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Marty Chilberg is a seasoned financial professional with over 30 years of executive leadership, board, consulting and advisory experience.  He began his career as a certified public accountant (CPA). He moved to Silicon Valley in 1981 to begin his career in the software industry, working for... More
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  • Natera: Morgan Stanley Initiates With Equal Weight Rating

    Morgan Stanley came out with their initial coverage report on Natera (NASDAQ:NTRA) today as the quiet period from their IPO ended. As lead investment bank on the offering, Morgan Stanley's insights are particularly insightful. Their report: Natera Inc Born Ready states: NTRA should grow at 25% CAGR over the next 3 years into a $15bn TAM despite price pressure on the core business. We believe the technology and commercial organization are top-tier but valuation suggests expectations are high. Equal-weight, PT $20.

    Morgan provides three valuation assessments as follows:

    • Bull Case $40 6.4x Bullish '17E Revenues Panorama differentiates capturing ~30% of average and low risk pregnancy market by '20E.
    • Base Case $20 3.6x Base '17E Revenues The transition to average and low risk pregnancies reimbursement begins in '16 reaching penetration of ~20% for NIPS in average & low risk pregnancies and ~7-8% in high risk by '20E in the US.
    • Bear Case $8 1.8x Bear '17E Revenues Payers hesitant to reimburse NIPS in average & low risk groups for NIPS with sub-10% penetration by '20E in the US.

    Exhibit 38 provides the trailing quarterly income statement and their projections looking forward

     Mar-2014Jun-2014Sep-2014Dec-2014Mar-2015Jun-2015Sep-2015Dec-2015
    Panorama Revenue19.527.833.835.034.8$32.330.425.9
    Total Revenue27.335.846.349.947.444.543.440.7
    Gross Profit %41.7%46.9%55.0%54.6%47.6%41.1%34.6%24.6%
    R&D %15.7%11.5%9.4%9.0%11.9%15.0%16.0%18.0%
    SG&A %52.6%38.8%35.3%36.7%49.0%62.0%63.0%67.0%
    Net Income$(9.6)$(0.5)$3.7$1.3$(10.0)$(16.9)$(20.2)$(25.2)
    Dil EPS$(0.31)$(0.02)$0.12$0.04$(0.27)$(0.44)$(0.42)$(0.44)

    Exhibit 18 was interesting as it provides a Sum-of-the-parts revenue analysis

     

    C14 $M

    C15 $M

    C16 $M

    C17 $M

    US NIPT High Risk39241615
    US NIPT Microdeletions28304256
    US NIPT Low Risk284582115
    Intl NIPT21253362
    All other43535765
    Total Revenue159176230313

    These tables do well to frame their investment thesis in Natera:

    1. The combined US high risk market (including Microdeletions) for Panorama is viewed to be a no growth business through C17.
    2. International NIPT is showing minimal growth until C17. This is a curious projection given how under-penetrated the international markets are and also that this line includes low risk outside the US.
    3. US low risk appears to be the only growth driver for Natera in C15 and C16 based upon average and low risk reimbursement in C16.
    4. Liquid Biopsy opportunity is large but not anticipated to generate any material revenues in the forecast period. They consider this to be worth approximately $100m in their valuation approach as a "call option" balancing execution risk and the size of the TAM.
    Jul 27 11:55 AM | Link | Comment!
  • Liquid Biopsy For Cancer Screening, Patient Stratification And Monitoring

     

    Graham Brock, Elena Castellanos-Rizaldos, Lan Hu, Christine Coticchia, Johan Skog

    Exosome Diagnostics, Cambridge, MA 02139, USA

    www.thetcr.org/article/view/4546/html


    Abstract: Molecular characterization of a patient's tumor to guide treatment decisions is increasingly being applied in clinical care and can have a significant impact on disease outcome. These molecular analyses, including mutation characterization, are typically performed on tissue acquired through a biopsy at diagnosis. However, tumors are highly heterogeneous and sampling in its entirety is challenging. Furthermore, tumors evolve over time and can alter their molecular genotype, making clinical decisions based on historical biopsy data suboptimal. Personalized medicine for cancer patients aims to tailor the best treatment options for the individual at diagnosis and during treatment. To fully enable personalized medicine it is desirable to have an easily accessible, minimally invasive way to determine and follow the molecular makeup of a patient's tumor longitudinally. One such approach is through a liquid biopsy, where the genetic makeup of the tumor can be assessed through a biofluid sample. Liquid biopsies have the potential to help clinicians screen for disease, stratify patients to the best treatment and monitor treatment response and resistance mechanisms in the tumor. A liquid biopsy can be used for molecular characterization of the tumor and its non-invasive nature allows repeat sampling to monitor genetic changes over time without the need for a tissue biopsy. This review will summarize three approaches in the liquid biopsy field: circulating tumor cells , cell free DNA (cfDNA) and exosomes. We also outline some of the analytical challenges encountered using liquid biopsy techniques to detect rare mutations in a background of wild-type sequences.

