Cancer-related biological materials in microvesicles

This application is a Continuation Application of U.S. application Ser. No. 13/819,539 filed Oct. 17, 2013 which is a 35 U.S.C. § 371 National Phase Entry Application of International Application No. PCT/US2011/050041 filed Aug. 31, 2011, which designates the U.S., and which claims the benefit of 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 61/378,860 filed Aug. 31, 2010; 61/421,421 filed Dec. 9, 2010; 61/437,547 filed Jan. 28, 2011; 61/438,199 filed Jan. 31, 2011; and 61/493,261 filed Jun. 3, 2011, the contents of each of which are incorporated herein by reference in their entirety.

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 29, 2016, is named Sequence_Listing_030258-069537-C and is 5,407 bytes in size.

This invention was made with Government support under grants CA86355, CA69246, CA141226, and CA141150 awarded by National Cancer Institute. The Government has certain rights in the invention.

The present invention relates to the fields of biomarker analysis, diagnosis, prognosis, patient monitoring, therapy selection, risk assessment, and novel therapeutic agents for human or other animal subjects, particularly the profiling of biological materials from a microvesicle fraction of a biological sample, and novel therapies related to microvesicles.

Increasing knowledge of the genetic and epigenetic changes occurring in cancer cells provides an opportunity to detect, characterize, and monitor tumors by analysing tumor-related nucleic acid sequences and profiles. Cancer-related changes include specific mutations in gene sequences (Cortez and Calin, 2009; Diehl et al., 2008; Network, 2008; Parsons et al., 2008), up- and down-regulation of mRNA and miRNA expression (Cortez and Calin, 2009; Itadani et al., 2008; Novakova et al., 2009), mRNA splicing variations, changes in DNA methylation patterns (Cadieux et al., 2006; Kristensen and Hansen, 2009), amplification and deletion of genomic regions (Cowell and Lo, 2009), and aberrant expression of repeated DNA sequences (Ting et al., 2011). Various molecular diagnostic tests such as mutational analysis, methylation status of genomic DNA, and gene expression analysis may detect these changes.

Research uncovering the molecular mechanisms underlying cancer improves our understanding of how to select and design optimal treatment regimes for a patient's disease based on the molecular makeup of his or her particular cancer. Over the past few years, this has led to a significant increase in the development of therapies specifically targeting gene mutations involved in disease progression. In parallel, the use of molecular diagnostic testing for cancer diagnosis, prognosis and treatment selection has expanded, driven by the need for more cost efficient applications of expensive therapies. Current molecular diagnostics has so far almost exclusively relied on assaying cancer cells from tissue biopsy by needle aspiration or surgical resection.

However, the ability to perform these tests using a blood sample is sometimes more desirable than using a tissue sample from a cancer patient because, frequently, fresh tissue samples are difficult or impossible to obtain, and archival tissue samples are often less relevant to the current status of the patient's disease. A less invasive approach using a more easily accessible biological sample, e.g., a blood sample, has wide ranging implications in terms of patient welfare, the ability to conduct longitudinal disease monitoring, and the ability to obtain expression profiles even when tissue cells are not easily accessible, e.g., in ovarian or brain cancer patients.

Currently, gene expression profiling of blood samples involves the analysis of RNA extracted from peripheral blood mononuclear cells (PBMC) (Hakonarson et al., 2005) or circulating tumor cells (CTC) (Cristofanilli and Mendelsohn, 2006).

Many types of cancer cells release an abundance of small membrane-bound vesicles, which have been observed on their surface in culture (Skog et al., 2008). These microvesicles are generated and released through several processes and vary in size (from about 30 nm to about 1 μm in diameter) and content (Simons and Raposo, 2009). Microvesicles can bud/bleb off the plasma membrane of cells, much like retrovirus particles (Booth et al., 2006), be released by fusion of endosomal-derived multivesicular bodies with the plasma membrane (Lakkaraju and Rodriguez-Boulan, 2008), or be formed as apoptotic bodies during programmed cell death (Halicka et al., 2000). In addition, defective (i.e., non-infectious without helper-virus) retrovirus particles derived from human endogenous retroviral (HERV) elements may be found within microvesicle populations (Voisset et al., 2008).

Microvesicles from various cell sources have been studied with respect to protein and lipid content (Iero et al., 2008; Thery et al., 2002; Wieckowski and Whiteside, 2006). They have also been observed to contain cellular RNAs and mitochondria DNA (Baj-Krzyworzeka et al., 2006; Guescini et al.; Skog et al., 2008; Valadi et al., 2007) and may facilitate the transfer of genetic information between cells and/or act as a “release hatch” for DNA, RNA, and/or proteins that the cell is trying to eliminate. Both mRNA and miRNA in microvesicles are observed to be functional following uptake by recipient cells (Burghoff et al., 2008; Deregibus et al., 2007; Ratajczak et al., 2006; Skog et al., 2008; Valadi et al., 2007; Yuan et al., 2009) and it has also been shown that apoptotic bodies can mediate horizontal gene transfer between cells (Bergsmedh et al., 2001).

Knowing the expression profile, mutational profile, or both expression and mutational profiles of individual cancer is helpful for personalized medicine as many drugs target specific pathways affected by the genetic status of the tumors. Detection of genetic biomarkers in blood samples from tumor patients is challenging due to the need for high sensitivity against a background of normal cellular nucleic acids found circulating in blood. Microvesicles released by tumor cells into the circulation can provide a window into the genetic status of individual tumors (Skog et al., 2008).

The present invention is directed to microvesicular nucleic acid profiles of microvesicle fractions obtained from a biological sample from a subject, methods for aiding in diagnosis, prognosis, patient monitoring, treatment selection, and risk assessment based on detecting the presence or absence of a genetic aberration in a nucleic acid profile, or changes in a polypeptide profile of a microvesicle fraction obtained from a biological sample from a patient, and therapeutic agents and methods of cancer treatment or prevention.

The present invention is based on the discovery of various types of cancer-related biological materials within microvesicles. The biological materials within microvesicles from a biological sample may be characterized and measured, and the results this analysis may be used to aid in biomarker discovery, as well as in diagnosis, prognosis, monitoring, treatment selection, or risk assessment for a disease or other medical condition.

In one aspect, the biological materials are nucleic acids and the invention is a method for assaying a biological sample comprising the steps of: a) obtaining or using a microvesicle fraction from a biological sample from a subject; b) extracting nucleic acid from the fraction; and c) detecting the presence or absence of a biomarker in the extracted nucleic acid. In a method for aiding in the diagnosis, prognosis or monitoring of a subject, the biomarker is a genetic aberration that is associated with the diagnosis, prognosis, or determination of the status or stage of a disease or other medical condition in the subject. In a method for aiding in treatment selection for a subject in need of or potentially in need of therapeutic treatment, the biomarker is a genetic aberration that is associated with a disease or other medical condition or with responsiveness to a specific therapy for the disease or other medical condition in the subject. In a method for aiding in a determination of a subject's risk of developing a disease or other medical condition, the biomarker is a genetic aberration that is associated with the subject's risk of developing a disease or other medical condition.

In some embodiments of the above methods, the genetic aberration is in or corresponds to a c-myc gene, a transposable element, a retrotransposon element, a satellite correlated gene, a repeated DNA element, a non-coding RNA other than miRNA, or a fragment of any of the foregoing.

In other embodiments of the above methods, the genetic aberration is in or corresponds to a transposable element listed in Table 4 or Table 5, or a fragment thereof. For one example, the genetic aberration is in or corresponds to retrotransposon elements including LINE, SINE or HERV, or a fragment thereof. For another example, the genetic aberration is in or corresponds to a retrotransposon element that is Line1 (L1), ALU, HERV-H, HERV-K, HERV-K6, HERV-W or HERV-C, or a fragment thereof.

In further embodiments of the above methods, the genetic aberration is in or corresponds to a satellite-correlated gene listed in Table 6, or a fragment thereof; a repeated DNA element listed in Table 8, or a fragment thereof; or a non-coding RNA listed in Table 9 (other than miRNA) or a fragment thereof. The non-coding RNA, for example, can be 7SL RNA.

In yet further embodiments of the above methods, the genetic aberration is in or corresponds to a cancer gene listed in Table 2 or 3, or a fragment thereof.

In another aspect, the biological material is protein or polypeptide and the invention is a method for assaying a biological sample from a subject comprising the steps of: a) obtaining or using a microvesicle fraction from a biological sample from a subject b) measuring a protein or polypeptide activity in the fraction; and c) determining whether the protein or polypeptide activity is higher or lower than a normal or average activity for the same protein or polypeptide. In a method for aiding in the diagnosis, prognosis or monitoring of a subject, an elevated or lowered activity is associated with a diagnosis, prognosis, status or stage of a disease or other medical condition in the subject. In a method for aiding in directing treatment of a subject, an elevated or lowered activity is associated with a disease or other medical condition or with the subject's responsiveness to a specific therapy for the disease or other medical condition. In a method in aid of a determination of a subject's risk of developing a disease or other medical condition, an elevated or lowered activity is associated with the subject's risk of developing a disease or other medical condition. In some embodiments of the foregoing methods, the polypeptide is an enzyme. For example, the polypeptide can be a reverse transcriptase and the method is to determine whether the reverse transcriptase activity is higher than a normal or average activity for reverse transcriptase.

In the present invention, the methods may further comprise a step of enriching the microvesicle fraction for microvesicles originating from a specific cell type. The enrichment may be achieved, for example, by affinity purification with antibody-coated magnetic beads.

In the present invention, the biological sample from a subject can be a bodily fluid, e.g., blood, serum, plasma, or urine. The subject can be a human subject. When the subject is a human, the disease or other medical condition may be brain cancer such as medulloblastoma and glioblastoma, or melanoma.

