CN104561322A - Quantitative gastric carcinoma patient survival prediction and individual follow-up schedule evaluation method based on helicobacter pylori DNA molecules - Google Patents

Abstract

The invention discloses a survival prediction and individualized follow-up time evaluation method for gastric cancer patients based on molecular quantification of Helicobacter pylori DNA, wherein the survival prediction comprises the following steps: 1) Detecting the relative infection amount of Helicobacter pylori (Hp) in gastric cancer tissue; 2) The prognosis is evaluated according to the relative infection amount of the detected Helicobacter pylori; wherein the degree of poor prognosis increases with the increase of the relative infection amount of the Helicobacter pylori. The method of the present invention estimates the prognosis of gastric cancer patients to determine the most reasonable follow-up time for the patients, and through the reasonable follow-up time, early detection of signs of metastasis or recurrence improves the quality of life and survival time of gastric cancer patients.

Description

Survival prediction and individualized follow-up time assessment method for gastric cancer patients based on molecular quantification of Helicobacter pylori DNA

technical field

The invention relates to the technical field of postoperative situation prediction for gastric cancer patients, in particular to a method for predicting the survival of gastric cancer patients and evaluating individualized follow-up time based on DNA molecular quantification of Helicobacter pylori.

Background technique

According to the latest statistics from the World Cancer Research Organization (IARC) Globocan, the number of new gastric cancer cases in the world in 2012 was more than 952,000, accounting for 6.8% of the global cancer incidence, ranking fifth; the global death toll of gastric cancer was 723,000 More than 100,000 people account for 8.8% of the total number of cancer deaths worldwide, ranking third. Cancer is a special type of disease. Cancer patients are cured and discharged after receiving effective standardized radical treatment in the hospital. However, cancer patients always have the possibility of recurrence or metastasis, and long-term follow-up is required for cancer patients. Therefore, the establishment of a reasonable follow-up time is particularly important: 1. Too short a follow-up interval will lead to unnecessary repeated inspections, wasting time and financial resources; Delays are detrimental to patient outcomes. The follow-up time of gastric cancer patients in the second edition of the National Comprehensive Cancer Network (NCCN) guidelines in 2013 is mentioned as follows: All gastric cancer patients should receive systematic follow-up. Follow-up visits include comprehensive medical history inquiry and physical examination, once every 3-6 months for a total of 1-2 years; then every 6-12 months for a total of 3-5 years; thereafter once a year; CBC, serum biochemical tests, imaging studies, or endoscopy were performed according to the clinical situation. The guidelines emphasize the importance of follow-up, but do not give an individualized follow-up plan based on the patient's own condition.

Contents of the invention

In view of this, the embodiment of the present invention provides a method for survival prediction and individualized follow-up time assessment of gastric cancer patients based on molecular quantification of Helicobacter pylori DNA. , and through follow-up with a reasonable follow-up time, early detection of signs of metastasis or recurrence can improve the quality of life and survival time of gastric cancer patients.

In order to achieve the above object, the present invention mainly provides the following technical solutions:

On the one hand, the embodiment of the present invention provides a method for predicting the survival of patients with gastric cancer based on molecular quantification of Helicobacter pylori DNA, comprising the following steps:

1) Detect the relative infection amount of Helicobacter pylori (Hp) in the gastric cancer tissue;

2) Carry out prognosis assessment according to the relative infection amount of detected Helicobacter pylori;

Among them, the degree of poor prognosis increased with the increase of the relative infection amount of Helicobacter pylori.

Preferably, the detection of the relative infection amount of Helicobacter pylori adopts real-time fluorescent quantitative PCR (qRT-PCR).

Preferably, the reaction conditions are 40 cycles of pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 30 seconds.

As preferably, the relative infection amount of Helicobacter pylori is obtained by the following formula, the relative infection amount of Helicobacter pylori=2 ^-ΔΔCt , wherein ΔΔCt=(Ct sample purpose-Ct sample internal reference)-(Ct blank group purpose- Ct blank group internal reference), select the blank control group and take the negative results of methylene blue staining and common PCR as the standard; the relative infection amount of the blank control group is 1, that is, ≤1 is HP negative, >1 is Hp positive, and the experiment is repeated 3 times. take the average.

On the other hand, the embodiment of the present invention provides a method for evaluating the individualized follow-up time of gastric cancer patients based on molecular quantification of Helicobacter pylori DNA, including the following steps:

1) Detect the relative infection amount of Helicobacter pylori in gastric cancer tissue;

2) Formulate the reference range for the relative infection amount of Helicobacter pylori with poor prognosis in gastric cancer patients;

3) Compare the detected relative infection amount of Helicobacter pylori with the obtained reference range, and determine the follow-up plan.

