample with a sensitivity, specificity, positive predictive value, negative predictive value, and / or overall accuracy of at least or about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0183] Other suitable statistical algorithms are well-known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets. In some embodiments, a single learning statistical classifier system such as a classification tree (e.g., random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (e.g., decision / classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, 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 algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, the method of the present invention further comprises sending the sample classification results to a clinician (a non-specialist, e.g., primary care physician; and / or a specialist, e.g., a histopathologist or an oncologist).

[0184] In some embodiments, the method of the present disclosure further provides a diagnosis in the form of a probability that the individual has a cancer. For example, the individual can have about a 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater probability of having the cancer. In yet another embodiment, the method of the present invention further provides a AOS-00725 prognosis of the cancer in the individual. In some instances, the method of classifying a sample as a cancer sample may be further based on the symptoms (e.g., clinical factors) of the individual from which the sample is obtained. The symptoms or group of symptoms can be, for example, lymphocyte count, white cell count, erythrocyte sedimentation rate, diarrhea, abdominal pain, bloating, pelvic pain, lower back pain, cramping, fever, anemia, weight loss, anxiety, depression, and combinations thereof. In some instances, the method of classifying a sample as a cancer sample may be further based on genetic mutations and / or predisposition to cancer, irrespective of the symptoms.

[0185] In some embodiments, the diagnosis of an individual as having a cancer is followed by administering to the individual a therapeutically effective amount of a cancer therapy (e.g., chemotherapeutic agents). In some embodiments, the diagnosis of an individual as having a cancer is followed by treating the individual with a cancer therapy.

[0186] In some embodiments, the methods further involve obtaining a control biological sample (e.g., biological sample from a subject who does not have a cancer), a biological sample from the subject during remission or before developing a cancer, or a biological sample from the subject during treatment for developing a cancer.

[0187] In some embodiments, the methods comprise analyzing the control sample to detect a biomarker (e.g., ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)), such that the presence, the level, the mutation status, and / or activity of said biomarker is detected in the biological sample, and comparing the presence, the level, and / or activity of the biomarker in the control with the same in the test sample.

[0188] A preferred biological sample is a serum, blood, saliva, tumor microenvironment, peritumoral cells / tissues, or intratumoral cells / tissues, isolated by conventional means from a subject. A person of ordinary skill in the art would understand that cell or tissue samples may need further processing (e.g., homogenize and / or partially purify the lipid fraction).

[0189] The presence, level, mutation status, and / or activity of a biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non- ganglioside biomarker (e.g., B3GALT4)), as determined by the methods of the present disclosure, correlate with different grades of a cancer. Accordingly, in some embodiments, the methods of the present disclosure can be used to determine a grade of a cancer, based on the presence, level, and / or activity of the biomarker determined as described herein. AOS-00725

[0190] A cancer’s grade describes how abnormal the cancer cells and tissue look under a microscope when compared to healthy cells. Cancer cells that look and organize most like healthy cells and tissue are low grade tumors. Doctors describe these cancers as being well differentiated. Lower grade cancers are typically less aggressive and have a better prognosis. The more abnormal the cells look and organize themselves, the higher the cancer’s grade. Cancer cells with a high grades tend to be more aggressive. They are called poorly differentiated or undifferentiated. Some cancers have their own system for grading tumors. Many others use a standard 1-4 grading scale.

[0191] • Grade 1: Tumor cells and tissue looks most like healthy cells and tissue. These are called well-differentiated tumors and are considered low grade.

[0192] • Grade 2: The cells and tissue are somewhat abnormal and are called moderately differentiated. These are intermediate grade tumors.

[0193] • Grade 3: Cancer cells and tissue look very abnormal. These cancers are considered poorly differentiated, since they no longer have an architectural structure or pattern. Grade 3 tumors are considered high grade.

[0194] • Grade 4: These undifferentiated cancers have the most abnormal looking cells.

[0195] These are the highest grade and typically grow and spread faster than lower grade tumors.

[0196] As used herein, low grade cancer refers to Grade I cancer; and high grade cancer refers to cancer of Grades 2-4.

[0197] Similarly, the presence, level, mutation status, and / or activity of a biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a nonganglioside biomarker (e.g., B3GALT4)), as determined by the methods of the present disclosure, correlate with different stages of a cancer. Accordingly, in some embodiments, the compositions and methods of the present disclosure can be used to determine a grade of a cancer, based on the level and / or heterogeneity of the ganglioside, one or more of its lipoforms, or lipid length of a ganglioside determined as described herein.

[0198] A cancer’s stage explains how large the primary tumor is and how far the cancer has spread in the patient’s body. There are several different staging systems. Many of these have been created for specific kinds of cancers. Others can be used to describe several types of cancer. One common system that many people are aware of puts cancer on a scale of 0 to IV. AOS-00725

[0199] • Stage 0 is for abnormal cells that haven’t spread and are not considered cancer, though they could become cancerous in the future. This stage is also called “in- situ.”

[0200] • Stage I through Stage III are for cancers that haven’t spread beyond the primary tumor site or have only spread to nearby tissue. The higher the stage number, the larger the tumor and the more it has spread.

[0201] • Stage IV cancer has spread to distant areas of the body.

[0202] As used herein, cancer at the early / low stage refers to cancer at Stage I; and cancer at the late / high / advanced stage includes cancer at Stage II to Stage IV. Cancer stages I, II, III, and IV are also referred to by arabic numbers, e.g., stage 1, stage 2, stage 3, or stage 4, respectively.

[0203] Neuroblastoma can be staged as follows.

[0204] Stage 1 : The cancer is still in the area where it started. It is on one side of the body (right or left). All visible tumor has been removed completely by surgery (although looking at the tumor’s edges under the microscope after surgery may show some cancer cells). Lymph nodes near the tumor are free of cancer (although nodes enclosed within the tumor may contain neuroblastoma cells).

[0205] Stage 2A: The cancer is still in the area where it started and on one side of the body, but not all of the visible tumor could be removed by surgery. Lymph nodes near the tumor are free of cancer (although nodes enclosed within the tumor may contain neuroblastoma cells). Stage 2B: The cancer is on one side of the body, and it may or may not have been removed completely by surgery. Nearby lymph nodes outside the tumor contain neuroblastoma cells, but the cancer has not spread to lymph nodes on the other side of the body or elsewhere. Stage 3: The cancer has not spread to distant parts of the body, but one of the following is true:

[0206] • The cancer can't be removed completely by surgery, and it has crossed the midline (defined as the spine) to the other side of the body. It may or may not have spread to nearby lymph nodes.

[0207] • The cancer is still in the area where it started and is on one side of the body. It has spread to lymph nodes that are relatively nearby but on the other side of the body.

[0208] • The cancer is in the middle of the body and is growing toward both sides (either directly or by spreading to nearby lymph nodes). AOS-00725

[0209] Stage 4: The cancer has spread to distant parts of the body such as distant lymph nodes, bones, liver, skin, bone marrow, or other organs (but the child does not meet the criteria for stage 4S).

[0210] Recurrent: While not a formal part of the staging system, this term is used to describe cancer that has come back (recurred) after it has been treated. The cancer might come back in the area where it first started or in another part of the body.

[0211] Likewise, the presence, level, mutation status, and / or activity of a biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a nonganglioside biomarker (e.g., B3GALT4)), as determined by the methods of the present disclosure, correlate with the tumor burden. Accordingly, in some embodiments, the compositions and methods of the present disclosure can be used to determine the tumor burden of a subject, based on the presence, level, and / or activity of a biomarker determined as described herein. Tumor burden (or tumor load) is defined as the total amount of tumor (cells / mass) distributed in the patients’ body, including bone marrow. In Response Evaluation Criteria in Solid Tumors (RECIST) analysis, tumor burden is considered the sum of the longest diameters of all measurable lesions. Various methods can be used to determine the tumor burden in a subject. For example, computed tomography (CT) and magnetic resonance (MR) imaging have been used to assess tumor response based on morphologic (size, location) criteria, specifically by using RECIST. RECIST classification describes lesions’ size and distinguishes 4 types of treatment response - stable disease (SD), partial response (PR), complete response (CR) or progressive disease (PD).

[0212] 3, Prognostic Assays

[0213] The term “prognosis” includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis of cancer in an individual. For example, the prognosis can be surgery, development of a clinical subtype of cancer (e.g., solid tumors, such as lung cancer, melanoma, and renal cell carcinoma), development of one or more clinical factors, development of intestinal cancer, or recovery from the disease.

[0214] The assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to to determine whether a subject can be administered a cancer therapy (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, immunotherapy, immune checkpoint inhibition therapy, or other drug candidate) to treat a cancer. For example, such methods can be used to determine whether a AOS-00725 subject can be effectively treated with one or a combination of agents. Thus, the present disclosure provides methods for determining whether a subject can be effectively treated with one or more agents for treating a cancer in which a test sample is obtained and a ganglioside is detected.

[0215] Other aspects of the present disclosure include uses of the methods described herein for association and / or stratification analyses in which a biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)) in biological samples from individuals with a cancer, are analyzed and the information is compared to that of controls e.g., individuals who do not have a cancer; ini some embodiments, controls may be also referred to as “healthy” or “normal” individuals or at early timepoints in a given time lapse study) who are preferably of similar age and race. The appropriate selection of patients and controls is important to the success of association and / or stratification studies. Therefore, a pool of individuals with well-characterized phenotypes is extremely desirable. Criteria for cancer diagnosis, cancer predisposition screening, predicting clinical outcomes, cancer prognosis, determining drug responsiveness (pharmacogenomics), drug toxicity screening, etc. are described herein.

[0216] Different study designs may be used for genetic association and / or stratification studies (Modem Epidemiology, Lippincott Williams & Wilkins (1998), 609-622). Observational studies are most frequently carried out in which the response of the patients is not interfered with. The first type of observational study identifies a sample of persons in whom the suspected cause of the disease is present and another sample of persons in whom the suspected cause is absent, and then the frequency of development of disease in the two samples is compared. These sampled populations are called cohorts, and the study is a prospective study. The other type of observational study is case-control or a retrospective study. In typical case-control studies, samples are collected from individuals with the phenotype of interest (cases) such as certain manifestations of a disease, and from individuals without the phenotype (controls) in a population (target population) that conclusions are to be drawn from. Then the possible causes of the disease are investigated retrospectively. As the time and costs of collecting samples in case-control studies are considerably less than those for prospective studies, case-control studies are the more commonly used study design in genetic association studies, at least during the exploration and discovery stage.

