WO2016186987A1

WO2016186987A1 - Biomarker micrornas and method for determining tumor burden

Info

Publication number: WO2016186987A1
Application number: PCT/US2016/032275
Authority: WO
Inventors: Arti B. Gaur
Original assignee: Trustees Of Dartmouth College
Priority date: 2015-05-15
Filing date: 2016-05-13
Publication date: 2016-11-24

Abstract

Methods for determining tumor burden, treatment toxicity and treatment efficacy using a panel of microRNAs as biomarkers in biological samples are described.

Description

BIO ARKER MICRORNAS AND METHOD FOR DETERMINING TUMOR BURDEN

Introduction

[0001] This application claims the benefit of priority from U.S. Patent Application Serial No. 62/162,133, filed May 15, 2015, the contents of which are incorporated herein by reference in their entirety.

Background

[0002] Every year, approximately 51,000 new cases of gliomas are diagnosed in the United States. They are the most common and aggressive type of primary brain tumors that originate and reside within the brain. Although a heterogeneous group of malignancies, gliomas are classified based on their histological characteristics into four grades by the World Health Organization: grade I (pilocytic astrocytoma) , grade II (diffuse astrocytoma) , grade III

(anaplastic astrocytoma) , and grade IV (glioblastoma) . Glioblastomas are among the most resistant of human cancers to currently available modalities of treatment and have the worst prognosis of any central nervous system malignancy with a median survival of only 14 months and a 5 year survival rate of less than 5%. Despite attempts to combine chemotherapy, surgery, and radiation, glioblastomas recur in more than 90% of cases, usually within 2 cm of the original site. Gliomas harbor diverse oncogenes and mutated tumor suppressor genes whose patterns of alteration and expression vary considerably from tumor to tumor making their routine, standardized molecular characterization challenging. EGF receptor amplification, p53 mutations, deletion of PTEN and pl6INK4A/pl4ARF and the activation of RAS-MAPK and PI3K-AKT signaling pathways have been frequently observed in glioblastomas. Recently mRNA expression profiling has been used to characterize glioblastoma, however these studies differ regarding the number of subclasses and the prognostic value regarding the correlation of the subclasses to patient survival (Furnari, et al. (2007) Genes Dev. 21:2683-2710; Dell'Albani (2008) Neurochem. Res. 33:2407-2415). The failure of multimodal therapies against glioblastomas and the resulting dismal prognosis has primarily been attributed to the paucity of knowledge on their cellular origin and the molecular mechanism that initiates and maintains gliomagenesis . Considering this, knowledge of molecular biomarkers that regulate normal development and contribute to malignancy is essential for designing effective strategies for glioblastoma treatment.

[ 0003 ] MicroRNAs (mxRNAs) are small non-coding RNAs that regulate gene expression following recognition of sequence- specific binding sites, typically within the 3 ' -UTR of target mRNAs, causing mRNA deadenylation and degradation or translational repression. They play a central role in diverse cellular and developmental processes and deregulated miRNA expression has been observed in numerous cancers. In glioblastoma tissue, characteristic miRNA expression patterns have been reported (Gaur, et al . (2007) Cancer Res. 67 : 2456-68 ; Chan, et al . (2005) Cancer Res. 65:6029-33; O'Donnell, et al . (2005) Nature 435:839-43; Johnson, et al . (2005) Cell 120:635-47; Godlewski, et al . (2008) 68:9125-9130; Silber, et al . (2008) BMC Med. 6:14; Kapsimali, et al . (2007) Genome Biol. R173; Ma, et al . (2010) Nat. Biotechnol. 28:341-7; WO 2012/114189). This analysis identified oncogenic miRNAs hsa-mir-21, hsa-mir- 196a, hsa-mir-lOa, hsa-mir-30a, and hsa-mir-451 in gliomas that are expressed at higher levels than in normal brain tissue. Further, miR-lOb has been associated with high- grade tumors (Gabriely, et al . (2011) Cancer Res. 71:3563- 72) and recent work has demonstrated that down regulation of miR-lOb inhibits metastasis in a mouse mammary tumor model as well as glioblastomas (Gabriely, et al . (2011) Cancer Res. 71:3563-72; Abdel-Fattah, et al . (2006) J^". Pathol. 209:15-24) . A validated target of miR-lOb is HOXD10, a homeobox (Hox) gene and transcription factor that regulates organogenesis and maintains differentiated tissue function. The loss of HOXD10 results in increased invasiveness, angiogenesis , and metastasis in cancer. miR- 10b- induced glioma cell invasion occurs by modulating tumor invasion factors MMP-14 and uPAR expression via the direct target, HOXD10.

Summary of the Invention

[0004] This invention is a method for determining tumor burden in a subject with glioma by (a) providing a biological sample obtained from a subject with glioma; (b) determining the level of expression of at least one biomarker microRNA (miRNA) in said biological sample from the subject, wherein the at least one biomarker miRNA is selected from a miRNA in Table 1; and (c) comparing the level of expression determined in step (b) to a reference level of the same biomarker miRNA, wherein differences in the expression level of the biomarker miRNA are indicative of tumor burden. In one embodiment, the biological sample in step (a) is obtained from the subject after the subject has received treatment for glioma. In another embodiment, the reference level of the same biomarker miRNA in step (c) is obtained from a second biological sample from the subject before the subject received treatment for glioma. In further embodiments, the treatment is surgery, chemotherapy, radiation therapy or a combination thereof and the subject has glioblastoma.

Brief Description of the Drawings

[0005] Figure 1 shows the timepoints for the four blood draws scheduled during standard treatment post -surgical resection for gliomas. Blood draw 1 took place before surgical resection.

[0006] Figure 2 shows the timepoints for seven blood draws scheduled during standard treatment post -surgical resection for gliomas to monitor treatment toxicity/efficacy . Blood draw 1 took place before surgical resection.

Detailed Description of the Invention

[0007] Glioma biomarkers have been conventionally developed through comparison between primary tissues with and without cancer, which requires invasive biopsy procedures. In contrast, the present invention is based on a detailed analysis and discovery that profiles of circulating miRNAs and their mRNA targets in glioma patients from time of diagnosis, post surgical resection, through end of treatment and progression free survival or death are indicative of tumor burden, treatment toxicity and treatment efficacy. Thus, compared to conventional biomarkers, the biomarker miRNAs of the present invention can be readily measured by noninvasive procedures and used in methods for determining tumor burden, treatment toxicity and treatment efficacy in glioma patients. Furthermore, development of non-radiographical response measures for glioma is useful in preventing a pseudoprogression or pseudoresponse that is known to be associated with standard of care agents. Accordingly, the circulating biomarker miRNAs of this invention provide a clinician with information useful for screening, examination, surveillance, diagnosis and/or treatment options.