Preferably, the reference range is as follows: patients with negative Helicobacter pylori infection, that is, when the relative infection amount of Helicobacter pylori is less than or equal to 1, no special follow-up time is required, according to international guidelines: follow-up once every 3-6 months, a total of 1 -2 years; then follow-up every 6-12 months for a total of 3-5 years; then once a year;

For patients with positive H. pylori infection, that is, when the relative infection amount of H. shorten accordingly:

1<When the relative infection amount of Helicobacter pylori<15, follow-up visits are made every 3-4 months within 3 years after surgery, and at least 3 times a year; follow-up visits are made every six months in the 4th-5th year;

The relative infection rate of Helicobacter pylori ≥ 15, follow-up every 2-3 months within 3 years after operation, at least 4 times a year; follow-up every 4 months in 4-5 years.

Preferably, the radical treatment regimen for Helicobacter pylori is as follows: standard triple therapy, ie PPI+ clarithromycin+ amoxicillin or PPI+ clarithromycin+ metronidazole, is used for 10 days or 14 days.

Preferably, the detection of the relative infection amount of Helicobacter pylori adopts real-time fluorescent quantitative PCR (qRT-PCR).

Preferably, the reaction conditions are 40 cycles of pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 30 seconds.

As preferably, the relative infection amount of Helicobacter pylori is obtained by the following formula, the relative infection amount of Helicobacter pylori=2 ^-ΔΔCt , wherein ΔΔCt=(Ct sample purpose-Ct sample internal reference)-(Ct blank group purpose- Ct blank group internal reference), select the blank control group and take the negative results of methylene blue staining and common PCR as the standard; the relative infection amount of the blank control group is 1, that is, ≤1 is HP negative, >1 is Hp positive, and the experiment is repeated 3 times. take the average.

Compared with prior art, the beneficial effect of the present invention is:

1. In the prediction method of the embodiment of the present invention, the real-time fluorescence quantitative detection technology of the relative infection amount of Hp is mature, and has been widely used in clinical detection and laboratory research in countries all over the world;

2. In the prediction method of the embodiment of the present invention, patients who detect Hp negative (about 25% according to our research) do not need special follow-up, which can greatly reduce the psychological burden of patients;

3. In the prediction method of the embodiment of the present invention, the patient’s follow-up time is adjusted according to the relative infection amount of H. , so the treatment effect is not good, leading to the deterioration of the condition, which is not conducive to the prognosis of patients, and realizes the high cost performance of tumor prevention and treatment;

4. In the prediction method of the embodiment of the present invention, the detection of Hp-positive patients can remind the patients to pay attention to their own physical changes, so as not to delay the disease, and lay the foundation for early detection and treatment of these diseases;

5. In the prediction method of the embodiment of the present invention, patients are divided into different grades according to Hp negative and positive, and the amount of infection, and different follow-up times are formulated according to different grades, that is, the higher the amount of Hp infection, the higher the follow-up interval. The shorter the time, not only can timely detect changes in the patient's condition, prolong the life of the patient, but also save a lot of manpower, material and financial resources for the individual and the country.

Description of drawings

FIG. 1 is a schematic flowchart of a follow-up example of an embodiment of the present invention.

Fig. 2A-Fig. 2D show the influence of different Hp detection methods on the prognosis of patients with gastric cancer by detecting the relative infection amount of Hp.

Figure 3A is the cumulative survival curve of patients with gastric cancer, and Figure 3B is the relationship between the amount of Hp infection in survival and death patients in different periods.

Detailed ways

The present invention will be described in further detail below in conjunction with specific examples, but not as a limitation of the present invention. In the following description, different "one embodiment" or "embodiment" do not necessarily refer to the same embodiment. Furthermore, the particular features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.

Referring to Figure 1, the method for predicting the survival of gastric cancer patients based on molecular quantification of Helicobacter pylori DNA includes the following steps:

1) Detect the relative infection amount of Helicobacter pylori (Hp) in the gastric cancer tissue;

2) Carry out prognosis assessment according to the relative infection amount of detected Helicobacter pylori;

Among them, the degree of poor prognosis increased with the increase of the relative infection amount of Helicobacter pylori.