[0217] After all relevant phenotypic and / or genotypic information has been obtained, AOS-00725 statistical analyses are carried out to determine if there is any significant correlation between the presence of an allele or a genotype with the phenotypic characteristics of an individual. Preferably, data inspection and cleaning are first performed before carrying out statistical tests for genetic association. Epidemiological and clinical data of the samples can be summarized by descriptive statistics with tables and graphs well-known in the art. Data validation is preferably performed to check for data completion, inconsistent entries, and outliers. Chi-squared tests and t-tests (Wilcoxon rank-sum tests if distributions are not normal) may then be used to check for significant differences between cases and controls for discrete and continuous variables, respectively.

[0218] An important decision in the performance of genetic association tests is the determination of the significance level at which significant association can be declared when the p-value of the tests reaches that level. In an exploratory analysis where positive hits will be followed up in subsequent confirmatory testing, an unadjusted p-value <0.2 (a significance level on the lenient side), for example, may be used for generating hypotheses for significant association of a ganglioside level with certain phenotypic characteristics of a cancer. It is preferred that a p-value <0.05 (a significance level traditionally used in the art) is achieved in order for the level to be considered to have an association with a cancer. When hits are followed up in confirmatory analyses in more samples of the same source or in different samples from different sources, adjustment for multiple testing will be performed as to avoid excess number of hits while maintaining the experiment-wise error rates at 0.05. While there are different methods to adjust for multiple testing to control for different kinds of error rates, a commonly used but rather conservative method is Bonferroni correction to control the experiment-wise or family -wise error rate (Multiple comparisons and multiple tests, Westfall et al, SAS Institute (1999)). Permutation tests to control for the false discovery rates, FDR, can be more powerful (Benjamini and Hochberg, Journal of the Royal Statistical Society, Series B 57, 1289-1300, 1995, Resampling-based Multiple Testing, Westfall and Young, Wiley (1993)). Such methods to control for multiplicity would be preferred when the tests are dependent and controlling for false discovery rates is sufficient as opposed to controlling for the experiment-wise error rates.

[0219] Once individual risk factors, genetic or non-genetic, have been found for the predisposition to disease, a classification / prediction scheme can be set up to predict the category (for instance, disease or no-disease) that an individual will be in depending on his phenotype and / or genotype and other non-genetic risk factors. Logistic regression for AOS-00725 discrete trait and linear regression for continuous trait are standard techniques for such tasks (Applied Regression Analysis, Draper and Smith, Wiley (1998)). Moreover, other techniques can also be used for setting up classification. Such techniques include, but are not limited to, MART, CART, neural network, and discriminant analyses that are suitable for use in comparing the performance of different methods (The Elements of Statistical Learning, Hastie, Tibshirani & Friedman, Springer (2002)).

[0220] 4. Prophylactic Methods

[0221] In one aspect, the present invention provides a method for preventing in a subject, a cancer. Subjects at risk for a cancer that would benefit from treatment with the certain agents or methods can be identified, for example, by any or a combination of diagnostic or prognostic assays known in the art. Administration of a prophylactic agent can occur prior to the manifestation of symptoms associated with a cancer. The appropriate cancer therapy used for treatment (e.g. antibodies, peptides, fusion proteins or small molecules) can be determined based on clinical indications.

[0222] 5. Therapeutic Methods

[0223] Another aspect encompassed by the present disclosure pertains to therapeutic methods. The therapeutic compositions described herein (e.g., cancer therapies described herein or those known in the art) can be used in a variety of in vitro, ex vivo, and in vivo therapeutic applications using the formulations and / or combinations described herein. In some embodiments, a cancer therapy can be used to treat cancers determined to be responsive thereto. For example, single or multiple cancer therapies can be used to treat cancers in subjects identified as likely responders thereto.

[0224] Modulatory methods encompassed by the present invention involve contacting a cell, such as an immune cell with a cancer therapy. A cancer therapy or a combination of one or more cancer therapies described herein or those known in the art can be administered in vitro or ex vivo (e.g., by contacting the cell with a cancer therapy) or, alternatively, in vivo (e.g., by administering a cancer therapy to a subject). As such, the present invention provides methods useful for treating an individual afflicted with a cancer.

[0225] The duration and / or dose of treatment with therapies may vary according to the particular therapeutic agent or combination thereof. An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan. The present disclosure contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined by AOS-00725 the methods encompassed by the present invention is a factor in determining optimal treatment doses and schedules.

[0226] Accordingly, various diagnostic and / or prognostic methods of the present disclosure can be followed by treating the subject whose sample was tested in said methods. For example, a subject who was diagnosed with cancer using a method described herein can be treated with cancer therapy (e.g., the standard of care or cancer therapy described herein or those known in the art).

[0227] Monitoring of Effects During Clinical Trials

[0228] Monitoring the influence of agents e.g., compounds, drugs or small molecules, immunotherapy, cancer therapy described herein or those known in the art) on the presence, level, mutation status, and / or activity of a biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay to decrease the level of a biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)) can be monitored in clinical trials of subjects, detectable by any methods described herein or those known in the art. In such clinical trials, the presence, level, mutation status, and / or activity of a biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)) and / or symptoms or other markers of the cancer, can be used as a “read out” or marker of the phenotype of a particular cell, tissue, or system.

[0229] In preferred embodiments, the present disclosure provides a method for monitoring the effectiveness of treatment of a subject with an agent e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, immunotherapy, immune checkpoint inhibition therapy, or other drug candidate) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the presence, level, mutation status, and / or activity of at least one biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)) in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the presence, level, mutations staus, and / or activity of the biomarker (e.g., a ganglioside or a AOS-00725 lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)) in the post-administration samples; (v) comparing the presence, level, and / or activity of the at least one biomarker in the pre-administration sample with the same in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase or decrease the level of at least one biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)), z.e., to increase the effectiveness of the agent. According to such embodiments, at least one biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non- ganglioside biomarker (e.g., B3GALT4)) may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response. Similarly, at least one biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)) analysis, such as any one or more methods described herein or those known in the art, can also be used to select patients who will receive a cancer therapy.

[0230] Clincal Efficacy / Response to a Therapy

[0231] Clinical efficacy can be measured by any method known in the art. For example, the response to a therapy relates to any response of the cancer, e.g., a tumor, to the therapy, preferably to a change in tumor mass and / or volume after initiation of neoadjuvant or adjuvant chemotherapy. Tumor response may be assessed in a neoadjuvant or adjuvant situation where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation and the cellularity of a tumor can be estimated histologically and compared to the cellularity of a tumor biopsy taken before initiation of treatment. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or cellularity or using a semi-quantitative scoring system 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) in a qualitative fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive AOS-00725 disease” (cPD) or other qualitative criteria. Assessment of tumor response may be performed early after the onset of neoadjuvant or adjuvant therapy, e.g. , after a few hours, days, weeks or preferably after a few months. A typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and / or the tumor bed.

[0232] In some embodiments, clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy. The shorthand for this formula is CBR=CR+PR+SD over 6 months. In some embodiments, the CBR for a particular anti-immune checkpoint therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.

[0233] Additional criteria for evaluating the response to a cancer therapy are related to “survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); “recurrence -free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point e.g., time of diagnosis or start of treatment) and end point e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0234] For example, in order to determine appropriate threshold values, a particular anticancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any cancer therapy. The outcome measurement may be pathologic response to therapy given in the neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free survival can be monitored over a period of time for subjects following the cancer therapy for whom biomarker measurement values are known. In certain embodiments, the same doses of anti-cancer agents are administered to each subject. In related embodiments, the doses administered are standard doses known in the art for anti- AOS-00725 cancer agents. The period of time for which subjects are monitored can vary. For example, subjects may 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. Biomarker measurement threshold values that correlate to outcome of a cancer therapy can be determined using methods such as those described in the Examples section.

[0235] Sample

[0236] Biological samples can be collected from a variety of sources from a subject including a body fluid sample, cell sample, or a tissue sample. Body fluids refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ej aculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In some embodiments, the subject and / or control sample is selected from the group consisting of cells, cell lines, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, and bone marrow. In some embodiments, the samples can contain live cells / tissue, fresh frozen cells, fresh tissue, biopsies, fixed cells / tissue, cells / tissue embedded in a medium, such as paraffin, histological slides, or any combination thereof. In some embodiments, the samples can contain live cells / tissue, fresh frozen cells, fresh tissue, and / or biopsies.

[0237] The samples can be collected from individuals repeatedly over a longitudinal period of time (e.g. , once or more on the order of days, weeks, months, annually, biannually, etc.).

[0238] Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and / or analysis of biomarker measurement(s). Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, 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.

[0239] A sample may comprise a fixed molecule. A molecule is “fixed” or “affixed” to a substrate if it is covalently or non-covalently associated with the substrate such the AOS-00725 substrate can be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.

[0240] As described herein, in some embodiments, the presence, level, mutation status, and / or activity of at least one biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)) measurement(s) in a sample from a subject is compared to a control, which includes but not limited to e.g., a control biological sample from a subject who does not have a cancer, a control biological sample from the subject during remission or before developing a cancer, or a control biological sample from the subject during treatment for developing a cancer. In some embodiments, a control biological sample is from a subject prior to treatment with a certain therapy. In some embodiments, wherein a subject is treated with multiple rounds of one or more therapies, a control biological sample may be from an earlier or later time point with respect to the subject sample during such treatment. For example, a subject sample after third rounds of therapy may be compared with a control subject sample after the first round of therapy.

[0241] In some embodiments, the presence, level, mutation status, and / or activity of at least one biomarker (e.g., a ganglioside or a lipoform thereof, a ratio of gangliosides or lipoforms thereof, or a non-ganglioside biomarker (e.g., B3GALT4)) measurement(s) in a sample from a subject is compared to a predetermined control (standard) sample. The sample from the subject is typically from a diseased tissue, such as cancer cells or tissues. The control sample can be from the same subject or from a different subject. The control sample is typically a normal, non-diseased sample. However, in some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, the control sample can be from a diseased tissue. The control sample can be a combination of samples from several different subjects. In some embodiments, the biomarker amount and / or activity measurement(s) from a subject is compared to a pre -determined level. This pre-determined level is typically obtained from normal samples.

