(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2006/0216714 A1
`(43) Pub. Date:
`Sep. 28, 2006
`Kanaoka
`
`US 20060216714A1
`
`(54) METHOD OF DETECTING COLON CANCER
`MARKER
`
`(75) Inventor: Shigeru Kanaoka, Shizuoka (JP)
`Correspondence Address:
`FRISHAUF, HOLTZ, GOODMAN & CHICK,
`PC
`220 Fifth Avenue
`16TH Floor
`NEW YORK, NY 10001-7708 (US)
`(73) Assignee: Hamamatsu Foundation for Science
`and Technology Pr, Shizuoka (JP)
`(21) Appl. No.:
`10/549,389
`
`(22) PCT Filed:
`Sep. 19, 2003
`(86). PCT No.:
`PCT/UP03/11972
`(30)
`Foreign Application Priority Data
`
`Mar. 19, 2003 (JP)......................................... 2003-75552
`
`Publication Classification
`
`
`
`(51) Int. Cl.
`(2006.01)
`CI2O I/68
`(2006.01)
`CI2P 19/34
`(2006.01)
`CI2N I/08
`(52) U.S. Cl. .............................. 435/6: 435/91.2: 435/270
`(57)
`ABSTRACT
`It is intended to provide a non-invasive and convenient
`method of detecting a tumor marker for diagnosing colon
`cancer which is Superior in sensitivity and specificity to the
`existing fecal occult blood test. More specifically speaking,
`a method of detecting a tumor marker for diagnosing colon
`cancer which comprises collecting biological sample which
`is immediately frozen using liquid nitrogen in some cases,
`homogenizing the sample in the presence of an inhibitor of
`an RNA digesting enzyme to give a suspension, extracting
`RNA from the obtained suspension, subjecting the extracted
`RNA to reverse transcription to give cDNA, amplifying the
`obtained cDNA and then detecting the thus amplified cDNA.
`This method is characterized by involving no procedure of
`separating cell components from the biological sample.
`
`Geneoscopy Exhibit 1007, Page 1
`
`

`

`Patent Application Publication Sep. 28, 2006
`Fig. 1
`
`US 2006/0216714 A1
`
`< - 28S rRNA
`
`d
`x, Wis
`
`
`
`Geneoscopy Exhibit 1007, Page 2
`
`

`

`US 2006/0216714 A1
`
`Sep. 28, 2006
`
`METHOD OF DETECTING COLON CANCER
`MARKER
`
`FIELD OF THE INVENTION
`0001. The present invention relates to a tumor marker
`detecting method for diagnosing colon cancer comprising a
`process of extracting RNA from a biological sample, char
`acterized by involving no procedure of separating cell
`components from the biological sample.
`
`BACKGROUND OF THE INVENTION
`0002 The deaths by colon cancer are increasing. The
`number of deaths by colon cancer is the fourth large among
`male, and the second large among female deaths in all cancer
`deaths (Statistics of Japanese cancer deaths in 1999).
`According to an estimation of cancer patients in 2015 in
`Japan, number of colon cancer patients is estimated to be the
`first in both male and female. Global measures to counter
`colon cancer including secondary prevention are thus
`required, and mass Screening of cancer may be one of the
`most effective methods.
`0003 For the mass screening of cancer, it is important
`that the detection method is easy and non-invasive. The only
`non-invasive method now available is the method to exam
`ine existence of occult blood in feces, that is, the fecal occult
`blood test, and is used extensively as a standard method of
`the mass screening of colon cancer.
`0004. However, the fecal occult blood test has rather low
`sensitivity and specificity (the sensitivity: 30 to 90%, the
`specificity: 70 to 98%), because appearance of hemoglobin
`in feces is not specific to tumor. Therefore, there is a
`shortcoming that quite a few false negatives and false
`positives exist.
`0005 Also, in the diagnosis of colon cancer, after or in
`parallel with the screening by the immunological fecal
`occult blood test, total colonoscopy or a combination of
`Ba-enema and sigmoidoscopy has been adopted. There is
`thus a shortcoming that it needs much time and effort.
