Interviews with Doctors

1.Dr.Li Wenjin, Hematology Department, First Xiangya Affiliated Hospital of CSU

(1)Procedure of Current Cancer Detection

Dr.Li mentions the procedure of current cancer detection,including extracting DNA from blood cells, amplifying it with PCR and then getting its sequences.Detection varies from diseases and gene chip(detection for all genes) also could be applied with enough money.However, only some genes are of necessity to be detected for individuals.

(2)Gap Exists Between Clinical Application and Basic Research

There was distance between clinical application and basic research in early detection of cancer. For instance, some means of early cancer detection still had a long way to go before proved efficacious---commonly 5-10 years experimental validation phase is integral to clinical application.

(3)Stability of MiRNA in Blood is Doubted

Dr.Li shows her concern about the instability of miRNA when the topic is involved with miRNA and its potential application in early detection of cancer.However,she also mentions that as a clinical doctor,she doesn’t have much research time so that she is not expert in miRNA field.Meanwhile,Dr.Li says that they clinical doctors usually pay more attention on developed technology instead of the developing one and her knowledge about miRNA comes from papers.

(4)Efficiency of MiRNA Detection is Not Guaranteed

Dr.Li says that they clinician commonly assess the severity not only by the gene detection,but also by patients’ clinical symptoms,chromosome. The oncologists even need to do the Immunohistochemical checker and pathology section to make sure.So they suspect a bit about the efficiency of early cancer detection due to the lack of pathological symptoms and currently so-so specified biomarkers in patients’ early stage.

(5)Prospect of MiRNA Is Expected

Dr.Li hopes that the team of NUDT_CHINA would find a way to detect cancer, with much greater specificity and fewer side effects, so that more targeted medicine could be invented to kill cancer tumors. She also figures that early cancer detection could help people carried with household hereditary disease factors.Additionally, the index of miRNA could help alert people to pay more attention to their living habits and health condition as well.

2.Dr.Hou Tao, Oncology Department，Second Xiangya Affiliated Hospital of CSU

(1)Current situation of tumor biomarker test

We asked Dr.Hou about some details about current tumor biomarker test the hospital usuallly suggested patients to take. And this helped us learn more about the current situation of tumor biomarker test.

a.Biomarker tests in health exams

In a normal health exam, there is an optional choice of tumor biomarker test including tests for 12 biomarkers of most frequent cancers, which is called ‘set 12’ by doctors. Talking about taking the biomarker tests, Dr.Hou said that the popularity of biomarker tests was increasing in oncology department in hospitals in China. And with more people getting familiar with tumor biomarker test, it was gaining increasing attention, and a great number of healthy people requested tumor biomarker test in their health exams, especially the elder. Tumor biomarker tests are gaining increasing attention from the crowd.

b.Details of a normal biomarker test in health exams

Contents of ‘set 12’ biomarker test in a normal health exam:

Cost of the test for 12 biomarkers, or the ‘set 12’ is ￥500, including biomarkers of breast cancer, liver cancer and so on. Dr.Hou also told us that the validness of biomarkers was not so optimistic, thus could only work as a reference for diagnosis of cancers.

(2)Advice for The Project---SPLIT-GFP, SPLIT LUCIFERASE, OR SPLIT HRP?

To improve the sensitivity and specificity of RCA output signal and to visualize the RCA outputs in the wet-lab experiments, we designed a single guide RNA mediated dCas9 binding system and a conjugated reporting system. However, dilemma came that split-luciferase, split-HRP and split-GFP all seemed to meet our requirements of reporting the signal. Which was the best fit?

Firstly we searched for information of the three reporting systems:

Then we turned to our medical consultant Dr.Hou and asked him about suggestions of our reporting system.

Sample Collection for Wet Lab

Since our project this year was to develop a blood-microRNA detection system as a novel method for early cancer detection, and results of previous wet lab all confirmed the effect of our detection system, it was necessary to apply it to serum as a final confirmation. We came to the health exam center of Affiliated Hospital of NUDT and talked with doctors and 5 non-small cell lung cancer(NSCLC) patients about our need for serum samples to and got their support and permission. Also we had 30 healthy postgraduates from NUDT as our volunteer of serum samples. And we collected the blood samples with the help of doctors.

1.Approve of the Informed Consent Form

We submitted the Informed Consent Form to the department of ethics in NUDT. After days of checking it was approved to use.