In the present invention, the presence or absence of a biomarker in the extracted nucleic acid can be determined by various techniques, e.g., microarray analysis, PCR, quantitative PCR, Digital Gene Expression, or direct sequencing.

In yet another aspect, the present invention is a kit for genetic analysis of a microvesicle fraction obtained from a body fluid sample from a subject, comprising, in a suitable container, one or more reagents capable of hybridizing to or amplifying a nucleic acid corresponding to one or more of the genetic aberrations referenced above.

In yet another aspect, the present invention is an oligonucleotide microarray for genetic analysis of a microvesicle preparation from a body fluid sample from a subject, wherein the oligonucleotides on the array are designed to hybridize to one or more nucleic acids corresponding to one or more of the genetic aberrations referenced above.

In yet another aspect, the present invention is a profile of microvesicular nucleic acid derived from a bodily fluid sample from a subject. The profile may be a genetic aberration in or corresponding to: a) cancer gene listed in Table 2 or 3, or a fragment thereof; b) a transposable element from the subject's genome, preferably an element listed in Table 4 or 5, or a fragment of any of the foregoing; c) a retrotransposon element from the subject's genome, preferably LINE, SINE or HERV, more preferably LINE1 (L1), ALU, HERV-H, HERV-K, HERV-K6, HERV-W or HERV-C, or a fragment of any of the foregoing; d) a satellite correlated gene from the subject's genome, preferably a satellite correlated gene listed in Table 6, or a fragment of any of the foregoing; e) an element of repeated DNA from the subject's genome, preferably an element listed in Table 8, or a fragment of any of the foregoing; or f) a non-coding RNA other than miRNA, preferably a species listed in Table 9, or a fragment of any of the foregoing. In one embodiment, the profile is a genetic aberration in the cancer gene c-myc. In another embodiment, the profile is a genetic aberration in the non-coding 7SL RNA.

In all of the foregoing nucleic acid-related embodiments of the invention, the genetic aberration can be a species of nucleic acid, the level of expression of a nucleic acid, a nucleic acid variant; or a combination of any of the foregoing. For example, the genetic aberration may be an RNA expression profile. For another example, the genetic aberration may be a fragment of a nucleic acid, and in some instances, the fragment contains more than 10 nucleotides.

In yet another aspect, the present invention is a method of identifying a potential new nucleic acid biomarker associated with a disease or other medical condition, status or stage of disease or other medical condition, a subject's risk of developing a disease or other medical condition, or a subject's responsiveness to a specific therapy for a disease or other medical condition. The method comprises the steps of: a) obtaining or using a microvesicle fraction from a biological sample from a subject; b) extracting nucleic acid from the fraction; c) preparing a profile according to any of the above-described profiles; and d) comparing the profile of step c) to a control or reference profile and selecting one or more potential new biomarkers based on one or more differences between the profile of step c) and the control or reference profile.

In yet anther aspect, the present invention is a method of treating a subject having a form of cancer in which cancer cells secrete microvesicles. The method comprises administering to the subject a therapeutically effective amount of a composition including an inhibitor of microvesicle secretion; an inhibitor of a reverse transcriptase; a microvesicle neutralizer that neutralizes the pro-tumor progression activity of tumor microvesicles; or any combination of the forgoing. In some embodiments, the inhibitor of microvesicle secretion is an inhibitor of RAB GTPase which may be Rab 27a, Rab 27b or Rab 35. In other embodiments, the inhibitor of a reverse transcriptase is a nucleoside analog selected from the group comprising 3′-azido2′,3′-dideoxythymidine (AZT); 2′,3′-dideoxyinosine (ddT), 2′,3′-didehyro-3′-deoxythymidine (d4T); nevirapine and efavirenz. In further embodiments, the inhibitor of a reverse transcriptase is RNAi targeting the reverse transcriptase gene. In still further embodiments, the microvesicle neutralizer is a biological agent that binds microvesicles and destroys the integrity of the microvesicles.

In yet another aspect, the present invention is a pharmaceutical composition comprising, in a suitable pharmaceutical carrier: a) an inhibitor of microvesicle secretion, particularly an inhibitor of RAB GTPase, and more particularly Rab 27a, Rab 27b or Rab 35); b) an inhibitor of reverse transcriptase, particularly a nucleoside analog, more particularly 3′-azido2′,3′-dideoxythymidine (AZT); 2′,3′-dideoxyinosine (ddI), 2′,3′-didehyro-3′-deoxythymidine (d4T); nevirapine, or efavirenz, or an RNAi targeting the reverse transcriptase gene; c) a microvesicle neutralizer that neutralizes the pro-tumor progression activity of tumor microvesicles, particularly a biological agent that binds microvesicles and destroys the integrity of the microvesicles; or d) a combination of any of the foregoing.

FIG. 1 shows a graph depicting the quantification, size distribution and RNA yield of microvesicles purified from the medulloblastoma cell line D384. Each bar represents the number of particles of a certain size that are present in the media and are released by one cell over 48 hours (hrs). The sum refers to the total number of particles released by one cell over 48 hrs. ExoRNA refers to the total RNA yield in microvesicles from 1×10 6 cells over 48 hrs. The result is presented as the mean±SEM (n=3).

FIG. 2 shows a graph depicting the quantification, size distribution and RNA yield of microvesicles purified from the medulloblastoma cell line D425 in the same manner as in FIG. 1 .

FIG. 3 shows a graph depicting the quantification, size distribution and RNA yield of microvesicles purified from the medulloblastoma cell line D458 in the same manner as in FIG. 1 .

FIG. 4 shows a graph depicting the quantification, size distribution and RNA yield of microvesicles purified from the melanoma cell line Yumel 0106 in the same manner as in FIG. 1 .

FIG. 5 shows a graph depicting the quantification, size distribution and RNA yield of microvesicles purified from the glioblastoma cell line 20/3 in the same manner as in FIG. 1 .

FIG. 6 shows a graph depicting the quantification, size distribution and RNA yield of microvesicles purified from the glioblastoma cell line 11/5 in the same manner as in FIG. 1 .

FIG. 7 shows a graph depicting the quantification, size distribution and RNA yield of microvesicles purified from the normal fibroblast cell line HF19 in the same manner as in FIG. 1 .

FIG. 8 shows a graph depicting the quantification, size distribution and RNA yield of microvesicles purified from the normal fibroblast cell line HF27 in the same manner as in FIG. 1 .

FIG. 9 shows a graph depicting the c-Myc gene yields in terms of genomic DNA extracted from cells of the following cell lines: one normal human fibroblast line (HF19), one GBM line (11/5), one atypical teratoid rhabdoid tumor (AT/RT) line (NS224) and three medulloblastoma (MB) lines (D425, D458 and D384). Quantitative PCR was used to obtain c-Myc Ct values, which were normalized to GAPDH Ct values in the same preparation. The X-axis lists the names of the cell lines tested. The Y-axis is the fold change, represented as the ratio of the Ct value for each cell line to the Ct value for the normal fibroblast cell line HF19. In all cases, the Ct values are expressed as mean±SEM (n=3) and analyzed by a two-tailed t-test.

FIG. 10 shows a graph depicting the c-Myc gene yields in terms of RNA extracted from microvesicles secreted by cells of the same cell lines and in the same manner as in FIG. 9 . Quantitative Reverse Transcription PCR was used to obtain c-Myc RNA Ct values.

FIG. 11 shows a graph depicting the c-Myc gene yields in terms of DNA extracted from microvesicles secreted by cells of the same cell lines and in the same manner as in FIG. 9 . Quantitative PCR was used to obtain c-Myc DNA Ct values.

FIG. 12 shows a graph depicting the c-Myc gene yields in terms of RNA extracted from xenograft subcutaneous tumor cells. The subcutaneous tumors were generated by xenografting medulloblastoma cells (MBT; D425 cell line) or epidermoid carcinoma (ECT; A431 cell line) cells in nude mice. The X-axis refers to the different tumor-bearing mice characterized by the type of tumor cell and the tumor mass weight at sacrifice. MBT tumor mass weights are as follows: MBT 1: 3.4 g; MBT 2: 1.7 g; MBT 3: 2.4 g; MBT 4: 2.9 g; and MBT 5: 1.7 g. ECT tumor mass weights are as follows: ECT 1: 1.7 g; ECT 2: 2.3 g; ECT 3: 3.1 g; ECT 4: 1.9 g; and ECT 5: 2.2 g. Ct values were normalized to GAPDH. The Y-axis refers to the Ct values generated by quantitative reverse transcription PCR of the extracted RNA in each sample. For each RNA extract, two replicate qPCR were performed.

FIG. 13 shows a gel picture depicting the c-Myc gene yields in terms of RNA extracted from serum microvesicles from mice that bear subcutaneous tumors. The subcutaneous tumors were generated by xenografting medulloblastoma cells (MBT; D425 cell line) in nude mice. C-Myc product was amplified by reverse transcription PCR method using human c-Myc specific primers and the RNA extracted from serum microvesicles as templates. The amplified c-Myc product should be 89 bp in length. The amplified c-Myc products were resolved by electrophoresis in a 2% agarose gel and visualized with ethidium bromide staining. The arrow points to the position where an 89 bp product appears on the agarose gel. The lanes are referenced as follows: MW: DNA size marker; 1: MBT tumor mass weight of 3.4 g; 2: MBT tumor mass weight of 1.7 g; 3: MBT tumor mass weight of 2.4 g; 4: MBT tumor mass weight of 2.9 g; 5: MBT tumor mass weight of 1.7 g; NC: negative control where no RNA/cDNA was used.