`0006. As alternative methods to the fecal occult blood
`test, methods using DNA are reported, such as detection of
`mutations in K-ras, p-53, or APC genes, or detection of
`microsatellite instability in feces (D. Sidransky, et al., Sci
`ence, 256, Apr. 3, 1992, 102-105: S. M. Dong, et al., Journal
`of the National Cancer Institute, 93 (11), Jun. 11, 2001,
`858-865; G. Traverso, et al., The New England Journal of
`Medicine, 346 (5), Jan. 31, 2002, 311-320; G. Traverso, et
`al., The Lancet, 359, Feb. 2, 2002, 403-404).
`0007. These methods using DNA are non-invasive and
`can capture the direct changes in cancer cells, and have
`characteristics of having high specificity, and so are consid
`ered to be a hopeful method in the future. However, it has a
`demerit that the sensitivity is lower compared to the fecal
`occult blood test, a prior art, and is rather time and effort
`consuming.
`0008 Further, as an alternative method to the fecal occult
`blood test, in order to detect gene expression more directly,
`a method for detecting mRNA of protein kinase C (PKC) or
`the like in the feces has been developed (L. A. Davidson, et
`al., Carcinogenesis, 19(2), 1998, 253-257; R. J. Alexander
`
`and R. F. Raicht, Digestive Diseases and Sciences, 43(12),
`1998, 2652-2658; T. Yamao, et al., Gastroenterology, 114(6),
`1998, 1198-1205).
`0009. However, the method making use of RNA
`described above could not have the sensitivity exceeding
`that of the fecal occult blood test method, because it was
`impossible to extract RNA easily and efficiently from a small
`amount of feces.
`0010. A method to detect RNA qualitatively and quanti
`tatively by combining the PCR method with the reverse
`transcriptase reaction (RT), has been known. This RT-PCR
`method is superior to Northern blot technique in the high
`sensitivity to be able to detect trace molecules, and is more
`advantageous than the in situ hybridization technique in
`speed and easiness of manipulation.
`0011. However, since RNA is more unstable compared
`with DNA and is always subjected to a danger of decom
`position by RNA digesting enzymes (RNases) which are
`ubiquitous in all the biological samples and very stable,
`strict control to avoid contamination of RNases is necessary
`in the RT-PCR method, during and after purification pro
`cesses of RNA.
`0012. Therefore, when RNA is extracted from the feces,
`which is a biologically very crude sample, a process to
`separate the cell fraction in advance has been necessary, to
`exclude effects of RNases.
`0013. Accordingly, it has been considered impossible to
`extract RNA directly from feces containing enormous
`amount of RNases derived from very large amount of
`microorganisms, and, a separation of the cell fraction has
`been considered to be essential for removing at least exog
`enous RNases derived from microorganisms or the like.
`0014 Surprisingly, however, the inventor of the present
`invention found that in Some cases, homogenization of
`frozen biological materials in the presence of RNase inhibi
`tors can resolve the problems described above, and has
`completed the present invention.
`
`SUMMARY OF THE INVENTION
`0015. It is therefore an object of the present invention to
`provide a non-invasive and convenient tumor marker detect
`ing method for diagnosing colon cancer, which is Superior in
`sensitivity and specificity to the existing fecal occult blood
`teSt.
`0016. The present invention is a method to prepare a
`sample for extracting RNA used to detect a tumor marker for
`diagnosing colon cancer, comprising following process;
`0017 a) a process to homogenize the collected biological
`sample in the presence of an RNase inhibitor to prepare a
`Suspension thereof; characterized by involving no procedure
`of separating cell components from the biological sample.
`0018. Here, said collected biological sample is preferably
`frozen.
`0019 Moreover the present invention is the method
`described above, wherein the RNase inhibitor is guanidine
`thiocyanate.
`0020. Also, the present invention is the method described
`above, wherein the biological sample is feces.
`
`Geneoscopy Exhibit 1007, Page 3
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`US 2006/0216714 A1
`
`Sep. 28, 2006
`
`Further, the present invention is a tumor marker
`0021
`detecting method for diagnosing colon cancer, comprising
`the following processes in addition to the process described
`above:
`0022 b) a process to extract RNA from the obtained
`sample for extracting RNA;
`0023 c) a process to reverse transcribe the extracted
`RNA to give cDNA;
`0024 d) a process to amplify the obtained cDNA; and,
`e) a process to detect the amplified cDNA.