[0008] The methods of this invention include the steps of

(a) providing a biological sample obtained from a subject;

(b) determining the level of expression of at least one biomarker miRNA in said biological sample from the subject; and (c) comparing the level of expression determined in step (b) to a reference level of the same biomarker miRNA, wherein differences in the expression level of the biomarker miRNA are indicative of tumor burden, treatment toxicity and treatment efficacy. In certain embodiments, the biological sample in step (a) is obtained from the subject after the subject has received treatment for glioma, e.g., surgery, chemotherapy and/or and radiation therapy. In another embodiment, the reference level of the same biomarker miRNA in step (c) is obtained from a biological sample from the subject before the subject received treatment for glioma. A "subject" or "patient" in accordance with the methods of this invention is meant to be any single subject, including humans, cattle, dogs, guinea pigs, rabbits, chickens, and so on, that has been diagnosed with a glioma, e.g., glioblastoma, anaplastic astrocytoma, anaplastic oligoastrocytoma, low-grade astrocytoma, oligodendroglioma, or pilocytic astrocytoma. Also intended to be included as a subject are any subjects involved in clinical research trials or subjects involved in epidemiological studies for glioma.

[0009] As used herein, the term "biological sample" refers to a fluid containing cells and compounds of biological origin, and may include blood, stool or feces, lymph, urine, serum, plasma, pus, saliva, seminal fluid, tears, urine, bladder washings, colon washings, sputum or fluids from the respiratory, alimentary, circulatory, or other body systems. In particular embodiments, the "biological sample" is blood, e.g., whole blood, plasma or serum. For the purposes of the present invention the biomarker miRNA in the biological sample may be present in a circulating cell or may be present in cell-free circulating nucleic acids. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Alternatively, the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[ 0010 ] A "biomarker" refers to a molecular indicator that is associated with a particular pathological or physiological state. The biomarker as used herein is a molecular indicator for glioma, more specifically an indicator for tumor burden, treatment toxicity and treatment efficacy in glioma. The term "biomarker miRNA" refers to a miRNA whose expression level is an indicator for, or predictive of, tumor burden, treatment toxicity and treatment efficacy in glioma. Biomarker miRNAs are short RNA molecules (e.g., 16-29 nucleotides in length) that arise from longer precursors, which are transcribed from non-protein coding genes . The precursors are processed by cellular proteins to generate short double-stranded miRNA. One of the miRNA strands is incorporated into a complex of proteins and miRNA called the RNA-induced silencing complex (RISC) . The miRNA guides the RISC complex to a target mRNA, which is then cleaved or translationally silenced, depending on the degree of sequence complementarity of the miRNA or its target mRNA. [ 0011] In accordance with the methods of this invention, the level of expression of at least one biomarker miRNA in a biological sample is determined or measured. Measuring or determining expression levels of a miRNA can be accomplished by a variety of different chemical and/or enzymatic reactions. In certain embodiments, methods may involve, but not be limited to, next generation sequencing, single-molecule real-time sequencing, mass spectrometry, digital color-coded barcode technology analysis, microarray expression profiling, quantitative PCR, reverse transcriptase PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end-point PCR, multiplex end- point PCR, cold PCR, ice-cold PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HP A) , branched DNA (bDNA) assay, rolling circle amplification (RCA) , single molecule hybridization detection, invader assay, and/or Bridge Litigation Assay. Other non-limiting amplification methods may include real-time PCR (quantitative PCR (q-PCR) ) , digital PCR, nucleic acid sequence-base amplification (NASBA) , ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave) , rolling circle amplification, in vitro transcription (IVT) , strand displacement amplification, transcription- mediated amplification (TMA) , RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art.

[ 0012 ] The methods may involve isolating nucleic acids such as ribonucleic acids from the biological sample, amplifying the nucleic acids and/or contacting or hybridizing one or more probes to an amplified or non-amplified nucleic acid. In certain embodiments, a microarray may be used to measure or determine the level of miRNA expression in a sample. Methods and compositions for isolating, enriching, and/or labeling miRNA molecules and for preparing and using probes, primer and/or arrays or other detection techniques for miRNA analysis are described in the art and may be found in US 7,919,245 (incorporated herein by reference in its entirety) .

[ 0 013 ] In some embodiments, the level of expression of at least one biomarker miRNA is determined or measured by contacting the biomarker miRNA with a binding agent (e.g., a probe or primer, or microarray containing a probe) and detecting binding between the miRNA and the binding agent. In other embodiments, the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or more biomarker miRNAs is determined or measured by contacting the biomarker miRNA with a binding agent and detecting binding between the miRNA and the binding agent . Biomarker miRNAs of use in the methods of this invention are preferably selected from the group of miRNA listed in Table 1.

