WO2015114641A1

WO2015114641A1 - Detection of cancer using pcr method

Info

Publication number: WO2015114641A1
Application number: PCT/IN2014/000064
Authority: WO
Inventors: Adhiraj SINGH
Original assignee: Resource Life Sciences Private Limited
Priority date: 2014-01-28
Filing date: 2014-01-28
Publication date: 2015-08-06

Abstract

The invention presents a novel molecular biology approach and a Plasma DNA based kit for detection of cancer. It is driven to meet the needs for cancer detection, whereby a non-invasive test that can detect any form of cancer and not specific to a particular tumor type that can drive quick and affordable results without requiring hospitalization of the patients. Fig 3 depicts that the cfDNA from an individual with cancer has a different DNA integrity because the cfDNA is a result of necrosis that yields larger DNA fragments. Primer P80F and 80R in combination will yield 80bp product as normal individual, in addition primer P80F and P400R will also yield a 400bp product because of the presence of intact β-Actin gene in cfDNA from cancer patient. Thus the appearance of the 400bp PCR product will be hallmark for the detection of cancer in plasma.

Description

DETECTION OF CANCER USING PCR METHOD

BACKGROUND Field of the invention

[0001] The present invention relates to the molecular architecture of the human genome in cancer and its intervention by polymerase chain reaction method to differentiate the genomic architecture between a healthy individual and a patient with cancer. Description of the related Art

[0002] Cancer is the world's second killer after cardiovascular disease. Higher incidence of non-communicable diseases, especially cancer is positively associated with work related stress, modern food habits, lack of leisure time and percentage of aged population of a country. The World Cancer Report documents that cancer rates are set to increase at an alarming rate globally. Cancer rates could increase by 50% new cases for the year 2020. Details published by Indian Council of Medical Research (ICMR) indicates that the age adjusted incidence of gall bladder cancer in women in New Delhi is ^ 0.6 per 100 000 population and is the world's highest rate for women. It is possible to stop this loss of life by 40% through implementation of cancer prevention program and early diagnosis. With these numbers on the forefront it is required that tools that can detect cancer are becoming more and more demanding. The geo-economic and geographical scenario demand early cancer detection tools that can be made available and affordable to the low income group which also encompasses a large portion of the population that are susceptible to cancer. It is therefore necessary to develop a robust too! for cancer detection that can be used in remote areas with minimum utilities and expenses. Thus the present invention is useful and offers a cancer detection kit based on molecular approaches to fulfill the needs of demanding cancer patients in developed and underdeveloped countries.

[0003] Cancer is a class of diseases characterized by out-of-control cell growth. There are over 100 different types of cancer, and each is classified by the type of cell that is initially affected. Cancer harms the body when damaged cells divide uncontrollably to form lumps or masses of tissue called tumors (except in the case of leukemia where cancer prohibits normal blood function by abnormal cell division in the blood stream). Tumors can grow and interfere with the digestive, nervous, and circulatory systems and they can release hormones that alter body function. Tumors that stay in one spot and demonstrate limited growth are generally considered to be benign. Formation of malignant tumors is due to the two following causes:

A) cancerous cell manages to move throughout the body using the blood or lymph systems, destroying healthy tissue in a process called invasion that cell manages to divide and grow, making new blood vessels to feed itself in a process called angiogenesis.

B) When a tumor successfully spreads to other parts of the body and grows, invading and destroying other healthy tissues, it is said to have metastasized. This process itself is called metastasis, and the result is a serious condition that is very difficult to treat.