[0242] As described herein, a “pre-determined” biomarker amount measurement(s) may be a biomarker amount measurement(s) used to, by way of example only, evaluate a subject that may be selected for treatment, evaluate a response to a cancer therapy, and / or evaluate a response to a combination of anti-cancer therapies. A pre-determined biomarker amount and / or activity measurement(s) may be determined in populations of patients with or without cancer. The pre-determined biomarker amount measurement(s) can be a single AOS-00725 number, equally applicable to every patient, or the pre-determined biomarker amount measurement(s) can vary according to specific subpopulations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount measurement(s) of the individual. Furthermore, the pre-determined biomarker amount can be determined for each subject individually. In some embodiments, the amounts determined and / or compared in a method described herein are based on absolute measurements.

[0243] In some embodiments, the amounts determined and / or compared in a method described herein are based on relative measurements, such as ratios (e.g., biomarker level before a treatment vs. after a treatment, such biomarker measurements relative to a spiked or man-made control, such biomarker measurements relative to the expression of a housekeeping gene, and the like). For example, the relative analysis can be based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement. Pre-treatment biomarker measurement can be made at any time prior to initiation of anti-cancer therapy. Post-treatment biomarker measurement can be made at any time after initiation of anti-cancer therapy. In some embodiments, post-treatment biomarker measurements are made 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more after initiation of anti-cancer therapy, and even longer toward indefinitely for continued monitoring. Treatment can comprise one or more anti-cancer therapies, e.g., immune checkpoint inhibitors.

[0244] The pre-determined biomarker amount measurement(s) can be any suitable standard. For example, the pre-determined biomarker amount measurement(s) can be obtained from the same or a different human for whom a patient selection is being assessed. In some embodiments, the pre-determined biomarker amount measurement(s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time. In addition, the control can be obtained from an assessment of another human or multiple humans, e.g., selected groups of humans, if the subject is a human. In such a manner, the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g., other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same condition(s) and / or of the same ethnic group.

[0245] In some embodiments of the present disclosure the change of biomarker amount measurement(s) from the pre-determined level is at least or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, AOS-00725

[0246] 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 fold or greater, or any range in between, inclusive. Such cutoff values apply equally when the measurement is based on relative changes, such as based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement.

[0247] Cancer

[0248] Cancer, tumor, or hyperproliferative disorder refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, 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 adenocarcinomas, 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 carcinoma, small cell lung carcinoma (SCLC), AOS-00725 bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, 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 still other embodiments, the epithelial cancer is non-smallcell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0249] The compositions and methods of the present inventions may be used to detect various cancers, including ovarian cancer, neuroblastoma, small cell lung cancer (SCLC), or melanoma.

[0250] Cancer therapy

[0251] The methods of the present disclosure (e.g., diagnostic and / or prognostic methods) can be followed by treating the patients using cancer therapy described herein or those known in the art, e.g., standard-of-care treatments for cancer well-known to the skilled artisan, chemotherapeutic agents, hormones, antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy, immunotherapy, cancer vaccine, immune cell engineering (e.g., CAR-T), and / or radiotherapy. The preceding treatment methods can be administered in conjunction with other forms of cancer therapy, either consecutively with, pre- or post-said cancer therapy. For example, immunotherapy can be administered with a therapeutically effective dose of chemotherapeutic agent, e.g., immunotherapy can be administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The Physicians’ Desk Reference (PDR) discloses dosages of AOS-00725 chemotherapeutic agents that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art, and can be determined by the physician.

[0252] Immunotherapy is a targeted therapy that may comprise, for example, the use of cancer vaccines and / or sensitized antigen presenting cells. For example, an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site. The immunotherapy can involve passive immunity for shortterm protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). For example, anti-VEGF is known to be effective in treating renal cell carcinoma. Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and / or pathology of a tumor or cancer.

[0253] Immunotherapy also encompasses immune checkpoint modulators. Immune checkpoints are a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well-known in the art and include, without limitation, 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, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR (see, for example, WO 2012 / 177624). Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer. In some embodiments, the 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. AOS-00725

[0254] Adoptive cell-based immunotherapies can be combined with the therapies of the present invention. Well-known adoptive cell-based immunotherapeutic modalities, including, without limitation, irradiated autologous or allogeneic tumor cells, tumor lysates or apoptotic tumor cells, antigen-presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell transfer, adoptive CAR T cell therapy, autologous immune enhancement therapy (AIET), cancer vaccines, and / or antigen presenting cells. Such cellbased immunotherapies can be further modified to express one or more gene products to further modulate immune responses, such as expressing cytokines like GM-CSF, and / or to express tumor-associated antigen (TAA) antigens, such as Mage-1, gp-100, and the like.

[0255] The term “chimeric antigen receptor” or “CAR” refers to engineered T cell receptors (TCR) having a desired antigen specificity. T lymphocytes recognize specific antigens through interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, naive T cells are dependent on professional antigen-presenting cells (APCs) that provide additional co-stimulatory signals. TCR activation in the absence of costimulation can result in unresponsiveness and clonal anergy. To bypass immunization, different approaches for the derivation of cytotoxic effector cells with grafted recognition specificity have been developed. CARs have been constructed that consist of binding domains derived from natural ligands or antibodies specific for cell-surface components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric antigen receptors link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. Since the first reports on chimeric antigen receptors, this concept has steadily been refined and the molecular design of chimeric receptors has been optimized and routinely use any number of well-known binding domains, such as scFV and another protein binding fragments described herein.

[0256] In other embodiments, immunotherapy comprises non-cell-based immunotherapies. In some embodiments, compositions comprising antigens with or without vaccineenhancing adjuvants are used. Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, recombinant antigen comprising fusion proteins, and the like. In some embodiments, immunomodulatory cytokines, such as interferons, G- CSF, imiquimod, TNFalpha, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In some embodiments, immunomodulatory interleukins, such as IL-2, IL-6, IL-7, IL-12, IL-17, IL-23, and the AOS-00725 like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In some embodiments, immunomodulatory chemokines, such as CCL3, CCL26, and CXCL7, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In some embodiments, immunomodulatory molecules targeting immunosuppression, such as STAT3 signaling modulators, NFkappaB signaling modulators, and immune checkpoint modulators, are used.

[0257] In still other embodiments, immunomodulatory drugs, such as immunocytostatic drugs, glucocorticoids, cytostatics, immunophilins and modulators thereof (e.g., rapamycin, a calcineurin inhibitor, tacrolimus, ciclosporin (cyclosporin), pimecrolimus, abetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, a non-glucocorticoid steroid, a pyrimidine synthesis inhibitor, leflunomide, teriflunomide, a folic acid analog, methotrexate, anti-thymocyte globulin, antilymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, an opioid, an IMPDH inhibitor, mycophenolic acid, myriocin, fmgolimod, an NF- xB inhibitor, raloxifene, drotrecogin alfa, denosumab, an NF-xB signaling cascade inhibitor, disulfiram, olmesartan, dithiocarbamate, a proteasome inhibitor, bortezomib, MG132, Prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, non-steroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethylepoxy quinomycin (DHMEQ), I3C(indole-3-carbinol) / DIM(di- indolmethane) (13C / DIM), Bay 11-7082, luteolin, cell permeable peptide SN-50, IKBa.- super repressor overexpression, NFKB decoy oligodeoxynucleotide (ODN), or a derivative or analog of any thereo, are used. In yet other embodiments, immunomodulatory antibodies or protein are used. For example, antibodies that bind to CD40, Toll-like receptor (TLR), 0X40, GITR, CD27, or to 4- IBB, T-cell bispecific antibodies, an anti-IL-2 receptor antibody, an anti-CD3 antibody, OKT3 (muromonab), otelixizumab, teplizumab, visilizumab, an anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, an anti-CD I l a antibody, efalizumab, an anti-CD 18 antibody, erlizumab, rovelizumab, an anti-CD20 antibody, afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, an anti-CD23 antibody, lumiliximab, an anti-CD40 antibody, teneliximab, toralizumab, an anti-CD40L antibody, ruplizumab, an anti-CD62L antibody, aselizumab, an anti-CD80 antibody, AOS-00725 galiximab, an anti-CD147 antibody, gavilimomab, a B-Lymphocyte stimulator (BLyS) inhibiting antibody, belimumab, an CTLA4-Ig fusion protein, abatacept, belatacept, an anti- CTLA4 antibody, ipilimumab, tremelimumab, an anti-eotaxin 1 antibody, bertilimumab, an anti-a4-integrin antibody, natalizumab, an anti-IL-6R antibody, tocilizumab, an anti-LFA-1 antibody, odulimomab, an anti-CD25 antibody, basiliximab, daclizumab, inolimomab, an anti-CD5 antibody, zolimomab, an anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atorolimumab, cedelizumab, dorlimomab aritox, dorlixizumab, fontolizumab, gantenerumab, gomiliximab, lebrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab, aflibercept, alefacept, rilonacept, an IL- 1 receptor antagonist, anakinra, an anti-IL-5 antibody, mepolizumab, an IgE inhibitor, omalizumab, talizumab, an IL 12 inhibitor, an IL23 inhibitor, ustekinumab, and the like.

[0258] Nutritional supplements that enhance immune responses, such as vitamin A, vitamin E, vitamin C, and the like, are well-known in the art (see, for example, U.S. Pat. Nos. 4,981,844 and 5,230,902 and PCT Publ. No. WO 2004 / 004483) can be used in the methods described herein.

[0259] Similarly, various agents or a combination thereof can be used to treat a cancer. For example, chemotherapy, radiation, epigenetic modifiers (e.g., histone deacetylase (HDAC) modifiers, methylation modifiers, phosphorylation modifiers, and the like), targeted therapy, and the like are well-known in the art.