`0025)
`0026. The present invention is a tumor marker detecting
`method for diagnosing colon cancer, wherein said tumor
`marker is COX-2.
`0027. The present invention is also a kit for preparing a
`sample to extract RNA used in the tumor marker detecting
`method for diagnosing colon cancer, comprising the follow
`ing means;
`0028 a) a means to homogenize the collected biological
`sample in the presence of an RNase inhibitor, to prepare a
`Suspension thereof.
`said kit being characterized by involving no means of
`separating cell components from the biological sample.
`0029. Also, the kit of the present invention preferably
`contains a means to freeze said collected biological sample.
`0030 The present invention is the kit described above,
`wherein the RNase inhibitor is guanidine thiocyanate.
`0031. The present invention is also the kit described
`above, wherein the biological sample is feces.
`0032 Moreover, the present invention is a tumor marker
`detecting kit for diagnosing colon cancer, comprising the
`following means:
`0033 b) a means to extract RNA from the obtained
`sample for extracting RNA;
`0034 c) a means to reverse transcribe the extracted RNA
`to give cDNA;
`0035) d) a means to amplify the obtained cDNA; and,
`e) a means to detect the amplified cDNA.
`0036)
`0037. Further, the present invention is the kit described
`above, wherein said tumor marker is COX-2.
`
`BRIEF DESCRIPTION OF DRAWINGS
`0038 FIG. 1 shows a result of an electrophoresis in
`Example 2. The lane 1 shows total RNA extracted from
`human feces with the method by Alexander et al. The lane
`2 shows total RNA extracted from human feces with the
`method of the present invention. The lane 3 shows total RNA
`extracted from a human colon cancer tissue. The lane M
`shows the molecular weight markers.
`
`THE BEST MODE FOR CARRYING OUT THE
`INVENTION
`0039. As for the RNase inhibitors of the present inven
`tion, guanidine thiocyanate, Isogene, Ultraspec II (a regis
`tered trade mark) and the like are included.
`
`0040. The biological samples of the present invention are
`tissues of animals and plants, body fluids, excrements and
`the like, and preferably are feces, and more preferably are
`human feces.
`0041. The biological samples of the present invention can
`be used as they are, or, in Some cases, after frozen.
`0042. Freezing methods can be any conventional meth
`ods, and preferably a method using liquid nitrogen. The
`freezing (and preserving) temperature is -1 to -196° C.
`preferably -20 to -196° C., preferably -75 to -196° C.,
`more preferably -110 to -196° C., and most preferably
`-196° C.
`0043. The frozen sample may be preserved in frozen
`state. The preservation temperature is -75 to -196° C.,
`preferably -110 to -196° C., and more preferably -196° C.
`The preservation period is one day to 10 years, preferably
`one day to 3 years, and more preferably one day to one year.
`0044) The tumor marker used in the present invention is
`COX-2, matrixmetalloprotease (MMP), c-met, CD44 vari
`ants, EGF-R, EF-1, Wnt-2, Bradeion, SKP2, KPC-1, KPC-2,
`PRL-3, Angiogenin, Integrin, Snail, Dysadherin, or the like,
`and is preferably COX-2.
`0045. The processes c) to e) described above are called as
`the RT-PCR method, and can be carried out, for example,
`according to the description by T. Sekiya et al. eds. Fore
`front of PCR method, 1997, Kyoritu Pub., 187-196.
`0046 Extraction of RNA from the suspension can be
`carried out using methods well known in the art, and using
`commercially available kits, for example, RNeasy Mini
`(QIAGEN) or RNA Extraction Kit (Pharmacia Biotech).
`0047. “Reverse transcription” in the present invention
`means conversion of RNA to the complementary DNA
`(cDNA) using a reverse transcriptase. The reverse transcrip
`tion reaction is usually conducted using a solution contain
`ing a buffer, salts such as MgCl2, KC1, and the like,
`dithiothreitol (DTT), a primer, kinds of deoxyribonucle
`otides, RNase inhibitors, and a reverse transcriptase. The
`salts described above can be appropriately replaced by other
`salts after testing. Proteins such as gelatine, albumin or the
`like, or detergents can also be added.