TABLE 1

hsa-miR-4302 150 hsa-miR-4516 277 hsa-miR-450a-5p hsa-miR-9-5p 151 hsa-miR-4732-5p 278 hsa-miR-183-3p hsa-miR-624-5p 152 hsa-let-7g-5p 279 hsa-miR-485-3p hsa-miR-99b~5p 153 hsa-miR-652-3p 280 hsa-miR-222-3p hsa-miR~338-3p 154 hsa-miR-27b-3p 281 hsa-miR-211-5p sa-miR-17-5p 155 hsa-miR-142-3p 282 hsa-miR-126-3p hsa-miR-29b-3p 156 hsa-miR-454-3p 283 hsa-miR-26a-5p hsa-miR-3183 157 hsa-miR-4505 284 hsa-miR- 664a-3p hsa-miR-106b-5p 158 hsa-miR-96-5p 285 hsa-miR-199b-3p hsa-miR-720 159 hsa-miR-550a-5p 286 hsa-miR- 203a- 3p hsa-let-7i-5p 160 hsa-miR-23b-3p 287 hsa-miR-1180-3p hsa-miR-20b-5p 161 hsa-miR-433-3p 288 hsa-miR-1287-5p hsa-miR-194-5p 162 hsa-miR-487b-3p 289 hsa-miR-3653-3p hsa-miR-486-3p 163 hsa-miR-25-3p 290 hsa-miR-4538 hsa-miR-181c-5p 164 hsa-miR-330-3p 291 hsa-miR-487a-3p hsa-miR-20a-5p 165 hsa-miR-374c-5p 292 hsa-miR-4687-5p hsa-miR-885-5p 166 hsa-miR-215-5p 293 hsa-miR-379-5p hsa-miR-4289 167 sa-miR-629-5p 294 hsa-miR-1307-3p hsa-miR-144-5p 168 hsa-miR-627-5p 295 hsa-miR-1233-3p hsa-miR-1207-5p 169 hsa-miR-340-5p 296 hsa-miR-19b-l-5p hsa-miR-195-5p 170 hsa-miR-422a 297 hsa-miR-151a-3p hsa-miR-3200-3p 171 hsa-miR-378e 298 hsa-miR-145-5p hsa-miR-192-5p 172 hsa-miR-4296 299 hsa-miR-1247-5p hsa-miR-93-3p 173 hsa-miR-423-5p 300 hsa-miR-1203 sa-miR-1280 174 hsa-miR-485-5p 301 hsa-miR-548o-5p hsa-miR-500a-5p 175 hsa-let-7a-3p 302 hsa-let-7f -2-3p hsa-miR-16-5p 176 hsa-miR-339-5p 303 hsa-miR- 675 -3p hsa-miR-214-3p 177 hsa-miR-483-5p 304 hsa-miR-199a-5p hsa-miR-4306 178 hsa-miR-320a 305 hsa-miR-3923 hsa-miR-497-5p 179 hsa-miR-590-3p 306 hsa-miR-425-3p hsa-miR-424-5p 180 hsa-miR-22 -5p 307 hsa-miR-3159 hsa-miR-193b-3p 181 hsa-miR-296-5p 308 hsa-miR-628-3p hsa-miR-140-5p 182 hsa-miR-502-3p 109 hsa-miR-324-3p hsa-miR-3911 183 hsa-miR-424-3p 310 hsa-miR- 23 a- 3p sa-miR-101-3p 184 hsa-miR-1976 311 hsa-miR-1260b hsa-miR-15b-5p 185 hsa-miR-30e-3p 312 hsa-miR-202-3p hsa-miR-103a-3p 186 hsa-miR-378h 313 hsa-miR-197-3p hsa-miR-185-5p 187 hsa-miR-5095 314 hsa-miR-4323 hsa-miR-18a-5p 188 hsa-miR-139-5p 315 hsa-miR-576-5p hsa-miR-942-5p 189 hsa-miR-369-3p 316 hsa-miR-2276-3p hsa-miR-3141 190 hsa-miR-l-3p 317 hsa-miR-1237-3p hsa-miR-590-5p 191 hsa-miR-548d-5p 318 hsa-miR-31-5p hsa-miR-183-5p 192 hsa-miR-378g 319 hsa-miR-551a hsa-miR-181b-5p 193 hsa-miR-1909-5p 330 hsa-miR-499a-5p hsa-miR-503 -5p 194 sa-miR-4454 331 hsa-miR-4688 sa-miR-15a-5p 195 hsa-miR-155-5p 332 hsa-miR-4267 hsa-miR-210-3p 196 hsa-miR- 7 - 1 - 3p 333 hsa-miR-375 hsa-miR-542-3p 197 hsa-miR-181c-3p 334 hsa-miR- 30e-5p sa-let-7f -5p 198 hsa-miR- 596 335 hsa-miR- 615 -5p hsa-miR-874-3p 199 hsa-miR-186-5p 336 hsa-miR-637 hsa-miR-19b-3p 200 hsa-miR-378b 337 hsa-miR-4422 74 hsa-miR-18b-5p 201 hsa-miR-103a-2 -5p 338 hsa-miR-22-3p