[0260] In some embodiments, chemotherapy is used. Chemotherapy includes the administration of a chemotherapeutic agent. Such a chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: 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 rhizoxin. Compositions comprising one or more chemotherapeutic agents AOS-00725

[0261] (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiments, 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-lOOl (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001; Pacher et al., 2002b); 3 -aminobenzamide (Trevigen); 4-amino- 1,8-naphthalimide; (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re. 36,397); and NU1025 (Bowman et al ). The mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity. PARP catalyzes the conversion of .beta. -nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly-ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard V. J. et.al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 Jun. 2001, pp. 97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule in the repair of DNA singlestrand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame J C, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang Z Q, et al. (1997) Genes Dev 11:2347-2358). Knockout of SSB repair by inhibition of PARP 1 function induces DNA double-strand breaks (DSBs) that can trigger synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant H E, et al. (2005) Nature 434:913-917; Farmer H, et al. (2005) Nature 434:917-921). The foregoing examples of chemotherapeutic agents are illustrative, and are not intended to be limiting.

[0262] In other embodiments, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J. B. Lippencott Company, Philadelphia. The radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. The radiation treatment AOS-00725 can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfm (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy -hypocrellin A; and 2BA-2-DMHA.

[0263] In other embodiments, hormone therapy is used. Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide 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, progestins), vitamin A derivatives (e.g., all -trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0264] In other embodiments, photodynamic therapy (also called PDT, photoradiation therapy, phototherapy, or photochemotherapy) is used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light.

[0265] In yet other embodiments, laser therapy is used to harness high-intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It may also be used to treat cancer by shrinking or destroying tumors.

[0266] Exemplary Embodiments

[0267] The present disclosure describes the use of at least one biomarker (e.g., one or more gangliosides or one or more lipoforms thereof; ratio of one or more gangliosides or lipoforms thereof; inverse ratio of one or more gangliosides or lipoforms thereof; and / or one or more non-ganglioside biomarkers) in varioius diagnostic and prognostic methods.

[0268] In certain aspects, provided herein is a method of diagnosing a cancer in a subject, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer, wherein the at least one AOS-00725 biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

[0269] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or (viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a cancer.

[0270] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, AOS-00725

[0271] GM2, GA2, and a lipoform thereof; (vii) an amount of GDI a over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (viii) an amount of GM lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0272] (ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (x) an amount of GQ lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0273] (xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or (xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof. In some embodiments, a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a cancer.

[0274] In some embodiments, the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a cancer.

[0275] In some embodiments, the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1. In some embodiments, a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a cancer.

[0276] In certain aspects, provided herein is a method of determining a stage of a cancer, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the control indicates the stage of a cancer, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

[0277] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, AOS-00725

[0278] GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or (viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates that the cancer is of stage I, II, Ila, lib, III, or IV.

[0279] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (viii) an amount of GMlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; AOS-00725

[0280] (ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (x) an amount of GQ lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0281] (xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or (xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof. In some embodiments, a significantly lower level of the ratio in the subject sample as compared to the control indicates that the cancer is of stage I, II, Ila, lib, III, or IV.

[0282] In some embodiments, the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates that the cancer is of stage I, II, Ila, lib, III, or IV.

[0283] In some embodiments, the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1. In some embodiments, a significantly lower level of the ratio in the subject sample as compared to the control indicates that the cancer is of stage I, II, Ila, lib, III, or IV.

[0284] In certain aspects, provided herein is a method of detecting a recurrence of a cancer in a subject, the method comprising: a) determining the level of at least one biomarker in a sample from a subject whose cancer has regressed after receiving a cancer therapy; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control indicates a recurrence of a cancer in the subject, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

[0285] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a AOS-00725 lipoform thereof; (iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;

[0286] (iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or (viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

[0287] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (viii) an amount of GMlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0288] (ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (x) an amount of GQ lb over an AOS-00725 amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0289] (xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or and / or (xiii) an amount of GD 1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof. In some embodiments, a significantly lower level of the ratio in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

[0290] In some embodiments, the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

[0291] In some embodiments, the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1. In some embodiments, a significantly lower level of the ratio in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

[0292] In certain aspects, provided herein is a method for predicting the clinical outcome of a subject afflicted with a cancer, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b); wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control is an indication that the subject has a poor clinical outcome, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

[0293] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iii) an amount of GM3 or a lipform thereof over an amount of any one AOS-00725 selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;

[0294] (iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or (viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

[0295] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (viii) an amount of GMlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0296] (ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (x) an amount of GQ lb over an AOS-00725 amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0297] (xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or (xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof. In some embodiments, a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

[0298] In some embodiments, the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

[0299] In some embodiments, the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1. In some embodiments, a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

[0300] In certain aspects, provided herein is a method of monitoring the progression of a cancer in a subject, the method comprising: a) determining in a subject sample at a first point in time the level of at least one biomarker; b) repeating a) at a subsequent point in time; and c) comparing the level of the at least one biomarker determined in a) and b) to monitor the progression of the cancer in the subject, optionally wherein the subject is at risk for developing a cancer, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

[0301] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iv) an amount of lactosylceramides (LacCer) or a lipform AOS-00725 thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or (viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof.

[0302] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (viii) an amount of GMlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0303] (ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (x) an amount of GQ lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0304] (xi) an amount of GTla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a AOS-00725 lipoform thereof; and / or (xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof.

[0305] In some embodiments, the at least one biomarker comprises (a) a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1; and / or (b) a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1.

[0306] In some embodiments, between the first point in time and the subsequent point in time, the subject has received a cancer therapy.

[0307] In certain aspects, provided herein is a method of assessing the efficacy of a cancer therapy in a subject afflicted with a cancer, the method comprising: a) determining the level of at least one biomarker in a first sample obtained from a subject; b) repeating step a) during at least one subsequent point in time after administration of the cancer therapy; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the at least one subsequent sample, relative to the first sample, is an indication that the cancer therapy is or is not efficacious to treat a cancer in the subject, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

[0308] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, AOS-00725

[0309] GDlc, and a lipoform thereof; and / or (viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof. In some embodiments, a significantly lower level of the ratio in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

[0310] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (viii) an amount of GMlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (xi) an amount of GTla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or (xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof. In some embodiments, a significantly higher level of the ratio in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

[0311] In some embodiments, the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1. In some embodiments, a significantly lower level of the ratio in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject. AOS-00725

[0312] In some embodiments, the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1. In some embodiments, a significantly higher level of the ratio in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

[0313] In some embodiments, the first and / or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.

[0314] In certain aspects, provided herein is a method of detecting a minimal residual disease in a subject, the method comprising: a) determining the level of at least one biomarker in a sample from a subject in remission; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control indicates that the subject has a minimal residual disease, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

[0315] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; (vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or (viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, AOS-00725

[0316] GPlc, GQlb, GTla, GDlc, and a lipoform thereof. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

[0317] In some embodiments, the at least one biomarker comprises a ratio of: (i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vi) an amount of GT lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (vii) an amount of GDI a over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (viii) an amount of GM lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (xi) an amount of GTla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; (xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or (xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof. In some embodiments, a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

[0318] In some embodiments, the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1. In some embodiments, a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

[0319] In some embodiments, the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1. In some embodiments, a significantly lower level AOS-00725 of the ratio in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

[0320] In certain aspects, provided herein is a method of diagnosing a cancer in a subject, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

[0321] In some embodiments, the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2

[0322] 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3

[0323] 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3

[0324] 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1. In some embodiments, a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer.

[0325] In some embodiments, the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1. In some embodiments, a significantly lower level of of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer.

[0326] In certain aspects, provided herein is a method of determining a stage of a cancer, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the control indicates the stage of a cancer, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

[0327] In some embodiments, the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2

[0328] 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3

[0329] 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3

[0330] 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1. AOS-00725

[0331] In some embodiments, the at least one biomarker comprises GD2, GD3, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1, optionally wherein

[0332] (i) a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the cancer is of stage lib, III, or IV (e.g., stage 2b of neuroblastoma, stages III and / or IV of ovarian cancer); and / or (ii) a significantly lower level of the at least one biomarker in the subject sample as compared to the control indicates that the cancer is of stage II (e.g., stage II of ovarian cancer).

[0333] In some embodiments, the at least one biomarker comprises GDI, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, and / or GDI 42:2, optionally wherein (i) a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the cancer is of stage lib (e.g., neuroblastoma stage 2b); and / or (ii) a significantly lower level of the at least one biomarker in the subject sample as compared to the control indicates that the cancer is of stage II or Ila (e.g., stage II of ovarian cancer, stage 2a of neuroblastoma).

[0334] In certain aspects, provided herein is a method of detecting a recurrence of a cancer in a subject, the method comprising: a) determining the level of at least one biomarker in a sample from a subject whose cancer has regressed after receiving a cancer therapy; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control indicates a recurrence of a cancer in the subject, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

[0335] In some embodiments, the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2

[0336] 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3

[0337] 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3

[0338] 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1. In some embodiments, a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

[0339] In some embodiments, the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1. In some embodiments, a significantly lower level of the at least one AOS-00725 biomarker in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

[0340] In certain aspects, provided herein is a method for predicting the clinical outcome of a subject afflicted with a cancer, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b); wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control is an indication that the subject has a poor clinical outcome, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

[0341] In some embodiments, the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1. In some embodiments, a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

[0342] In some embodiments, the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1. In some embodiments, a significantly lower level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

[0343] In certain aspects, provided herein is a method of monitoring the progression of a cancer in a subject, the method comprising: a) determining in a subject sample at a first point in time the level of at least one biomarker; b) repeating a) at a subsequent point in time; and c) comparing the level of the at least one biomarker determined in a) and b) to monitor the progression of the cancer in the subject, optionally wherein the subject is at risk for developing a cancer, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

[0344] In some embodiments, the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, GD3 39: 1, GDI 36: 1, GDI 36:2, and / or GD3 44: 1. AOS-00725

[0345] In some embodiments, between the first point in time and the subsequent point in time, the subject has received a cancer therapy.

[0346] In certain aspects, provided herein is a method of assessing the efficacy of a cancer therapy in a subject afflicted with a cancer, the method comprising: a) determining the level of at least one biomarker in a first sample obtained from a subject; b) repeating step a) during at least one subsequent point in time after administration of the cancer therapy; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the at least one subsequent sample, relative to the first sample, is an indication that the cancer therapy is or is not efficacious to treat a cancer in the subject, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

[0347] In some embodiments, the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2

[0348] 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3

[0349] 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3

[0350] 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1. In some embodiments, a significantly lower level of the at least one biomarker in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

[0351] In some embodiments, the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1. In some embodiments, a significantly higher level of the at least one biomarker in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

[0352] In some embodiments, the first and / or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.

