O PPORTUNITIES FOR B IONANOMATRIX IN THE G ENETIC T ESTING M ARKET

Penn Biotechnology Group

Roy Amariglio Daniel Chen Daniel Hussey Santhosh Palani Jason Ruth Najaf A. Shah Jonathan Zucker Fall 2009

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OPPORTUNITIES FOR BIONANOMATRIX IN THE GENETIC TESTING MARKET

Contents
Prenatal Genetic testing ................................................................................................. 3 Background .................................................................................................................. 3 Table 1. Pregnancy distribution in the United States and European Union. ...... 3 Karyotyping.................................................................................................................. 4 FISH ............................................................................................................................. 4 Screening...................................................................................................................... 5 Future trends ............................................................................................................ 6 Table 2. Selected companies developing noninvasive prenatal genetic diagnostics methods. (Adapted from IN VIVO, Vol. 27, No. 10, Nov. 2009). ........ 7 Scientific Exploration...................................................................................................... 8 Overview of chromosomal abnormalities ................................................................... 8 Down Syndrome ....................................................................................................... 8 Sex chromosome aneuploidies ................................................................................. 9 Neural tube defects (NTDs) ..................................................................................... 9 Cardiac chromosomal abnormalities....................................................................... 9 Deletions ................................................................................................................. 10 Potential application areas ....................................................................................... 10 Table 3. Disorders associated with aneuploidy .................................................... 11 Table 4. Disorders associated with large chromosomal deletions ....................... 11 Table 5. Disorders associated with gene deletions ............................................... 12 Diseases and their corresponding genetic loci ...................................................... 12 Intellectual Property ..................................................................................................... 17 Potential issues .......................................................................................................... 17 Market Analysis ............................................................................................................ 19

Fall 2009 Table 7. Global market for gene/chromosome diagnostics by segment (Source BCC)........................................................................................................................ 20 Table 8. Demand for DNA microarray by end use. .............................................. 21 Forces driving growth in the DNA microarray market ........................................... 21 Table 9. Market distribution for molecular diagnostics for inherited genetic disease testing in the US, ($ millions, estimates). ............................................... 22 Table 10. Market driving forces. ........................................................................... 22 Competitive Analysis .................................................................................................... 23 Cost per use (reagents, labor/technical staff requirements, time additional per run).......................................................................................................................... 23 Advantages, limitations, efforts to improve that currently used technology...... 23 Table 11. Summary Table of Competitive Technologies ...................................... 25 Risks of addressing this market at this time........................................................ 25 Cancer Genetic Testing ................................................................................................. 27 Background ................................................................................................................ 27 All cancers .................................................................................................................. 27 Leukemia.................................................................................................................... 27 Colorectal cancer........................................................................................................ 28 Breast cancer ............................................................................................................. 30 Table 12. Currently available cancer genetic tests. ............................................ 31 Bibliography .................................................................................................................. 33

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PRENATAL GENETIC TESTING Background

Annually, there are roughly 6 million pregnancies in the United States, approximately 4 million of which result in live births, and the remaining 2 million ending in pregnancy losses (1), with induced abortion and fetal losses each contributing approximately 1 million (2). Among the overall population of pregnant women, approximately 23% are above the age of 30 (225 in 1000, year 2004), and approximately 9% are over the age of 35 (93 in 1000, year 2004) (2).

Table 1. Pregnancy European Union.

distribution

in

the

United

States

and

US
Total Pregnancies Induced Abortion Fetal loses Live birth Less than 30 Above 30 Above 35 6,000,000 1,000,000 1,000,000 4,000,000 3,100,000 900,000 372,000

EU
7,300,000 1216667 1216667 4866667 3,309,334 1119333 438000

To check for chromosomal abnormalities (of which Down syndrome is most common), women over the age of 35 are offered amniocentesis, which is performed at 15-20 weeks. Women who have previously experienced pregnancies with chromosomal or other genetic abnormalities are also offered this test. The amniocentesis procedure involves the use of a needle guided by ultrasound to withdraw 1-2 tbsp of amniotic fluid from which fetal cells are separated and cultured in the lab for 10-12 days, in order to perform chromosomal and genetic analyses. Amniotic fluid is also used to look for aberrant expression of various proteins, which may be indicative of disease 3

Fall 2009 (3). The amniocentesis procedure carries a risk of miscarriage, which is estimated to between 1-2 out of 400 procedures performed; in contrast, chorionic villus sampling (CVS), is typically performed at 10-12 weeks, carries a miscarriage risk of roughly 1% (1). In the United States, amniocentesis is performed roughly 200,000 times a year; this may be a good estimate of the size of the prenatal genetic testing market.

Karyotyping
To screen for chromosomal aberrations, the fluid obtained via amniocentesis is analyzed using karyotyping, a low-resolution (detection of changes larger than 5MB) method that has been the de-facto standard, and which is now being increasingly supplanted by higher resolution technologies. A quasi-reliable reference suggests that approximately 400,000 karyotypes are performed each year in the United States and Canada (this number seems plausible with respect to the number of amniocentesis procedures performed per year; however, karyotyping is the standard analysis method for a number of other genetic disorders. Although low in resolution, karyotyping remains a reliable method for detecting aneuploidy, and other gross chromosomal aberrations such as breaks, fusions, and rearrangements. The cost associated with performing a karyotype test mainly involves the cost of technician labor since reagent and other associated costs are relatively low.

FISH
Fluorescence in situ hybridization (FISH) is often used to provide rapid and fairly accurate determination of chromosomal aberrations. FISH uses chromosomespecific DNA probes and is performed when cells are in metaphase and chromosomes are condensed and can be individually distinguished.