FIG. 14 shows a gel picture depicting the c-Myc gene yields in terms of RNA extracted from serum microvesicles from mice that bear subcutaneous tumors in the same manner as in FIG. 13 except that the subcutaneous tumors were generated by xenografting epidermoid carcinoma (ECT; A431 cell line) in nude mice. The lanes are referenced as follows: MW: DNA size marker; 1: ECT tumor mass weight of 1.7 g; 2: ECT tumor mass weight of 2.3 g; 3: ECT tumor mass weight of 3.1 g; 4: ECT tumor mass weight of 1.9 g; 5: ECT tumor mass weight of 2.2 g; NC: negative control where no RNA/cDNA was used.

FIG. 15 shows a MA plot depicting relative levels of all represented RNA sequences (using 44,000 RNA probes on the Agilent microarray chip) in cells and microvesicles derived from the cells. The levels of transposon and retrotransposon sequences were compared to the rest of the RNA transcriptome in cells and microvesicles. ExoRNA and cellular RNA were isolated from GBM 20/3 cells and analyzed on an Agilent two-color 44 k array. Y-axis (M)=log 2 Exo−log 2 Cell, X-axis (A)=0.5×(log 2 Exo+log 2 Cell).

FIG. 16 shows a MA plot similar to the plot in FIG. 15 except that the present plot only depicts relative levels of the following four HERV family sequences: HERV-H, HERV-K6, HERV-W and HERV-C, all of which are enriched in microvesicles more than 16-fold as compared to the host cells, i.e., M≥4.

FIG. 17 shows a MA plot similar to the plot in FIG. 15 except that the present plot only depicts relative levels of DNA transposons.

FIG. 18 shows a MA plot similar to the plot in FIG. 15 except that the present plot only depicts relative levels of L1 sequences.

FIG. 19 shows a MA plot similar to the plot in FIG. 15 except that the present plot only depicts relative levels of HERV sequences with HERV-H, HERV-C, HERV-K6 and HERV-W being more than 16 fold enriched.

FIG. 20 shows a MA plot similar to the plot in FIG. 15 except that the present plot only depicts relative levels of Alu sequences.

FIGS. 21A, 21B and 21C show MA plots depicting relative expression levels of L1 ( FIG. 21A ), ALU ( FIG. 21B ) and HERV-K ( FIG. 21C ) RNA in cells and microvesicles derived from the cells. qRT-PCR was carried out for retrotransposon elements in cell RNA and exoRNA from three medulloblastoma (D425, D384 and D458), one GBM (11/5), one melanoma (0106) and one human fibroblast (HF19) line. The RNA expression levels were measured and normalized to GAPDH. HERV-K RNA was not detectable in exoRNA from normal human fibroblasts (HF19), so it was given a Ct value of 36 (below detection limit).

FIG. 22 shows a chart depicting the expression levels of HERV-K at different time points in HUVEC cells. The HUVEC cells were exposed to medulloblastoma D384 microvesicles and their expression level of HERV-K RNA was analyzed by qRT-PCR over 72 hrs following exposure. MOCK is non-exposed cells. HERV-K was normalized to GAPDH. P values were calculated using the two-tailed t-test, comparing levels to MOCK infected cells.

FIGS. 23A, 23B and 23C show MA plots depicting relative levels of L1 ( FIG. 23A ), ALU ( FIG. 23B ) and HERV-K ( FIG. 23C ) DNA in cells and microvesicles derived from the cells. q-PCR was carried out for retrotransposon elements with cell genomic DNA and microvesicle DNA from three medulloblastoma (D425, D384 and D458), one GBM (11/5), one melanoma (0106) and one human fibroblast (HF19) line. The DNA levels were measured and normalized to GAPDH. Results are expressed as average±SEM (n=3).

FIG. 24 shows a chart depicting the Reverse Transcriptase (RT) activity in microvesicles secreted by three medulloblastoma (D425, D384 and D458), one GBM (11/5), one melanoma (0106) and one human fibroblast (HF19) line. The RT activity was measured in the microvesicles using the EnzChek RT Assay Kit (Invitrogen) and normalized to protein content. The RT activity is measured as RT units calculated based on the standard curve generated using SuperScript III (Invitrogen). Results are expressed as average±SEM (n=3).

FIGS. 25A, 25B, 25C and 25D show charts depicting Bioanalyzer profiles of exoRNA and exoDNA from tumor or normal cell. FIG. 25A depicts the profile of exoRNA from GBM 11/5 cells. Both 18S and 28S rRNA peaks are detectable (arrowheads). FIG. 25B depicts the profile of exoDNA GBM 11/5 cells. Sizes ranged from 25 to 1000 nucleotides with a peak at 200 nt. FIG. 25C depicts the profile of ExoRNA from human fibroblasts HF19, which was extracted and analyzed as in FIG. 25A . The RNA yield was too low to yield distinct 18S and 28S rRNA peaks. After concentration, these peaks were visible (data not shown). FIG. 25D depicts the profile of ExoDNA from human fibroblasts HF19, which was not readily detectable on the Bioanalyzer even after it was concentrated 30 times. Bioanalyzer profiles were generated using the RNA Pico Chip (Agilent).

FIGS. 26A and 26B show charts depicting the Bioanalyzer profiles of exoDNA from microvesicles isolated from medulloblastoma D384 cells. FIG. 26A depicts the profile of exoDNA purified from externally DNase-treated microvesicles using the Agilent DNA 7500 bioanalyzer chip (Agilent Technologies Inc., Santa Clara, Calif. Cat. Number 5067-1506) that detects dsDNA. FIG. 26B depicts the profile of exoDNA after a second-strand synthesis treatment. Here the same sample as in (A) was subjected to second strand synthesis with Superscript Double-Stranded cDNA synthesis kit (Invitrogen) according to manufacturer's recommendation.

FIG. 27 is an agarose gel picture depicting electrophoresis of GAPDH (112 bp) PCR products using templates from different samples. The different samples were exoDNA samples extracted from microvesicles isolated from three medulloblastoma cell lines (D425, D384 and D556) and genomic DNA extracted from L2132 normal fibroblasts as a control double stranded DNA, all four of which were mock treated or treated with S1 nuclease enzyme which degrades single-stranded nucleic acids.

FIG. 28 depicts representative bioanalyzer profiles of exoDNA extracted from medulloblastoma cell line D384 before and after S1 nuclease treatment.

FIGS. 29A and 29B show charts depicting quantitative PCR results of c-Myc and POU5F1B, respectively, using as templates genomic DNA from cells or exoDNA extracted from microvesicles isolated from cells. FIG. 29A depicts the results for c-Myc gene. FIG. 29B depicts the results for POU5F1B, which gene sequence (AF268618) is found 319 kb upstream of the c-Myc gene in the genome, but still within the commonly amplified region in tumor cells. The cell lines are medulloblastoma cell lines D458 and D384, glioblastomas (11/5), and fibroblasts HF19.

FIG. 30 illustrates the c-Myc copy number analysis results in tumor cell lines using an Affymetrix 250K SNP array. The c-Myc genomic region was analyzed in medulloblastoma lines, D425, D458 and D384, as well as rhabdoid tumor line, NS224.

FIGS. 31A and 31B show charts depicting the qPCR results of the n-Myc gene in cells lines medulloblastoma D425, D458 and D384, rhabdoid tumor, GBM, and normal fibroblasts using genomic DNA FIG. 31A or exoDNA FIG. 31B extracted from microvesicles isolated from the cells as templates.

FIG. 32 shows a chart depicting the amount of exoDNA extracted from microvesicles isolated from medulloblastoma D384 cell culture media. D384 cells were seeded in 6-well plates and treated with increasing dosages of L-mimosine (200, 400 and 600 μM) or mock treated. Microvesicles were isolated from the medium after 48 hrs and ssDNA was extracted using the Qiagen PCR purification kit. Single-stranded DNA yields were quantified using the Bioanalyzer and the yields were compared to mock treated cells (normalized to 1.0).

FIG. 33 depicts the results of quantitative RT-PCR analysis of the expression levels of 7SL RNA, EGFR and GAPDH in microvesicles isolated from serum samples obtained from a GBM patient or a normal individual. The X-axis is the number of PCR cycles. The Y-axis is the fluorescent intensity (delta Rn) measured by the ABI7500 machine.

FIG. 34 depicts a series of signaling pathways related to cell proliferation, growth and/or survival.

As described above, cell-derived vesicles are heterogeneous in size with diameters ranging from about 10 nm to about 1 μm. For example, “exosomes” have diameters of approximately 30 to 100 nm, with shedding microvesicles and apoptotic bodies often described as larger (Orozco and Lewis, 2010). Exosomes, shedding microvesicles, microparticles, nanovesicles, apoptotic bodies, nanoparticles and membrane vesicles co-isolate using various techniques and will, therefore, collectively be referred to throughout this specification as “microvesicles” unless otherwise expressly denoted.

The present invention is based on the discovery that cancer-related biological materials such as transposable elements, oncogenes, and reverse transcriptase (RT) can be detected in microvesicles.

The biological materials in microvesicles can be genetic materials, protein materials, lipid materials, or any combination of genetic, protein and lipid materials.

Genetic materials include nucleic acids, which can be DNA and its variations, e.g., double-stranded DNA (“dsDNA”), single-stranded DNA (“ssDNA”), genomic DNA, cDNA; RNA and its variations, e.g., mRNA, rRNA, tRNA, microRNA, siRNA, piwi-RNA, coding RNA, non-coding RNA, transposons, satellite repeats, minisatellite repeats, microsatellite repeats, Interspersed repeats such as short interspersed nuclear elements (SINES), e.g. but not limited to Alus, and long interspersed nuclear elements (LINES), e.g. but not limited to LINE-1, human endogenous retroviruses (HERVs), e.g. but not limited to HERV-K; or any combination of any of the above DNA and RNA species.