`0048 For amplification of cDNA carried out subsequent
`to the reverse transcription, the PCR technique is usually
`adopted. The PCR reaction mixture usually contains a
`buffer, salts such as MgCl, and KC1, primers, kinds of
`deoxyribonucleotides, and a heat resistant polymerase. The
`salts described above can be appropriately replaced by other
`salts after testing. Proteins such as gelatine, albumin or the
`like, dimethylsulphoxide, detergents, or the like, can also be
`added.
`0049. For amplification of cDNA, the LAMP method
`(Japan Patent No. 33.13358) or the ICAN method (Japan
`Patent Laid-Open No. 2001-136965) can be used.
`0050. A "primer' in the present invention means an
`oligonucleotide which works as a synthesis initiation point
`in the case of cDNA synthesis or polynucleotide amplifica
`
`Geneoscopy Exhibit 1007, Page 4
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`

`

`US 2006/0216714 A1
`
`Sep. 28, 2006
`
`tion. The primer is preferably a single-strand, but a double
`strand can also be used. When the primer is a double-strand,
`it is preferable to make it single-stranded prior to the
`amplification reaction. The primer can be synthesized
`according to a method well known in the art, or can be
`isolated from the living organisms.
`0051. The reverse transcriptase used in the reverse tran
`Scription reaction is an enzyme capable of reverse transcrib
`ing RNA to cDNA. As for the reverse transcriptase, there are
`reverse transcriptases derived from retroviruses such as
`RAV (Rous associated virus), AMV (Avian myeloblastosis
`virus) and the like, and reverse transcriptases derived from
`mouse retroviruses such as MMLV (Moloney murine leu
`kemia virus) and the like, but it is not limited to the aboves.
`0.052 As the heat resistant polymerase used for PCR, Taq
`polymerase can be nominated, but it is not confined to this.
`0053 As the detection method of amplified DNA, elec
`trophoresis using agarose gel can be used, but the method
`may not be confined to this.
`0054 Further, the kit according to the present invention
`may contain an instruction describing the methods of the
`present invention.
`
`EXAMPLE 1.
`0.055 The following examples illustrate the present
`invention, but do not limit the invention.
`0056 Among patients hospitalized in the First Depart
`ment of Internal Medicine of Hamamatsu University School
`of Medicine for detailed examination and therapy, 30 cases
`confirmed to have colon cancer and 22 cases to have no
`tumor or inflammatory alteration in their colon (non colon
`disorder) by the total colonoscopy were selected as the
`subject of the study. Informed consents of all the cases had
`been obtained.
`0057. As soon as possible after sampling feces, the feces
`were separated into 5 ml tubes about 1 g each, were frozen
`using liquid nitrogen, and were stored at -80° C. Also, for
`comparison and reference, human hemoglobin (Hb) in the
`feces of each sample was measured by the immunological
`fecal occult blood test. Tissue biopsy specimen, taken both
`from the cancer and the normal parts when the endoscopy
`was carried out before the therapy, were frozen by liquid
`nitrogen and stored at -80° C. Then, feces were homog
`enized using a homogenizer, guanidine salt, and phenol, and
`whole RNA was extracted using chloroform and ethanol.
`0.058. One ug of the obtained RNA was reverse tran
`scribed using ReverScript II (a registered trade mark),
`(reaction mixture volume: 20 ul, Wako Pure Chemical
`Industries) to give cDNA. A part thereof was amplified by
`means of nested PCR using GeneTaq (Wako). The PCR
`product obtained was electrophresed on 4% agarose gel, and
`stained by ethidium bromide.
`0059 Here, the primers used were: the random primers in
`reverse transcription, and in PCR, were those reported by
`Gerhard (JJCO, 1994) for CEA, and were originally
`
`designed for COX-2. The first round of PCR was executed
`20 cycles, and the second round 25 cycles.
`0060. The followings indicate the primers used.
`0061 <CEA->
`
`TABLE 1.