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Screening
In the past decade, prenatal screening has been introduced to screen pregnancies, prior to invasive prenatal genetic diagnosis (i.e. amniocentesis or CVS), in order to both reduce the number of unnecessary invasive procedures, and expand the age range of women who are able to reasonably receive invasive prenatal diagnosis. The consequence of the success of screening has been a precipitous drop in the number of genetic prenatal diagnoses. In 2002, amniocentesis was performed in 1.9% of pregnancies, while in 2003 it was performed in 1.7% of pregnancies (1). In 2007, The American College of Obstetricians and Gynecologists (ACOG) published new guidelines for prenatal screening which recommend that age not be the primary screening technique, and that combined ultrasonography and serum screening methods be used (4). Likely due in large part to this report, our research indicates that the level of amniocentesis performed may have fallen by 80%, down to 0.36% of all pregnancies [primary research]. Genetic diagnoses with amniocentesis or CVS are currently performed in either pregnancies with Down Syndrome which have undergone successful prenatal screening, or non-Down Syndrome pregnancies which have resulted in false positive screening results. In 2007 when the new ACOG guidelines were published, a 5% false positive rate was considered acceptable (4). In 2009, false positive rates for screening tests at 2.29% were considered acceptable (5). As the false positive rate is related to the number of invasive screenings performed and thereby the number of procedure-related fetus losses in normal pregnancies, great effort is underway to decrease the false positive rate while maintaining a high (> 90%) true positive rate, which would further decrease the number of genetic diagnoses performed in the future (5).

*Projected trend for 2009.

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Future trends
In the future, noninvasive methods of detecting fetal DNA, such as either nucleated fetal erythrocytes or fetal DNA in maternal blood, may essentially eliminate the risk of attaining fetal DNA. If this occurs, then it is probable that genetic methods currently used for prenatal diagnosis will obviate serum marker screening due to their higher sensitivity and specificity. This assumes that: 1. The sensitivity and specificity remain high with the new sources of DNA (i.e. assuming that sufficient DNA is attainable by these noninvasive methods to maintain a sensitivity and specificity with genetic testing which are equivalent to the sensitivity and specificity provided by the current amount of DNA from invasive testing) 2. This is a more cost effective method than the current screening method, from the standpoint of public health. While circulating fetal DNA is an area of current interest, and epigenetic markers are being used to distinguish fetal from paternal DNA, this DNA is fractionated, and therefore not ideal for the BNM platform. Fetal nucleated red blood cells (fNRBCs), however, are a promising noninvasive source of the entire genetic sequence of a fetus, which would be suitable for BNMs platform. Though nucleated fetal progenitor cells can persist for long periods after pregnancy (6), NRBCs have a limited life span and therefore only represent a current pregnancy (7). Additionally, a promising new publication reveals that spectral scattering characteristics can be used to reliably differentiate fNRVCs from adult NRBCs, using only the endogenous properties of the microstructure of the cells and thereby not requiring any additional imaging contrast agents (8). While not currently used, there is movement in clinical research to refine maternal blood methods to allow for noninvasive screening, such as the NIH funded multicenter NIFTY trial (9). fNRBCs occur at a rate of 1 in 109 maternal blood cells. Regarding our finding that fetal nucleated cells are identifiable in the cervical mucus plug which develops during pregnancy, other than these cells and fetal DNA in maternal blood there are likely no other sources of fetal DNA available in a manner less invasive than amniocentesis or CVS (10). No research has been published which regards to attaining fetal DNA from cervical mucus plugs since 2006.

Fall 2009 It is expected that the non-invasive methods for harvesting fetal DNA that are currently in development will yield smaller sample size than amniocentisis and CVS. It is our recommendation that BNM develop its technology in conjunction with these new methods in order to capitalize on this trend. As BNMs platform will theoretically require less fetal DNA, it is well suited to fill in this emerging market need. Listed below, in Table 2, are several companies in this emerging field which BNM could potentially collaborate with.

Table 2. Selected companies developing noninvasive prenatal genetic diagnostics methods. (Adapted from IN VIVO, Vol. 27, No. 10, Nov. 2009).
Company Genetic material/Source Cell free nucleic acid from blood Comments

Sequenom

Developing down syndrome test using mass spec and sequencingbased detection technology. Initially developing whole fetal cell separation methods using microfluidics. In January 2009 added a second program to develop technology for sequencing-based detection of fetal nucleic acids for down syndrome. In clinical trials of its PloidYX array based technology using DNA methylation differences to detect trisomies. Treat blood samples with formaldehyde to boost proportion of fetal DNA in the samples. Published results in Lancet

Artemis Health

Cell free nucleic acids / Whole fetal cells from blood

Lenetix

Cell-free nucleic acids from blood

Ravgen

Cell free nucleic acids from blood

Zoragen

Cell free nucleic acids

Detection of unpaired nucleic acids for prenatal diagnostics Isolation of cells partly based on cell sorting technology in licensed from Genoptix.

Celuda

Fetal cells from blood

Fall 2009 Ikonisys Fetal cells cervical smear from Digital microscopy imaging technology-using FISH probes on fetal cells isolated from cervical smear. Device to separate fetal cells from 1.5 ml of whole blood

Parsortix

Fetal Cells from blood

SCIENTIFIC EXPLORATION
Within the context of the prenatal genetic testing market, the BNM core technology in its present state is most suited to detecting gross chromosomal aberrations including deletions, duplications, and translocations. The following section presents an overview of high-prevalence chromosomal abnormalities and the diseases associated with them, where BNM technology can potentially be applied. The core BNM protocol involves an initial nickase labelling step that generates a signal which is then read by NanoAnalyzer device. Specifically, Sample DNA is nicked by enzymes (nickases) in a sequence-specific manner, and these nicks are then repaired by incorporating fluorescent nucleotides which can be detected by the NanoAnalyzer. In its current form the technology platform achieves a resolution of 2.5 kilobases between motifs, and can process 10 genomes in 30 minutes. The nicklabeling scheme provides a very specific readout due to the requirement of two enzymatic reactions (DNA nicking by nickase, and fluorescent nucleotide incorporation by polymerase) for a signal output.

Overview of chromosomal abnormalities

Down Syndrome
Down syndrome is a chromosomal abnormality characterized by the presence of an extra copy of genetic material on the 21st chromosome (Trisomy 21). Trisomy 21 is usually caused by nondisjunction in the gametes prior to conception, and all cells in the body are affected. However, when some of the cells in the body are normal and other cells have trisomy 21, it is called mosaic Down syndrome. The effects of the extra copy vary greatly among people, depending on the extent of the extra copy, genetic history, and pure chance. The incidence of Down syndrome is estimated at one per 800 to one per 1000 births. In 2006, the Centers for Disease Control and Prevention estimated the rate as one per 733 live births in the United States (5429 new cases per year). Approximately 95% of these are trisomy 21. Down syndrome occurs in all ethnic groups and among all economic classes.