75 hsa-miR-16-2-3p 202 hsa-miR-542 -5p 339 hsa-miR-3191-3p

76 hsa~miR-365b-3p 203 hsa-miR-378a-3p 330 hsa-miR-3689e

77 hsa-miR-421 204 hsa-miR-484 331 hsa-miR-26b-3p

78 hsa-miR- 15a-3p 205 hsa-miR-28-3p 332 hsa-miR-126-5p

79 hsa-miR-362 -3p 206 hsa-miR-331-3p 333 hsa-miR-184

80 hsa-miR-769-5p 207 hsa-miR-4732-3p 334 hsa-miR-372-3p

81 hsa-miR-29a-3p 208 hsa-let-7g-3p 335 hsa-miR-1290

82 hsa-miR-301b-3p 109 hsa-miR-3131 336 hsa-miR-1286

83 hsa-miR-34a-5p 210 hsa-miR-342-3p 337 hsa-miR-3120-3p

84 hsa-miR- 15b-3p 211 hsa-miR-423-3p j 338 hsa-miR-133b

85 hsa-miR-17-3p 212 hsa-miR-339-3p 339 hsa-miR-373-3p

86 hsa-miR-1183 213 hsa-miR-124-3p 340 hsa-miR-1193

87 hsa-miR-19a-3p 214 hsa-miR-21-3p 341 hsa-let-7f-l-3p

88 hsa-miR-188-5p 215 hsa-miR-877-5p 342 hsa-miR-548e-3p

89 hsa-miR- 1260a 216 hsa-miR-361-3p 343 hsa-miR-412-3p

90 hsa-miR- 152 -3p 217 hsa-miR-34c-3p 344 hsa-miR-221-3p

91 hsa-miR-4770 218 hsa-miR-154-5p 345 hsa-miR-133a-3p

92 hsa-miR-140-3p 219 hsa-miR-127-3p 346 hsa-miR-141-3p

93 hsa-miR-138-l-3p 220 hsa-let-7d-3p 347 hsa-miR-208a-3p

94 hsa-miR-29c-3p 221 hsa-miR-142 -5p 348 hsa-miR-4524a-3p

95 hsa-miR-324-5p 222 hsa-miR-342-5p 349 hsa-miR-23b-5p

96 hsa-miR-377-3p 223 hsa-miR-98-5p 350 hsa-miR-2355 -5p

97 hsa-miR-2110 224 hsa-miR-574-3p 351 hsa-miR-200a-3p

98 SNORD61 225 hsa-miR-1539 352 hsa-miR-206

99 hsa-miR-145-3p 226 hsa-miR-136-5p 353 hsa-miR-200c-3p

100 hsa-miR- 13 Ob-3p 227 hsa-miR-335-3p 354 hsa-miR-190a-5p

101 hsa-miR-495-3p 228 hsa-miR-1910-5p 355 hsa-miR-1277-3p

102 hsa-miR-196a-5p 229 hsa-miR-1225-3p 356 hsa-miR-2467-3p

103 hsa-miR-451a 230 hsa-miR-30a-3p 357 hsa-miR-2355-3p

104 hsa-miR-18a-3p 231 hsa-miR-150-5p 358 hsa-miR-200b-3p

105 hsa-miR-320e 232 hsa-miR-21-5p 359 SNORD72

106 hsa-miR-93-5p 233 hsa-miR-3651 360 RNU6-2

107 hsa-miR-374a-5p 234 hsa-miR-4291 361 hsa-miR-30a-5p

108 hsa-miR-125a-5p 235 hsa-miR-3655 362 hsa-miR-490-3p

109 hsa-miR-329-3p 236 hsa-miR-26b-5p 363 hsa-miR-224-5p

110 hsa-miR-598-3p 237 hsa-miR-13 Ob~5p 364 hsa-miR-489-3p

111 hsa-miR- 10b- 5p 238 hsa-miR-1231 365 hsa-miR-3185

112 hsa-miR-1587 239 hsa-miR-631 366 hsa-miR-7-2-3p

113 hsa-miR-148a-3p 240 hsa-miR-3176 367 hsa-miR-337-3p

114 hsa-miR-151a-5p 241 hsa-miR-3622a~5p 368 hsa-miR-409-3p

115 hsa-miR-193a-5p 242 hsa-miR-3613 -3p 369 hsa-miR-223-3p

116 hsa-miR-130a-3p 243 hsa-miR-337-5p 370 hsa-miR-30b-5p

117 hsa-miR-4274 244 hsa-miR-1301-3p 371 hsa-miR-326

118 hsa-miR-151b 245 hsa-miR-523 -5p 372 hsa-miR-30d-5p

119 hsa-miR-328-3p 246 hsa-miR-338-5p 373 hsa-miR-1281

120 hsa-miR-4689 247 hsa-miR-24-3p 374 hsa-miR- 146a-5p

121 hsa-miR-486-5p 248 hsa-miR-382-5p 375 hsa-miR-223-5p

122 hsa-miR-660-5p 249 hsa-miR-92b-3p 376 hsa-miR-1913

123 hsa-miR-4651 250 hsa-miR-28-5p 377 hsa-miR-205-5p

124 hsa-miR-532-3p 251 hsa-miR-146b-5p 378 hsa-miR-122-5p 125 hsa-miR-378a-5p 252 hsa-miR-134-5p

126 hsa-miR-128-3p 253 hsa-miR-3646

127 hsa-miR-501-5p 254 hsa-miR-3610

[ 0014 ] Once measured, the methods further include the step of comparing the level of expression of at least one biomarker miRNA in the biological sample to a reference level of the same biomarker miRNA in the subject before the subject received treatment for glioma to determine a biomarker difference value.

[ 0015 ] A difference between expression levels or between or among weighted comparisons may be at least, or be at most, about 10-, 20-, 30-, 40-, 50-, 60-, 80-, 90-, 100-, 110-, 120-, 130-, 140-, 150-, 160-, 170-, 180-, 190-, 200-, 250- 300-, 350-, 400-, 450-, 500-, 600-, 700-, 800-, 900-, or 1000 times or -fold (or any range derivable therein) . In particular embodiments, a level of miRNA is increased or decreased in the biological sample after treatment compared to a reference level if it is at least 20-, 30-, 40-, 50-, 60-, 80-, 90-, 100-, 110-, 120-, 130-, 140-, 150-, 160-, 170-, 180-, 190-, 200-, 250- 300-, 350-, 400-, 450-, 500-, 600-, 700-, 800-, 900-, or 1000-fold higher or lower (or any range derivable therein) , respectively, than the reference level. This may or may not include using a standardized or normalized level of expression in determining whether there is an increase or decrease. In one embodiment, the expression level of the biomarker miRNA exhibits an increase in the biological sample compared to the reference level. In another embodiment, the expression level of the biomarker miRNA exhibits a decrease in the biological sample compared to the reference level . In other embodiments, the expression levels of two or more biomarker miRNAs exhibit an increase in the biological sample compared to reference levels. In yet other embodiments, the expression levels of two or more biomarker miRNAs exhibit a decrease in the biological sample compared to reference levels. In further embodiments, the expression level at least one biomarker miRNA exhibits an increase in the biological sample compared to reference level and at least one biomarker miRNA exhibits a decrease in the biological sample compared to reference level. In some embodiments, A human biomarker miRNA exhibiting at least a 20-fold decrease in expression level is miR-122-5p. Human biomarker miRNAs exhibiting at least a 20-fold, 30-fold, 40-fold, 50- fold, or more than 100-fold increase in expression level are provided in Table 2. Preferably said at least one biomarker miRNA is hsa-miR-204-5p, hsa-let-7c-5p, hsa-miR- 125b-5p, hsa-miR-373-5p, hsa-miR-3907 , hsa-let-7e-5p, hsa- miR-3135b, hsa-miR-370-3p, hsa-miR-99a-5p, hsa-miR-100 -5p and/or hsa-miR-122-5p .

TABLE 2

[0016] Previous analyses have demonstrated a correlation between smaller post -operative tumor burden and improved patient outcome in glioblastoma (Keles, et al . (1999) Surg. Neurol. 52:371-9) . In particular, it has been shown that patients with a residual tumor postoperatively had a 6.595- times higher risk of death in comparison to patients without a residual tumor (Albert, et al . (1994) Neurosurgery 34:45-60). In addition, residual tumor burden of less than 45 mm diameter on postoperative CT scans was associated with 70% chance of long-term survival (Andreou, et al . (1983) AJNR Am. J. Neuroradiol . 4:488-90) . Moreover, a significant survival advantage has been associated with resection of 98% or more of the tumor volume (median survival 13 months, 95% confidence interval 11.4-14.6 months), compared with 8.8 months (95% confidence interval 7.4-10.2 months; p < 0.0001) for resections of less than 98% (Lacroix, et al . (2001) J^". Neurosurg. 95:190-8). Accordingly, a determination of tumor burden (i.e., tumor volume) in glioma after treatment {e.g., surgery, chemotherapy and/or and radiation therapy) provides the clinician with valuable information concerning outcome including treatment efficacy and long-term survival .