`
`Forward 1 : 5'-TCTGGAACTTCTCCTGGTCTCTCAGCTGG-3'
`
`Forward 2: 5'-GGGCCACTGCTGGCATCATGATTG-3'
`
`Reverse:
`
`5'-TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC-3'
`
`0062 COX-2>
`
`TABLE 2
`
`Forward 1:
`
`5'-CTGAAAACTCCAAACACAG-3'
`
`Forward 2:
`
`5'-GCACTACATACTTACCCACTTCAA-3'
`
`Reverse:
`
`5'-ATAGGAGAGGTTAGAGAAGGCT-3'
`
`Results
`0063) Feces from 30 colon cancer cases (3 early cancer
`and 27 advanced cancer cases) and from 22 cases in the
`control group were examined by RT-PCR, in order to detect
`CEA and COX-2, and the following results were obtained.
`0064 CEA was detected in all cases among the 30 colon
`cancer cases, and in 21 among 22 cases in the control group.
`Also, it turned out that RNA suitable for RT-PCR amplifi
`cation could be extracted from both samples.
`0065 COX-2 was detected in 27 cases among the 30
`colon cancer cases (caeca: 2/2, ascending colon: 3/5.
`descending colon: 1/1, sigmoid colon: 7/7, rectum: 12/13;
`early cancer: 2/3, advanced cancer: 25/27), but was not
`detected in any of 22 cases in the control group (sensitivity:
`90%, specificity: 100%).
`0066.
`In the immunological fecal occult blood test, 23
`among 28 colon cancer cases and 3 among 22 control cases
`were positive (sensitivity: 82.1%, specificity: 86.3%).
`0067. Among three COX-2 negative colon cancer cases,
`one was positive in the immunological fecal occult blood
`test, and 2 were negative.
`0068 COX-2 was detected in 3 among 5 colon cancer
`cases negative to the immunological fecal occult blood test,
`
`EXAMPLE 2
`0069. The amount and the distribution of molecular
`weights of total RNA obtained from human feces according
`to the method of the present invention were compared with
`those obtained according to Alexander's method (R. J.
`Alexander and R. F. Raicht, Digestive Diseases and Sci
`ences, 43(12), 1998, 2652-2658). As a control, total RNA
`was extracted from the human colon cancer tissues using a
`commercially available RNA extraction reagent (ISOGEN,
`Wako)
`0070 The same amount of total RNA extracted from each
`sample was electrophoresed on an agarose gel.
`
`Geneoscopy Exhibit 1007, Page 5
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`

`US 2006/0216714 A1
`
`Sep. 28, 2006
`
`0071. Two main bands recognized on the lane 3 (RNA
`derived from human colon cancer tissues) show 28s and 18s
`rRNAs. Smeared parts thereon indicate that various kinds of
`high molecular weight RNAs are contained in the obtained
`total RNA.
`0072. Two main bands recognized on the lane 2 (RNA
`derived from feces obtained by the method of the present
`invention) show 23s and 16S rRNAs derived from enteric
`bacteria. Since Smeared parts were also recognized thereon
`similarly to the lane 3, the total RNA obtained from the feces
`by the method of the present invention is considered to
`contain also various kinds of high molecular weight RNAS.
`0.073
`Contrarily, any bands and smears were not detected
`at all in the lane 1, showing that high molecular weight
`RNAs were not contained in the extract of the sample.
`0074. In fact, the desired products were obtained from the
`sample of lane 2 by the RT-PCR technique, but no PCR
`products were obtained from the sample of lane 1.
`0075 From the results of the present studies, it became
`obvious that the RNA extracted from the human feces by the
`method of the present invention can be amplified by means
`
`of the RT-PCR technique. Also, the detection of COX-2
`from the feces by the RT-PCR technique had 90% sensitivity
`and 100% specificity, and it is proved that the present
`invention is Superior to a conventional technique of the
`immunological fecal occult blood test.
`0076 Further, since the method of the present invention
`needs Smaller amount of feces for detection and has higher
`detection sensitivity compared to the detection of the gene
`mutation of APC, K-ras, or p53, it can save largely the time
`and effort needed for the detection.
`0077. While the conventional technique of the fecal
`occult blood test targets a general and indirect event, "bleed
`ing from the lesion, the method of the present invention
`targets a specific and direct event, the expression of a marker
`of carcinogenesis, COX-2. Therefore, the data obtained by
`the method of the present invention provide diagnosis with
`higher quality.
`0078. Accordingly, the method of the present invention is
`clinically very useful as a novel non-invasive screening
`method with high specificity and high sensitivity.