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Sex chromosome aneuploidies

Chromosome 23 is susceptible to a variety of aneuploidies resulting from nondisjunction during meiosis or mitosis. The most common sex chromosome aneuploidies are Turner's Syndrome (XO), Kleinfleter's (XXY), XYY, and XXX with a prevalence of ~1/1000 live births, comparable to Down's Syndrome. Pre-natal screening for sex chromosome aneuploidies is identical to Down's Syndrome (i.e. fetal cell extraction via amniocentesis or CVS followed by karyotyping or FISH).

Neural tube defects (NTDs)

Neural Tube Defects (NTDs) are caused by the neural tube not fully closing in development. The neural tube forms 28 days after conception, so this is the earliest that this problem can be identified. There are many different manifestations, depending on the manner in which the tube does not close. One out of every 1000 babies have a neural tube defect. However, many manifestations of NTDs cause the baby to be stillborn. For babies that are born alive, they are critically disabled, and almost always unable to live a normal life. The tests for neural tube defects are identical to those used for prenatal genetic testing (maternal serum alpha feroprotein testing and ultrasound, follow by amniocentesis if it is determined there is a high probably of a defect). NTDs are caused by a mixture of environmental and genetic factors. High folic acid intake by a mother can decrease the likeliness of a defect, while maternal insulin dependent diabetes and the use of anti-seizure medication increases the chances of a defect. The genetic impact can be seen because normally there is only a 0.1% chance of a defect in a first child, but the chances increase to 2-5% if a couple's first child had a defect. NTDs are common affects of Trisomy 13, Trisomy 18, Mecket-Groubert Syndrome, and more. Duke University is currently doing SNP research, genome wide, to determine which genes are involved in NTDs. Duke University currently doing SNP research, genome wide, to determine which genes are involved.

Cardiac chromosomal abnormalities

The congenital cardiac diseases include congenital restrictive cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy. If the disease has manifested sufficiently in a patient, it can be screened for with a thorough physical exam or an echocardiogram, which costs approximately $130 for a hospital to perform, and diagnosed with echocardiography. Long QT syndrome may also be congenital, in which case it is a congenital cardiac channelopathy. All of these diseases are very rare, and represented by a wide range of genetic mutations, predominantly point mutations. Additionally, these diseases have variable penetrance so that some people may have one of these diseases but never have clinical manifestations, while other individuals may be more severely affected. Congenital restrictive cardiomyopathy is by far the least common of these diseases. The disease itself represents an inability of the heart to stretch out during its relaxation phase (diastole). It is diagnosed by echocardiogram. 9

Fall 2009 Congenital hypertrophic cardiomyopathy is a disease in which the ventricular walls are thickened, leading to mitral prolapase, poor cardiac output, and potentially fatal arrhythmias. This is the most common cause of sudden death of young athletes who die during an athletic event (e.g. young basketball star who suddenly dies in the middle of a game). A physician may first suspect the disease during the physical exam, and may confirm the diagnosis with an ECG and echocardiogram. There are individual point mutations which can determine whether the disease phenotype is mild or carries a high chance of sudden death. Early diagnosis allows physicians to recommend that the affected individual avoid potentially dangerous activities. A list of the known genetic mutations can be found at (11). Congenital dilated cardiomyopathy is a disease in which all four chambers of the heart are expanded and have thin walls. This may also be related to neuromuscular disorders, mitochondrial disorders, carnitine deficiency, Barth syndrome, or Naxos disease. This is normally diagnosed by ultrasound after detected on physical exam by a physician. Long QT syndrome is an electrical conduction disorder of the heart which makes individuals susceptible to Torsade de Pointes when placed on certain drugs. This is generally diagnosed by ECG.

Deletions
The most common deletions are DiGeorge (22q), Smith-Magenis (17p), WolfHirschorn (4p), Angelmann (15q), Prader-Willi (15q), Williams (7q), Miller-Dieker (17p), Cri-du-chat (5p), Rubenstein-Taybi (16p or 16q). Generally these deletions manifest early on in child development as cognitive delays and in some specific instances can be correlated with e.g. cardiac abnormalities as in the case of DiGeorge's. Currently, these deletions are not screened for pre-natally as their prevalence is low. With the exception of DiGeorge (~1/4000 live births) the deletions are in the < 1/20,000 prevalence range.

Potential application areas

The tables below list the diseases (classified based on the size of structural aberration) and their corresponding genetic abnormality, prevalence and the potential of BNMs technology to diagnose the condition. To show proof of principle, only the recognition sequence of nickase BtsI1 was tested. As seen from the following tables, the technology in its current form, is sufficient to diagnose diseases arising from chromosomal duplications to small structural abnormality at the gene level.

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Table 3. Disorders associated with aneuploidy

Disease Down syndrome Klinefelter's syndrome XYY syndrome Triple X syndrome Turner syndrome Edwards syndrome Patau syndrome Tetrasomy Abnormality Trisomy 21 XXY XYY XXX XO Trisomy 18 Trisomy 13 XXXX, XXXY Prevalence 1 in 800 1 in 1000 1 in 1000 1 in 1000 1 in 2500 1 in 3000 1 in 25000 Extremely rare

Table 4. Disorders associated with large chromosomal deletions

Diseases DiGeorge syndrome Williams syndrome Prader-Willi syndrome Smith Magenis syndrome Cri du chat syndrome Wolf-Hirschhorn syndrome Rubenstein-Taybi syndrome Langer-Giedion syndrome Abnormality 22q11.2 7q11.23 15q11-13 17p11.2 5p15.2 4p16.3 Prevalence 1 in 4000 1 in 10000 1 in 15000 1 in 25000 1 in 30000 1 in 50000

16q13 8q23.2 to 8q24.1

1 in 125000 Extremely rare

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Table 5. Disorders associated with gene deletions

Diseases Fragile X syndrome Angelman syndrome Alagille syndrome Miller-Dieker syndrome Abnormality FMR1 gene (Xq28) UBE3A gene (15q11-13) JAG1 gene (20p12.2) Prevalence 1 in 5000 1 in 15000 1 in 100000

LIS1 gene (17p13.3)

Extremely rare

Diseases and their corresponding genetic loci

1. Down syndrome Trisomy 21 Also known as Trisomy 21, a condition in which one has an extra 21 chromosome .

2. Klinefelter's syndrome XXY A chromosomal abnormality in males due to the presence of an extra X chromosome.

3. XYY syndrome XYY It is an aneuploidy in which males have an extra Y chromosome.

4. Triple X syndrome XXX Also known as Trisomy X, a condition in which females have an extra X chromosome.

5. Turner syndrome XO Turner syndrome results from the absence of part or all of the X chromosome.

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6. Edwards syndrome Trisomy 18 Also known as Trisomy 18, a condition in which one has an extra chromosome 18.