In the embodiment of the present invention, the prognosis of gastric cancer patients is estimated by detecting the relative infection amount of Helicobacter pylori (Hp) in gastric cancer tissues. It can predict the survival rate and poor prognosis of gastric cancer patients more accurately. And on this basis, formulate the most reasonable follow-up time for patients, and through follow-up with reasonable follow-up time, early detection of signs of metastasis or recurrence can improve the quality of life and survival time of gastric cancer patients.

As a preference of the above embodiment, the detection of the relative infection amount of Helicobacter pylori adopts real-time fluorescent quantitative PCR (qRT-PCR). The method uses a fluorescent chemical substance to measure the total amount of products after each polymerase chain reaction (PCR) cycle, and has strong anti-interference and accurate prediction. This method is relatively mature and easy to operate.

The reaction system of this method is as follows:

The reaction conditions of the method are as follows: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 10s, 60°C annealing and extension for 30s, 40 cycles. The relative infection amount of Helicobacter pylori is obtained by the following formula, the relative infection amount of Helicobacter pylori=2- ^△△Ct , where △△Ct=(Ct sample object-Ct sample internal reference)-(Ct blank group object-Ct blank group internal reference), select the blank control group and take the negative results of methylene blue staining and common PCR as the standard; the relative infection amount of the blank control group is 1, that is, ≤1 is HP negative, >1 is Hp positive, and the experiment is repeated 3 times to take the average value.

On the other hand, referring to FIG. 1 , the embodiment of the present invention provides a method for evaluating the individualized follow-up time of gastric cancer patients based on molecular quantification of Helicobacter pylori DNA, including the following steps:

1) Detect the relative infection amount of Helicobacter pylori in gastric cancer tissue;

2) Formulate the reference range for the relative infection amount of Helicobacter pylori with poor prognosis in gastric cancer patients;

3) Compare the detected relative infection amount of Helicobacter pylori with the obtained reference range, and determine the follow-up plan.

The detection of the relative infection amount of Helicobacter pylori adopts the method of the above-mentioned examples.

As a preference for the above embodiment, the reference range is as follows: for patients with negative Helicobacter pylori infection, that is, when the relative infection amount of Helicobacter pylori is less than or equal to 1, no special follow-up time is required, according to international guidelines: follow-up every 3-6 months 1 once every 6-12 months for 3-5 years; then once a year;

For patients with positive H. pylori infection, that is, when the relative infection amount of H. shorten accordingly:

1<When the relative infection amount of Helicobacter pylori<15, follow-up visits are made every 3-4 months within 3 years after surgery, and at least 3 times a year; follow-up visits are made every six months in the 4th-5th year;

The relative infection rate of Helicobacter pylori ≥ 15, follow-up every 2-3 months within 3 years after operation, at least 4 times a year; follow-up every 4 months in 4-5 years.

As a preference of the above embodiment, the radical treatment plan for Helicobacter pylori is as follows: standard triple therapy, ie PPI+ clarithromycin+ amoxicillin or PPI+ clarithromycin+ metronidazole, the course of treatment is 10 days or 14 days.

Figure 2A-Figure 2D shows the influence of different Hp detection methods on the prognosis of gastric cancer patients by detecting the relative infection amount of Hp, in which Figure 2A is the methylene blue staining method; Figure 2B is the ordinary PCR detection method; Figure 2C and Figure 2D are the real-time fluorescent quantitative PCR detection As can be seen from the figure, there are many drawbacks such as the instability of Methylene blue staining and the many interference factors of ordinary PCR bands, so the embodiment of the present invention selects the real-time fluorescent quantitative PCR detection method as the Hp infection detection method. Figure 2C shows that the prognosis of Hp positive patients is worse than that of negative patients; Figure 2D shows that the higher the amount of Hp infection, the poorer the prognosis of the patients.

Take 118 Hp-positive patients with gastric cancer as an example:

1. First follow-up (3-5 years) Hp infection-positive patients with gastric cancer after gastric cancer surgery at a certain clinical stage (several years in a row) to obtain follow-up data.

2. Then enter the patient follow-up data and the measured Hp infection into the SPSS 17.0 statistical analysis software to make a database of the sample.

3. In the database, the data of half of the patients who died first (59 cases) were screened out, and the mean (mean, M) and standard deviation (standard deviation, SD) of the Hp infection amount of the 59 patients were analyzed in SPSS software. ) and the distribution of each Hp infection grade (Hp≤1, 1<Hp<15, Hp≥15). The prediction range of the Hp infection amount used to formulate the estimation of postoperative prognosis of gastric cancer patients was M=9.75, SD=6.45 in the 59 patients, and the obtained Hp infection amount ranged from 0.12 to 26.76.