`
`SEQUENCE LISTING
`
`<160> NUMBER OF SEQ ID NOS: 6
`
`<210> SEQ ID NO 1
`&2 11s LENGTH 29
`&212> TYPE DNA
`<213> ORGANISM: Artificial
`&22O > FEATURE
`<223> OTHER INFORMATION: designed primer
`
`<400 SEQUENCE: 1
`
`totggaactt citcctggtot citcagotgg
`
`<210> SEQ ID NO 2
`<211& LENGTH 24
`&212> TYPE DNA
`<213> ORGANISM: Artificial
`&22O > FEATURE
`<223> OTHER INFORMATION: designed primer
`
`<400 SEQUENCE: 2
`
`gggcc actoc togcatcat g attg
`
`<210> SEQ ID NO 3
`&2 11s LENGTH 32
`&212> TYPE DNA
`<213> ORGANISM: Artificial
`&22O > FEATURE
`<223> OTHER INFORMATION: designed primer
`
`<400 SEQUENCE: 3
`
`tgtagctgtt goaaatgctt taaggaagaa go
`
`<210> SEQ ID NO 4
`&2 11s LENGTH 19
`&212> TYPE DNA
`<213> ORGANISM: Artificial
`&22O > FEATURE
`
`29
`
`24
`
`32
`
`Geneoscopy Exhibit 1007, Page 6
`
`

`

`US 2006/0216714 A1
`
`Sep. 28, 2006
`
`-continued
`
`<223> OTHER INFORMATION: designed primer
`
`<400 SEQUENCE: 4
`
`citgaaaacto caaacacag
`
`<210 SEQ ID NO 5
`<211& LENGTH 24
`&212> TYPE DNA
`<213> ORGANISM: Artificial
`&220s FEATURE
`<223> OTHER INFORMATION: designed primer
`
`<400 SEQUENCE: 5
`
`gcactacata cittacccact tcaa
`
`<210> SEQ ID NO 6
`<211& LENGTH 22
`&212> TYPE DNA
`<213> ORGANISM: Artificial
`&220s FEATURE
`<223> OTHER INFORMATION: designed primer
`
`<400 SEQUENCE: 6
`
`ataggagagg ttagagaagg ct
`
`19
`
`24
`
`22
`
`1. A method for preparing a sample to extract RNA used
`in a tumor marker detecting method for diagnosing colon
`cancer, comprising the following process:
`a) a process to homogenize the collected biological
`sample in the presence of an RNase inhibitor, to prepare
`a Suspension thereof;
`characterized by involving no procedure of separating cell
`components from the biological sample.
`2. A method according to claim 1, wherein the collected
`biological sample is frozen.
`3. A method according to claim 1
`4. A method according to claim 1, wherein the biological
`sample is feces.
`5. A tumor marker detecting method for diagnosing colon
`cancer, comprising the following processes:
`b) a process to extract RNA from the obtained sample for
`extracting RNA;
`c) a process to reverse transcribe the extracted RNA to
`give cDNA;
`d) a process to amplify the obtained cDNA; and
`e) a process to detect the amplified cDNA,
`in addition to the method according to claim 1.
`6. A method according to claim 1, wherein the tumor
`marker is COX-2.
`
`7. A kit for preparing a sample to extract RNA used in a
`tumor marker detecting method for diagnosing colon cancer,
`comprising the following means:
`a) a means to homogenize a collected biological sample in
`the presence of an RNase inhibitor, and prepare a
`Suspension thereof;
`characterized by involving no means for separating cell
`components from the biological sample.
`8. A kit according to claim 7, further comprising a means
`to freeze the collected biological sample.
`9. A kit according to claim 7, wherein the RNase inhibitor
`is guanidine thiocyanate.
`10. A kit according to claim 7, wherein the biological
`sample comprises feces.
`11. A tumor marker detecting kit for diagnosing colon
`cancer, comprising the following means:
`b) a means to extract RNA from the obtained sample for
`extracting RNA;
`c) a means to reverse transcribe the extracted RNA to give
`cDNA;
`d) a means to amplify the obtained cDNA; and
`e) a means to detect the amplified cDNA.
`12. A kit according to claim 7, wherein the tumor marker
`is COX-2.
`
`Geneoscopy Exhibit 1007, Page 7
`
`