Protein materials can be any polypeptides and polypeptide variants recognized in the art. For convenience, “polypeptide” as disclosed in this application refers to both a polypeptide without modifications and a polypeptide variant with modifications. Polypeptides are composed of a chain of amino acids encoded by genetic materials as is well known in the art. For example, a reverse transcriptase is a polypeptide that can function as an enzyme to transcribe RNA into DNA. Polypeptide variants can include, e.g. polypeptides modified by acylation, ubiquitination, SUMOYlation, alkylation, amidation, glycosylation, hydroxylation, carboxylation, phosphorylations, oxidation, sulfation, selenoylation, nitrosylation, or glutathionylation.

Lipid materials include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides.

Microvesicles may be isolated from tissue, cells or other biological samples from a subject. For example, the biological sample may be a bodily fluid from the subject, preferably collected from a peripheral location. Bodily fluids include but are not limited to blood, plasma, serum, urine, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid and combinations thereof. In some embodiments, the preferred bodily fluid for use as the biological sample is urine. In other embodiments, the preferred bodily fluid is serum.

The term “subject” is intended to include all animals shown to or expected to harbor nucleic acid-containing microvesicles. In particular embodiments, the subject is a mammal, e.g., a human or nonhuman primate, a dog, cat, horse, cow, other farm animal, or rodent (e.g. a mouse, rat, guinea pig, etc.). In one embodiment, the subject is an avian, amphibian or fish. The terms “subject,” “individual” and “patient” are used interchangeably herein.

Methods for isolating microvesicles from a biological sample and extracting biological materials from the isolated microvesicles are described in this application as well as in scientific publications and patent applications, e.g. (Chen et al., 2010; Miranda et al., 2010; Skog et al., 2008). See also WO 2009/100029, WO 2011/009104, WO 2011/031892 and WO 2011/031877. These publications are incorporated herein by reference for their disclosure pertaining to isolation and extraction methods and techniques.

A profile, as used herein, refers to a set of data or a collection of characteristics or features, which can be determined through the quantitative or qualitative analysis of one or more biological materials, particularly biological materials contained in microvesicles isolated from a subject. The biological materials, extraction of the biological materials, and various types of analysis of the biological materials are described herein. A control or reference profile is a profile obtained from the literature, from an independent subject or subjects, or from the same subject at a different time point.

In one aspect, the present invention includes a profile of one or more nucleic acids extracted from microvesicles. The nucleic acids include both RNA and DNA. A nucleic acid profile may be an RNA profile, a DNA profile, or may include profiles of both RNA and DNA. In other aspects, the present invention includes a profile of one or more protein or polypeptide species extracted from microvesicles, particularly, a level of protein activity.

In all of the various aspects of the invention described herein in relation to RNA, the RNA can be coding RNA, e.g., messenger RNA. The RNA can also be non-coding RNA (ncRNA), e.g., ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA, and other non-coding transcripts that may originate from genomic DNA. See Table 9 for more examples of non-coding RNA. Non-coding RNA transcripts may include transcripts from satellite repeats or from transposons, which may be Class I retrotransposons or Class II DNA transposons.

In all of the various aspects of the invention described herein in relation to DNA, the DNA can be single-stranded DNA, e.g., cDNA, which is reverse transcribed from RNA. Reverse transcription is usually mediated by reverse transcriptase encoded by a reverse transcriptase gene in a cell. The DNA can also be single stranded DNA generated during DNA replication. Genomic DNA replicates in the nucleus while the cell is dividing. Some of the replicated DNA may come off its template, be exported out of the nucleus, and packaged into microvesicles. The DNA can further be fragments of double-stranded DNA.

In addition, the DNA can be non-coding DNA (ncDNA). The human genome contains only about 20,000 protein-coding genes, representing less than 2% of the genome. The ratio of non-coding to protein-coding DNA sequences increases as a function of developmental complexity (Mattick, 2004). Prokaryotes have less than 25% ncDNA, simple eukaryotes have between 25-50%, more complex multicellular organisms like plants and animals have more than 50% ncDNA, with humans having about 98.5% ncDNA (Mattick, 2004)

Some of the ncDNA from the genome is transcribed into ncRNA. NcRNAs have been implicated in many important processes in the cell, e.g., enzymes (ribozymes), binding specifically to proteins (aptamers), and regulating gene activity at both the transcriptional and post-transcriptional levels. Examples of ncRNA classes and examples of their functions are shown in Table 9.

Many of the ncRNA species have multiple functions. For example, Ribonuclease P (RNase P) is a ribozyme which is involved in maturation of tRNA by cleaving the precursor tRNA, and nuclear RNaseP can also act as a transcription factor (Jarrous and Reiner, 2007). In addition, bifunctional RNAs have also been described that function both as mRNA and as regulatory ncRNAs (Dinger et al., 2008) or have two different ncRNA functions (Ender et al., 2008).

One example of the many long ncRNAs is the X-inactive specific transcript (Xist) expressed by the inactive X-chromosome, which is used to silence the extra X-chromosome in females (Ng et al., 2007). This RNA transcript binds to and inactivates the same X chromosome from which it is produced.

Another example is the HOX antisense intergenic RNA (HOTAIR) (Rinn et al., 2007). This RNA is expressed from chromosome 12, but controls gene expression on chromosome 2, affecting the skin phenotype on different parts of the body surface (Rinn et al., 2007) and also being involved in cancer metastasis (Gupta et al., 2010).

Yet another example of ncRNA is PCA3, a biomarker for prostate cancer (Day et al., 2011). PCA3 can be readily measured in the RNA from urine microvesicles which can be extracted using a rapid filtration concentrator method (Miranda et al., 2010; Nilsson et al., 2009). Another biomarker for prostate cancer is PCGEM1, which is an ncRNA transcript over-expressed in prostate cancer (Srikantan et al., 2000).

Yet another example of ncRNA is NEAT2/MALAT1, which has been found to be upregulated during metastasis of non-small cell lung cancer, and was correlated with poor patient survival (Ji et al., 2003).

Microvesicles contain a substantial array of the cellular gene expression profile from the cells from which they originate (their parent cells) at any given time. That is, substantially all the RNAs expressed in the parent cell are present within the microvesicle, although the quantitative levels of these RNAs may differ in the microvesicle compared to the parent cell. Substantially all the genes from the parent cell can, therefore, be tracked in the microvesicle fraction. In addition, microvesicles contain DNA from the parent cell, which corresponds to diagnostically relevant aspects of the subject's genome. Therefore, a nucleic acid profile from microvesicles may be associated with a disease or other medical condition.

In one embodiment, the disease is a neurological disease or other medical condition, e.g., Alzheimer's disease. The nucleic acid profile for Alzheimer's disease may be a profile of early-onset familial Alzheimer's disease, associated genes including, but not limited to, amyloid beta (A4) precursor protein gene, presenilin 1 and presenilin 2.

In another embodiment, the disease is a cancer. The microvesicular nucleic acid profile for cancer may, e.g., include nucleic acids of one or more cancer-related genes (e.g., known or suspected oncogenes or tumor suppressor genes; or genes whose expression levels correlate with the expression levels of nearby satellites). The determination of a cancer nucleic acid profile, including such cancer related genes, can aid in understanding the status of the cancer cells. In one embodiment, the oncogenes or tumor suppressor genes are one or more of those listed in Tables 2 and 3. In another embodiment, the cancer-related genes are one or more of those genes whose expression levels correlate with the expression levels of nearby satellites, such as but not limited to the satellite correlated genes listed in Table 6.

In some instances, the cancer-related gene is c-myc. The copy number of c-myc oncogene is usually increased in tumor cells, e.g., medullablastoma cells. The detection of increased c-myc gene copy number in microvesicles indicates an increased c-myc copy number in tumor cells that secret the microvesicles.

In other instances, the cancer-related gene is one or more members in the signaling pathways depicted in FIG. 34 . These signaling pathways control the growth, proliferation and/or survival of cells (Alessi et al., 2009; Dowling et al.; Hanahan and Weinberg, 2000; Sarbassov et al., 2006). These pathways are sometimes cross-linked to each other, and thus enable extracellular signals to elicit multiple biological effects. For example, the growth promoting Ras protein interacts with the survival promoting PI3K and thus growth signals can concurrently evoke survival signals in the cell (Hanahan and Weinberg, 2000).

For one example, the member is from the RAS/RAF/MEK/MAPK pathway related to melanoma, brain and lung cancers. The MAP kinase is a convergence point for diverse receptor-initiated signaling events at the plasma membrane. The RAS/RAF/MEK/MAPK pathway regulates cell proliferation, differentiation, migration and invasion (Hanahan and Weinberg, 2000). In addition, extracellular signal-regulated kinases (ERKs) become activated upon integrin ligation and, thereby, regulate cell migration (Klemke et al., 1997).

For another sample, the member is from the PI3K/PTEN/AKT pathway related to prostate, bladder and kidney cancers. The PI3K/PTEN/AKT pathway is responsible for regulating cell survival (Cheng et al., 2008). Genetic variations in AKT1, AKY2, PIK3CA, PTEN, and FRAP1 are associated with clinical outcomes in patients who receive chemoradiotherapy (Hildebrandt et al., 2009). Therefore, the determination of genetic variations in members of the pathway may help evaluating cancer treatment efficacy.

The microvesicular nucleic acid profile of the present invention may also reflect the nucleic acid profile of DNA repeats and/or transposable elements in cells from which the microvesicles originate.