[0004] Cancer is ultimately the result of cells that uncontrollably grow and do not die. Normal cells in the body follow an orderly path of growth, division, and death. Programmed cell death is called apoptosis. and when this process breaks down, cancer begins to form. Unlike regular cells, cancer cells do not experience programmatic death and instead continue to grow and divide. This leads to a mass of abnormal cells that grows out of control. [0005] Cell is controlled by genes in the chromosomes which is the master controller of all the processes of any living cell. The root of cancer is malfunction of the DNA. Since the completion of the human genome project much has been know about the complexities of the genome and its behavior in cancer. The abnormal growth of cells in cancer is therefore attributed to loss or gain in the chromosomal regions harboring the genes that control cell growth. Mutations, Methylation or amplification of genes damages the DNA (deoxyribonucleic acid) architecture; hence leads to uncontrolled cell growth culminating to tumor. The tumor cells then grow as a lump of tissue and will be defined by the cells of origin. Any tissue with epithelial cells is the source of tumor generation, where the genetic changes take place and the cancer cells starts growing.

[0006] Our bodies are made up of millions of cells. Within almost every cell is 46 rod-like structures called chromosomes. Chromosomes are the packages of genetic information that make each person unique. There are 23 pairs of chromosomes. One chromosome of each pair comes from each of our parents. Along the chromosomes are thousands of genes, which are made up of DNA. There are approximately 30,000 different genes and each has an important role in the body. Genes also play an important role in inherited disease conditions for example cystic fibrosis, cancer and sickle cell anemia is caused by defects in our genes. Such gene defects are called mutations, and these mutations can be passed down from a parent to a child.

[0007] Over the past ten years, the location and the role of hundreds of genes have been determined. Through research, we now know that there are some genes that, when mutated, can give a person an increased risk for cancer. These genes are called cancer susceptibility genes or "cancer genes." Over 100 different cancer genes have been described, and some of these have been shown to cause an increased risk for different types of cancer including breast cancer, ovarian cancer, colon cancer, thyroid cancer and uterine cancer. [0008] Cells can experience uncontrolled growth if there are damages or mutations to DNA, of the genes involved in cell division. Four key types of gene are responsible for the cell division process: oncogenes tell cells when to divide, tumor suppressor genes tell cells when not to divide, suicide genes control apoptosis and tell the cell to kill itself if something goes wrong, and DNA-repair genes instruct a cell to repair damaged DNA. Cancer occurs when the cells fail to correct the mutations and the damage is linked to failure in apoptosis.

[0009] DNA sequence information has taken a major leap after the human genome project. The sequence information of the DNA from cancer patients have been used to define the risk of a population for susceptibility towards particular cancer, for cancer diagnosis and therapy. Sequence information of gene for mutation has long been used in patients to define a cancer. There are now lots of diagnostic kits available from commercial sources and being used by clinicians to confirm the pathological symptoms for cancer. The demand for such approaches has multiplied in recent years for more biomarkers for various types of cancer. DNA based kits detecting mutations for APC, BRCA1 , and p53 have been used to detect colorectal, breast, ovarian and lung cancers. These kits are based on genetic changes such as mutations, and polymorphism in these specific genes that leads to predictive indicator for the presence of specific tumors. While all these tests are in the market more and more biomarkers are being developed to broaden the choice for cancer diagnostic using molecular tools. The DNA is therefore a vital source to identify the malignancy and the type of malignancy of human tissues.

[0010] The molecular approaches that are exploited to develop kits for the identification of cancer are specific for a particular type of cancer and have been very successful in predicting the tumor type in laboratory and clinical settings. Information regarding the cancer detection tests can be found at www.cancure.org/tests, wherein most of the recent tests for detection of cancer are highlighted. All these tests are antigen specific, cancer imaging, endoscopy or ultrasound. The drawbacks of all these tests are:

a) Expensive

b) Some of the tests are positive for cancer as well as other altered pathology

c) The tests are time consuming.

d) Computed Tomography Scan or CT scans exposes patients to more than 100 times the radiation of a typical chest X-ray. Moreover the contrast used in this procedure may cause allergic reactions.

e) Biopsy needs special procedures and sometimes temporary hospitalization.- While this test is very specific, detailed and confirmatory tests for the presence cancer but is only test after the physical exam, imaging, endoscopy, and laboratory tests for the patient is indicative.