7. Patau syndrome Trisomy 13 Also known as Trisomy 13, a condition in which one has an extra chromosome 13.

8. Tetrasomy XXXX, XXXY Aneuploidies arising from tetrasomy in sex chromosomes.

9. DiGeorge Syndrome Deletion 22q11.2 A genetic disorder that arises from the deletion of q11.2 region in chromosome 22, resulting in congenital heart disease and neuromuscular problems.

10. Williams Syndrome Deletion 7q11.23 Williams syndrome is a neuro-developmental disorder caused by the deletion of q11.23 in the long arm on chromosome 7.

11. Prader-Willi syndrome Deletion 15q11-13

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Fall 2009 Prader-Willi syndrome is a very rare chromosomal disorder that causes several growth defects such as low muscle tone, short stature and mild mental retardation. It is often misdiagnosed as Down syndrome.

12. Smith Magenis Syndrome Deletion 17p11.2 Smith-Magenis Syndrome (SMS) is a genetic disorder that impairs development resulting in mental retardation, sleep disturbances and distinctive facial features.

13. Cri du chat Loss of a small region in band 5p15.2 Cri du chat syndrome arises from the deletion of region p15.2 in chromosome 5 that results in impaired cerebral development.

14. Wolf-Hirschhorn Deletion 4p16.3 Wolf-Hirschorn syndrome is caused by the deletion of region p16.3 in chromosome 4 resulting in mental retardation, microcephaly and poor muscle tone.

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15. Rubinstein-Taybi Syndrome Deletion 16p13 Rubinstein-Taybi Syndrome is a genetic disorder that results in short stature, learning difficulties and skeletal abnormalities.

16. Langer-Giedion syndrome Deletion 8q23.2 to q24.1 Occurs from the loss of region q23.2-24.1 in chromosome 8 and results in learning disabilities and impaired skeletal development.

17. Fragile X syndrome FMR1 gene (Xq28) Fragile X syndrome is a genetic disorder arising from the expansion of single trinucleotide (CGG) sequence on the X chromosome that results in a failure to express the FMR1 gene required for normal neural development.

18. Angelman syndrome UBE3A gene (15q11-13) A genetic disorder that arises from the deletion of q11-13 region of chromosome 15 that causes an absence in UBE3A gene expression required for normal brain development. 15

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19. Alagille syndrome JAG1 gene (20p12.2) A genetic disorder that arises from the microdeletion of 20p12 chromosome corresponding to the JAG1 gene required for normal embryonic development.

20. Miller-Dieker syndrome Deletion of part of 17p (which includes both the LIS1 and 14-3-3 epsilon gene). Miller-Dieker syndrome is a developmental defect caused by incomplete neuronal migration.

Detailed information for the above mentioned 20 diseases are available through the following references. The reference numbers are matched up to the disease numbers.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. http://emedicine.medscape.com/article/943216-overview http://emedicine.medscape.com/article/945649-overview http://ghr.nlm.nih.gov/condition=47xyysyndrome http://ghr.nlm.nih.gov/condition=triplexsyndrome http://emedicine.medscape.com/article/949681-overview http://emedicine.medscape.com/article/943463-overview http://emedicine.medscape.com/article/947706-overview http://en.wikipedia.org/wiki/Tetrasomy http://emedicine.medscape.com/article/886526-overview http://emedicine.medscape.com/article/893149-overview

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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. http://emedicine.medscape.com/article/947954-overview http://ghr.nlm.nih.gov/condition=smithmagenissyndrome http://emedicine.medscape.com/article/942897-overview http://emedicine.medscape.com/article/950480-overview http://emedicine.medscape.com/article/948453-overview http://ghr.nlm.nih.gov/condition=langergiedionsyndrome http://emedicine.medscape.com/article/943776-overview http://ghr.nlm.nih.gov/condition=angelmansyndrome http://ghr.nlm.nih.gov/condition=alagillesyndrome http://ghr.nlm.nih.gov/condition=millerdiekersyndrome

INTELLECTUAL PROPERTY
Potential issues
There are three potential IP issues that need to be examined. They are the method being used, the materials being used, and the particular DNA sequences being identified. The first two issues are not of particular concern. The method being used is a unique process created by Bionanomatrix. The materials being used, such as nickases, are also not of concern. While these materials potentially are patented, as they are purchased this satisfies the patent concerns. The primary IP issue pertains to the specific DNA sequences and aberrations being detected and identified. DNA sequences can be patented. The rationale behind this is that the normal method of patenting methods is not sufficient with DNA. This is because there are numerous methods of detecting the presence of genes. Suppose if patents for DNA sequences did not exist, and in this setting, Company A identifies the link between a gene and a disease. If Company A is only able to patent their method for detecting for the presence of the gene, Company B could develop a new method, and take control of the market. The idea behind allowing for DNA sequences to be patented is that it will provide incentive to discover the links between genetics and diseases. However, this incentive comes at a price as it discourages research into additional effects of patented genes. To combat this reaction, royalties associated with DNA sequence patents are kept low. This therefore provides incentive to initial research, while not making future research prohibitively expensive. The genetic diseases being screened for all involve specific DNA sequences that have been linked to a disease. Therefore, it is likely that all of these sequences have been patented. However, as is evidenced by the fact that there is currently a large market for these tests, the royalty cost is not prohibitively expensive. There are two possible explanations for this. First, for genetic links that have been known for a while, the patents may have expired. This is likely true for diseases which the