    Keywords: Liquid biopsy; exosome; circulating tumor cell (NYSE:CTC); cell free DNA (cfDNA); nucleic acids


    IntroductionOther Section

    The science of noninvasive disease monitoring has advanced greatly since circulating cell free DNA (cfDNA) was first reported in body fluids by Mandel and Metais (1). Since then, the evolution of sensitive cfDNA detection technologies has enabled the development of liquid biopsies with many clinical applications. For example, in oncology, the use of liquid biopsy allows for patient stratification (companion diagnostics), screening, monitoring treatment response and detection of minimal residual disease after surgery/recurrence.

    Liquid biopsies have grown in importance because, the genetic profile of tumors can affect how well they respond to a certain treatment. However, this characterization is currently achieved through a biopsy despite the inherent problems in procurement of tissue samples and the limitations of tumor analyses. For example, the invasive nature of a biopsy poses a risk to patients and can have a significant cost. Tumor sampling from some cancer types also remains difficult resulting in inadequate amount of tissue for genetic testing. In the case of advanced or metastatic non-small cell lung cancers (NSCLC) as many as 31% of cases do not have accessible tissue (2). Even when tissue can be collected, preservation methods such as formalin fixation can cause C > T transitions through deamination of cytosine, potentially leading to false positive results for genetic tests (3). Finally, due to tumor heterogeneity, biopsies often suffer from sample bias (4).

    More concerning with respect to guiding treatment decisions; biopsies will only inform of the genotype at that time-point. However, it is known that tumors are very dynamic and can change their dominant mutation pattern or acquire new mutations, especially after the selective pressure of drug treatment. This could be particularly unfavorable when stratifying patients to a specific targeted therapy based on historical mutation profiles of past tumor biopsies. In another example, approximately 50% of NSCLC patients become resistant to tyrosine kinase inhibitor therapy through an epidermal growth factor receptor (EGFR) T790M mutation (5,6), significantly only <5% of NSCLC patient have this mutation detectable in the primary biopsy (7). Another study showed that 38% of colorectal cancers with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS) developed mutations in this gene after anti-EGFR therapy as rapidly as 6 months after treatment (8).

    Several reports have indicated there are difficulties in detecting tumor derived mutations in plasma, while others have been able to efficiently isolate circulating tumor derived nucleic acid in both metastatic and non-metastatic disease (9-11). This discrepancy is likely due to the methodologies used for detection of the mutation, as the allelic fraction of tumor derived circulating DNA varies from less than 0.01% (or undetectable) to over 90% (12,13). In addition, the amount of recoverable DNA varies significantly (over 3 logs) between patients with an average of about 17 ng of DNA per mL of plasma from advanced-stage cancers (14), corresponding to roughly 5,000 haploid genome equivalents.

    Recent technological developments and the downstream analytics being applied to liquid biopsies are now capable of reproducibly detecting mutations at very low allelic frequencies. Advances have also been made in droplet digital PCR (ddPCR) (15), next-generation sequencing (NYSE:NGS) (16), beads, emulsion, amplification and magnetics (BEAMing) (13), amplification of refractory mutation system (ARMS) (17), co-amplification at lower denaturation temperature-PCR (COLD-PCR) and its derivatives (18,19) and PointMan™ DNA enrichment technology (20), to name but a few.

    Ultimately the choice of platforms and required detection limit will depend on the clinical sample being analyzed, as the most sensitive methods are reported to detect allelic frequencies of as little as 0.01%, providing a theoretical lower limit to detect one mutated copy in a background of 10,000 wild-type alleles (13). Thus, this level of sensitivity requires samples/patients where at least 10,000 target alleles enter the downstream analytical assay.