[0011] Non-invasive and cost effective tests for cancer detection have therefore become very necessary to combat the above mentioned drawbacks particularly in a country where medical tests are not only expensive but also not readily available.

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BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Aspect of the present invention is to detect cancer of any origin in human body. [0013] Another aspect of the present invention to enable screen for presence of cancer in a population based study.

[0014] One more aspect of the invention is to observe the recovery of cancer after chemotherapy or resection of the tumor.

[0015] Another aspect of the present invention is to monitor the re- occurrence of the tumor post therapy or surgery.

[0016] One more aspect of the present invention is to identify the efficacy of the best chemotherapy treatment that is responded by the patient.

[0017] Other ^' aspects, advantages, and salient features of the invention will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, disclose exemplary embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] So that the manner in which the .above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawmgs illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

[0019] Figure 1 shows the formation of blood vessels in and around a tumor, according to one or more embodiments of the present invention;

[0020] Figure 2 depicts the β-actin gene DNA obtained from normal individual is fragmented to size not more than 80bp as a result of apoptosis, according to one or more embodiments of the present invention;

[0021] Figure 3 depicts the β-actin gene DNA obtained from cancer patient, according to one or more embodiments of the present invention. [0022] Figure 4 is the representative PCR Product of normal patients and tumor patients, according to one or more embodiments of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS [0023] Cancer cells initiates and promotes formation of new blood vessels and this process is typically called Angiogenesis. Tumor cells cannot grow beyond 1-2 mm without developing its own blood supply and this allows the tumor cells to procure its entire nutrient for growth. Fig.1. shows the formation of blood vessels in and around a tumor. The formation of new blood vessels allows the cancer cells to flow in the blood stream for metastasis. This also allows the necrotic tumor cells to release its DNA in the blood stream.

[0024] Number of reports have suggested detection of tumor DNA in serum and have proclaimed for tumor diagnosis. Moreover DNA extracted from plasma of cancer patients showed the same characteristics as the tumor cell DNA. Furthermore, the mean DNA levels in benign neoplasms differed significantly to that found in aggressive tumors. Interestingly, a correlation between free DNA levels and advanced disease was found in lung cancer patients. Thus based on evidences it is likely that tumor DNA found in blood of cancer patients significantly differs from the blood of a healthy individual and can be exploited to develop molecular markers for cancer detection.

[0025] Plasma is long known to have cell-free circulating DNA in minute amount but is increased in patients with autoimmune disorders, trauma and cancer. Particularly in cancer the very vasculature of the blood vessels resulting from angiogenesis keeps the tumor tissue in close contact with blood circulation. Therefore the debris of cell death by necrosis from a tumor tissue will eventually drain in the blood stream and that should contain DNA.

[0026] Cell death is a frequent event both in normal and tumor tissues. The only difference between these events is that in normal tissue cell death the fragmented DNA released by the cells in the serum are small and uniform with approximately the size of 185-200 bp. While the same DNA released in the serum by tumor tissues through necrosis generates a spectrum of DNA fragments with large sized strand lengths. The differences in the DNA strand length has been used as a template in the polymerase chain reaction (PCR). The other major difference that has impacted in the invention is the amounts of cell free DNA in plasma or serum. The mean concentration of cell free DNA in plasma from a healthy population amounts to 14 nanograms/milliliter and in patients with different cancers the mean concentration detected is 118 nanograms/milliliters. This significantly different amount of DNA between a healthy and diseased individual has also been exploited in the invention process.