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Fall 2009 genetic markers have been know for a long period of time, such as is true for Downs Syndrome. Second, for genetic links that have been discovered more recently and are currently tested for, the royalties must be reasonable. There are two potential areas of concern. One is diseases that are not currently tested for that are still patented. As Bionanomatrixs technology will be able to test for a much larger number of diseases than previous tests, it is possible that some of these new sequences have high royalties. However, this is unlikely because of the practice of keeping royalties on DNA sequences low. The second potential problem is adding up royalties. Although Bionanomatrix may be able to test for an unlimited number of diseases at one time, if too many are still patented, this cost may add up. This may provide a limit as to the number of diseases able to be tested for at one time. However, as SNP Chip and CGH test for a similarly large number of diseases, it is unlikely this will be a major problem. For an additional analysis for this problem, please consult (12). In conclusion, although there are potential IP obstacles, they are the same problems competitors currently in the market face. If Bionanomatrix is going to be testing for the same diseases as its competitors do, then patents of DNA sequences will not be an issue. If Bionanomatrix is planning on testing for different DNA sequences, then it will be necessary to determine if that sequence is covered by a patent, and the cost of the royalty if a patent does exist. If a patent does exist, it will then have to be determined in light of the royalty if it is still profitable to test for this additional DNA sequence.

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MARKET ANALYSIS
Global market for molecular diagnostics
The most common assays in the overall molecular diagnostics market are those for genetic/chromosomal tests, including various cancer-related tests, karyotyping, genetic disease carrier testing, prenatal diagnosis, sex determination, and susceptibility testing. This currently accounts for about 60% of all molecular diagnostic testing. The remainder of the total molecular diagnostics arena includes infectious disease testing with 36.3%, and other applications like identity/blood bank screening.

Table 6. Distribution of molecular diagnostics testing market in 2009 (Source BCC).

Application Infectious disease diagnostics Percent 36.3

Oncology diagnostics

31.8

Prenatal and newborn screening Identity/Blood bank/other

25.6 6.3

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Global gene/chromosome diagnostics market

In 2008, the global gene chromosome (DNA) diagnostics market was valued at approximately $10.6 billion. The total market is projected to grow at a compound annual growth rate (CAGR) of 13.7% to reach $20.1 billion by 2013. Within the global DNA diagnostics market, the market for PCR-based diagnostic assays claimed the largest share in 2008 amounting to an estimated $5.39 billion. The PCR diagnostics market is projected to grow at a compound annual growth rate (CAGR) of 11.9% to reach nearly $9.5 billion by 2013. The market for microarray diagnostic assays was valued at $2.4 billion in 2008 and is projected to grow at a compound annual growth rate (CAGR) of 13.8% to reach $4.6 billion by 2013. The market for in situ hybridization diagnostic assays was valued at $1.9 billion in 2008, and is projected to increase at a compound annual growth rate (CAGR) of 15.3% to reach $3.9 billion by 2013.

Table 7. Global market for gene/chromosome diagnostics by segment ($ Millions, Source BCC)

PCR-based assays DNA microarrays FISH diagnostics Other

5,389 2,396 1,890 945

Global market for DNA microarrays

The global market for DNA Microarray or clinical diagnostics was $2.4 billion in 2009 and estimated to grow at a rate of 13.8%. The global biochip market by end use is shown in the table below (Table 8). Drug discovery and development and diagnostics are large consumers of biochips. Whole genome association studies and next-generation DNA sequencing market segments are important drivers of future growth in these markets. Diagnostics applications for biochips are rapidly expanding, and this market segment is expected to experience high future growth. By 2014, drug discovery and development, and diagnostics applications are expected to represent more than 71% of the overall biochip market, emphasizing the future importance of these devices to the medical and pharmaceutical industries.

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Table 8. Demand for DNA microarray by end use ($milions).

Drug Discovery and Development Diagnostics Research Tool Others (Bio-defense, forensics)

781.2 627.3 614.3 372.7

Forces driving growth in the DNA microarray market

The main factors for high growth in the DNA microarray industry are listed in the table below. The drug discovery and diagnostics sectors are important users of biochips and will have significant effects on the future growth of the biochip business. The main driving forces for the favorable growth projections in DNA microarray market include the following: Growth in life-science R&D fundingsince the beginning of the Human Genome Project, funding for life science R&D has remained at a high level. The 2009 Stimulus package passed by the Obama Administration provided $8.5 billion in stimulus spending for NIH, with a high proportion of these funds going toward genetic analysis. Additional funds from the stimulus package will be spent over fiscal years 2009 and 2010. Research in genomics and proteomics are driving the need for high multiplex, high- throughput DNA microarray technologies.

Market distribution for molecular diagnostics for inherited genetic disease testing
The four basic applications of molecular diagnostics for inherited genetic disease testing are heterozygote screening, presymptomatic genetic screening (genetic susceptibility testing), prenatal diagnosis, and newborn screening. Exhibits 2 and 3 present lists of key pathologies that result from genetic and chromosomal mutations and for which molecular testing is of value in their analyses.

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Table 9. Market distribution for molecular diagnostics for inherited genetic disease testing in the US, ($ millions)

Prenatal Newborn Hetrozygote Presymptomatic Total

240 53.7 15.8 6.32 315.8

Table 10. Market driving forces

Market Driving Force Significance for Biochip Demand

Growth in life science R&D funding

Drives demand for biochips in research tools, drug discovery and development

Growth in large-scale biochip, genomic and proteomic initiatives Need for more efficient drug discovery and development Development of personalized medicines Need for early cancer detection Aging populations in U.S., Europe, and Japan New discoveries in genomics and proteomics Disposability, small sample sizes Rapid decline in genetic analysis costs

Standardizes biochip products across platforms and provides high visibility Testing sites for biochip products Creates incentive for novel biochips technologies that provide high throughput and multiplex capability at lower prices Creates strong demand for pharmacogenetic (PGx) tests that use biochips

Drives biomarker discovery and diagnostic applications for biochips

Increases demand for MDx tests in cancer and other diseases

Increases growth opportunities in novel biochip products

Drives growth for miniaturized biochips in diagnostics market segment

Enables widespread adoption of biochips by research and drug discovery markets

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Discovery of more gene and/or SNP- disease associations Drives demand for biochip consumables and instruments in research and diagnostics applications

Competitive Analysis
Currently the standard technologies used for pre-natal diagnostics are karyotyping and FISH. For post-natal screening, the current dominant technologies are arrayCGH and SNP chips. To determine the practical details of the currently employed technologies as well as to gain insights into emerging market trends, we have conducted extensive literature and primary surveys of technicians and clinicians at CHOP (Childrens Hospital of Philadelphia), HUP (Hosptial of the University of Pennsylvania), Johns Hopkins Hospital. The results are discussed below and summarized in the Table 11.