[0353] In certain aspects, provided herein is a method of detecting a minimal residual disease in a subject, the method comprising: a) determining the level of at least one biomarker in a sample from a subject in remission; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control indicates that the subject has a minimal residual disease, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof. AOS-00725

[0354] In some embodiments, the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2

[0355] 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3

[0356] 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3

[0357] 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1. In some embodiments, a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

[0358] In some embodiments, the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1. In some embodiments, a significantly lower level of of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

[0359] In certain aspects, provided herein is a method of diagnosing a cancer in a subject, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a subject sample; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer, wherein the at least one biomarker comprises B3GALT4.

[0360] In certain aspects, provided herein is a method of determining a stage of a cancer, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a subject sample; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the control indicates the stage of a cancer, optionally a stage I, II, Ila, lib, III, or IV of a cancer, wherein the at least one biomarker comprises B3GALT4.

[0361] In certain aspects, provided herein is a method of detecting a recurrence of a cancer in a subject, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a sample from a subject whose cancer has AOS-00725 regressed after receiving a cancer therapy; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the level in the control indicates a recurrence of a cancer in the subject, wherein the at least one biomarker comprises B3GALT4.

[0362] In certain aspects, provided herein is a method for predicting the clinical outcome of a subject afflicted with a cancer, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a subject sample; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b); wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the level in the control is an indication that the subject has a poor clinical outcome, wherein the at least one biomarker comprises B3GALT4.

[0363] In certain aspects, provided herein is a method of monitoring the progression of a cancer in a subject, the method comprising: a) determining in a subject sample at a first point in time the presence of at least one mutation, level, and / or activity of at least one biomarker; b) repeating a) at a subsequent point in time; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b) to monitor the progression of the cancer in the subject, optionally wherein the subject is at risk for developing a cancer, wherein the at least one biomarker comprises B3GALT4.

[0364] In some embodiments, between the first point in time and the subsequent point in time, the subject has received a cancer therapy.

[0365] In certain aspects, provided herein is a method of assessing the efficacy of a cancer therapy in a subject afflicted with a cancer, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a first sample obtained from a subject; b) repeating step a) during at least one subsequent point in time after administration of the cancer therapy; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), AOS-00725 wherein the absence of at least one mutation, a significantly higher level, and / or a significantly higher activity of the at least one biomarker in the at least one subsequent sample, relative to the first sample, is an indication that the cancer therapy is efficacious to treat a cancer in the subject, wherein the at least one biomarker comprises B3GALT4.

[0366] In some embodiments, the first and / or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.

[0367] In certain aspects, provided herein is a method of detecting a minimal residual disease in a subject, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a subject sample; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a minimal residual disease, wherein the at least one biomarker comprises B3GALT4.

[0368] Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the level of B3GALT4 is an RNA level, a cDNA level, a DNA level, and / or a protein level of B3GALT4.

[0369] In some embodiments, the at least one mutation reduces the activity of B3GALT4.

[0370] In some embodiments, the activity of B3GALT4 comprises the enzymatic activity of B3GALT4.

[0371] In some embodiments, the level of the at least one biomarker is determined using mass spectrometry, Western blot, radioimmune assay (RIA), immunohistochemistry (IHC), thin layer chromatography (TLC), flow cytometry, RT-PCR, PCR, and / or enzyme linked immunosorbent assay (ELISA).

[0372] In some embodiments, the level of the at least one biomarker is determined using mass spectrometry, optionally wherein the mass spectrometry is selected from LC-ESI- MS / MS, LC-ESI-CID-MS / MS, nanobore LC-ESI-MS, and nanobore LC-ESI-MS / MS.

[0373] In some embodiments, the level of the at least one biomarker is determined using ELISA, optionally wherein the ELISA is selected from sandwich ELISA or competitive ELISA. AOS-00725

[0374] In some embodiments, the level of the at least one biomarker is determined using IHC.

[0375] In some embodiments, the cancer is selected from ovarian cancer, neuroblastoma, lymphoma, leukemia, melanoma, glioma, small cell lung cancer, breast carcinoma, soft tissue sarcomas, osteosarcoma, Ewing’s sarcoma, desmoplastic round cell tumor, rhabdomyosarcoma, retinoblastoma, non-small cell lung cancer, renal cell cancer, Wilms tumor, prostate cancer, gastric cancer, endometrial cancer, pancreatic cancer, and colon cancer.

[0376] In preferred embodiments, the cancer is ovarian cancer or neuroblastoma.

[0377] In some embodiments, the sample comprises cells, serum, blood, peritumoral tissue, and / or intratumoral tissue.

[0378] In preferred embodiments, the sample comprises serum or blood.

[0379] In some embodiments, the significantly higher level of the at least biomarker comprises at least, no more than, or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%,

[0380] 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%,

[0381] 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%,

[0382] 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%,

[0383] 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%,

[0384] 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%,

[0385] 1000% increase compared to the control.

[0386] In some embodiments, the significantly lower level of the at least biomarker comprises at least, no more than, or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%,

[0387] 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%,

[0388] 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%,

[0389] 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%,

[0390] 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%,

[0391] 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%,

[0392] 1000% decrease compared to the control. AOS-00725

[0393] In some embodiments, the control comprises: (a) the level of the at least one biomarker (e.g., ratio, level) in a sample from a cancer-free subject; (b) the level of the at least one biomarker (e.g., ratio, level) in a sample from a subject afflicted with a cancer; (c) the level of the at least one biomarker (e.g., ratio, level) in a sample from a subject afflicted with a cancer, wherein the cancer is of stage I, II, Ila, lib, III, or IV ; or (d) the level of the at least one biomarker (e.g., ratio, level) in a portion of a single sample or pooled samples from any one of (a)-(c).

[0394] In some embodiments, the control is from the subject (e.g., first sample collected from the subject in longitudinal collections, to evaluate changes over time of the level of the at least one ganglioside).

[0395] In some embodiments, the method further comprises treating the subject. For example, in some embodiments, the method further comprises recommending, prescribing, and / or administering to the subject a cancer therapy. In some embodiments, the method comprises diagnosing or prognosing a subject and treating said subject based on the diagnosis or prognosis.

[0396] In some embodiments, the cancer therapy is a surgery, chemotherapy, cancer vaccines, chimeric antigen receptors, radiation therapy, immunotherapy, a modulator of expression of immune checkpoint inhibitory proteins or ligands, or any combination thereof. In some embodiments, the immunotherapy inhibits an immune checkpoint. In some embodiments, the immune checkpoint is selected from CTLA-4, PD-1, VISTA, B7-H2, B7- H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.

[0397] In some embodiments, the subject is a mammal.

[0398] In some embodiments, the subject is afflicted with a cancer, suspected of being afflicted with a cancer, or at risk for developing a cancer.

[0399] In some embodiments, the subject is an animal model of cancer, a dog, a cat, or a human, preferably a human.

[0400] EXAMPLES

[0401] Example 1: Materials and methods

[0402] Patients AOS-00725

[0403] Patients samples (liquid biopsies (serum or plasma); TMA can also be used) were obtained from the LDI / JHG biobank under ethical approval. The samples from the following patients were used:

[0404] • Diagnosis

[0405] • Normal (n = 70)

[0406] • Neuroblastoma (n = 15)

[0407] • Ovarian Cancer (n = 35)

[0408] • Stage

[0409] • Stage 1 OC (n = 5)

[0410] • Stage 2a Neuroblastoma (n = 10)

[0411] • Stage 2b Neuroblastoma (n = 5)

[0412] • Stage 2 OC (n = 3)

[0413] • Stage 3 OC (n = 21)

[0414] • Stage 4 OC (n = 6)

[0415] • Age (n = 119)

[0416] Extraction and isolation of gangliosides from tissues

[0417] Total lipids from tissues (e.g., tumors) (500 mg) that have been homogenized are extracted with 9 ml chloroform-methanol (2: 1; v / v) and then with 7.6 ml chloroformmethanol-water (1:2:0.8; v / v / v). The extraction is dried under a gentle stream of nitrogen, re-dissolved with 7.5 ml chloroform-methanol-water (30:60:8; v / v / v), and applied to a DEAE-Sephadex A-25 column (6 x 45 mm, acetate form) (Amersham Biosciences AB, Sweden). After washing neutral lipids from the column with 15 ml chloroform-methanol- water (30:60:8; v / v / v), the acidic glycolipid mixtures containing gangliosides are eluted with 7.5 ml chloroform-methanol-0.8 M sodium acetate (30:60:8; v / v / v).

[0418] Extraction and isolation of gangliosides from liquid biopsy

[0419] Monophasic lipid extraction of polar gangliosides with a solvent volumes ratio of 0.85 : 2 : 1 : 0.75 MeOH : EtOH : CHC13 : H2O. The method has been adapted from Svennerhohn and Fredman (1980) Biochim Biophys Acta 617:97-109; and Lydic et al. (2014) J Lipid Res 55: 1797-809, both of which are incorporated by reference in their entirety.

[0420] Liquid chromatography / electrospray ionization-MS AOS-00725

[0421] Liquid chromatography (LC) separations are achieved by using an octadecyl silane (ODS) reverse-phase LC (RPLC) column (150 x 0.3 mm inner diameter) (LC PACKINGS; Amsterdam, The Netherlands) at 45°C. The mobile phase is prepared by methanol (A), isopropanol (B), and water (containing 200 mM ammonium formate) (C), and the composition is produced by mixing these solvents. The gradient consist of holding solvent (A / B / C 55:25:20) (D) for 5 min, then linearly converting solvent D to solvent (A / B 60:40) (E) for 50 min, and holding solvent E for 35 min. The mobile phase is pumped at a flow rate of 7.5 pl / min for the gradient elution. Typically, 3 pl of sample solution is applied.

[0422] Electrospray ionization-tandem mass spectrometry analysis

[0423] The electrospray ionization (ESI)-MS analyses are performed by 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) combined with an HTS PAL autosampler (CTC Analytics AG, Zwingen, Switzerland).