4. Make the overall survival curve of gastric cancer patients (118 cases) in SPSS software, and find out the time when the remaining 50% surviving patients are the predicted half of the patients with Hp infection, that is, the 50% death time.

Figure 3A is the cumulative survival curve of gastric cancer patients, and Figure 3B is the relationship between the amount of Hp infection in survival and death patients in different periods, and Figure 3A is the cumulative survival (CumSurvival) curve of 118 gastric cancer patients, in which 50 patients were left at 24 months % of Hp infection-positive patients survived, and the range of Hp infection amount was (0.12,-26.76). Among the half patients who died first, the Hp infection positive rate was 79.4%, and the negative rate was 20.3% (while in the remaining half patients , Hp infection positive rate was 71.2%, negative rate was 28.8%). The significance of this prediction method is: if the Hp infection in a patient with gastric cancer is positive, the possibility of death within 24 months after surgery for the gastric cancer patient is 79.4%.

As shown in Figure 3B, among the 118 patients with gastric cancer, by the end of follow-up, the amount of Hp infection in the surviving patients was significantly lower than that in the dead patients (P=0.004, t test), and the mean ± standard deviation (M ± SD) of the survival group was 5.9 ± SD. 6.0, and the death group was 8.7±6.4, indicating that the higher the level of Hp infection in gastric cancer patients, the worse the survival of the patients. When 50% of patients died after operation, that is, at 24 months after operation, the M±SD of survival and death patients were 7.7±6.4 and 9.8±6.4, respectively, and there was no difference in the amount of Hp infection between the two groups at 24 months after operation; From the last 24 months to the end of the follow-up period, the M±SD of Hp infection in the survival group and the death group were 5.9±6.0 and 9.7±6.5, respectively, and the amount of Hp infection in the death group was significantly higher than that in the survival group (P=0.021).

Significant differences in Hp infection between survivors and dead patients appeared in the two periods of "0-124 months" and "24-124 months", indicating that before 24 months after surgery, there are many factors that affect the life and death of patients, including The severity of the disease, whether there is metastasis, whether the operation is thorough, the general physical condition of the patient, and even the toxic effects of radiotherapy and chemotherapy and the patient's treatment response, etc., Hp infection is one of them. At 24 months, patients who died due to multiple factors all died one after another. After 24 months, the influence of Hp infection gradually became apparent. Hp may be used as a marker to highlight the role of Hp-related lethal factors, and Hp may affect survival by interfering with immune responses. For example, the microenvironment formed by Hp-Stat3-IL17 may be more conducive to the growth, spread, and metastasis of cancer cells, which may affect survival. The analysis results of this design plan suggest that Hp infection as a marker may have a greater effect on the prognosis after 24 months, and the follow-up interval should be reasonably arranged.

Reference time frame for follow-up of patients with gastric cancer and suggested anti-Hp infection program

1. Patients with negative Hp infection do not need special follow-up time, according to international guidelines: follow-up once every 3-6 months, a total of 1-2 years; after that, follow-up once every 6-12 months, a total of 3-5 years; 1 time per year;

2. Hp infection-positive patients are recommended to undergo radical treatment of Hp after chemotherapy, and to monitor C14 breath test regularly. This plan proposes that the prognosis of Hp infection patients is poor, and it is related to the amount of Hp infection (as shown in Figure 2). According to our According to the follow-up data, the number of patients who died within 3 years accounted for 82.8% (72/87) of the total deaths. We suggest that the follow-up of patients with gastric cancer should be strengthened within 3 years after surgery, and the follow-up time should be shortened according to the amount of Hp infection: (1) 1<Hp<15, follow-up every 3-4 months within 3 years after operation, at least 3 times a year; follow-up every 6 months in the 4th-5 years; (2) Hp≥15, follow-up every 2-3 months within 3 years after operation Monthly follow-up, at least 4 times a year; 4-5 years follow-up every 4 months (as shown in Figure 1).

3. Suggested Hp eradication therapy for gastric cancer patients with positive Hp infection: the eradication rate of standard triple therapy (PPI+ clarithromycin+amoxicillin or PPI+ clarithromycin+metronidazole) has been lower or far lower than 80% , this program also recommends extending the course of standard triple therapy from 7 days to 10 or 14 days.