Cost per use (reagents, labor/technical staff requirements, time additional per run)
The cost of the molecular markers used in FISH is ~ $20 - $30 per marker [primary survey of HUP clinician]. Hybridization requires overnight incubation of the cells with the molecular markers and quantification of the results is done manually, with the goal of observing ~ 20 labelled cells out of ~200 cells in a typical amniotic fluid extraction sample. The cost of Illumina's 610 beadchip analysis is $400/ sample, as determined via a consensus survey of online academic sequencing centers. The costs of other comparable chip-based platforms such as those of Affymetrix and Agilent can also be found at (13). The total time for a run on the Illumina platform is ~ 6 days with a breakdown of 1-2 days to isolate DNA from blood and 3-4 days of runtime on the beadchip [primary survey of CHOP technician].

Advantages, limitations, efforts to improve that currently used technology

Whereas the accuracy of karyotyping is quite high (> 99%) a fact which has no doubt contributed to its longevity as the "gold standard" in cytogenetic screening of large scale chromosomal aberrations, the accuracy of FISH is relatively poorer (~ 70%) (13). This is due in large part to human error, as nearly overlapping fluorescent spots must be discriminated manually under a microscope in typical FISH protocols [primary survey]. This relatively large error rate could be the basis of a competitive advantage for BNM's technology platform over FISH. FISH is a highly accurate test for detecting the standard 22q11.2 deletion when a couple has had a previous child with the deletion or a parent has the deletion.

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Fall 2009 However, DiGeorges (DGS) is a heterogeneous disorder and 1015% of individuals with the characteristic features do not have the standard 22q11.2 deletion (14) . These atypical deletions of 22q11 have not been detected by FISH using commercially available probes. Therefore, FISH has an estimated false negative rate of 10-15% for DGS. For a pre-implantation genetic screen (PGS) done prior to IVF, it was found that some abnormalities could not be detected by the seven-probe panel (13, 16, 18, 21, 22, X and Y) used in fluorescence in-situ hybridization that were detected with CGH. This data was generated from patients with reoccurring IVF failure due to aneuploidy. FISH has been used with probes for up to 15 chromosomes. The primary advantage of BNM technology over karyotyping is speed. Karyotyping requires cells to be in metaphase, and this fact requires the amniotic sample to be cultured for 1 week in order to achieve a sufficient yield of ~ 20-50 metaphasic cells for fixing and displaying analysis. In principle, BNM technology which requires only ~ 10-100 cells worth of DNA, would obviate the need for culturing. As an indicator of current trends in post-natal genetic screening, Children's Hospital of Philadelphia (CHOP) uses SNP chips almost exclusively to screen for developmental disorders in infants [primary research]. They perform the testing in-house and utilize the Illumina 610 quad bead chip. The Illumina 610 quad beadchip is capable of producing genotype calls in over 590,000 SNPs, an average genomic marker spacing of 1 SNP per 6 kbp. The average call rate, the proportion of all SNPs called across each sample, for Illumina's 610 quad beadchip platform is > 99% [primary survey of CHOP technician and (15)]. The false positive/false negative rates are dependent on the specific characteristics of the genetic disorder and are therefore not quoted as an intrinsic spec in the platform's technical performance literature. However, interviews with a CHOP technician indicated that accuracy is not an issue of concern in analyses carried out on the Illumina platform. The potential advantages of BNM's platform over chip-based technologies such as Illumina 610 platform are speed, and sensitivity to certain types of mutations such as balanced translocations and insertions of tandem repeats. Illumina 610 requires 3-4 days of runtime per sample [primary survey CHOP technician], whereas BNM is potentially shorter. Detection of balanced translocations and tandem repeats is not straightforward using SNP, FISH, or array-CGH, whereas it is an intrinsic strength of BNM's platform. For the Illumina 610 platform used at CHOP, the analysis is done on DNA isolated from blood. Typically, 1-2 mL of blood is extracted, yielding ~ 1 million white blood cells from which DNA is extracted using Qiagen's PureGene kit. This step takes about a day of technician labor [primary survey of CHOP technician]. Typically, array-CGH procedures use between 50,000 to 500,000 cells of specimen DNA in the labeling reaction (16) . If BMN's application can perform analysis on

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Fall 2009 smaller samples, than it will have a sizable advantage over array CGH applications, which are often limited by the amount of specimen DNA. This advantage can be realized in a faster turnaround time and lower cost, as other competing applications require cell culturing and expansion when the sample size too small. BNM's application may also be better equipped to detect and quantify copy-number changes than array CGH platforms. It has been found for array CGH that the signal intensity element is affected by a number of factors, including base composition, proportion of repetitive sequence content and amount of hybridizable DNA in the array element (16) . This has resulted in intensities varying by a factor of 30 or more with no change in copy-number. This feature may create opportunity for BNM's technology to fulfill unmet detection needs in diseases copy number alterations.

Table 11. Summary Table of Competitive Technologies

Sample Requirements Time Requirements Total time (days)
a

Cost Breakdown

Consumer Cost Labor

Accuracy

type

amount

hands on (hours) 6
a

Equipment

Reagents

Karyotype

Fetal cells

20-50 cells (metaphase)

7 -10

Standard

~$200

$700

~ 99%

FISH

Fetal cells

20-50 cells

1-2

Standard

$30/probe

~$200

$1000 [5 a diseases]

~ 70% (13)

(interphase) ArrayCGH Blood (WBC) 50,000500,000 cells (16) 200 ng b DNA 3-4 4 Standard, Reader, chip
a,b

Label Kit

~$120

SNP

Blood (WBC)

3-4

Standard, Reader, chip

Label Kit

~$120

$372

> 99% (15)

a.b

Sources: a: Primary Research b: http://www.solexa.co.uk/downloads/Illumina2008ProductGuideUpdate.pdf c: http://www.broadinstitute.org/gen_analysis/genotyping/pricing.html

Risks of addressing this market at this time.