    Although technically challenging, an inherent advantage of liquid biopsies over other traditional tissue-based methodologies is the enablement of longitudinal monitoring which could help clinical oncologists gain a broader molecular understanding of the disease. This review will focus on the application of genetic profiling of tumor associated RNA and DNA derived from biofluids.


    Approaches to liquid biopsy analysisOther SectionCirculating tumor cells

    CTCs are cells shed into the vasculature from a primary tumor and may constitute seeds for subsequent growth of additional tumors (metastasis) in distant organs. They have been detected in various metastatic carcinomas for example breast, prostate, lung, and colorectal cancer (21,22) but are extremely rare in healthy subjects and patients with nonmalignant diseases (23). Clinical evidence indicates that patients with metastatic lesions are more likely to have CTCs amenable to isolation but their frequency is low, often ~1-10 CTCs per mL of whole blood (24). As 1 mL of blood contains ~7×10e6 white blood cells and ~5×10e9 red blood cells (25), technologies capable of reproducibly isolating a single CTC from the background of all other blood components are fundamental. While such levels of sensitivity are challenging, there are several novel developments in this area. These include positive selection, negative selection, physical properties or even enrichment-free assays to efficiently isolate these rare CTCs (26,27).

    Typically, CTCs are defined as cells with an intact viable nucleus, cytokeratin positive, epithelial cell adhesion molecule (EpCAM) positive and with the absence of CD45. Unfortunately EpCAM and other markers are not always expressed on CTCs and are down-regulated by processes such as epithelial to mesenchymal transition (28). In addition, non-tumor epithelial cells are known to circulate in the blood of patients with prostatitis (29) or patients undergoing surgery (30). From a technical standpoint, the heterogeneity of CTCs is a major challenge and this has led to alternative strategies of CTC enrichment, such as the CTC-iChip (31), which do not rely on tumor antigen expression.

    Sequencing the genetic material from CTCs has demonstrated that, even when the isolated cell(s) fit the phenotypic criteria of being a CTC, the majority are not cancer cells. One study developed a protocol to recover the CTC enriched samples from the cartridge of the Veridex platform and found that from 37 NSCLC patients, the mutation allele abundance ranged between 0.02% and 24.79% with a mean of 6.34% (32). The number of CTCs found in the blood is therefore highly dependent on how the platform defines a cell as a CTC.

    Currently, most CTC isolation platforms require that the whole blood is processed soon after collection, negating the option of long-term bio-banking. In addition, CTCs are fragile and tend to degrade when collected in standard evacuated blood collection tubes. The CellSearch CTC test, a Food and Drug Administration (FDA) approved actionable CTC test, requires that samples are processed within 96 hours of collection after being drawn into the Cellsave preservative tube. This test does not analyze the molecular genetics of the tumor; ratherCellsave is a platform for CTC numeration. A positive test (more than five detected CTCs for metastatic breast and prostate cancer and more than three CTCs for metastatic colorectal cancer per 7.5 mL of blood) is associated with decreased progression-free survival and decreased overall survival in these patients (33-37).

    Cell free DNA (cfDNA)

    There is currently an intensive research effort to understand the utility of cfDNA in various clinical fields such as cancer research (38,39), non-invasive prenatal testing (40) and transplant rejection diagnostics (41). Initial studies in cancer patients reported that cfDNA concentration in serum was significantly increased in comparison to healthy individuals (42), and it was suggested that this correlated with malignancy (43).

    Most cfDNA in plasma is reportedly fragmented, around 150-180 bp in length (44) with a higher prevalence of tumor associated mutations in the shorter fragments (9). In fact, when analyzing the mutation abundance with massively parallel sequencing a significant correlation was found between mutations and fragments less than 150 bp (44). Notably, the size of the majority of cfDNA fragments overlaps well with the size of histone DNA (45).

    The entry of cfDNA into the bloodstream is thought to originate from a cell following apoptosis or necrosis. Late stage cancer patients also have an increased level of cfDNA in plasma, however, most of this DNA is wild-type and believed to be from non-malignant cells and tumor stroma (9). It has also been suggested that the mutant fraction of cfDNA is derived from necrotic neoplastic cells phagocytized by macrophages, which then release digested DNA, a phenomena not seen in macrophages that engulf apoptotic cells (14). The extensive background of wild-type DNA limits the ability of downstream analytical platforms to detect tumor-derived mutation, presenting technical challenges for the use of cfDNA in liquid biopsies. While cell-free tumor DNA analyses are capable of examining the genetic or epigenetic changes that originate in tumor DNA (such as mutations, translocations, amplifications, indels and methylation abnormalities), they cannot analyze the tumor RNA transcriptome or proteome.