[0017] Accordingly, in certain embodiments, the method of the invention involves the steps of (a) providing a blood sample (e.g., whole blood, serum or plasma) from a subject after the subject has received treatment for a glioma; (b) determining the level of expression of at least one biomarker miRNA presented in Table 2, optionally in combination with hsa-miR-122-5p, in said blood sample from the subject; (c) comparing the level of expression determined in step (b) to a reference level of the same biomarker miRNA in a second blood sample from the subject that was obtained prior to the treatment for the glioma; and (d) determining that the subject's tumor burden is indicative of a positive or favorable outcome if the expression level of the at least one biomarker miRNA presented in Table 2 has increased by at least 20-fold and optionally the expression level of hsa-miR-122 -5p has decreased by at least 50-fold in the first blood sample as compared to the second blood or serum sample.

[0018] For the purposes of the present invention, a positive or favorable outcome may be an overall response rate, overall survival rate, overall complete response rate, duration of response, longer time to next therapy, treatment free interval, positive response to treatment, a longer time-to-progression, longer term survival and/or longer progression- free survival. Indeed, given that the biomarker miRNAs of this invention are predictive of tumor burden, they can be used to monitor treatment thereby reducing treatment toxicity. The favorable outcome may be dose-dependent or dose-independent . The favorable outcome may favorable be in comparison to no treatment, or in comparison to another cancer treatment or cancer treatment (s) .

[0019] Methods of this invention may further involve recording the expression levels or tumor burden in a tangible medium; reporting the expression levels or tumor burden to the patient, a health care payer, a physician, an insurance agent, or an electronic system; monitoring the patient for recurrence of glioma; or treatment of the patient based on the expression levels or tumor burden within one hour, one day, one week, one month, one year, two years, three years, four years, five years of the measuring or determining of expression levels or within any intermediate time values or ranges. The treatment may include altering the expression levels of the biomarker miRNAs or any traditional cancer therapies, such as surgery, chemotherapy, radiation, gene therapy, or immunotherapy for patients with risks determined based on the biomarker miRNA expression levels.

[0020] Also provided are kits containing the disclosed compositions or compositions used to implement the disclosed methods. In some embodiments, kits can be used to evaluate one or more miRNA molecules. The kits may include any one or more of the following materials: enzymes, reaction tubes, buffers, detergent, primers and probes. In a particular embodiment, these kits allow a practitioner to obtain samples of blood or serum. In another embodiment, these kits include the needed apparatus for performing RNA extraction, RT-PCR, and gel electrophoresis. Instructions for performing the assays can also be included in the kits.

[ 0021 ] In certain embodiments, a kit contains, contains at least, or contains at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more, or any range and combination derivable therein, miRNA probes or primers including those that may specifically hybridize under stringent conditions to miRNAs disclosed herein.

[ 0022 ] In certain embodiments, the kit includes miRNA arrays or miRNA probe arrays, which are ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of miRNA molecules or precursor miRNA molecules and are positioned on a support or support material in a spatially separated organization. Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted .

[ 0023 ] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be included in a vial . The kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale.

[0024] The following non-limiting examples are provided to further illustrate the present invention.

Example 1 : MicroRNAs for Determining Tumor Burden

[0025] Blood Plasma Preparation . Patient blood samples (5-8 ml) were drawn from consented patients at four specific time points: once before surgery and at three scheduled time points post -surgery and during treatment with temozolomide (TMZ) and radiation therapy. The post-surgery time points were weeks 0, 6 and the last week of undergoing standard treatment (Figure 1) . Blood was placed in VACUTAINER tubes containing sodium citrate, inverted carefully 10 times, and subsequently stored on ice. Samples underwent centrifugation for a minimum of 10 minutes at 1000-2000 RCF (generally 1300 RCF) at 4°C. The blood separated into three layers: (from top to bottom) plasma, leucocytes (buffy coat), and erythrocytes.

[0026] RNA Sample Preparation . RNA was liberated from plasma samples using the FASTLANE^® Cell One-Step Buffer Set

(QIAGEN) . Briefly, 3 μΐ of plasma was combined with 3 μΐ of FASTLA E processing mix (Buffer FCPL + gDNA Wipeout Buffer 2) . Each mixture was incubated at room temperature (15- 25°C) for 10 minutes followed by 75°C for 5 minutes.

[0027] miRNA Expression Analysis . Stabilized RNA samples were evaluated using the miScript PCR System (QIAGEN) . Briefly, 3 μΐ of each sample were reverse-transcribed into cDNA using the miScript II RT Kit with miScript HiSpec Buffer. The reactions were incubated for 60 minutes at 37°C followed by a heat inactivation step for 5 minute at 95 °C. Each cDNA synthesis was diluted 5-fold and one -tenth was preamplified using the miScript PreAMP PCR Kit in combination with a Human Serum & Plasma 384HC (for human samples) or Mouse Serum & Plasma (for mouse samples) miScript PreAMP Primer Mix. Twelve cycles of preamplification were performed using the QIAGEN recommended 384-plex preamplification protocol (for human samples) or 96-plex preamplification protocol (for mouse samples) . Amplified cDNA was diluted to a final volume of 125 μΐ using RNase-free water and assessed in real-time PCR using the miScript SYBR Green PCR Kit and Human Serum & Plasma 384HC (for human samples) or Mouse Serum & Plasma (for mouse samples) miScript miRNA PCR Array. The Human Serum & Plasma 384HC miScript miRNA PCR Array targets the 372 most routinely detectable miRNAs in serum or plasma samples . The Mouse Serum & Plasma miScript miRNA PC Array targets the 84 miRNAs most widely published to be expressed in serum or plasma. Real-time PCR was performed on an ABI- 7900HT (Applied Biosystems) using the miScript cycling program (hot start at 95°C for 15 minutes and 40 cycles of 94°C for 15 seconds, 55°C for 30 seconds, and 70°C for 30 seconds) . A miRNA was deemed to be expressed if its C_T value was less than 30 with a single, sharp melt peak.

[ 0028 ] Normalization of Expression. A mean expression value of all expressed microRNAs in a given sample, i.e., a "C_T Mean of Expressed miRNAs," was used as a normalization factor for microRNA real-time quantitative PCR data. It has been shown that the mean expression value outperforms conventional normalization strategies in terms of better reduction of technical variation and more accurate appreciation of biological changes (Mestdagh, et al . (2009) Genome Biol. 10:R64; D'haene, et al . (2012) Methods Mol . Biol . 822 :261-72) .