[0027] The Polymerase chain reaction (PCR) is a powerful methods to amplify tiny amounts of DNA and has now long been used to characterize DNA and also for diagnostic purposes. As shown in Fig2 the β-actin gene DNA obtained from normal individual is fragmented to size not more thanl 100bp as a result of apoptosis. Primer P80F-(5'- GGC ATC CTC ACC CTG AAG TA-3') at 342 bp downstream to the start site can efficiently yield a PCR product of 80bp when combined with primer P80R (5'- AGG TGT GGT GCC AGA TTT TC-3'). However, if Primer P400R (5 - TGT CAC GCA CGA TTT CCC -3') is combined with Primer P80F the PCR reaction will fail to yield any product due to the nick in the DNA strand of the β-Actin gene. Therefore cfDNA (cell free DNA from healthy individual will produce only on PCR product from primer P80F and P80R.

[0028] cfDNA from an individual with cancer has a different DNA integrity because the cfDNA is a result of necrosis that yields larger DNA fragments (Fig.3). Primer P80F and 80R in combination will yield 80bp product as normal individual, in addition primer P80F and P400R will also yield a 400bp product because of the presence of intact β-Actin gene in cfDNA from cancer patient. Thus the appearance of the 400bp PCR product will be hallmark for the detection of cancer in plasma.

[0029] Cell Free DNA (cfDNA) Isolation from Plasma: Blood contains DNA at a very low concentration and has been a major target for genetic analysis for identifying genetic defects in the era after the sequencing of the human genome. The circulating DNA in the blood that is important from cancer the angle of cancer detection is the cells free DNA. The concentration of cell free DNA (cfDNA) is low in plasma or serum of healthy donors but is increased in patients with tumors. Mechanisms leading to the appearance of CFDNA in blood remain largely unknown to date. However, processes like apoptosis and necrosis has been shown to contribute to the generation of CFDNA. Apoptosis is a programmed cell death mechanism and occurs in normal epithelial cell of the intestine, colonic epithelium and uterine lining. These cells are replaced by the new and thus are a source of cfDNA at a minimum concentration in a healthy individual. The tumor tissue also 064 is a conglomerate of various mechanisms that leads to its uncontrolled growth in a very rapid pace. While growth of these cells are in progress many of those cells also die because of necrosis resulting in the release of cfDNA in the blood stream. Since this process is uncontrolled random and in massive amount the enzymes involved in the degradation of the nucleic acids become limiting leading to an increase in size of the fragmented DNA as well as in quantity.

[0030] There are currently number of methods to extract DNA from tissues, blood, plasma and serum. However, extraction of cfDNA is difficult because of the tiny amount present in the blood. Due to advancement of the DNA isolation technology it is now possible to purify tiny amounts of DNA from as low as 1ml of plasma using commercially available ^'kits. So the DNA isolation from the plasma is not included as a part of invention in this patent application. However, it is highly recommended to follow the precise protocols detailed to isolate the DNA from plasma. We have found that QIAamp blood kit (Qiagen Inc) is easier and yields consistently good amount of cfDNA and therefore recommended for use with this invention. The DNA obtained by the Qiagen kit is briefly dried in room temperature and then dissolved in 10- 20 μΙ of nuclease free water and stored at -20°C for further use. The DNA is quantified by spectroscopy to calculate the yield from 1.0 ml plasma. In brief 1 μΙ of the DNA is diluted in sterile water to 100μΙ and absorbance is measured at 260 and 280 nM. Based on the assumption that 1.0 OD₂6o equals to 50μg ml₁ the amount of DNA in each sample is calculated.

[0031] PCR Protocol and Interpretation of the results for read out of the cancer samples

[0032] The cfDNA purified will be used at concentration of 100pg. The following the PCR mixture is made by adding the reagents as shown in the chart for standard genomic DNA, 80 and 400bp product from plasma samples and non template control for 80 and 400 bp primers. The standard genomic DNA is isolated from discarded human placenta and purified using commercially available kits or is procured from commercial source. The genomic DNA will be used as a reference for intact DNA. The PCR mix for the real time PCR analysis is shown in chart and is standardized for amplification off 80bp and 400 bp of DNA fragment. 100pg of cfDNA is used for amplifying 80bp and 400bp of the plasma DNA from patients

Taa DNA Mix iS bergrec m

Non Template control is an additional reaction needs to be performed along with the positive and test samples. In negative control reactions the template is opted out of the reactions and is substituted by water only. The charts below show the reaction mix for a negative control reaction.