    However, an advantage of cfDNA is that it can be analyzed from bio-banked biofluids, such as frozen plasma. In addition, a direct comparison of mutation detection on cfDNA vs. CTCs showed a higher abundance of the mutation on the cfDNA from the same patient (39). Finally, recent large studies comparing the effectiveness of cfDNA analysis to tissue biopsy in NSCLC showed the clinical value of the liquid biopsy approach (46). This positive result led to an approval to use cfDNA analysis for EGFR mutation analysis for IRESSA® in Europe (in patients where a tumor sample was not evaluable), making it the first EGFR tyrosine kinase inhibitor for which cfDNA testing is included in the label.

    Although promising, challenges remain when using cfDNA to characterize the mutation status of a tumor. In addition to the low copy number of mutant alleles, the median half-life of cfDNA in circulation ranges from 15 minutes to a few hours (47). Also, the total concentration of cfDNA in the blood of cancer patients varies considerably (48) with tumor specific mutations ranging from undetectable (less than 1 copy per 5 mL of plasma) to patients with over hundred thousand copies of the mutation per ml of plasma (39). Thus, the challenge of how to maximize the yield of the cfDNA and pair this with a platform sensitive enough to detect rare variants in the background of wild-type DNA remains. Optimally, the ability to detect mutations in plasma should not be limited to a subpopulation of patients with very high mutant copy numbers in circulation. While many analytical platforms report the mutation load with an allelic frequency compared to the wild-type DNA, platforms relying solely on the allelic frequency without recording the number of mutations have limitations. The allelic frequency is affected by the amount of wild-type DNA not related to the tumor. Therefore, it is important to consider the processes that affect the amount of wild-type DNA in circulation. For example, exercise increases cfDNA levels 10-fold (49) and other pre-analytical variables such as blood collection and extraction protocols affect the amount and size range of cfDNA fragments in a sample (50). Delays in blood processing, blood storage temperature, agitation of the sample and shipment can all cause wild-type cfDNA release from lysed nucleated blood cells and effect the allelic frequency (51). For the same reason, plasma is often preferred over serum because of the potential for cell lysis during blood coagulation (52).

    Exosomes

    The exosome field has grown exponentially the last few years impacting various areas of research. Studies demonstrating that exosomes are actively released vesicles (carrying RNA, DNA and protein) and can function as inter-cellular messengers, have contributed to their elevated recognition in the scientific community (53-64). A recent review outlining the biological properties of exosomes and other extracellular vesicles (EV's) highlights these developments (65). However, with respect to nomenclature, the exosome field still lags behind as the definition and characterization of EV types are not yet firmly established (66). The majority of exosomes range in size from 30-200 nanometer in diameter and are isolated from all biofluids, including serum (60), plasma, saliva, urine and cerebrospinal fluid (67).

    Exosomes and other EVs are particularly interesting as cancer biomarkers since they are stable carriers of genetic material and proteins from their cell of origin. They are also thought to be part of the disease process, for example, tumor exosomes have been shown to stimulate tumor cells growth, suppress the immune response and induce angiogenesis (60,68) and even be part of the metastatic process (63,69). Exosome release is also an active process and tumor cells can shed tens of thousands of vesicles per day resulting in hundreds of billions of vesicles per mL of plasma (55). The two mechanisms by which exosomes are released, either involve the formation of multivesicular bodies (MVB) and direct budding at the plasma membrane, or a process more akin to a retrovirus particle leaving the cell (Figure 1) (70).

    (click to enlarge)
    Figure 1 Exosome/microvesicle biogenesis. The classical exosome biogenesis pathway begins with the formation of an endosome, followed by inward budding of the endosome resulting in MVB with ILV. These ILV contain a sample of the cell's cytoplasm, including nucleic acids. (NYSE:A) The ILV are then liberated by fusion of the MVB to the plasma membrane; (NYSE:B) the second way of exosome/microvesicle biogenesis is through direct budding at the plasma membrane. MVB, multivesicular bodies; ILV, intraluminal vesicles.