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Fall 2009 Using the microarray technology market as an indicator, there has been slowerthan-expected growth with new applications using unconventional approaches. This market resistance to new technologies may be a barrier to BNM technology. It is expected that the research markets will grow much faster than clinical applications (17).

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Fall 2009

CANCER GENETIC TESTING

Background
Cancer genetic testing represents a potentially significant market opportunity for Bionanomatrix technology. The various types of cancers are collectively of enormous public health significance. In 2002, approximately 1.3 million new cases of cancer were diagnosed in the United States, and about 0.6 million deaths were attributed to cancer, making it the second leading cause of death (18). Currently, genetic tests are used in the diagnosis and management of only a small number of cancers. This is primarily due to the fact that the molecular mechanisms of most cancers are not known, or do not cluster consistently into a small number of categories, and due to the difficulty in obtaining high-resolution genetic information from patient DNA. However, progress in both areas is continually increasing the scope of genetic testing in the diagnosis and management of cancer, as is already the case in some cancers such as leukemia, where genetic testing constitutes an integral part of the treatment and management program (19). Patients for whom cancer genetic testing is performed can be roughly divided into two main categories: patients actively manifesting symptoms, and patients not manifesting symptoms but considered to be at risk due to family history. Genetic tests based on Bionanomatrix technology can potentially cater to both groups.

All cancers
Since all cancerous cells share some general features, certain genetic tests can be of use in all cancers. Aneuploidy in cells (i.e. cells having an abnormal number of chromosomes) is often indicative of cancer, and is almost always observed in all cancers (20). Furthermore, in some cancers such as ovarian cancer, there is a strong association between the extent of aneuploidy and the progress of the disease, and aneuploidy tests are an integral component of the information considered to assess a patients prognosis, and are also being used in therapy planning (21). Since fast, inexpensive, and sensitive DNA aneuploidy tests based on staining and flow cytometry already exist, it would be hard for Bionanomatrix technology to compete in this space, unless it is offered in a basket of multiple tests.

Leukemia
Leukemia is the term used for a group of cancers of the blood and bone marrow which are typically characterized by abnormally high levels of leukocyte

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Fall 2009 proliferation. In 2002 there were approximately 31,000 new cases of leukemia, and 22,000 deaths attributed to leukemia, in the United States. Acute myeloid leukemia, or AML, is a leukemia subtype in which abnormal proliferation of leukocytes in the bone marrow hinders hematopoiesis, the production of normal blood cells; in 2002, approximately 11,000 new cases of AML were diagnosed, and approximately 7,000 deaths were attributed to AML. AML is perhaps one of the best studied cancers, and genetic lesions associated with AML usually involve large-scale translocations or inversions. Genetic testing plays an integral role in the contemporary management of AML; the National Comprehensive Cancer Network considers cytogenetics to be the single most important prognostic factor for predicting remission rate, relapse, and overall survival. (19) Currently, genetic testing is being used to diagnose AML, to classify patients into AML subtypes for purposes of targeted treatment, and assess prognosis (survival and relapse rates can vary dramatically depending on subtype), to predict how a patient with a particular genetic lesion will respond to a specific treatment, and also to monitor the status of the disease (active versus latent). These tests have for some time been performed with genome scale low-resolution assays such as karyotyping and FISH. However, since approximately half of adult AML patients have a normal karyotype, high-resolution assays such as RT-PCR, expression profiling via microarrays, and sequencing are becoming increasingly popular. A recent review (22) highlights the added benefits of obtaining high-resolution genetic information from AML patients, and outlines a protocol in which increasingly higher resolution assays are used to place patients into specific disease subtypes; the review also states the future prospect of monitoring the state of the disease at a very high resolution using arrays, sequencing, etc. Since prohibitively high costs and sample requirements (number of cells, extraction, etc) are considered to be the primary barriers to adoption of high-resolution assays, this can be a potential market opportunity for Bionanomatrix technology, especially if it can be used to perform these analyses at significantly lower costs than competing technologies such as SNP arrays.

Colorectal cancer
Colorectal cancers constitute the third-leading cause of cancer-related deaths in the United States; in 2002, approximately 150,000 patients were diagnosed, and 50,000 deaths were attributed to colorectal cancers (18). Patients diagnosed with colorectal cancer generally fall into two categories, those with strong family history of colorectal cancer comprise about 25% of the population, and the rest whose disease is considered to be sporadic. Colorectal cancer generally begins with the inactivation of the APC tumorsuppressor gene (adenomatous polyposis coli, which has been detected to be mutated

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Fall 2009 in 75% of sporadic colorectal cancers) as the initial neoplastic event, and progresses further with subsequent mutations in the oncogene KRAS, and tumor suppressor genes DCC and TP53. The genomic instability associated with colorectal cancer can be divided into two categories: microsatellite instability, which is most commonly associated with hereditary colorectal cancer, and chromosomal instability, which is most commonly associated with sporadic colorectal cancer. Microsatellites are repetitive sequences of DNA spread throughout the genome. Microsatellites can cause additions or deletions to be made during the copying process because the DNA polymerase can slip on repetitive sequences. These errors are usually corrected by the proteins involved in the mismatch repair system, but can accumulate and cause mutations in coding and non-coding regions if mismatch repair activity is reduced. Inactivation of one or more of the following genes has been observed to cause microsatellite instability in colorectal cancer: MLH1, MSH2, PMS1, PMS2, MSH6, EXO1 and MLH3 with the majority of sporadic colorectal tumors with microsatellite instability showing mutations in MLH1 (90%) or MSH2 (5%). Chromosome instability refers to the loss or gain of whole chromosomes (aneuploidy), or the loss of one of the parental alleles (loss of heterozygosity or LOH). Bionanomatrix has potential market opportunity in both familial and sporadic colorectal cancers. Individuals carrying a mutation in one of the aforementioned mismatch repair genes have a lifetime risk of developing colorectal cancer that is as high as 80%. Genetic tests usually involve the use of microsatellite instability analysis, and sequencing of specific regions to screen for mutations in mismatch repair genes. However, since these tests are expensive and time-consuming, they are offered only to candidates considered to be at high risk. Furthermore, this panel of genetic tests (detailed in (23)) fails to identify a significant fraction of genetic lesions that may eventually cause colorectal cancer in patients. The ability to interrogate the patients entire genome for microsatellite instability in a costeffective manner can significantly improve the quality of care. A market opportunity exists if Bionanomatrix technology can be utilized to offer existing genetic tests at significantly lower costs. Furthermore, if Bionanomatrix technology can be harnessed to detect microsatellite instability throughout the patients full genome, it can potentially improve the quality of care offered to patients. Microsatellite instability is not specific to colorectal cancer, and is generally considered a strong indicator of genomic instability, which in turn is a reliable predictor of neoplastic progression. Hence, fast and inexpensive microsatellite instability assays could be commercially viable in a number of different categories.