[0424] The scan range of the instrument is set from m / z 200 to 2,300 at a scan speed of 1,000 amu / s. The trap fill time is 10 ms in the positive-ion mode and 20 ms in the negativeion mode. Other MS scan conditions are operated with the ion spray voltage of 5,500 V, declustering potential (DP) of 80 V, and collision- induced dissociation (CID) of 10 V in the positive-ion mode, and with the ion spray voltage of -4,500 V, DP of -120 V, and CID of -5 V in the negative-ion mode. Tandem mass spectrometry (MS / MS) analyses of ceramide molecular species derived from in-source decay of gangliosides are performed under variable CID conditions.

[0425] Multiple reaction monitoring (MRM) analysis

[0426] MRM analyses are performed with the same instrument system used for the LC / ESLMS analysis. DP is set at -120 V, and CID conditions for each ganglioside are optimized as follows: GM1, GM2, and GM3 (-95 V), GDI, GD2, and GD3 (-55 V), GT1, GT2, GT3, and GQ1 (-60 V). The precursor and product ion pairs for the MRM analysis are selected by these MS / MS spectra. The following MRM m / z transitions are monitored: parent ions to sialic acid ions (SA-) or sialic acid-containing sugar chains for gangliosides. For the analysis of regioisomeric gangliosides, specific fragments derived from terminal regions of the sugar chain in these isomers are selected and operated under the same conditions of the MRM analysis for each ganglioside. AOS-00725

[0427] Example 2: Alteration of the level of ganglioside isoforms in cancer patients

[0428] Human serum from healthy individuals, neuroblastoma patients (stages 2a and 2b), and ovarian cancer patients (stages I, II, III, and IV) were analyzed using mass spectrometry coupled to ultra high-performance liquid chromatography (UHPLC-MS.)

[0429] Several species / lipoforms of disialogangliosides GDI, GD2, and GD3 were detected. It was observed that serum from cancer patients showed elevated levels of several individual species of GD2 and GD3, especially in the late stages of cancer (see e.g., Fig. 1- Fig. 2). In particular, serum from ovarian cancer patients, e.g., at stages II, III, and IV showed elevated levels of several individual species of GD2 (Fig. 2A-Fig. 2B). In addition, ovarian cancer (e.g., stage II) and neuroblastoma stage 2a showed a decreased level of GDI and its lipoforms, whereas neuroblastoma stage 2b showed an elevated level of GDI and its lipoforms (Fig. 3A). Fig. 3B further showed that neuroblastoma stage 2b and certain stages (e.g., stage I and late-stages) of ovarian cancer showed an elevated level of GDI and certain lipoforms.

[0430] Looking at all detected species of disialogangliosides, there was a clear metabolic difference between healthy and ovarian cancer by Partial Least Squares Discriminant Analysis (PLSDA), which was also observed in the heat map (Fig. 3) A volcano plot showed significant increases in GD2 and GD3 species as stated, but also showed the decreased levels of GDI species. (Fig. 4) The enzyme B3GALT4 catalyzes the conversion of GD2 to GD 1 , thus the ratio of GD2 36 : 1 to GD 1 36 : 1 was determined to approximate the metabolic activity of this enzyme. B3GALT4 also showed the decreased level of protein expression in ovarian cancer tissue. A significant increase in the GD2 36: 1 to GDI 36: 1 ratio was observed, indicating that B3GALT4 had a decreased level and / or activity (Fig. 5).

[0431] To evaluate the ability of L CMS -derived tumor-marker gangliosides (TMG) profiles to distinguish ovarian cancer (OC) from non-cancer, LCMS data were generated from serum for 70 non-cancer and 35 OC subjects (n=8 early stage, n=27 late stage), with 31 distinct TMG species measured (n=8 GDI, n=6 GD2, n=17 GD3). Samples were profiled with 3 technical replicates per specimen and averaged for comparative statistical analysis. Evaluating OC vs. non-cancer groups, we found a significant reduction in the level of GD1(36: 1) (adj.P < 0.0076) in OC, as well as a significant increase in multiple GD2 and GD3 species including GD3(41:2), GD3(39: 1), GD2(34: 1), GD2(38: 1), GD2(36: 1), and GD3(35: 1) (adj.P < 0.01 for all comparisons, see Fig. 12A (Volcano plot)). Different AOS-00725 strategies for normalizing the LCMS data were further explored, emphasizing sample measures relative to the corresponding level of GD1(36: 1). This approach enhanced the signal between OC and non-cancer subjects, such that there was an improvement in effect size between OC and non-cancer subjects as well as an increase in statistical significance for several quantified species and improved directional trends from non-cancer, to early - stage and finally late-stage cancer (see Fig. 12B (Boxplot)). As an example, after normalization by GD1(36: 1), receiver operating characteristic (ROC) curve analysis found that GD2(36: 1) could distinguish normal from OC subjects with an area under the curve (AUC) of 92% (95CI 86-98%), as well as early-stage and late-stage cancers with AUCs of 86% and 94%, respectively (see Fig. 12C (ROC plot)).

[0432] Example 3: The ratio of GD2 36:1 to GDI 36:1 is increased in all stages of neuroblastoma and ovarian cancer

[0433] The relative abundance of GD2 36: 1 and GDI 36: 1 was detected using UHPLC-MS in human serum from healthy controls, neuroblastoma patients, and ovarian cancer patients (Fig. 5A-5C). Fig. 6A shows the ratio of GD2 36: 1 to GDI 36: 1 that was determined in serum from healthy individuals (n=70), all neuroblastoma patients (n=15), and all ovarian cancer patients (n=35). Fig. 6B shows the ratio of GD2 36: 1 to GDI 36: 1 that was determined in serum from healthy individuals (n=70), stage 2a neuroblastoma patients (n=10), stage 2b neuroblastoma patients (n=5), early-stage ovarian cancer patients (n=8), and late-stage ovarian cancer patients (n=27). Fig. 6C shows the ratio of GD2 36: 1 to GDI 36: 1 that was determined in serum from healthy individuals (n=70), stage 2a neuroblastoma patients (n=10), stage 2b neuroblastoma patients (n=5), and ovarian cancer patients from stage I (n=5), stage II (n=3), stage III (n=21), and stage IV (n=6). ns p > 0.05, * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0434] Example 4: The ratio of GDI 36:1 to GD2 36:1 is decreased in all stages of neuroblastoma and ovarian cancer

[0435] The relative abundance of GDI 36: 1 and GD2 36: 1 was detected using UHPLC-MS in human serum from healthy controls, neuroblastoma patients, and ovarian cancer patients. Fig. 7A shows the ratio of GDI 36: 1 to GD2 36: 1 in serum from healthy individuals (n=70), all neuroblastoma patients (n=15), and all ovarian cancer patients (n=35). Fig. 7B shows the ratio of GDI 36: 1 to GD2 36: 1 in serum from healthy individuals (n=70), stage 2a neuroblastoma patients (n=10), stage 2b neuroblastoma patients (n=5), early-stage AOS-00725 ovarian cancer patients (n=8), and late-stage ovarian cancer patients (n=27). Fig. 7C shows the ratio of GDI 36: 1 to GD2 36: 1 in serum from healthy individuals (n=70), stage 2a neuroblastoma patients (n=10), stage 2b neuroblastoma patients (n=5), and ovarian cancer patients from stage I (n=5), stage II (n=3), stage III (n=21), and stage IV (n=6). ns p > 0.05,

[0436] * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0437] Example 5: The ratio of certain gangliosides is decreased or increased in cancer

[0438] The ratio of various gangliosides are determined. For example, the ratio of:

[0439] (i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;

[0440] (ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;

[0441] (iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;

[0442] (iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;

[0443] (v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;

[0444] (vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;

[0445] (vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or

[0446] (viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof, AOS-00725 is determined and the ratio may be higher in cancer patients compared with the ratio of the same gangliosides in healthy patients.

[0447] The ratio of:

[0448] (i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0449] (ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0450] (iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0451] (iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0452] (v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0453] (vi) an amount of GT lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0454] (vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0455] (viii) an amount of GM lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0456] (ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0457] (x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0458] (xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;

[0459] (xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof, and / or

[0460] (xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof is determined and the ratio may be lower in cancer patients compared with the ratio of the same gangliosides in healthy patients. AOS-00725

[0461] Example 6: The level of GD3 and certain lipoforms of GD3 is increased in neuroblastoma and ovarian cancer

[0462] The GD3 level in cancer patients was determined (Fig. lA-Fig. IB). The relative abundance of various individual GD3 species was detected using UHPLC-MS in human serum from healthy controls (n=70), stage 2a (n=10) and stage 2b (n=5) neuroblastoma, early-stage ovarian cancer (n=8), and late-stage ovarian cancer (n=27). The total relative abundance of all detected GD3 species was summed to approximate total GD3 levels (bottom right).

[0463] The total level of GD3 or the level of each of certain lipoforms, e.g., GD3 (42:2), GD3 (40: 1), GD3 (38: 1), GD3 (36: 1), GD3 (32: 1), was increased in neuroblastoma stage 2b patients and certain stages of ovarian cancer patients compared to healthy patients.

[0464] Example 7: The level of GD2 and certain lipoforms of GD2 is increased in neuroblastoma stage 2b and ovarian cancer II, III and IV

[0465] The GD2 level in cancer patients was determined (Fig. 2A-Fig. 2B). The relative abundance of various individual GD2 species was detected using UHPLC-MS in human serum from healthy controls (n=70), stage 2a (n=10) and stage 2b (n=5) neuroblastoma, early-stage ovarian cancer (n=8) and late-stage ovarian cancer (n=27). The total relative abundance of all detected GD2 species was summed to approximate total GD2 levels (bottom right).

[0466] The total level of GD2 or the level of each of certain lipforms, e.g., GD2 (42:2), GD2 (40: 1), GD2 (38: 1), GD2 (36: 1), GD2 (34: 1), was increased in neuroblastoma stage 2b patients and certain stages of ovarian cancer stages patients compared to healthy patients.