    In the early decades of exosome research, it was thought that they contained only protein and lipids. However, it has since been shown that exosomes are highly stable packages of RNA from the cell of origin (61). The finding that exosomes contain RNA with tumor specific mutations, can be isolated from biofluid samples and stored for many years in the freezer has opened up new opportunities in the field of diagnostics (60,71). Recent publications have also examined the DNA associated with exosomes and shown its utility for detection of gene amplifications as well as mutations (55,64,72).

    Due to the size of an exosome, on average just over 100 nanometers, the entire transcriptome cannot be packaged inside every vesicle. By way of comparison, retrovirus particles with a similar size can package only around 10 kb (73), so it is likely that a single vesicle of that size carries only a limited number of transcripts. However, exosomes are extremely abundant (10e11 per mL of plasma) and when isolating the vesicle fraction, most of the transcriptome can be detected (74). Exosomal RNA can be used for mutation detection (55,60,71,72) as well as global profiling of most types of RNA (74), and the profile alone (without mutation characterization) can be utilized for diagnostics (58,75,76).

    The precipitous release of exosomes by cancer cells seems to correspond to activation of the mitogen-activated protein kinases (MAPK) pathway frequently upregulated in tumor cells (77). Tumor derived mutations can be detected in exosomes from cerebrospinal fluid (67), serum (60), plasma (64) as well as in urine (71). However, as exosomes are released by all cells, they are particularly useful to profile not only mutations in cancer but also RNA profiles in inflammatory (78), metabolic (79), cardiovascular (80), neurodegenerative (81) and other disease processes.

    Exosomes also carry surface markers from the cell of origin, which can be used for enrichment strategies, similar to CTCs (75). For example, characterization and analysis of exosome surface proteins hold great promise for the ability to identify, separate, sort and enrich exosomes originating from diverse cell sources. While the development of methods that allow for the routine analysis of exosome surface proteins has been a challenge, a number of recent advances have demonstrated potential. Immunoaffinity-bead based capture methods, microfluidic chip methods and antibody-based exosome arrays using both label and label-free detection platforms have all successfully exploited specific exosome surface proteins. This has enabled the capture, enrichment and characterization of unique populations of exosomes in the blood of healthy donors and of patients with pancreatic cancer (82), ovarian cancer (83), lung cancer (84,85). Surface protein-based exosome isolation methods combined with exosomal RNA extraction and qPCR detection assays have proven to be rapid and sensitive enough to monitor therapeutic response and resistance using exosomes from the blood of patients with glioblastoma (86,87).

    In addition, the rapid advancement of a novel method of nanoscale fluorescence activated cell sorting call nanoFACS has further advanced methods of exosome isolation and sorting and allowed for the study of discrete, free, individual exosomes from body fluids (88). This technique and variants thereof hold great promise for future diagnostic applications where isolation and examination of individual exosomes is paramount. Finally, in addition to proteins, analysis of exosome protein-to-lipid ratios can be used to further isolate and characterize subpopulations of exosomes in body fluids (89).

    Exosome investigations have focused on the important physiologic and pathophysiologic functions of these vesicles in micro-metastasis, angiogenesis and immune modulation (63,90) and as a means for detection of tumor specific mutations in biofluids. Consequently, in 2012, interest in this new field increased when the National Institute of Health (NIH) dedicated the large strategic Common Fund to study these new entities of extracellular RNA. The goal of this effort is to better understand how exosomes can be utilized for biomarkers and therapeutics as well as understanding this new mechanism of intercellular communication (http://commonfund.nih.gov/Exrna/index).


    Mutation detection and RNA profilingOther Section

    Analysis of nucleic acids present in bodily fluids can provide a better understanding of the disease, as summarized in Table 1.

    (click to enlarge)
    Table 1 Comparison of the analysis capability of CTC's, cfDNA and exosomes
    Full table

    Mutation detection in biofluids is a challenging task and requires highly sensitive analytical platforms. As this field has evolved, the clinical applications of liquid biopsies have improved significantly. Examples of these analytical platforms include BEAMing (13), ARMS (17), and ddPCR (15). These platforms were developed specifically for the detection of extremely rare alleles and are used when the mutation type and position is known. Other platforms such as ice-COLD PCR and targeted resequencing using NGS platforms can detect rare allelic frequencies even when the type and location of the mutation in the gene is undefined. Targeted resequencing is becoming increasingly popular since it can easily accommodate larger panels of genes to cover the actionable mutations in cancer that have significant diagnostic, prognostic or therapeutic implications for a specific therapy. Initially, the inherent error rate of NGS platforms made it difficult to identify very rare alleles (<1%), but strategies using paired-end sequencing and background correction have enabled detection of allelic frequencies at or below 0.1% (91). Incorporation of unique identifiers to each target enables highly sensitive digital sequencing capable of quantifying the number of mutated reads as well as their allelic frequency (92,93).