DNA repeats include one or more repeated DNA elements that are composed of arrays of tandemly repeated DNA with the repeat unit being a simple or moderately complex sequence. The array of tandemly repeated DNA can be of varying size, thereby giving rise to categories of megasatellite, satellite, minisatellite and microsatellite repeats. See Table 7. Repeated DNA of this type is not transcribed and accounts for the bulk of the heterochromatic regions of the genome, being notably found in the vicinity of the centromeres (i.e., pericentromeric heterochromatin). The base composition, and therefore density, of such DNA regions is dictated by the base composition of constituent short repeat units and may diverge from the overall base composition of other cellular DNA. The nucleic acid profiles of the present invention comprising satellite repeats may include profiles of satellite repeat DNA and/or profiles of transcripts that are transcribed from satellite repeats.

DNA repeats may serve as biomarkers of cancer cells. For example, some satellite repeats like HSATII are over-expressed in many types of cancers including pancreatic, lung, kidney, ovarian and prostate cancers (Ting et al., 2011). The RNA expression level of such satellite repeats correlates with cancer disease status. DNA repeats encompassed within the scope of the present invention can be one or more of those recited in Table 8. In some embodiments, the DNA repeats may be HSATII, ALR, (CATTC) n , or a combination of the HSATII, ALR, and (CATTC) n .

Transposable elements encompassed within the scope of the present invention may be one or more DNA transposons and/or retrotransposons. The retrotransposon can be one or more of those recited in Tables 3 and 4. In other embodiments, the retrotransposon can be one or more LINEs, Alus, HERVs or a combination of the LINES, Alus and HERVs.

Transposable elements can serve as biomarkers of cancer cells. These repetitive elements constitute almost 50% of the human genome and include: half a million LINE-1 (L1) elements, of which about 100 are transcriptionally active and encode proteins involved in retrotransposition, including reverse transcriptase (RT) and integrase; a million Alu elements, which depend on L1 functions for integration; and thousands of provirus HERV sequences, some of which contain near-to-full length coding sequences (Goodier and Kazazian, 2008; Vois set et al., 2008). Without being bound by theory, increased expression of retrotransposon elements in cancer appears to result in part from overall hypomethylation of the genome, which is also associated with genomic instability (Daskalos et al., 2009; Estecio et al., 2007) and tumor progression (Cho et al., 2007; Roman-Gomez et al., 2008).

Increased transcription of retrotransposon elements in the human genome has been noted in a number of cancer cell types. For example, increased expression of L1 and HERV, as well as formation of retrovirus-like particles, has been reported in tumor tissue from breast cancer, melanoma, germ cell carcinoma and prostate cancer. See U.S. Pat. No. 7,776,523 and Bratthauer et al., 1994; Golan et al., 2008; Ruprecht et al., 2008. Retrotransposon RNA and proteins, as well as antibodies against HERV proteins and virus-like particles, have also been found in blood of some cancer patients (Contreras-Galindo et al., 2008; Kleiman et al., 2004; Ruprecht et al., 2008; Wang-Johanning et al., 2008).

High level expression of retrotransposon genes and/or endogenous reverse transcriptase are sometimes associated with cancer. For example, human LINE-1 p40 protein is often expressed at a higher level in breast cancer than in normal mammary gland (Asch et al., 1996). Thus, the microvesicular nucleic acid profiles of retrotransposable elements are suitable for use in aiding the diagnosis, prognosis, and/or monitoring of medical conditions such as cancer, as well as for use in aiding in treatment selection for therapies whose efficacy is affected by the subject's genetic make-up.

In one embodiment of the present invention, the microvesicular profile(s) of retrotransposable element(s) are determined by analyzing the content of microvesicles originating from brain cancer, e.g., medullablastoma, glioblastoma, lymphoma, and breast cancer cells. In one instance, the profile comprises one or more RNA expression levels of L1, Alu and HERV elements. In another instance, the profile comprises one or more DNA levels of L1 and HERV elements.

In one embodiment, the profile comprises a profile of the HERV-K element. For example, the profile may comprise the expression of the HERV-K element in microvesicles isolated from plasma from a subject. The expression of the HERV-K element may be assessed by determining the expression of any gene that the HERV-K element may encode, e.g., the group-specific antigen gene (gag), the protease gene (prt), the polymerase gene (pol), and the envelope gene (env) (Lower et al., 1996).

In one instance, the present invention may comprise a profile of the expression of the gag gene in microvesicles. The gag gene is from the HERV-K element and the profile of gag expression reflects the profile of HERV-K expression. The expression of the gag gene can be measured by methods known in the art, e.g., quantitative reverse transcription PCR analysis.

In another instance, the present invention may comprise a profile of the expression of the env gene in microvesicles. The env gene is from the HERV-K element and the profile of env expression reflects the profile of HERV-K expression. The expression of env gene can be measured by methods known in the art, e.g., quantitative reverse transcription PCR analysis.

In addition to the mRNA expression levels of one or more nucleic acids, the nucleic acid profiles of the present invention may also comprise the copy number of one or more nucleic acids, the fusion of several nucleic acids, the mutations of one or more nucleic acids, the alternative splicing of one or more nucleic acids, the methylation of one or more nucleic acids, and the single nucleotide polymorphism of one or more nucleic acids. The nucleic acids may correspond to genes, repeats, transposable elements, or other non-coding parts of the genomes of various organisms, including human beings.

The present invention encompasses all forms of cancer and pre-cancerous conditions. For example, without limitation, the present invention encompasses cancer and pre-cancer cells in brain, esophagus, lung, liver, stomach, ovary, testicle, kidney, skin, colon, blood, prostate, breast, uterus, and spleen.

The profile of nucleic acids can be obtained through analyzing nucleic acids obtained from isolated microvesicles according to standard protocols in the art.

In one embodiment, the nucleic acid is DNA. The analysis of the DNA may be performed by one or more various methods known in the art, including microarray analysis for determining the nucleic acid species in the extract, Quantitative PCR for measuring the expression levels of genes, DNA sequencing for detecting mutations in genes, and bisulfite methylation assays for detecting methylation patterns of genes.

In some embodiments of the present invention, data analysis may be performed by any of a variety of methods know in the art, e.g., Clustering Analysis, Principle Component Analysis, Linear Discriminant Analysis, Receiver Operating Characteristic Curve Analysis, Binary Analysis, Cox Proportional Hazards Analysis, Support Vector Machines and Recursive Feature Elimination (SVM-RFE), Classification to Nearest Centroid, Evidence-based Analysis, or a combination thereof.

In another embodiment, the nucleic acid extracted and analyzed from the microvesicles is RNA. In some instance, the RNA may be subject to Digital Gene Expression (DGE) analysis (Lipson et al., 2009). In this method, the RNA may be digested and converted into single stranded cDNA which may then be subject to sequencing analysis on a DNA sequencing machine, e.g., the HeliScope™ Single Molecule Sequencer from Helicos BioSciences as described in a publication by Ting et al. (Ting et al., 2011).

In other instances, the RNA is preferably reverse-transcribed into complementary DNA (cDNA) before further amplification. Such reverse transcription may be performed alone or in combination with an amplification step. One example of a method combining reverse transcription and amplification steps is reverse transcription polymerase chain reaction (RT-PCR), which may be further modified to be quantitative, e.g., quantitative RT-PCR as described in U.S. Pat. No. 5,639,606, which is incorporated herein by reference for this teaching. Another example of the method comprises two separate steps: a first step of reverse transcription to convert RNA into cDNA and a second step of quantifying the amount of cDNA using quantitative PCR.

Nucleic acid amplification methods include, without limitation, polymerase chain reaction (PCR) (U.S. Pat. No. 5,219,727) and its variants such as in situ polymerase chain reaction (U.S. Pat. No. 5,538,871), quantitative polymerase chain reaction (U.S. Pat. No. 5,219,727), nested polymerase chain reaction (U.S. Pat. No. 5,556,773), self-sustained sequence replication and its variants (Guatelli et al., 1990), transcriptional amplification system and its variants (Kwoh et al., 1989), Qb Replicase and its variants (Miele et al., 1983), cold-PCR (Li et al., 2008), BEAMing (Li et al., 2006) or any other nucleic acid amplification methods, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. Especially useful are those detection schemes designed for the detection of nucleic acid molecules if such molecules are present in very low numbers. The foregoing references are incorporated herein for their teachings of these methods. In other embodiment, the step of nucleic acid amplification is not performed. Instead, the extracted nucleic acids are analyzed directly, e.g., through next-generation sequencing.

The analysis of nucleic acids present in the isolated microvesicles can be quantitative, qualitative, or both quantitative and qualitative. For quantitative analysis, the amounts (expression levels), either relative or absolute, of specific nucleic acids of interest within the isolated microvesicles are measured with methods known in the art (some of which are described below). For qualitative analysis, the species of specific nucleic acids of interest within the isolated particles, whether wild type or variants, are identified with methods known in the art.

The present invention further encompasses methods of creating and using the microvesicular nucleic acid profiles described herein. In one embodiment of a method for creating a microvesicular profile, the method comprises the steps of isolating microvesicles from a biological sample (e.g., from a body fluid) obtained from a subject or obtaining a microvesicle fraction isolated from a biological sample obtained from a subject, extracting nucleic acids from the isolated microvesicles or microvesicle fraction (or obtaining such as extraction), and determining the profile of the nucleic acids in the extract.

The microvesicular profiles of the present invention may be used in methods of aiding diagnosis, prognosis, monitoring, therapy selection, or risk assessment of a disease or other medical condition for a subject as described herein and in the claims.

In some embodiments of the present invention, the one or more nucleic acid(s) may be one or more genes listed in Table 2 (cancer genes), Table 3 (cancer-related somatic mutations) and Table 6 (satellite-correlated genes). In one embodiment, the one or more nucleic acid(s) may be a fragment of a c-myc gene, for example, a fragment of c-myc gene containing more than 10 nucleotides. The fragment may contain incrementally longer sequences of 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 nucleotides, up to the full length of the gene.