The method of the embodiment of the present invention is suitable for patients with gastric cancer to be divided into different grades according to Hp negative and positive, and the amount of infection, and to formulate different follow-up time according to different grades, that is, the higher the amount of Hp infection, the higher the follow-up interval. The shorter it is, the changes in the patient's condition can be detected in time, and the anti-Hp infection treatment in gastric cancer patients may improve the quality of life of patients to a certain extent and prolong the life span of patients. Individualized follow-up plans are proposed for patients with different Hp infection conditions, making full use of medical resources, avoiding waste, and maximizing medical cost-effectiveness. Therefore, in theory, it can save human, material and financial costs for individuals, the country and society. Proposing an individualized follow-up plan for patients with different Hp infection conditions can help gastric cancer patients detect disease progression in time to the greatest extent, receive timely treatment, prolong survival time, and improve quality of life.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (10)

1., based on the patients with gastric cancer Prediction of survival method that Hp DNA molecular is quantitative, it is characterized in that, comprise the steps:

1) the relative infective dose of Hp in stomach organization is detected;

2) prognosis evaluation is carried out according to the relative infective dose of the Hp detected;

Wherein prognosis mala degree increases with the rising of the relative infective dose of Hp.

2. method according to claim 1, is characterized in that, the detection of the relative infective dose of Hp adopts real-time fluorescence quantitative PCR.

3. method according to claim 1, is characterized in that, reaction conditions is, 95 DEG C of denaturation 5min, 95 DEG C of sex change 10s, and 60 DEG C of annealing extend 30s, 40 circulations.

4. method according to claim 1, is characterized in that, the relative infective dose of Hp is obtained by following formula, relative infective dose=2 of Hp ^{-△ △ Ct}, wherein △ △ Ct=(Ct sample object-Ct sample internal reference)-(Ct blank group object-Ct blank group internal reference), selects blank group to be negative findings for standard with methylene blue dyeing and regular-PCR; The relative infective dose of blank group is 1, namely≤1 is that HP is negative, and > 1 is that Hp is positive, and experiment repetition is averaged for 3 times.

5., based on the patients with gastric cancer individuation follow up time appraisal procedure that Hp DNA molecular is quantitative, it is characterized in that, comprise the steps:

1) the relative infective dose of Hp in stomach organization is detected;

2) term of reference of the relative infective dose of patients with gastric cancer prognosis mala Hp is formulated;

3) the relative infective dose of the Hp of detection and gained term of reference are compared, determine the scheme of following up a case by regular visits to.

6. method according to claim 5, is characterized in that, described term of reference is as follows: Helicobacter pylori infection negative patient, namely when the relative infective dose of Hp is less than or equal to 1, without the need to special follow up time, undertaken by international guidelines: within every 3-6 month, follow up a case by regular visits to 1 time, 1-2 altogether; Within every 6-12 month afterwards, follow up a case by regular visits to 1 time, 3-5 altogether; Annual 1 time later;

Helicobacter pylori infection positive patient, namely when the relative infective dose of Hp is greater than 1, after chemotherapy, recommend the radical cure treatment carrying out Hp, the experiment and periodic monitoring C14 exhales, and follow up time should carry out corresponding shortening with Hp relative to infective dose:

During 1 < Hp relative infective dose < 15, within postoperative 3 years every 3-4 month, follow up a case by regular visits to once, at least follow up a case by regular visits to 3 times every year; 4-5 follows up a case by regular visits to once every half a year;

Relative infective dose >=15 of Hp, follow up a case by regular visits to once, at least follow up a case by regular visits to 4 times every year in postoperative 3 years every 2-3 month; 4-5 follows up a case by regular visits to once for every 4 months.

7. method according to claim 6, is characterized in that, the radical cure treatment plan of Hp is as follows: adopt standard triple therapy, i.e. PPI+ clarithromycin+amoxycilline Trihydrate bp or PPI+ clarithromycin+metronidazole, the course for the treatment of is 10 days or 14 days.

8. method according to claim 5, is characterized in that, the detection of the relative infective dose of Hp adopts real-time fluorescence quantitative PCR.

9. method according to claim 8, is characterized in that, reaction conditions is, 95 DEG C of denaturation 5min, 95 DEG C of sex change 10s, and 60 DEG C of annealing extend 30s, 40 circulations.

10. method according to claim 8, is characterized in that, the relative infective dose of Hp is obtained by following formula, relative infective dose=2 of Hp ^{-△ △ Ct}, wherein △ △ Ct=(Ct sample object-Ct sample internal reference)-(Ct blank group object-Ct blank group internal reference), selects blank group to be negative findings for standard with methylene blue dyeing and regular-PCR; The relative infective dose of blank group is 1, namely≤1 is that HP is negative, and > 1 is that Hp is positive, and experiment repetition is averaged for 3 times.