    RNA profiling from biofluids also poses numerous challenges. However, the discovery that exosomes contained RNA made it possible to separate the fragile RNA from the large amounts of RNases and PCR inhibitors that are present in most biofluids. As cell-free RNA in blood is immediately degraded, RNAs in serum and plasma are either protected inside vesicles like an exosome, in protein complexes with the Ago2 protein (94) or associated with HDL particles (95) as outlined in Figure 2. Most of the early studies were limited to the more abundant short (~22 nt) regulatory microRNAs. The levels of these microRNAs are tightly regulated in normal cells and dysregulation has been implicated in a number of human diseases e.g., cardiovascular (96) neurological and is strongly linked to cancer development and progression as reviewed by Croce (97). However, although robust and readily detectable, microRNAs represent only a minor fraction of the transcriptome. By contrast, if the appropriate methods are used, the nucleic acids in exosomes can be isolated and the entire transcriptome interrogated for effective molecular profiling and mutation detection. Successful RNA profiling from biofluids requires that the contaminants, which could inhibit downstream analysis are removed. The effective purification of the exosomes can remove these contaminants making the exosome isolation platform scalable, where the sample volume input is linear to the RNA output and not affected by the increased amount of RNases that can co-purify (98). This feature is important, since scaling the volume appropriately will enable profiling also of low copy number RNAs.

    (click to enlarge)
    Figure 2 Circulating nucleic acids are coming from a wide range of cellular processes. It is important to optimize the sample processing for the particular target and understand where the RNA and DNA are coming from as well as their abundance. Whole blood as well as cell free plasma has multiple sources containing nucleic acids (shown in A and B respectively). Even components that lack a nucleus (like erythrocytes and thrombocytes) have been shown to carry RNA and can have cfDNA co-isolating in the preparations. *, based on a range of 0-50% of exosome RNA containing the tumor specific mutant allele (67) (and Exosome Diagnostics unpublished data).

    Finally, special precautions need to be taken to prevent degradation during the RNA extraction procedure, as the RNA purified from exosomes and the microRNAs from Ago2 complexes will now be exposed to RNases. Measuring integrity using an exogenous spiked-in sequence of similar size and structure as the RNAs in the exosomes is recommended. Ideally, the 'spike' should itself be protected from RNases, for example using a synthetic vesicle added directly into the biofluid as opposed to the lysed sample.


    DiscussionOther Section

    The most obvious hurdle for all forms of liquid biopsy remains the relative rarity of nucleic acid derived from a tumor against the background of normal material found in most patient samples. In fact, the majority of cell, cell free nucleic acids, microRNAs and exosomes in a liquid biopsy will have originated from normal cells with numbers fluctuating as a consequence of biological variations. Such challenges are addressed using the strategies highlighted in the methods described above. These methods are currently sensitive enough to detect very rare mutation events. However, it is critical that laboratories undertaking such methods must be scrupulous in their methodologies to avoid erroneous results. Although clichéd, the analogy of a needle in a haystack applies and is appropriate for each of these approaches.

    The analysis of CTCs and exosome has benefited from developments in the field of enrichment prior to the analytical readout. While still at an early stage, a number of studies have demonstrated that protein-based isolation and enrichment methods will be an important tool both in enhancing nucleic acid based assays and as stand-alone diagnostics in the future.

    Clearly, exosomes have a number of advantages for diagnostics. They enable high quality RNA to be extracted from fresh or frozen biofluids, thus increasing the scope of detectable mutations to include mutations, splice variants, fusions as well as expression based assays for mRNA, microRNA, lncRNA and other non-coding RNAs. They are also released from living cells as an active process, whereas cfDNA is released through the process of apoptosis and necrosis. On cfDNA, all genes are present at an equal level, whereas RNA originating from a highly expressed gene could occur in thousands of copies/cell. However, as mutations exist on both exosome RNA (living process) and cfDNA (dying process), utilizing a platform that can use both will have obvious advantages for detecting rare mutations. This is especially true in the case of patients who do not have an abundant amount of mutated nucleic acid in circulation.