In other embodiments, the one or more nucleic acids may be one or more sequences listed in Table 4 (GBM transposable elements), Table 5 (human transposable elements) and Table 8 (repeated DNA). In one embodiment, the one or more nucleic acids may be L1, Alu, HERV, fragments thereof, or any combination of any of the foregoing. The fragment may contain incrementally longer sequences of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 nucleotides up to the full length of each gene sequence.

In one embodiment, the invention comprises microvesicular profiles and methods based on microvesicular polypeptide species, polypeptide activities, or both the species and activities of polypeptides. The polypeptide may be any polypeptide in microvesicles. In some embodiments, the polypeptide is a reverse transcriptase. The activity of the reverse transcriptase (RT) can be measured by standard protocols known in the art. For example, the RT activity can be measured by the EnzChek RT Assay Kit (Invitrogen).

The human endogenous retrovirus K (HERV-K) reverse transcriptase may serve as a breast cancer prognostic marker (Golan et al., 2008). As such, one particular embodiment of the present invention encompasses profiles and related methods based on detecting the activity of HERV-K reverse transcriptase in microvesicles.

The present invention also includes a kit for genetic analysis of a microvesicle preparation from a biological sample (e.g., a bodily fluid sample) from a subject. The kit in a suitable container may include one or more reagents capable of hybridizing to or amplifying one or more nucleic acids extracted from microvesicles. In some embodiments, the nucleic acids correspond to one or more of those genes listed in Tables 2, 3, 4, 5, 6 and/or 8. In some further embodiments, the nucleic acids correspond to one or more RNA transcripts of one or more genes listed in Tables 2, 3, 4, 5, 6 and/or 8. In other further embodiments, the nucleic acid is DNA corresponding to one or more of the genes listed in Tables 2, 3, 4, 5, 6 and/or 8.

The present invention further includes an oligonucleotide microarray for genetic analysis of a microvesicle preparation from a body fluid sample from a subject, wherein the various oligonucleotides on the array are designed to hybridize exclusively to nucleic acids corresponding to one or more genes listed in Tables 2, 3, 4, 5, 6 and/or 8. The arrays can be made by standard methods known in the art. For example, SurePrint Technology (Agilent Technologies Corp.) may be used to make as many as 8 arrays on a single slide.

The present invention also includes a method of aiding the discovery of one or more biomarkers for a disease or other medical condition. The method may comprise, e.g., the steps of isolating microvesicles from subjects having a disease or other medical condition of interest and also from subjects who do not have the disease or other medical condition of interest; measuring the level of one or more target biological materials extracted from the isolated microvesicles from each of the subjects; comparing the measured levels of the one or more target biological materials from each of the subjects; and determining whether there is a statistically significant difference in the measured levels. The step of determination of a statistically significant difference in the measured levels identifies the one or more target biological materials as potential biomarkers for the disease or other medical condition. As an alternative to isolating microvesicles, the method may be carried out with pre-isolated microvesicle fractions.

The one or more biomarkers and nucleic acids in each of the various embodiments of the invention described herein can be one or a collection of genetic aberrations. The term “genetic aberration” is used herein to refer to the nucleic acid amounts as well as nucleic acid variants within the nucleic acid-containing particles. Specifically, genetic aberrations include, without limitation, over-expression of a gene (e.g., an oncogene) or a panel of genes, under-expression of a gene (e.g., a tumor suppressor gene such as p53 or RB) or a panel of genes, alternative production of splice variants of a gene or a panel of genes, gene copy number variants (CNV) (e.g., DNA double minutes) (Hahn, 1993), nucleic acid modifications (e.g., methylation, acetylation and phosphorylations), single nucleotide polymorphisms (SNPs) (e.g., polymorphisms in Alu elements), chromosomal rearrangements (e.g., inversions, deletions and duplications), and mutations (insertions, deletions, duplications, missense, nonsense, synonymous or any other nucleotide changes) of a gene or a panel of genes, which mutations, in many cases, ultimately affect the activity and function of the gene products, lead to alternative transcriptional splice variants and/or changes of gene expression level, or combinations of any of the foregoing.

Genetic aberrations can be found in many types of nucleic acids. The determination of such genetic aberrations can be performed by a variety of techniques known to the skilled practitioner. For example, expression levels of nucleic acids, alternative splicing variants, chromosome rearrangement and gene copy numbers can be determined by microarray analysis (see, e.g., U.S. Pat. Nos. 6,913,879, 7,364,848, 7,378,245, 6,893,837 and 6,004,755) and quantitative PCR. Particularly, copy number changes may be detected with the Illumina Infinium II whole genome genotyping assay or Agilent Human Genome CGH Microarray (Steemers et al., 2006).

Nucleic acid modifications can be assayed by methods described in, e.g., U.S. Pat. No. 7,186,512 and patent publication WO/2003/023065. Particularly, methylation profiles may be determined by Illumina DNA Methylation OMA003 Cancer Panel.

SNPs and mutations can be detected by hybridization with allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatched heteroduplex (Cotton et al., 1988), ribonuclease cleavage of mismatched bases (Myers et al., 1985), mass spectrometry (U.S. Pat. Nos. 6,994,960, 7,074,563, and 7,198,893), single strand conformation polymorphism (SSCP) (Orita et al., 1989), denaturing gradient gel electrophoresis (DGGE)(Fischer and Lerman, 1979a; Fischer and Lerman, 1979b), temperature gradient gel electrophoresis (TGGE) (Fischer and Lerman, 1979a; Fischer and Lerman, 1979b), restriction fragment length polymorphisms (RFLP) (Kan and Dozy, 1978a; Kan and Dozy, 1978b), oligonucleotide ligation assay (OLA), allele-specific PCR (ASPCR) (U.S. Pat. No. 5,639,611), ligation chain reaction (LCR) and its variants (Abravaya et al., 1995; Landegren et al., 1988; Nakazawa et al., 1994), flow-cytometric heteroduplex analysis (WO/2006/113590), nucleic acid sequencing, and combinations/modifications thereof.

Nucleic acid sequencing is to determine the base pair sequences of nucleic acids. Two traditional techniques for sequencing DNA are the Sanger dideoxy termination method (Sanger et al., 1977) and the Maxam-Gilbert chemical degradation method (Maxam and Gilbert, 1977). Both methods deliver four samples with each sample containing a family of DNA strands in which all strands terminate in the same nucleotide. Gel electrophoresis, or more recently capillary array electrophoresis is used to resolve the different length strands and to determine the nucleotide sequence, either by differentially tagging the strands of each sample before electrophoresis to indicate the terminal nucleotide, or by running the samples in different lanes of the gel or in different capillaries. Related methods using dyes or fluorescent labels associated with the terminal nucleotide have been developed, where sequence determination is also made by gel electrophoresis and automated fluorescent detectors. For example, the Sanger-extension method has recently been modified for use in an automated micro-sequencing system which requires only sub-microliter volumes of reagents and dye-labelled dideoxyribonucleotide triphosphates. U.S. Pat. No. 5,846,727.

More recently, high throughput DNA sequencing methods of various types have been developed and used to delineate nuclei acis sequences. These new methods are applied in sequencing machines including the 454 GenomeSequencer FLX instrument (Roche Applied Science), the lumina (Solexa) Genome Analyzer, the Applied Biosystems ABI SOLiD system, the Helicos single-molecule sequencing device (HeliScope), and the Ion semiconductor sequencing by Ion Torrent Systems Inc. See also US patent application publications No. 20110111401 and No. 20110098193. It is understood that as the sequencing technology evolves, the analysis of nucleic acids obtained in the invention may be performed using any new sequencing method as one skilled in the art sees appropriate.

Gene expression levels may be determined by the serial analysis of gene expression (SAGE) technique (Velculescu et al., 1995), quantitative PCR, quantitative reverse transcription PCR, microarray analysis, and next generation DNA sequencing as known in the art.

In general, the methods for analyzing genetic aberrations are reported in numerous publications, not limited to those cited herein, and are available to skilled practitioners. The appropriate method of analysis will depend upon the specific goals of the analysis, the condition/history of the patient, and the specific cancer(s), diseases or other medical conditions to be detected, monitored or treated. The forgoing references are incorporated herein for their teaching of these methods.

Many biomarkers may be associated with the presence or absence of a disease or other medical condition in a subject. Therefore, detection of the presence or absence of such biomarkers in nucleic acids extracted from isolated microvesicles, according to the methods disclosed herein, may aid diagnosis of the disease or other medical condition in the subject.

For example, as described in WO 2009/100029, detection of the presence or absence of the EGFRvIII mutation in nucleic acids extracted from microvesicles isolated from a patient serum sample aided in the diagnosis and/or monitoring of glioblastoma in the patient. This is so because the expression of the EGFRvIII mutation is specific to some tumors and defines a clinically distinct subtype of glioma (Pelloski et al., 2007).

For another example, as described in WO 2009/100029, detection of the presence or absence of the TMPRSS2-ERG fusion gene, PCA-3, or both TMPRSS2-ERG and PCA-3 in nucleic acids extracted from microvesicles isolated from a patient's urine sample may aid in the diagnosis of prostate cancer in the patient.

Further, many biomarkers may be associated with disease or medical status monitoring in a subject. Therefore, the detection of the presence or absence of such biomarkers in a nucleic acid extraction from isolated microvesicles, according to the methods disclosed herein, may aid in monitoring the progress or reoccurrence of a disease or other medical condition in a subject.