[0467] Example 8: The level of GDI and certain lipoforms of GDI is increased in cancer

[0468] Fig. 3A shows that ovarian cancer (e.g., stage II) and neuroblastoma stage 2a showed the decreased levels of GDI, and that neuroblastoma stage 2b showed the elevated levels of GD 1. The relative abundance of various individual GD2 species was detected using UHPLC-MS in human serum from healthy controls (n=70), stage 2a (n=10) neuroblastoma, stage 2b (n=5) neuroblastoma, early-stage ovarian cancer (n=8), and late- stage ovarian cancer (n=27). The total relative abundance of all detected GDI species was summed to approximate total GDI levels (bottom right). AOS-00725

[0469] Fig. 3B shows that neuroblastoma stage 2b and certain stages (e.g., stage I and latestage) of ovarian cancer showed the elevated levels of GDI. The relative abundance of various individual GDI species was detected using UHPLC-MS in human serum from healthy controls (n=70), stage 2a (n=10) neuroblastoma, stage 2b (n=5) neuroblastoma, stage I ovarian cancer (n=5), stage II ovarian cancer (n=3), stage III ovarian cancer (n=21), and stage IV ovarian cancer (n=6). The total relative abundance of all detected GDI species was summed to approximate total GDI levels (bottom right).

[0470] Example 9: The level of certain lipoforms of GDI, GD2, and GD3 are increased or decreased in the serum of patients with ovarian cancer compared to healthy patients

[0471] The level of various lipoforms of GDI, GD2, and GD3 was determined (Fig. 5 and Fig. 12A).

[0472] The level of GD2 34: 1, GD2 36: 1, GD2 38: 1, GD2 42:2, GD2 40: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, GD3 39: 1 was increased in ovarian cancer patients compared to healthy patients.

[0473] The level of GDI (36: 1), GDI (36:2), and GD3 (44: 1) was decreased in ovarian cancer pateints compared to healthy patients.

[0474] Example 10: The level of B3GALT4 is decreased in various cancers

[0475] Fig. 9 shows that the protein level of B3GALT4 was reduced in various cancers. Fig. 10 also shows that the RNA expression level of B3GALT4 was reduced in various cancers. 5.4 FPKM is the calculated median across all female tissue samples, and many cancers showed the RNA expression level that was lower than the median.

[0476] Fig. 1 lA-Fig. 11C show the correlation of the RNA expression level of B3GALT4 and survival rate. Fig. 11A shows that the high expression of B3GALT4 correlated with the decreased 5-year survival rate in ovarian cancer patients. Fig. 1 IB shows that the high expression of B3GALT4 correlated with the decreased 5-year survival rate in renal cancer. Fig. 11C shows that the high expression of B3GALT4 correlated with the decreased 5-year survival rate in glioma. AOS-00725

[0477] Incorporation by reference

[0478] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0479] Also incorporated by reference in their entirety are any polynucleotide and amino acid sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.

[0480] Equivalents

[0481] 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 present invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

AOS-00725What is claimed is:

1. A method of diagnosing a cancer in a subject, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

2. The method of claim 1, wherein the at least one biomarker comprises a ratio of:(i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / orAOS-00725(viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a cancer.

3. The method of claim 1 or 2, wherein the at least one biomarker comprises a ratio of:(i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(viii) an amount of GMlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xi) an amount of GTla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or(xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof,AOS-00725 optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a cancer.

4. The method of any one of claims 1-3, wherein the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a cancer.

5. The method of any one of claims 1-4, wherein the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1, optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a cancer.

6. A method of determining a stage of a cancer, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the control indicates the stage of a cancer, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

7. The method of claim 6, wherein the at least one biomarker comprises a ratio of:(i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;AOS-00725(iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or(viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates that the cancer is of stage I, II, Ila, lib, III, or IV.

8. The method of claim 6 or 7, wherein the at least one biomarker comprises a ratio of:(i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;AOS-00725(viii) an amount of GM lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or(xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof, optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates that the cancer is of stage I, II, Ila, lib, III, or IV.

9. The method of any one of claims 6-8, wherein the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates that the cancer is of stage I, II, Ila, lib, III, or IV.

10. The method of any one of claims 6-9, wherein the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1, optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates that the cancer is of stage I, II, Ila, lib, III, or IV.

11. A method of detecting a recurrence of a cancer in a subject, the method comprising: a) determining the level of at least one biomarker in a sample from a subject whose cancer has regressed after receiving a cancer therapy; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control indicates a recurrence of a cancer in the subject,AOS-00725 wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

12. The method of claim 11, wherein the at least one biomarker comprises a ratio of:(i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or(viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

13. The method of claim 11 or 12, wherein the at least one biomarker comprises a ratio of:AOS-00725(i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vi) an amount of GT lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(viii) an amount of GM lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or(xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof, optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

14. The method of any one of claims 11-13, wherein the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.AOS-0072515. The method of any one of claims 11-14, wherein the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1, optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

16. A method for predicting the clinical outcome of a subject afflicted with a cancer, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b); wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control is an indication that the subject has a poor clinical outcome, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

17. The method of claim 16, wherein the at least one biomarker comprises a ratio of:(i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;AOS-00725(vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GT la, GDlc, and a lipoform thereof;(vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or(viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

18. The method of claim 16 or 17, wherein the at least one biomarker comprises a ratio of:(i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(viii) an amount of GMlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;AOS-00725(x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or(xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof, optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

19. The method of any one of claims 16-18, wherein the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

20. The method of any one of claims 16-19, wherein the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1, optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

21. A method of monitoring the progression of a cancer in a subject, the method comprising: a) determining in a subject sample at a first point in time the level of at least one biomarker; b) repeating a) at a subsequent point in time; and c) comparing the level of the at least one biomarker determined in a) and b) to monitor the progression of the cancer in the subject, optionally wherein the subject is at risk for developing a cancer, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

22. The method of claim 21, wherein the at least one biomarker comprises a ratio of:AOS-00725(i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or(viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof.

23. The method of claim 21 or 22, wherein the at least one biomarker comprises a ratio of:(i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;AOS-00725(iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vi) an amount of GT lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(viii) an amount of GM lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or(xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof.

24. The method of any one of claims 21-23, wherein the at least one biomarker comprises(a) a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1; and / or(b) a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1.

25. The method of claim any one of claims 21-24, wherein between the first point in time and the subsequent point in time, the subject has received a cancer therapy.

26. A method of assessing the efficacy of a cancer therapy in a subject afflicted with a cancer, the method comprising: a) determining the level of at least one biomarker in a first sample obtained from a subject;AOS-00725 b) repeating step a) during at least one subsequent point in time after administration of the cancer therapy; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the at least one subsequent sample, relative to the first sample, is an indication that the cancer therapy is or is not efficacious to treat a cancer in the subject, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.

27. The method of claim 26, wherein the at least one biomarker comprises a ratio of:(i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / orAOS-00725(viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof, optionally wherein a significantly lower level of the ratio in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

28. The method of claim 26 or 27, wherein the at least one biomarker comprises a ratio of:(i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vi) an amount of GTlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(viii) an amount of GMlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xi) an amount of GTla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / orAOS-00725(xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof, optionally wherein a significantly higher level of the ratio in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

29. The method of any one of claims 26-28, wherein the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1, optionally wherein a significantly lower level of the ratio in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

30. The method of any one of claims 26-29, wherein the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1, optionally wherein a significantly higher level of the ratio in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

31. The method of any one of claims 21-30, wherein the first and / or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.

32. A method of detecting a minimal residual disease in a subject, the method comprising: a) determining the level of at least one biomarker in a sample from a subject in remission; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control indicates that the subject has a minimal residual disease, wherein the at least one biomarker comprises a ratio of the amount of a ganglioside or a lipoform thereof over the amount of another ganglioside or a lipoform thereof.AOS-0072533. The method of claim 32, wherein the at least one biomarker comprises a ratio of:(i) an amount of GT3 or a lipoform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(ii) an amount of GD3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iii) an amount of GM3 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(iv) an amount of lactosylceramides (LacCer) or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(v) an amount of GT2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vi) an amount of GD2 or a lipform thereof over an amount of any one selected from: GDI, GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof;(vii) an amount of GM2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof; and / or(viii) an amount of GA2 or a lipform thereof over an amount of any one selected from: GTlc, GDlb, GM1, GAI, GQlc, GTlb, GDla, GMlb, GPlc, GQlb, GTla, GDlc, and a lipoform thereof, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

34. The method of claim 32 or 33, wherein the at least one biomarker comprises a ratio of:(i) an amount of GTlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;AOS-00725(ii) an amount of GDlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iii) an amount of GM1 over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(iv) an amount of GAI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(v) an amount of GQlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vi) an amount of GT lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(vii) an amount of GDla over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(viii) an amount of GM lb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(ix) an amount of GPlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(x) an amount of GQlb over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xi) an amount of GT la over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof;(xii) an amount of GDlc over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof; and / or(xiii) an amount of GDI over an amount of any one selected from: GT3, GD3, GM3, LacCer, GT2, GD2, GM2, GA2, and a lipoform thereof, optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

35. The method of any one of claims 32-34, wherein the at least one biomarker comprises a ratio of an amount of GD2 36: 1 over an amount of GDI 36: 1, optionally wherein a significantly higher level of the ratio in the subject sample as compared to the control indicates that the subject has a minimal residual disease.AOS-0072536. The method of any one of claims 32-35, wherein the at least one biomarker comprises a ratio of an amount of GDI 36: 1 over an amount of GD2 36: 1, optionally wherein a significantly lower level of the ratio in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

37. A method of diagnosing a cancer in a subject, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

38. The method of claim 37, wherein the at least one biomarker comprises(a) GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD334:2, GD3 36:2, GD3 38:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD338: 1, GD3 36: 1, GD3 32: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1, optionally wherein a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer.

39. The method of claim 37 or 38, wherein the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1, optionally wherein a significantly lower level of of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer.

40. A method of determining a stage of a cancer, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the control indicates the stage of a cancer,AOS-00725 wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

41. The method of claim 40, wherein the at least one biomarker comprises(a) GD2, GD3, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1, optionally wherein(i) a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the cancer is of stage lib, III, or IV (e.g., stage 2b of neuroblastoma, stages III and / or IV of ovarian cancer); and / or(ii) a significantly lower level of the at least one biomarker in the subject sample as compared to the control indicates that the cancer is of stage II (e.g., stage II of ovarian cancer); and / or(b) GDI, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, and / or GDI 42:2, optionally wherein(i) a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the cancer is of stage lib (e.g., neuroblastoma stage 2b); and / or(ii) a significantly lower level of the at least one biomarker in the subject sample as compared to the control indicates that the cancer is of stage II or Ila (e.g., stage II of ovarian cancer, stage 2a of neuroblastoma)42. A method of detecting a recurrence of a cancer in a subject, the method comprising: a) determining the level of at least one biomarker in a sample from a subject whose cancer has regressed after receiving a cancer therapy; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control indicates a recurrence of a cancer in the subject, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.AOS-0072543. The method of claim 42, wherein the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1, optionally wherein a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

44. The method of claim 42 or 43, wherein the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1, optionally wherein a significantly lower level of the at least one biomarker in the subject sample as compared to the control indicates a recurrence of a cancer in the subject.