    Improvements to analytical sensitivity and specificity will address some of the current hurdles, for example, cancer patients who have very few mutations in their biofluids, likely due to biology rather than analytical sensitivity. In many cases, the mutated alleles can occur at less than 1 copy per mL of plasma. So, combining exosome RNA and cfDNA has the advantage of increasing the detection sensitivity for low frequency mutations.

    For the patient there is an obvious and clear advantage to a liquid biopsy in comparison to conventional surgical methods. However, most of the studies to date have focused on detection of actionable mutations in biofluids, and this is arguably only a fraction of the capability of liquid biopsies in enabling personalized medicine. As DNA mutations will only inform of some aspects of the disease, looking at RNA expression in biofluids can help further understand processes within the cancer patient.

    Cancer is a complex and dynamic disease that can change quickly. To fully deliver on the promise of personalized medicine, development of reliable and robust non-invasive platforms for the diagnosis, patient stratification and to monitor treatment response are paramount. The various liquid biopsy platforms described in this review have the potential to add tremendous value to the care of cancer patients.


    AcknowledgementsOther Section

    Disclosure: The authors are employees of Exosome Diagnostics.


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    Cite this article as: Brock G, Castellanos-Rizaldos E, Hu L, Coticchia C, Skog J. Liquid biopsy for cancer screening, patient stratification and monitoring. Transl Cancer Res 2015;4(3):280-290. doi: 10.3978/j.issn.2218-676X.2015.06.05

     

    Jul 26 12:48 PM | Link | Comment!
  • Illumina Comments On Liquid Biopsy

    Illumina has made a few comments about their intended path related to their OncoPanel and Liquid Biopsy plans. Here are a few from recent conference calls (links in header).

    Q2 2014 Conference Call

    • Importantly, NextSeq is to say the sweet spot with oncology, NIPT and microbiology customers to do the breadth applications and the flexibility of the workflow. As an illustration of this, half of orders came from commercial customers made which are exploring the use of NextSeq in a translational or clinical production setting.


    Q3 2014 Conference Call

    • NextSeq also continues to generate significant interest from the commercial segment which accounted for third of orders. These labs are focused on oncology, NIPT and microbiology and are choosing NextSeq due to the breadth of applications, the flexibility of the work flow and the attractive cost per base.

    • We announced strategic partnerships with AstraZeneca, Janssen, and Sanofi to develop a universal NGS-based oncology test system. This system will be used for clinical trials of targeted cancer therapies with a goal of developing and commercializing a multi gene panel for therapeutic selection. We hope to share additional partners with you in the coming quarters.

    • The second observation is that there's a general tendency toward increasing content on the panels and we're trying to figure out ways of making sure that we don't get gene creep, I guess is probably the right word to use where the panel just keeps getting perpetually larger because everybody wants their incremental five genes. And so locking down exactly what the content needs to be is challenging and an opportunity.


    Q4 2014 Conference Call

    • NextSeq continues to draw significant interest from the oncology market. In the fourth quarter, shipments to this segment grew 50% sequentially and included a multi-unit order for a reference laboratory that is scaled to production level, boding well for future consumable utilization
    • In 2015, we hope to further catalyze this market with programs such as our recently announced Actionable Genome Consortium and our development program in liquid biopsies. Liquid biopsies are expected to play a role across multiple segments of the oncology market. We are focused on first delivering an RUO kit into the market and expect to be in a position to present clinical utility data by year-end.

      We will run this test at Illumina's Redwood City CLIA lab to refine the product and develop data for FDA submission. Similar to NIPT, our strategy here is to provide the best technology into the market, as opposed to offering long-term lab tests or selling to physicians.

    • Clearly, Liquid biopsy is a breakthrough opportunity for anybody who is working in that area and our goal as I mentioned in the script to get some clinical data, some really important clinical data done by the end of 2015. So I think that will be critical as well.

    • I think it's what we've seen so far much more straightforward IP landscape than what we seen in the lot of other markets particularly in NIPT, clearly there will be companies when claim they have some fundamental IP around this as happens almost in every application area. I think when it all get sorted out these assay methods are pretty standard, there is not a lot of raw invention in the assay method just being used to sequence blood in a slightly different way. So we think it's a relatively open field from an intellectual property perspective.