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Fall 2009

Breast cancer
Breast cancer is the most frequently occurring cancer in women, with approximately 200,000 new cases, and 40,000 deaths attributed to it in 2002 (18). Despite its size, is not one Bionanomatrix technology can compete in, for the following reasons. First, breast cancer diagnostic testing is primarily done by non-genetic methods. A very small percentage of the population (less than 1%) harbors mutations in the BRCA1 or BRCA2 genes which significantly increase the likelihood of developing breast cancer. However, these mutations are at the nucleotide level, and thus cannot be resolved with Bionanomatrix technology; furthermore, inexpensive tests based on PCR and sequencing already exist for this market. Recent research has identified a number of rearrangements in the BRCA genes that are indicative of an increased risk of developing breast cancer. Hence, some have proposed the inclusion of tests of rearrangements in these genes as part of the diagnostic test panel. This may be a future market opportunity for Bionanomatrix technology.

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Fall 2009

Table 12. Currently available cancer genetic tests.

The following table contains information from a cancer test database published in a report commissioned by the Department of Health and Human Services. Only genetic tests for which Bionanomatrix technology in its current form can be employed are listed. This filters out the majority of entries since most cancer tests involve antibodies to detect specific molecular markers, and also since a large number of genetic tests involve sequence-level resolution.

Recurrence

Prognostic

Prevention Secondary Prevention

AML1/ETO translocation

t(8;21)

x x x x x x

Monitoring

Diagnostic

Name

Other Names
Primary

Specimen

blood, marrow blood, marrow, tissue

B-cell rearrangement

gene

BCL-1/JH rearrangement

gene

t(11;14)

x x

blood, marrow, tissue blood, marrow, tissue blood, marrow blood Blood, tissue Blood Blood

BCL-2 translocation

t(14;18)

x x x

BCR/ABL rearrangement BRCA Analysis

gene

Philadelphia chromosome

x x x x x x x x x x x x

BRCA1, BRCA2 18q/RER, DCC MLH1, MSH2 APC, FAP

Chromosome 18q assay Colaris Colaris AP FLT 3 mutation HER-2/neu

Blood Tissue

c-erbB-2, PathVysion, Herceptin eligibility

x x x

IgVH mutation analysis

Blood, marrow

Microsatellite instability MLH1, MSH2, mutations MSH6

MSI, BAT-26, RER+, HNPCC HNPCC mismatch repair gene

x x

x x

31 Blood, tissue
Blood

Fall 2009
IgVH mutation analysis

x
MSI, BAT-26, RER+, HNPCC HNPCC mismatch repair gene

Blood, marrow Blood, tissue Blood

Microsatellite instability MLH1, MSH2, mutations Oncotype Dx p53 tumor gene suppressor MSH6

x x

x x

Breast cancer assay p53

x x x x x x x x x x x

Tissue Tissue

PML/RARA translocation

t(15;17)

Blood, marrow Stool Stool Blood, marrow, tissue

PreGen-26 PreGen-Plus T-cell receptor rearrangement gene

MSI, BAT-26 k-ras, APC, p53

TEL/AML1 gene fusion

t(12;21)

x x

Blood, marrow

Genetic Tests for Cancer. Technology Assessment. January 2006. Rockville, MD: Agency for Healthcare Research and Quality. http://www.ahrq.gov/clinic/ta/gentests/.

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Fall 2009

BIBLIOGRAPHY
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Fall 2009 13. Interphase FISH for Prenatal Diagnosis of Common Aneuploidies in Methods in Molecular Biology. Feldman, B., Aviram-Goldring, A. and Evans, M.I. s.l. : Molecular Cytogenetics: Protocols and Applications, Humana Press., 2002, Vol. 204. 14. Prenatal diagnosis of the 22q11.2 deletion syndrome. Driscoll, Deborah. 1, s.l. : Genetics in medicine, 2001, Vol. 3. 15. Cost, risk and difficulty of the DNA isolation requirement. Illumina. [Online] http://www.illumina.com/Documents/products/datasheets/datasheet_infiniumhd.pdf. 16. Array comparative genomic hybridization and its applications in cance. Pinkel, D and Albertson, D. s.l. : Nat. gen, 2005, Vol. 37. 17. Analytical Chip Technology Markets. Kalorama. s.l. : Kalorama, 2007. 18. Cancer Statistics. Jemal, Ahmedin, et al. s.l. : CA Cancer J Clin, 2002, Vol. 52. 19. Genetic Tests To Evaluate Prognosis and Predict Therapeutic Response in Acute Myeloid Leukemia. Gulley, M, Shea, TC and Fedoriw, Y. s.l. : J. Mol. Diagn, 2009. 20. Aneuploidy and cancer. Rajagopalan, H and Lengauer, C. 7-15, s.l. : Nature, 2004. 21. Relevance of DNA-ploidy as a prognostic instrument for solid tumors. Silvestrini, R. s.l. : Ann Oncol, Vol. 11. 22. Genetic Tests To Evaluate Prognosis and Predict Therapeutic Response in Acute Myeloid Leukemia. Gulley, M, Shea, T and Fedoriw, Y. s.l. : J. Mol. Diagn., 2009. 23. Colorectal Cancer Screening and Surveillance, January 2006. Harford, W. 1, s.l. : Surgical Oncology Clinics of North America, Vol. 15.

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