[ 002 9 ] MicroRNA expression profiles were characterized in the blood collected from seven glioma patients pre- and post -treatment . Hierarchical clustering image analysis of this data indicated that certain plasma miRNA correlated with tumor burden. The average fold change in expression of miRNAs pre- and post-treatment is provided in Table 3.

TABLE 3

hsa-miR-1207-5p 24.8863 231 hsa-miR-150-5p 3.9103 hsa-miR-195-5p 24.7144 232 hsa-miR-21-5p 3.9103 sa-miR-3200-3p 24.2057 233 hsa-miR-3651 3.8833 sa-miR-192-5p 24.0385 234 hsa-miR-4291 3.8565 hsa-miR-93-3p 23.0593 235 hsa-miR-3655 3.8034 hsa-miR-1280 22.2738 236 hsa-miR-26b-5p 3.8034 sa-miR-500a-5p 21.2189 237 hsa-miR-130b-5p 3.7771 hsa-miR-16-5p 20.9268 238 sa-miR-1231 3.6738 hsa-miR-214-3p 20.7822 239 hsa-miR-631 3.5982 hsa-miR-4306 20.6387 240 hsa-miR-3176 3.5734 hsa-miR-497-5p 20.4961 241 hsa-miR-3622a-5p 3.5487 hsa-miR-424-5p 20.4961 242 hsa-miR-3613 -3p 3.4516 hsa-miR-193b-3p 20.4961 243 hsa-miR-337-5p 3.4041 hsa-miR-140-5p 20.3545 244 hsa-miR-1301-3p 3.3341 hsa-miR-3911 19.6612 245 hsa-miR-523-5p 3.3341 hsa-miR-101-3p 19.6612 246 hsa-miR-338-5p 3.2205 hsa-miR-15b-5p 19.5254 247 hsa-miR-24-3p 3.2205 hsa-miR-103a-3p 19.3905 248 hsa-miR-382-5p 3.1982 hsa-miR-185-5p 18.9914 249 hsa-miR-92b-3p 3.1542 hsa-miR-18a-5p 18.9914 250 hsa-miR-28 -5p 3.1108 hsa-miR-942-5p 18.9914 251 hsa-miR-146b-5p 3.0048 sa-miR-3141 18.4721 252 hsa-miR-134-5p 2.9635 hsa-miR-590-5p 18.4721 253 hsa-miR-3646 2.943 hsa-miR-183-5p 18.2178 254 hsa-miR-3610 2.9025 hsa-miR-181b-5p 18.092 255 hsa-miR-4301 2.8824 hsa-miR-503-5p 17.967 256 hsa-miR-92a-3p 2.8824 hsa-miR-15a-5p 17.967 257 hsa-miR-27a-3p 2.8427 hsa-miR-210-3p 17.4757 258 hsa-miR-340-3p 2.8231 hsa-miR-542-3p 16.9978 259 hsa-miR-4258 2.8036 hsa-let-7f -5p 16.533 260 hsa-miR-191-3p 2.765 hsa-miR-874-3p 16.533 261 h3a-miR-671-3p 2.7081 hsa-miR~19b-3p 16.533 262 hsa-miR-877-3p 2.6894 hsa-miR-18b-5p 16.3054 263 hsa-miR-625-3p 2.6524 hsa-miR-16-2-3p 16.3054 264 hsa-miR-199b-5p 2.5443 hsa-miR-365b- 3p 16.0809 265 hsa-miR-345-5p 2.5443 hsa-miR-421 15.9698 266 hsa-let-7i-3p 2.5093 hsa-miR-15a-3p 15.4258 267 hsa-miR~196b-3p 2.5093 hsa-miR-362-3p 15.2135 268 hsa-miR-629-3p 2.492 hsa-miR-769-5p 15.004 269 hsa-miR-33a-5p 2.4407 sa-miR-29a-3p 14.6952 270 SNORD96A 2.3904 hsa-miR-301b-3p 14.6952 271 hsa-miR-605-5p 2.3739 hsa-miR-34a-5p 14.3928 272 hsa-miR-766-3p 2.3251 lisa-miR-15b-3p 14.0966 273 hsa-miR-361 -5p 2.1845 hsa-miR-17-3p 13.9026 274 hsa-miR-219a-l-3p 2.1694 hsa-miR-1183 13.7112 275 hsa-miR-505-3p 2.1544 hsa-miR-19a-3p 13.429 276 hsa-miR-30c-5p 2.1395 hsa-miR-188-5p 13.3362 277 hsa-miR-450a-5p 2.1395 hsa-miR-1260a 13.2441 278 hsa-miR-183 -3p 2.0955 hsa-miR-152-3p 13.1526 279 hsa-miR-485-3p 2.0955 sa-miR-4770 13.1526 280 hsa-miR-222-3p 2.0955 hsa-miR-140-3p 12.8819 281 hsa-miR-211-5p 2.081 93 hsa-miR-138-l-3p 12.6168 282 hsa-miR-126-3p 2.0524