9S°C - 3.0 min

,9S°G - 0;5 min /;. ^* .^'„"T" ^'

; 72°cV o^" rrlin .,ί¹ &

[0033] The PCR reaction condition is adjusted to yield single product for both the pair of primers.

[0034] In a 45 cycles of PCR reactions the increase in product floursenee against background is set to 33 cycles. Any floursence greater than this cycle is considered formation of primer dimer or lack of appreciable templete to achieve detectable product. The cT obtained from the four reactions that include two of standards and two of patient samples are normalized and the ratio is calculated to assign a sample malignent or non-malignent. After careful analysis across several samples the cutoff value set for a sample to be positive if value is more than 3.00 based on the formula as described below. [0035] FORMULA

Q100bp= 2*^{cT Test 100 _cT G10}°⁾

cT Test 10Obp = cT 10Obp of sample

cT G100 = cT 10Obp of genomic DNA

Q400bp= 2^{(cT Test 400 _cT G 00)}

cT Test 400bp = cT 100bp of sample

cT G400 = cT 100bp of genomic DNA

Test (positive or negative) = Q400bp/ Q100bp

Ratio <3 is positive for malignancy

CT more than 33 considered not detectable for both fragments

[0036] METHOD & RESULTS

> PCR tubes equivalent to the number of samples are taken and labeled the tubes in a convenient way as the user wishes.

> Pipette μΙ of the EXTRACTED CELL FREE DNA (cfDNA equivalent to 100pg) to each PCR tubes.

> To this is added μΙ of H₂0 to the PCR MIX. (Primer + Taq DNA polymerase mix containing DNTP and MgCI₂) to the tube. The mixture is mixed by vortex followed by brief centrifugation on a Tabletop Centrifuge to settle the mixture. The mixture is set for

[0037] Our initial tests on cfDNA from colon cancer patients using conventional PCR have shown positive results in the samples tested. The representative PCR Products of blinded normal and tumor DNA from patients are shown in Fig. 4. cfDNA from normal individual yields product with primer pair P80F and P80R with a product size of 80bp and samples with primer P80F and P400R at 400bp (highlighted in red box) was observed. This technique is therefore able to purify the fragmented DNA from plasma and can be used to quantify the cfDNA in normal and cancer individuals. To further access the presence of fragmented cfDNA in cancer patient's similar technique was applied to plasma cfDNA obtained from various patients of colon cancer. The results indicate indeed the DNA purified was really fragmented but with larger size of the DNA as revealed in PCR with primer P80F and P400R. Therefore under similar conditions cfDNA purified from patients with colon rectal cancer showed two distinct PCR products with primer pair P80F&P80R and P80F&P400R. The primer pair (P80F&P400R) can be used to detect the presence of tumor in cancer patients based on the hypothesis made herein. The result shown is a representative of 17 colorectal samples analyzed by the invented technique. [0038] In an attempt to make this technology more sensitive and less technically cumbersome the same methodology was standardized for quantitative PCR using sybergreen dye to detect the product formed by the two pair of primers, The PCR conditions including the amount of DNA were similar as described for conventional PCR. The PCR platform can be any standard PCR machines but needs back ground corrections to replicate the test.