For example, as described in WO 2009/100029, the determination of matrix metalloproteinase (MMP) levels in nucleic acids extracted from microvesicles isolated from an organ transplantation patient may be used to monitor the post-transplantation condition, as a significant increase in the expression level of MMP-2 after kidney transplantation may indicate the onset and/or deterioration of post-transplantation complications. Similarly, a significantly elevated level of MMP-9 after lung transplantation, suggests the onset and/or deterioration of bronchiolitis obliterans syndrome.

Many biomarkers have also been found to influence the effectiveness of treatment in a particular patient. Therefore, the detection of the presence or absence of such biomarkers in a nucleic acid extraction from isolated microvesicles, according to the methods disclosed herein, may aid in evaluating the efficacy of a given treatment in a given patient. For example, as disclosed in Table 1 in the publication by Furnari et al. (Furnari et al., 2007), biomarkers, e.g., mutations in a variety of genes, affect the effectiveness of specific medicines used in chemotherapy for treating brain tumors. The identification of these and other biomarkers in nucleic acids extracted from isolated particles from a biological sample from a patient can guide the skilled practitioner in the selection of treatment for the patient.

Without limitation, all of the methods mentioned above may further comprise the step of enriching the isolated microvesicles for microvesicles originating from a specific cell type. For example, the cell can be a cancer or pre-cancer cell.

Another aspect of the present invention is a method of treating a subject suffering from a form of cancer in which the cancer cells secret microvesicles. The method comprises administering to the subject a therapeutically effective amount of a composition comprising: an inhibitor of microvesicle secretion; an inhibitor of a reverse transcriptase; another microvesicle neutralizer that neutralizes the pro-tumor progression activity of tumor microvesicles; or any combination of the inhibitors/neutralizers.

In one embodiment, the inhibitor of microvesicle secretion is an inhibitor of the Rab GTPase pathway (Ostrowski et al.).

In some instances, the Rab GTPases are Rab 27a and Rab 27b. The inhibition of the Rab 27a and Rab 27b can be effectuated by silencing the Slp4 gene (also known as SYTL4, synaptotagmin-like 4) and the Slac2b gene (also known as EXPH5, exophilin5), respectively. Gene silencing techniques are well known in the art. One example of such a gene silencing technique is an RNA interference technique that selectively silences genes by delivering shRNA with viral vectors (Sliva and Schnierle).

In other instances, the Rab GTPase is Rab35. The inactivation of Rab35 decreases microvesicle secretion. Therefore, silencing Rab35 may decrease the secretion of microvesicles by cells. Inactivation of Rab35 may be achieved by administering TBC1D10B (TBC1 domain family, member 10B) polypeptide (Sliva and Schnierle).

In another embodiment, instead of, or in addition to, inhibiting microvesicle secretion, the reverse transcriptase activity is inhibited by administration of an RT inhibitor. RT inhibitors may be any one of 3′-azido2′,3′-dideoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-didehyro-3′-deoxythymidine (d4T), nevirapine and efavirenz.

Further, a microvesicle neutralizer may be used to block the effects of microvesicles. For example, such neutralizer may bind to microvesicles and destroy the integrity of microvesicles so that the biological materials in microvesicles are not transferred to other intact cells.

It should be understood that this invention is not limited to the particular methodologies, protocols and reagents, described herein, which may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

The contents of earlier filed provisional applications U.S. Ser. No. 61/378,860, filed Aug. 31, 2010, U.S. Ser. No. 61/421,421, filed Dec. 9, 2010, U.S. Ser. No. 61/437,547, filed Jan. 28, 2011, U.S. Ser. No. 61/438,199, filed Jan. 31, 2011, and 61/493,261 filed Jun. 3, 2011 are herein incorporated by reference in their entirety.

All patents, patent applications, and publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies and techniques described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

The present invention may be as defined in any one of the following numbered paragraphs.

1. A method for assaying a biological sample from a subject in aid of diagnosis, prognosis or monitoring of a disease or other medical condition in the subject, comprising the steps of:

a. obtaining or using a microvesicle fraction from a biological sample from a subject; b. extracting nucleic acid from the fraction; and c. detecting the presence or absence of a biomarker in the extracted nucleic acid; wherein the biomarker is a genetic aberration associated with diagnosis, prognosis, status or stage of a disease or other medical condition, and wherein the genetic aberration is in or corresponds to:

i. a c-myc gene; ii. a transposable element; iii. a retrotransposon element; iv. a satellite correlated gene; v. a repeated DNA element; vi. non-coding RNA other than miRNA; or vii. a fragment of any of the foregoing.

2. The method of paragraph 1, wherein the genetic aberration is in or corresponds to a transposable element listed in Table 4 or Table 5, or a fragment thereof. 3. The method of paragraph 1, wherein the genetic aberration is in or corresponds to a retrotransposon element that is LINE, SINE or HERV, or a fragment thereof. 4. The method of paragraph 3, wherein the genetic aberration is in or corresponds to a retrotransposon element that is Line1 (L1), ALU, HERV-H, HERV-K, HERV-K6, HERV-W or HERV-C, or a fragment thereof. 5. The method of paragraph 1, wherein the genetic aberration is in or corresponds to a satellite correlated gene listed in Table 6, or a fragment thereof. 6. The method of paragraph 1, wherein the genetic aberration is in or corresponds to a repeated DNA element listed in Table 8, or a fragment thereof. 7. The method of paragraph 1, wherein the genetic aberration is in or corresponds to a non-coding RNA listed in Table 9 (or a fragment thereof), other than miRNA. 8. The method of paragraph 7, wherein the non-coding RNA is 7SL. 9. A method for assaying a biological sample from a subject in aid of directing treatment of the subject for a disease or other medical condition, comprising the steps of:

a. obtaining or using a microvesicle fraction from a biological sample from a subject; b. extracting nucleic acid from the fraction; and c. detecting the presence or absence of a biomarker in the extracted nucleic acid; wherein the biomarker is a genetic aberration associated with a disease or other medical condition or with responsiveness to a specific therapy for the disease or other medical condition, and wherein the genetic aberration is in or corresponds to:

i. a c-myc gene; ii. a transposable element; iii. a retrotransposon element; iv. a satellite correlated gene; v. a repeated DNA element; vi. non-coding RNA other than miRNA; or vii. a fragment of any of the foregoing.

10. The method of paragraph 9, wherein the genetic aberration is in or corresponds to a transposable element listed in Table 4 or Table 5, or a fragment thereof. 11. The method of paragraph 9, wherein the genetic aberration is in or corresponds to a retrotransposon element that is LINE, SINE or HERV, or a fragment thereof. 12. The method of paragraph 11, wherein the genetic aberration is in or corresponds to a retrotransposon element that is Line1 (L1), ALU, HERV-H, HERV-K, HERV-K6, HERV-W or HERV-C, or a fragment thereof. 13. The method of paragraph 9, wherein the genetic aberration is in or corresponds to a satellite correlated gene listed in Table 6, or a fragment thereof. 14. The method of paragraph 9, wherein the genetic aberration is in or corresponds to a repeated DNA element listed in Table 8, or a fragment thereof. 15. The method of paragraph 9, wherein the genetic aberration is in or corresponds to a non-coding RNA listed in Table 9 (or a fragment thereof), other than miRNA. 16. The method of paragraph 15, wherein the non-coding RNA is 7SL. 17. A method for assaying a biological sample from a subject in aid of a determination of the subject's risk of developing a disease or other medical condition, comprising the steps of:

a. obtaining or using a microvesicle fraction from a biological sample from a subject; b. extracting nucleic acid from the fraction; and c. detecting the presence or absence of a biomarker in the extracted nucleic acid, wherein the biomarker is a genetic aberration associated with a determination of the subject's risk of developing a disease or other medical condition, and wherein the genetic aberration is in or corresponds to:

i. a c-myc gene; ii. a transposable element; iii. a retrotransposon element; iv. a satellite correlated gene; v. a repeated DNA element; vi. non-coding RNA other than miRNA; or vii. a fragment of any of the foregoing.

18. The method of paragraph 17, wherein the genetic aberration is in or corresponds to a transposable element listed in Table 4 or Table 5, or a fragment thereof. 19. The method of paragraph 17, wherein the genetic aberration is in or corresponds to a retrotransposon element that is LINE, SINE or HERV, or a fragment thereof. 20. The method of paragraph 19, wherein the genetic aberration is in or corresponds to a retrotransposon element that is Line1 (L1), ALU, HERV-H, HERV-K, HERV-K6, HERV-W or HERV-C, or a fragment thereof. 21. The method of paragraph 17, wherein the genetic aberration is in or corresponds to a satellite correlated gene listed in Table 6, or a fragment thereof. 22. The method of paragraph 17, wherein the genetic aberration is in or corresponds to a repeated DNA element listed in Table 8, or a fragment thereof. 23. The method of paragraph 17, wherein the genetic aberration is in or corresponds to a non-coding RNA listed in Table 9 (or a fragment thereof), other than miRNA. 24. The method of paragraph 23, wherein the non-coding RNA is 7SL. 25. A method for assaying a biological sample from a subject in aid of diagnosis, prognosis or monitoring of a disease or other medical condition in the subject, comprising the steps of:

a. obtaining or using a microvesicle fraction from a biological sample from a subject; b. extracting nucleic acid from the fraction; and c. detecting the presence or absence of a biomarker in the extracted nucleic acid; wherein the biomarker is a genetic aberration associated with diagnosis, prognosis, status or stage of a disease or other medical condition, and wherein the genetic aberration is in or corresponds to a cancer gene listed in Table 2 or 3, or a fragment thereof.