94 hsa-miR-29c-3p 12.6168 283 hsa-miR-26a-5p 2.0241

95 sa-miR-324-5p 12.5297 284 hsa-miR- 664a-3p 2.0101

96 hsa-miR-377-3p 12.4431 285 hsa-miR-199b-3p 1.9687

97 hsa-miR-2110 12.1871 286 hsa-miR- 203a-3p 1.9416

98 SNORD61 12.1871 287 hsa-miR-1180-3p 1.9017

99 hsa-miR-145-3p 12.0193 288 hsa-miR-1287-5p 1.8885

100 hsa-miR-130b-3p 11.8538 289 hsa-miR-3653 -3p 1.8497

101 hsa~miR-495-3p 11.7719 290 hsa-miR-4538 1.8369

102 hsa-miR-196a-5p 11.7719 291 hsa-miR-487a-3p 1.8369

103 hsa-miR-451a 11.6099 292 hsa-miR-4687-5p 1.8116

104 hsa-miR-18a-3p 11.2924 293 hsa-miR-379-5p 1.7378

105 hsa-miR~320e 11.2144 294 hsa-miR-1307-3p 1.7258

106 hsa-miR-93-5p 11.1369 295 hsa-miR-1233-3p 1.7139

107 hsa-miR-374a-5p 10.9836 296 hsa-miR- 19b- l-5p 1.6903

108 hsa-miR-125a-5p 10.4634 297 hsa-miR-151a-3p 1.6786

109 hsa-miR-329-3p 10.3911 298 hsa-miR-145-5p 1.6786

110 hsa-miR-598-3p 10.3911 299 hsa-miR-1247-5p 1.667

111 hsa-miR-10b-5p 10.3515 300 hsa-miR-1203 1.6327

112 hsa-miR-1587 10.2481 301 hsa-miR- 548o-5p 1.5771

113 hsa-miR-148a-3p 10.1773 302 hsa-let-7f -2-3p 1.5662

114 hsa-miR-151a-5p 9.8306 303 hsa-miR- 675 -3p 1.5554

115 hsa-miR-193a-5p 9.4301 304 hsa-miR-199a-5p 1.5446

116 hsa-miR-130a-3p 9.365 305 hsa-miR-3923 1.5024

117 hsa-miR-4274 9.3003 306 hsa-miR-425-3p 1.4715

118 hsa-miR-151b 9.2361 307 hsa-miR-3159 1.4313

119 hsa-miR-328-3p 9.2361 308 hsa-miR-628-3p 1.4115

120 hsa-miR-4689 9.1723 109 hsa-miR-324-3p 1.3825

121 sa-miR-486-5p 9.1089 310 hsa-miR-23a-3p 1.3635

122 hsa-miR-660-5p 8.9835 311 hsa-miR-1260b 1.3447

123 hsa-miR-4651 8.9835 312 hsa-miR-202-3p 1.3262

124 hsa-miR-532-3p 8.9835 313 hsa-miR-197-3p 1.3262

125 hsa-miR-378a- 5p 8.9835 314 hsa-miR-4323 1.2546

126 hsa-miR-128-3p 8.9214 315 hsa-miR-576-5p 1.2546

127 hsa-miR~501-5p 8.8598 316 hsa-miR-2276-3p 1.246

128 hsa-miR-378i 8.6175 317 hsa-miR-1237-3p 1.2288

129 SNORD95 8.6175 318 hsa-miR-31-5p 1.1706

130 hsa-miR-3200-5p 8.558 319 hsa-miR-551a 1.1307

131 hsa-miR-139-3p 8.4989 330 hsa-miR-499a-5p 1.1307

132 SNORD68 8.4402 331 hsa-miR-4688 1.0998

133 hsa-miR- 143 -3p 8.3819 332 hsa-miR-4267 1.0922

134 hsa-raiR-148b-3p 8.2665 333 hsa-miR-375 1.0847

135 hsa-miR-425-5p 8.2665 334 hsa-miR-30e-5p 1.0847

136 hsa-miR-55lb-3p 8.0964 335 hsa-miR- 615 -5p 1.0772

137 hsa-miR-1537-3p 8.0405 336 hsa-miR-637 1.0772

138 hsa-miR-320b 7.875 337 hsa-miR-4422 1.0624

139 hsa-miR-301a-3p 7.875 338 hsa-miR-22-3p 1.055

140 hsa-miR-744-5p 7.875 339 hsa-miR-3191-3p 1.0333

141 hsa-miR-376c-3p 7.7666 330 hsa-miR-3689e 1.0333

142 hsa-miR- 191 -5p 7.7129 331 hsa-miR-26b-3p 1.0051

143 hsa-miR-196b-5p 7.6067 332 hsa-miR- 126 -5p -1.0301 144 hsa-miR-4286 7.6067 333 hsa-miR-184 -1.0517