[0039] A typical real time PCR run is shown. The primer pair P80F- P80R used for genomic DNA as template usually shows 17-18 cT and primer pair P80F-P400R 17-19 yielding roughly a ratio close to 1.0. This value matches with the theoretical prediction because in an intact DNA both the fragments is amplified from one intact piece of DNA. Therefore such representation of template can have the same amount at the start of the DNA. TABLE-II i

SN# j Sample Name i 400bp/1 OObj; Comments

1 '7381 #VALUE! i ot_tDetectable cT<33

2 *7380 9.37 Cancer Suspected

3 "7379 ^' #VALUE ! Ϊ Not Detectable ^■ cT<33

4 "7378 #VALUE ! ; Not Detectable

5 '7377 2 92 I Normal

6 "7376 109.45 ! False positive

7 '7373 3.1 7 ; Normal

8 '7374 12.66 Cancer S uspected

9 "7375 1 .84 • Normal

1 0 7372 1 99 : Normal

1 1 "7371 1 .33 ( Normal

1 2 "7367 i 1 0.75 I Cancer S uspected 15 ;

[0040] A read out of the assay from a real time PCR analysis will be presented in the format as shown in Table I. In the NTC control there is no detectable fluorescence which typically arises due to primer dimer formation. RLS designed primers and the conditions are standardized not to give any background fluorescence. The calculation of such analysis is shown in Table II and based on the cutoff value set at <3 the results will be categorized as positive, negative or undetermined.

[0041] Based on the hypothesis and experimental evidences the invention for the use of cfDNA in detecting cancer using PCR technique is subject matter of the patent. The primer sequences can serve as a universal tool for detecting presence of any kind of tumor in human using PCR and to amplify cfDNAs obtained from any form of cancer. This technology is very well suited for development of a kit that can be used in clinical practice as means of quick and cheap test for detection of human cancer.

[0042] While the foregoing is directed to embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

CLAIMS:

What is claimed is: . A method for targeting cell free dna in plasma for detection of human cancer using Polymerase Chain Reaction (PCR), comprising the steps of:

a) Obtaining blood sample of the patient to be tested;

b) Obtaining the cell free DNA from serum;

c) Determining the mean concentration of Cell free DNA in

blood plasma; and

d) Comparing the said mean concentration with that of cancer free patients wherein a high mean concentration indicates the probability of cancer

2. The method as claimed in Claim 1 , wherein the said mean

concentration of a healthy person is 14 nanograms/millilitre

3. The method as claimed in Claim 1 , wherein the said mean

concentration of a person suffering from cancer is of the range of 118 nanograms/millilitre

4. A method for diagnosing cancer in humans comprising the steps of:

a) Obtaining blood sample of the patient and isolating the cell free DNA from serum

b) Performing the PCR on the isolated Cell free DNA to which PCRMIX containing primers P1 , P2 and P3 and other regents are added;

c) Analysing products corresponding to primers where products . from primers PI and P3 in addition to the product from P and P2 indicates high probability of cancer wherein the said PCRMIX comprises H20, 10X PCR Buffer, Mgcl2, DNTP, primers and Taq Enzyme

5. * A method as claimed in claim 4, wherein the product obtained by primer P80F and P80R is of 80 bp.

6. A method as claimed in claim 4, wherein the product obtained by primer P80F and P3 is of 400bp.

7. A method as claimed in claim 4, wherein the primers are

specifically designed for B-actin gene present in human genome

8. A method as claimed in claim 4, wherein the primer P80F is having sequence P80F-(5'- GGC ATC CTC ACC CTG AAG TA- 3') on β-actin gene.

9. A method as claimed in claim 4, wherein the primer P80R is having sequence P80R (5'- AGG TGT GGT GCC AGA TTT TC- 3'). on β-actin gene.

10. A method as claimed in claim 4, wherein the primer P400R is having sequence P400R (5'- TGT CAC GCA CGA TTT CCC -3') on β-actin gene.

11. A method as claimed in claim 4, wherein the said method can be performed using any gene of the human genome

12. A kit for practicing the method of claim 1 , comprising of

a) Reagents suitable for performing polymerase chain reaction b) Primer specific for performing PCR reaction on β-actin gene; and

c) Instruction material