45. A method for predicting the clinical outcome of a subject afflicted with a cancer, the method comprising: a) determining the level of at least one biomarker in a subject sample; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b); wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control is an indication that the subject has a poor clinical outcome, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

46. The method of claim 45, wherein the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1, optionally wherein a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.AOS-0072547. The method of claim 45 or 46, wherein the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1, optionally wherein a significantly lower level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a poor clinical outcome.

48. A method of monitoring the progression of a cancer in a subject, the method comprising: a) determining in a subject sample at a first point in time the level of at least one biomarker; b) repeating a) at a subsequent point in time; and c) comparing the level of the at least one biomarker determined in a) and b) to monitor the progression of the cancer in the subject, optionally wherein the subject is at risk for developing a cancer, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

49. The method of claim 48, wherein the at least one biomarker comprises GDI, GD2,GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI42:2, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD338:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD332: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, GD3 39: 1, GDI 36: 1, GDI 36:2, and / or GD3 44: 1.

50. The method of claim 48 or 49, wherein between the first point in time and the subsequent point in time, the subject has received a cancer therapy.

51. A method of assessing the efficacy of a cancer therapy in a subject afflicted with a cancer, the method comprising: a) determining the level of at least one biomarker in a first sample obtained from a subject; b) repeating step a) during at least one subsequent point in time after administration of the cancer therapy; and c) comparing the level of the at least one biomarker determined in a) and b),- I l l -AOS-00725 wherein a significantly lower level or higher level of the at least one biomarker in the at least one subsequent sample, relative to the first sample, is an indication that the cancer therapy is or is not efficacious to treat a cancer in the subject, wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

52. The method of claim 51, wherein the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1, optionally wherein a significantly lower level of the at least one biomarker in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

53. The method of claim 51 or 52, wherein the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1, optionally wherein a significantly higher level of the at least one biomarker in the at least one subsequent sample, relative to the first sample, indicates that the cancer therapy is efficacious to treat a cancer in the subject.

54. The method of any one of claims 48-53, wherein the first and / or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.

55. A method of detecting a minimal residual disease in a subject, the method comprising: a) determining the level of at least one biomarker in a sample from a subject in remission; b) determining the level of the at least one biomarker in a control; and c) comparing the level of the at least one biomarker determined in a) and b), wherein a significantly lower level or higher level of the at least one biomarker in the subject sample as compared to the level in the control indicates that the subject has a minimal residual disease,AOS-00725 wherein the at least one biomarker comprises at least one ganglioside or a lipoform thereof.

56. The method of claim 55, wherein the at least one biomarker comprises GDI, GD2, GD3, GDI 32: 1, GDI 34: 1, GDI 34:2, GDI 36: 1, GDI 36:2, GDI 38: 1, GDI 40:2, GDI 42:2, GD2 42:2, GD2 40: 1, GD2 38: 1, GD2 36: 1, GD2 34: 1, GD3 34:2, GD3 36:2, GD3 38:2, GD3 40:2, GD3 41: 1, GD3 44:2, GD3 42:2, GD3 40: 1, GD3 38: 1, GD3 36: 1, GD3 32: 1, GD3 35: 1, GD3 37: 1, GD3 41:2, and / or GD3 39: 1, optionally wherein a significantly higher level of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

57. The method of claim 55 or 56, wherein the at least one biomarker comprises GDI 36: 1, GDI 36:2, and / or GD3 44: 1, optionally wherein a significantly lower level of of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a minimal residual disease.

58. A method of diagnosing a cancer in a subject, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a subject sample; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a cancer, wherein the at least one biomarker comprises B3GALT4.

59. A method of determining a stage of a cancer, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a subject sample; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; andAOS-00725 c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the control indicates the stage of a cancer, optionally a stage I, II, Ila, lib, III, or IV of a cancer, wherein the at least one biomarker comprises B3GALT4.

60. A method of detecting a recurrence of a cancer in a subject, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a sample from a subject whose cancer has regressed after receiving a cancer therapy; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the level in the control indicates a recurrence of a cancer in the subject, wherein the at least one biomarker comprises B3GALT4.

61. A method for predicting the clinical outcome of a subject afflicted with a cancer, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a subject sample; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b); wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the level in the control is an indication that the subject has a poor clinical outcome, wherein the at least one biomarker comprises B3GALT4.AOS-0072562. A method of monitoring the progression of a cancer in a subject, the method comprising: a) determining in a subject sample at a first point in time the presence of at least one mutation, level, and / or activity of at least one biomarker; b) repeating a) at a subsequent point in time; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b) to monitor the progression of the cancer in the subject, optionally wherein the subject is at risk for developing a cancer, wherein the at least one biomarker comprises B3GALT4.

63. The method of claim 54, wherein between the first point in time and the subsequent point in time, the subject has received a cancer therapy.

64. A method of assessing the efficacy of a cancer therapy in a subject afflicted with a cancer, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a first sample obtained from a subject; b) repeating step a) during at least one subsequent point in time after administration of the cancer therapy; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), wherein the absence of at least one mutation, a significantly higher level, and / or a significantly higher activity of the at least one biomarker in the at least one subsequent sample, relative to the first sample, is an indication that the cancer therapy is efficacious to treat a cancer in the subject, wherein the at least one biomarker comprises B3GALT4.

65. The method of any one of claims 62-64, wherein the first and / or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.AOS-0072566. A method of detecting a minimal residual disease in a subject, the method comprising: a) determining the presence of at least one mutation, level, and / or activity of at least one biomarker in a subject sample; b) determining the presence of at least one mutation, level, and / or activity of the at least one biomarker in a control; and c) comparing the presence of at least one mutation, level, and / or activity of the at least one biomarker determined in a) and b), wherein the presence of at least one mutation, a significantly lower level, and / or a significantly lower activity of the at least one biomarker in the subject sample as compared to the control indicates that the subject has a minimal residual disease, wherein the at least one biomarker comprises B3GALT4.

67. The method of any one of claims 58-66, wherein the level of B3GALT4 is an RNA level, a cDNA level, a DNA level, and / or a protein level of B3GALT4.

68. The method of any one of claims 58-67, wherein the at least one mutation reduces the activity of B3GALT4.

69. The method of any one of claims 58-68, wherein the activity of B3GALT4 comprises the enzymatic activity of B3GALT4.

70. The method of any one of claims 1-69, wherein the level of the at least one biomarker is determined using mass spectrometry, Western blot, radioimmune assay (RIA), immunohistochemistry (IHC), thin layer chromatography, flow cytometry, RT-PCR, PCR, and / or enzyme linked immunosorbent assay (ELISA).

71. The method of claim 70, wherein the the level of the at least one biomarker is determined using mass spectrometry, optionally wherein the mass spectrometry is selected from LC-ESLMS / MS, LC-ESI-CID-MS / MS, nanobore LC-ESLMS, and nanobore LC- ESI-MS / MS.AOS-0072572. The method of claim 70, wherein the level of the at least one biomarker is determined using ELISA, optionally wherein the ELISA is selected from sandwich ELISA or competitive ELISA.

73. The method of claim 70, wherein the level of the at least one biomarker is determined using IHC.

74. The method of any one of claims 1-73, wherein the cancer is selected from ovarian cancer, neuroblastoma, lymphoma, leukemia, melanoma, glioma, small cell lung cancer, breast carcinoma, soft tissue sarcomas, osteosarcoma, Ewing’s sarcoma, desmoplastic round cell tumor, rhabdomyosarcoma, retinoblastoma, non-small cell lung cancer, renal cell cancer, Wilms tumor, prostate cancer, gastric cancer, endometrial cancer, pancreatic cancer, and colon cancer.

75. The method of any one of claims 1-74, wherein the cancer is ovarian cancer or neuroblastoma.

76. The method of any one of claims 1-75, wherein the sample comprises cells, serum, blood, peritumoral tissue, and / or intratumoral tissue.

77. The method of any one of claims 1-76, wherein the sample comprises serum or blood.

78. The method of any one of claims 1-77, wherein the significantly higher level of the at least biomarker comprises at least about 20% increase.

79. The method of any one of claims 1-77, wherein the significantly lower level of the at least biomarker comprises at least about 20% decrease.

80. The method of any one of claims 1-79, wherein the control comprises:(a) the level of the at least one biomarker (e.g., ratio, level) in a sample from a cancer-free subject;AOS-00725(b) the level of the at least one biomarker (e.g., ratio, level) in a sample from a subject afflicted with a cancer;(c) the level of the at least one biomarker (e.g., ratio, level) in a sample from a subject afflicted with a cancer, wherein the cancer is of stage I, II, Ila, lib, III, or IV; or(d) the level of the at least one biomarker (e.g., ratio, level) in a portion of a single sample or pooled samples from any one of (a)-(c).

81. The method of any one of claims 1-80, wherein the control is from the subject (e.g., first sample collected from the subject in longitudinal collections, to evaluate changes over time of the level of the at least one ganglioside).

82. The method of any one of claims 1-81, further comprising treating the subject, optionally recommending, prescribing, and / or administering to the subject a cancer therapy.

83. The method of any one of claims 1-82, wherein the cancer therapy is a surgery, chemotherapy, cancer vaccines, chimeric antigen receptors, radiation therapy, immunotherapy, a modulator of expression of immune checkpoint inhibitory proteins or ligands, or any combination thereof.

84. The method of claim 83, wherein the immunotherapy inhibits an immune checkpoint.

85. The method of claim 84, wherein the immune checkpoint is selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX- 40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.

86. The method of any one of claims 1-85, wherein the subject is a mammal, optionally wherein the subject is afflicted with a cancer, suspected of being afflicted with a cancer, or at risk for developing a cancer.AOS-0072587. The method of any one of claims 1-86, wherein the subject is an animal model of cancer, a dog, a cat, or a human, preferably a human.

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