145 hsa-miR-532-5p 7.4502 334 hsa-miR-372-3p -1.0517

146 hsa-miR-29c-5p 7.4502 335 hsa-miR-1290 -1.0517

147 hsa-miR~381-3p 7.4502 336 hsa-miR-1286 -1.0517

148 hsa-miR-136-3p 7.3476 337 hsa-miR-3120-3p -1.0517

149 hsa-miR-335-5p 7.2969 338 hsa-miR-133b -1.0517

150 hsa-miR-4516 7.2465 339 hsa-miR-373 -3p -1.0517

151 hsa-miR-4732-5p 7.1467 340 hsa-miR-1193 -1.0517

152 hsa-let-7g-5p 7.0973 341 hsa-let-7f-l-3p -1.0517

153 hsa-miR-652 -3p 7.0973 342 hsa-miR-548e-3p -1.0517

154 hsa-miR-27b-3p 7.0483 343 hsa-miR-412-3p -1.0517

155 hsa-miR-142-3p 6.7612 344 hsa-miR-221-3p -1.0517

156 hsa-miR-454-3p 6.7145 345 hsa-miR-133a-3p -1.0517

157 hsa-miR-4505 6.7145 346 hsa-miR-141-3p -1.0517

158 hsa-miR-96-5p 6.7145 347 hsa-miR-208a-3p -1.0517

159 hsa-miR-550a-5p 6.622 348 hsa-miR-4524a-3p -1.0517

160 hsa-miR-23b-3p 6.5763 349 hsa-miR-23b-5p -1.0517

161 hsa-miR-433 -3p 6.5763 350 hsa-miR-2355-5p -1.0517

162 hsa-miR-487b-3p 6.5763 351 hsa-miR-200a-3p -1.0517

163 hsa-miR-25-3p 6.5309 352 hsa-miR-206 -1.0517

164 hsa-miR-330-3p 6.441 353 hsa-miR-200c-3p -1.0517

165 hsa-miR-374c-5p 6.3965 354 hsa-miR-190a-5p -1.0517

166 hsa-miR-215 -5p 6.3523 355 hsa-miR-1277-3p -1.0517

167 hsa-miR-629-5p 6.3084 356 hsa-miR-2467-3p -1.0517

168 hsa-miR-627-5p 6.1786 357 hsa-miR-2355 -3p -1.0517

169 hsa-miR-340-5p 6.1359 358 hsa-miR-200b-3p -1.0517

170 hsa-miR-422a 6.0935 359 SNORD72 -1.0517

171 hsa-miR-378e 6.0935 360 RNU6-2 -1.0517

172 hsa-miR-4296 6.0935 361 hsa-miR-30a-5p -1.0888

173 hsa-miR-423-5p 6.0514 362 hsa-miR-490-3p -1.0964

174 hsa-miR-485-5p 6.0514 363 hsa-miR-224-5p -1.104

175 hsa-let -7a-3p 6.0096 364 hsa-miR-489-3p -1.1915

176 hsa-miR-339-5p 6.0096 365 hsa-miR-3185 -1.2165

177 hsa-miR-483-5p 5.8453 366 hsa-miR-7-2-3p -1.2858

178 hsa-miR-320a 5.8049 367 hsa-miR-337-3p -1.3129

179 hsa-miR-590-3p 5.7648 368 hsa-miR-409-3p -1.3312

180 hsa-miR-22-5p 5.725 369 hsa-miR-223 -3p -1.4267

181 hsa-miR-296-5p 5.6462 370 hsa-miR-30b-5p -1.4567

182 hsa-miR-502-3p 5.6072 371 hsa-miR-326 -1.5186

183 hsa-miR-424-3p 5.6072 372 hsa-miR-30d-5p -1.685

184 hsa-miR-1976 5.6072 373 hsa-miR-1281 -2.0317

185 hsa-miR-30e-3p 5.5685 374 hsa-miR-146a~5p -2.27

186 hsa-miR-378h 5.53 375 hsa-miR-223-5p -2.6624

187 hsa-miR-5095 5.53 376 hsa-miR-1913 -7.9048

188 hsa-miR-139-5p 5.4162 377 hsa-miR-205-5p -9.9364

189 hsa-miR-369-3p 5.3788 378 hsa-miR-122-5p -78.9421

[ 003 0 ] To further validate the role of these circulating miRNAs, the expression levels of verified targets regulated by these miRNAs (that are correlated to disease) are determined in resected tumor tissue of the same patient. This will also elucidate molecular pathways involved in glioma pathology. In addition, correlations between expression levels of specific miRNAs and their targets to time of tumor recurrence can be determined to provide a precise assessment of disease progression and insights into the molecular basis of gliomas. Moreover, to provide a complete picture of toxicity, efficacy and tumor burden, the four blood draws are increased to seven blood draws (Figure 2) , wherein miRNA expression is determined over the course of treatment .

Example 2 : MicroRNAs for Determining Treatment Toxicity

[0031] Blood samples from two patients were used to establish a noninvasive assay that uses microRNAs for monitoring treatment toxicity in glioma patients. Blood samples were collected as described in Example 1 at the four specific time points. MicroRNA expression profiles in blood collected from glioma patients were analyzed pre- and post - treatment . The data showed that miRNA profiles could be correlated to decreases in total lymphocytic counts. Hierarchical clustering image analysis of this data indicated that both patients had a similar profile pre- and post-treatment. In particular, microRNAs that were correlated to lymphocyte numbers included miR-181, the miR- 125bl/let-7a-2/miR-100 cluster and the miR- 99a/let-7c/miR- 125b-2 cluster. Furthermore, mir-122-5p, an indicator of normal liver function was drastically reduced after surgery during undergoing treatment, indicating most probably liver functions are being affected by therapy. Accordingly, the present invention also provides a method for determining treatment toxicity by measuring the expression of miR-181, the miR-125bl/let-7a-2/miR-100 cluster, the miR-99a/let- 7c/miR-125b-2 cluster and mir-122-5p in a blood or plasma sample from a subject.

[0032] To further validate the role of these circulating miRNAs in treatment toxicity, a determination is made of the expression levels of verified targets regulated by these miRNAs that are correlated to normal physiological function that is being disrupted by the chemotherapy. This will also elucidate molecular pathways/organ functions that are involved in the use of specific drugs for chemotherapy. In addition, correlations between expression levels of specific miRNAs and their targets are made to the precise time of onset of any adverse effects as correlated by clinical assessment of patient symptoms as well as read outs from routine laboratory blood tests for complete blood counts, liver and kidney function.

[0033] It is important to note that microRNAs are being used in glioma patients as a model to study adverse effects of therapy. However, these microRNA profiles can be used as readouts for treatment of not just oncogenic diseases, but also for treatments of any disease as well as pathology.

Example 3 : MicroRNAs for Determining Treatment Efficacy

[0034] Patient blood samples were used to establish a noninvasive assay that uses microRNAs for monitoring treatment efficacy in glioma patients. Blood samples were collected from two patients as described in Example 1 at the four specific time points. MicroRNA expression profiles in blood collected from glioma patients were analyzed pre- and post- treatment . The data showed that miRNA profiles could be correlated to tumor ablation after surgery and treatment. Hierarchical clustering image analysis of this data indicated that both patients had a similar profile pre- and post-surgery and treatment. In particular, microRNAs that enhanced glioma invasiveness and growth included miR-9, miR-lOa, miR-21, miR-30e, miR-125, and miR- 151, which were very highly expressed before Standard care of treatment (radiation therapy and temazolomide/chemotherapy) and showed a very significant decrease after treatment. Accordingly, the present invention also provides a method for determining treatment efficacy by measuring the expression of miR-9, miR-lOa, miR-21, miR-30e, miR-125, and miR-151 in a blood or plasma sample from a subject.

Claims

What is claimed is:

1. A method for determining tumor burden in a subject with glioma comprising

(a) providing a biological sample obtained from a subject with glioma;

(b) determining the level of expression of at least one biomarker microRNA (miRNA) in said biological sample from the subject, wherein the at least one biomarker miRNA is selected from miRNA in Table 1; and

(c) comparing the level of expression determined in step (b) to a reference level of the same biomarker miRNA, wherein differences in the expression level of the biomarker miRNA are indicative of tumor burden.

2. The method of claim 1, where the biological sample in step (a) is obtained from the subject after the subject has received treatment for glioma.

3. The method of claim 1, wherein the reference level of the same biomarker miRNA in step (c) is obtained from a second biological sample from the subject before the subject received treatment for glioma.

4. The method of claim 2, wherein the treatment comprises surgery, chemotherapy, radiation therapy or a combination thereof.

5. The method of claim 1, wherein the subject has glioblastoma .

6. A method for determining tumor burden in a subject with glioma comprising (a) providing a first blood sample from a subject after the subject has received treatment for a glioma;

(b) determining the level of expression of at least one biomarker miRNA of Table 2 in said first blood sample from the subject;

(c) comparing the level of expression determined in step (b) to a reference level of the same biomarker miRNA in a second blood sample from the subject that was obtained prior to the treatment for the glioma; and

(d) determining that the subject's tumor burden is indicative of a positive outcome if the expression level of the at least one biomarker miRNA of Table 2 has increased by at least 20-fold in the first blood sample as compared to the second blood sample.

7. The method of claim 6, wherein the treatment comprises surgery, chemotherapy, radiation therapy or a combination thereof.

8. The method of claim 6, wherein the subject has glioblastoma .