2. Consultation with doctors

Firstly we found Dr.Tan, one of the principles in the department of clinical lab of the health center. He introduced the formal process of collecting blood samples to us and approved our Informed Consent Form. Dr.Tan also helped us with the collecting work.

3.Meeting with sample groups

We talked to 5 cancer patients in different stages of NSCLC as well as 30 healthy volunteers. We introduced what we had been working on and explained what we would do with their blood samples. We also made a security guarantee that we would keep their personal information as secret.

4.Processes of sample gathering

a. Determination of sample group of patients and healthy volunteers and register their information.

b. Filling in Informed Consent Form and diagnostic information.

c.Doctors collected 5L blood sample/person with coagulation tube and match each with the person’s information(including name, age and extra information as AD, stages of NSCLC, etc from cancer patients).

d.Samples transmitted to professional operators and serum was separated.

e.Registering samples in the information management system and setting up links with the Informed Consent Form.

f.Sample serum was transmitted to cryopreserve tubes and placed in -80℃ in the lab.

PART II

PRODUCT EVALUATION

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Market Demands

Previously we did surveys in hospitals and health center about current early cancer detection methods. To gather more information, we searched the Internet about the same issue. Here is the summary.

Cost Evaluation

We made an evaluation of efficiency and price of our detection system in this part to see how much our product may decrease the cost of biomarker detection for cancer, or to say how much our project could help the world with.

The other reason we made this cost evaluation was to make it a feedback for the project and find other aspects it may develop itself in.

The following is the evaluation of our expected product cost.(Click here to view details of contents, instrument, reagents and experimental procedure of our potential product.)

1.Project Contents

miRNA extraction; Incubation from protein and sgRNA; Fluorescence coloration

2.Major Instruments and Reagents

(1)Instruments

Centrifugal machine, Freeze drying machine, 37 degree constant temperature box, Filter paper and other laboratory supplies, Computers and printers

(2)Reagents

Ribonuclease inhibitor, Double distilled water, Special probe , Phi29 DNA polymerase, Buffer phi29, BSA, dNTP, Cas9 protein,and sgRNA

3.Experimental Procedure

Firstly,freeze-dry the required reagents (such as enzymes, proteins, buffer, etc.) in a solid carrier (filter paper, etc.).

Then,get serum from provided blood, boil to destroy binding between miRNA and protein and extract miRNA.

Finally,add extracted miRNA to the solid carrier and wait for coloration,compare with the standard color card to estimate contents of miRNA.

1.Cost Evaluation

Assuming that the protein can be made to the level of industrial production, the total reagent cost is about $2.672.

All of these machines can be used for long and we don’t need to take them while testing miRNA outside. So we consider loss only. Centrifugal machine is used to extract serum while freeze dryer is used to freeze-dry relative reagents in the test paper. We apply HP color laser integrated machine to make colorimetric card.Briefly,the loss price is about $0.3.

2.Interpretation of Result

(1)The total price of our project is around $3.

During the experiment, we found that this method can achieve requirements of high sensitivity, low cost and easy operation.

(2)Comparisons with some other quantitative miRNA detection methods.

a.miRNA Real-time qPCR

miRNA CHIP

From the comparisons, we can easily draw the conclusion that:

a.Our product-to-be costs only $3 in each round, it has an overwhelming advantage in cost.

b.The reporting system of our project is the split-HRP, which satisfied the need of a new visible signal showing method. Thus, we eliminated heavy instruments and strict environment requests, finally making it possible to put it into wide use in remote areas which lack qualified conditions for current early cancer detection methods.

c.Our project is expected to solve the problem of lack of early cancer detection methods in remote areas as well as places bad in medical conditions.

PART III

RAISING AWARENESS

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In the project this year, the aim of our creation of the product was to detect blood-microRNA thus to promote early cancer detection and then to help the world.

However, to realize helping the world, this is far from enough. Thus we scheduled a series of Human Practice works for all ages to expand our project and ideas out of the lab---we raised people’s awareness of cancer&early cancer detection.

Internet Survey For The Public

In this survey about cancer and early cancer detection, we we chose netizens as our respondent, thus the results would be more objective and random. And we may get more comprehensive and true answers. We designed an Internet survey and put it on social media. Until we stopped the Internet survey, there were 524 netizens who took part in it.

The survey mainly involved the three aspects:

(1)How much the public knew about early cancer detection.