    • And I mean, first half I would say we are not planning to do kind of standalone basis (Liquid Biopsy). Many of the companies that have announced projects and begin to show some clinical data in a NIPT, most them use sequencing as their underlying method, there are few other alternatives. But most of them use sequencing. And most of them use our platform and we will continue to encourage that, those are going to be great customers for us because remember in the US even though we are developing our own method.

      We don't have the ability to go out to the labs and to teach those labs how to use that method because home [indiscernible]. So in the US we will go through the FDA and try to get an approved test, we will run LDTs necessary to gather data to build clinical evidence and encourage the overall market to grow with lots of different methods here. It's going to be a diverse set of methods that we believe for NIPT that is for local biopsy. There is a number of different ways to go about it, there will be some companies which use to have very targeted tests that have limited indications. There will be others that go after much more universal methods. Our option that we have chosen is to go toward the more universal test, even though we march through some more targeted collections of variants on the way.


    Q1 2015 Conference Call

    • We remain focused on delivering sample to answer solutions to address more than $12 billion oncology market opportunity and continue to make progress towards this goal.

      Product development activities for both versions of our onco panel remain on track. During the quarter we announced that Merck Serono has joined the onco panel program adding to the partnerships we previously announced. Our liquid biopsy project continues to progress well and we remain on track to launch a targeted RUO kit by year end.

    • Yes, there is a bunch of sub segments here the attributable part of the panel we expect to have available in an RUO partnership mode by the end of the year what most of the pharma partners want is very often a subset of that panel in some cases so those are truly companion diagnostic contracts. And some of the other cases they want to use the larger universal panel in the clinical trial setting to understand what genes might be relevant for them as an ultimate companion diagnostic and that would largely be a subset and so I think the fast forward to couple of years there will be a series of various combinations of these panels driven by specific pharma interest as well as the migration of knowledge about what genes are increasingly important in oncology. So, we will be adding more to the attributable panel overtime.

      In the cell free area, I think that's a little - its distinct in fact from what we are doing in the onco panel part there I think the focus on particular sub disease categories is driven by the fact that you want to create diagnostics where there is clear alternatives in the clinical decision making and that's why there is so much focus in areas like lung cancer. Its prevalent there is a lot known about the potential markers there and they are various treatment options. And so, we and others I think will focus initially on a couple of key cancer types because you could do the clinical validation more narrowly on those and then continue to add genes overtime to make the panel to increasingly universal.


    Q2 2015 Conference Call

    • Over the past several quarters, we've shared some of our strategies to penetrate this market which include our onco panel and liquid biopsy programs. We're on track to deliver - release one of the onco panel as an RUO product by the end of the third quarter.

      This initial release includes 15 genes critical in oncology and sets the groundwork for our onco panel products that will be CE-IVD marked and FDA approved.

      We're committed to delivering the enhanced 170-gene version of the onco panel to address the needs of the research market early next year. We will also be providing this product for use by our pharma partners in their clinical trial.

      Our liquid biopsy program continued to make progress during the second quarter. As part of this effort, a study was recently published in the Journal of the American Medical Association which showed specific examples where discordant NIPT results were detecting maternal cancer. Our development teams are optimizing assays and exploring their clinical power for use in this exciting frontier of genomics.

    • Aneuploidy will pick up some types of cancers. Nobody quite knows yet the fundamentals of the biology, so we don't know exactly which ones would be picked up by that method versus permutations versus fusions versus methylation. So there's many things we're exploring there, and I think lots of companies are working on this to determine what the best methods might be. And I suspect the market is going to wind up as a whole host of different tests that will be complementary to each other.

    • Ultimately, the goal here would be to create as general a panel as you can but likely this whole market will begin with more targeted panel types from us and from others that have specific knowledge of the variants that are known in particular cancer types.

    • In the case of NIPT, it became a marketplace that exploded because the clinical data came out and replaced an invasive test. And so in oncology, we're going to see that inflection point happen. Is it going to happen a year from now or two years from now, it's hard to predict. But there will be an inflection point in the oncology market where that market will explode. And that would be around perhaps the time when in-practice guidelines it becomes standard of care to sequence every tumor when it's biopsied, and that will cause the market to grow very, very quickly.

    Jul 24 12:17 PM | Link | Comment!
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