26. A method for assaying a biological sample from a subject in aid of directing treatment of the subject for a disease or other medical condition, comprising the steps of:

a. obtaining or using a microvesicle fraction from a biological sample from a subject; b. extracting nucleic acid from the fraction; and c. detecting the presence or absence of a biomarker in the extracted nucleic acid; wherein the biomarker is a genetic aberration associated with a disease or other medical condition or with responsiveness to a specific therapy for the disease or other medical condition, and wherein the genetic aberration is in or corresponds to a cancer gene listed in Table 2 or 3, or a fragment thereof

27. A method for assaying a biological sample from a subject in aid of a determination of the subject's risk of developing a disease or other medical condition, comprising the steps of:

a. obtaining or using a microvesicle fraction from a biological sample from a subject; b. extracting nucleic acid from the fraction; and c. detecting the presence or absence of a biomarker in the extracted nucleic acid; wherein the biomarker is a genetic aberration associated with a determination of the subject's risk of developing a disease or other medical condition, and wherein the genetic aberration is in or corresponds to a cancer gene listed in Table 2 or 3, or a fragment thereof.

28. A method for assaying a biological sample from a subject in aid of diagnosis, prognosis or monitoring of a disease or other medical condition in the subject, comprising the steps of:

a. obtaining or using a microvesicle fraction from a biological sample from a subject; b. measuring a polypeptide activity in the fraction; and c. determining whether the polypeptide activity is higher or lower than a normal or average activity for the polypeptide; wherein an elevated or lowered activity is associated with diagnosis, prognosis, status or stage of a disease or other medical condition.

29. The method of paragraph 28, wherein the polypeptide is an enzyme. 30. The method of paragraph 29, wherein the enzyme is reverse transcriptase. 31. The method of paragraph 30, wherein step (c) involves determining whether the reverse transcriptase activity is higher than a normal or average activity for reverse transcriptase. 32. A method for assaying a biological sample from a subject in aid of directing treatment of the subject for a disease or other medical condition, comprising the steps of:

a. obtaining or using a microvesicle fraction from a biological sample from a subject; b. measuring a polypeptide activity in the fraction; and c. determining whether the polypeptide activity is higher or lower than a normal or average activity for the same polypeptide; wherein an elevated or lowered activity is associated with a disease or other medical condition or with responsiveness to a specific therapy for the disease or other medical condition.

33. The method of paragraph 32, wherein the polypeptide is an enzyme. 34. The method of paragraph 33, wherein the enzyme is reverse transcriptase. 35. The method of paragraph 34, wherein step (c) involves determining whether the reverse transcriptase activity is higher than a normal or average activity for reverse transcriptase. 36. A method for assaying a biological sample from a subject in aid of a determination of the subject's risk of developing a disease or other medical condition, comprising the steps of:

a. obtaining or using a microvesicle fraction from a biological sample from a subject; b. measuring a polypeptide activity in the fraction; and c. determining whether the polypeptide activity is higher or lower than a normal or average activity for the same polypeptide; wherein an elevated or lowered activity is associated with a subject's risk of developing a disease or other medical condition.

37. The method of paragraph 36, wherein the polypeptide is an enzyme. 38. The method of paragraph 37, wherein the enzyme is reverse transcriptase. 39. The method of paragraph 38, wherein step (c) involves determining whether the reverse transcriptase activity is higher than a normal or average activity for reverse transcriptase. 40. The method of any of paragraphs 1-27, wherein the genetic aberration is:

a. a species of nucleic acid; b. the level of expression of a nucleic acid; c. a nucleic acid variant; or d. a combination of any of the foregoing.

41. The method of any of paragraphs 1-27, wherein the nucleic acid is RNA and the genetic aberration is an expression profile. 42. The method of any of paragraphs 1-27, wherein the fragment contains more than 10 nucleotides. 43. The method of any of paragraphs 1-39, wherein the biological sample is a bodily fluid. 44. The method of paragraph 43, wherein the bodily fluid is blood, serum, plasma, or urine. 45. The method of any of paragraphs 1-39, wherein the subject is a human subject. 46. The method of paragraph 45, wherein the disease or other medical condition is brain cancer. 47. The method of paragraph 46, wherein the brain cancer is medulloblastoma or glioblastoma. 48. The method of paragraph 45, wherein the disease or other medical condition is melanoma. 49. The method of any of paragraphs 1-27, wherein the step of detecting the presence or absence of a biomarker in the extracted nucleic acid comprises microarray analysis, PCR, quantitative PCR, Digital Gene Expression, or direct sequencing. 50. The method of any of paragraphs 1-39, further comprising the step of enriching the microvesicle fraction for microvesicles originating from a specific cell type. 51. A kit for genetic analysis of a microvesicle fraction obtained from a body fluid sample from a subject, comprising, in a suitable container, one or more reagents capable of hybridizing to or amplifying a nucleic acid corresponding to one or more of the genetic aberrations referenced in any of paragraphs 1-27. 52. An oligonucleotide microarray for genetic analysis of a microvesicle preparation from a body fluid sample from a subject, wherein the oligonucleotides on the array are designed to hybridize to one or more nucleic acids corresponding to one or more of the genetic aberrations referenced in any of paragraphs 1-27. 53. A profile of microvesicular nucleic acid derived from a bodily fluid sample from a subject, wherein the profile comprises a genetic aberration in or corresponding to a cancer gene listed in Table 2 or 3, or a fragment thereof. 54. The profile of paragraph 53, wherein the cancer gene is a c-myc gene. 55. A profile of microvesicular nucleic acid derived from a bodily fluid sample from a subject, wherein the profile comprises a genetic aberration in or corresponding to transposable element from the subject's genome, preferably an element listed in Table 4 or 5, or a fragment of any of the foregoing. 56. A profile of microvesicular nucleic acid derived from a bodily fluid sample from a subject, wherein the profile comprises a genetic aberration in or corresponding to a retrotransposon element from the subject's genome, preferably LINE, SINE or HERV, more preferably LINE1 (L1), ALU, HERV-H, HERV-K, HERV-K6, HERV-W or HERV-C, or a fragment of any of the foregoing. 57. A profile of microvesicular nucleic acid derived from a bodily fluid sample from a subject, wherein the profile comprises a genetic aberration in or corresponding to a satellite correlated gene from the subject's genome, preferably a satellite correlated gene listed in Table 6, or a fragment of any of the foregoing. 58. A profile of microvesicular nucleic acid derived from a bodily fluid sample from a subject, wherein the profile comprises a genetic aberration in or corresponding to an element of repeated DNA from the subject's genome, preferably an element listed in Table 8, or a fragment of any of the foregoing. 59. A profile of microvesicular nucleic acid derived from a bodily fluid sample from a subject, wherein the profile comprises a genetic aberration in or corresponding to non-coding RNA other than miRNA, preferably a species listed in Table 9, or a fragment of any of the foregoing. 60. The profile of paragraph 59, wherein the non-coding RNA is 7SL. 61. The profile of any of paragraphs 53-60, wherein the genetic aberration is:

a. a species of nucleic acid; b. the level of expression of a nucleic acid; c. a nucleic acid variant; or d. a combination of any of the foregoing.

62. A method of identifying a potential new nucleic acid biomarker associated with a disease or other medical condition, status or stage of disease or other medical condition, a subject's risk of developing a disease or other medical condition, or a subject's responsiveness to a specific therapy for a disease or other medical condition, comprising the steps of:

(a) obtaining or using a microvesicle fraction from a biological sample from a subject; (b) extracting nucleic acid from the fraction; (c) preparing a profile according to any of paragraphs 53-60; and (d) comparing the profile of step (c) to a control or reference profile and selecting one or more potential new biomarkers based on one or more differences between the profile of step (c) and the control or reference profile.

63. A method of treating a subject having a form of cancer in which cancer cells secrete microvesicles, the method comprising administering to the subject a therapeutically effective amount of a composition comprising:

a. an inhibitor of microvesicle secretion; b. an inhibitor of a reverse transcriptase; c. a microvesicle neutralizer that neutralizes the pro-tumor progression activity of tumor microvesicles; or d. any combination of the forgoing.

64. The method of paragraph 63, wherein the inhibitor of microvesicle secretion is an inhibitor of RAB GTPase. 65. The method of paragraph 64, where in the Rab GTPase is Rab 27a, Rab 27b or Rab 35. 66. The method of paragraph 63, wherein the inhibitor of a reverse transcriptase is a nucleoside analog selected from the group comprising 3′-azido2′,3′-dideoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-didehyro-3′-deoxythymidine (d4T), nevirapine and efavirenz. 67. The method of paragraph 63, wherein the inhibitor of a reverse transcriptase is RNAi targeting the reverse transcriptase gene. 68. The method of paragraph 63, wherein the microvesicle neutralizer is a biological agent that binds microvesicles and destroys the integrity of the microvesicles. 69. A pharmaceutical composition comprising, in a suitable pharmaceutical carrier: (a) an inhibitor of micro vesicle secretion, particularly an inhibitor of RAB GTPase, and more particularly Rab 27a, Rab 27b or Rab 35); (b) an inhibitor of reverse transcriptase, particularly a nucleoside analog, more particularly 3′-azido2′,3′-dideoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-didehyro-3′-deoxythymidine (d4T), nevirapine, or efavirenz, or an RNAi targeting the reverse transcriptase gene; (c) a microvesicle neutralizer that neutralizes the pro-tumor progression activity of tumor microvesicles, particularly a biological agent that binds microvesicles and destroys the integrity of the microvesicles; or (d) a combination of any of the foregoing.

1.

The invention is further illustrated by the following examples, which should not be construed as further limiting. Examples of the disclosed subject matter are set forth below. Other features, objects, and advantages of the disclosed subject matter will be apparent from the detailed description, figures, examples and claims. Methods and materials substantially similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter. Exemplary methods and materials are now described as follows.

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