imaging examination and tumor biopsy are the main methods of clinical tumor diagnosis and monitoring. These examinations are indeed very effective before treatment, but frequent and repeated imaging examinations and biopsy for postoperative monitoring are impractical. Liquid biopsy (Liquid biopsy) technology refers to a technology that realizes tumor monitoring by collecting and testing human body fluid samples. Although urine, feces, cerebrospinal fluid, saliva, ascites and other body fluids are potential sources of tumor-derived substances, blood is still It is the main liquid biopsy sample.
Compared with traditional tumor detection methods, liquid biopsy technology has the characteristics of flexible sampling method, non-invasive and convenient, repeated sampling, and high compliance. Therefore, it may be used in tumor screening , efficacy testing, medication Guidance and other aspects play an important role.
Liquid biopsy and HPV virus
Liquid biopsy samples contain a variety of substances that carry tumor-related information, including circulating tumor cells, circulating DNA, and ) and RNA , exosomes released by tumor cells, etc. In fact, normal people’s blood also contains some fragmented DNA, which we call cfDNA. Scientists believe that this cfDNA is released into the blood through apoptosis or necrosis of cells. After entering the blood, cfDNA will be quickly released. Cleared from the blood, half-life is less than 1 hour. The cfDNA content of normal cells in the plasma of healthy people is relatively low.
and ctDNA refers to the DNA released from tumor cells,There are some differences in sequence characteristics from normal cfDNA. Therefore, by sequencing ctDNA in plasma, you can learn a lot of tumor-related information. , especially for tumors caused by certain virus infections, often contains a lot of cfDNA. Virus-specific genetic information.
Human papillomavirus (human papillomavirus, HPV ) is a virus that infects humans widely. Most HPV infections are harmless to the human body and can be cleared by the body’s immune system within two years. However, continuous high-risk HPV infection can cause a variety of cancers, posing a serious threat to the health of all human beings.
In the United States, 14 million people are infected with HPV every year. Most people will be infected with HPV at least once in their lives. The total medical expenses related to HPV-related diseases (warts, precancerous lesions, cancer) are as high as 8 billion U.S. dollars. In 2009, there were an estimated 34,788 new HPV-related cancers in the United States, including cervical cancer, anal cancer, oropharyngeal cancer and vulvar cancer, among which the most common are oropharyngeal cancer and cervical cancer. A large part of it is caused by HPV types 16 and 18.
Figure 1 HPV 16 virus particle structure
In tumors caused by HPV virus, cfDNA can be distinguished from normal cells through the virus genome. Therefore, cfDNA can be distinguished from normal cells. The cfDNA in the patient’s plasma is tested to understand the patient’s tumor burden.
ctDNA can be used as an indicator of tumor monitoring caused by HPV.The treatment effect of HPV-induced oral squamous cell carcinoma is far better than that of non-HPV-infected oral squamous cell carcinoma. However, there are still 10%-25% of patients who will relapse after treatment. The dynamic changes of CtDNA levels are related to tumor metastasis or the patient's response to treatment.
A study enrolled 115 patients with HPV-related oral squamous cell carcinoma, all patients received therapeutic radiotherapy and chemotherapy (CRT), and the patients received 3 months of therapeutic radiotherapy and chemotherapy after PET-CT , after For 1 to 2 years, clinical evaluations will be conducted every 2 to 4 months, and evaluations will be conducted every 6 months for 3 to 5 years. Collect blood samples from patients every 6 to 9 months, and use multiple analysis digital PCR to analyze HPV-related ctDNA in plasma. The median follow-up time for all patients was 23 months.
Figure 2 DOI: 10.1200/JCO.19.02444
Among them, 15 patients had no recurrence of the disease after treatment, and 87 patients had no recurrence after treatment. ctDNA was positive, none of these patients had recurrence. 28 patients showed ctDNA positive in the post-treatment monitoring, 15 of them relapsed, and 16 patients showed positive blood tests for 2 consecutive times, and all 15 patients who relapsed were among them. The average time between HPV-related ctDNA positive and biopsy confirmed recurrence was 3.9 months.
This study confirms that HPV-related ctDNA detection is feasible for monitoring the recurrence of HPV-related tumors. Other similar studies have also verified this conclusion.
However, the current ctDNA detection mostly uses quantitative PCR (qPCR) and digital PCR (dPCR), and there is a problem of insufficient accuracy. When the number of DNA copies is less than 1,There are some difficulties in using these methods to detect. How to improve the sensitivity of detection is a problem that scientists have been paying attention to.
Ultra-high HPV ctDNA detection helps tumor liquid biopsy An article entitled HPV sequencing facilitates ultrasensitive detection of HPV circulating tumor DNA. This study provides an ultra-sensitive HPV-specific ctDNA detection method. HPV-seq greatly improves the sensitivity of detection and can be negative in dPCR CtDNA as low as 0.03 copies/ml was detected in samples after treatment.
Figure 3 DOI: 10.1158/1078-0432.CCR-19-2384
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HPV-seq uses a double-strand hybrid capture method to enrich viral DNA from the sequence library, which can provide qualitative and quantitative information about the ctDNA of HPV-related tumor patients. Sensitive, and can provide ctDNA fragment length and HPV genotype information.Each full-length viral genome can produce approximately 50 different cfDNA fragments.
The results show that HPV-seq results are highly correlated with dPCR, and ctDNA that cannot be detected by dPCR can be detected. HPV ctDNA detected after treatment is related to poor disease-free survival, Its sensitivity to disease recurrence prediction is 100%, and its specificity is 67%. The accuracy of HPV type identification reaches 100%. HPV ctDNA fragments are always shorter in size than non-cancer-derived cfDNA fragments, and a fixed cfDNA fragment group pattern is observed in the HPV genome.
In short, the HPV-seq proposed in this study is indeed an effective ctDNA quantification method, superior to traditional dPCR.
Through this ultra-sensitive detection method, it is possible to quickly find out whether the patient is prone to tumor recurrence, and to take action earlier is of great significance for improving the prognosis of the patient. At the same time, this technology can also be used for the detection and treatment of other cancers caused by specific pathogens, benefiting more cancer patients.
Author: Yin Qilei
Source: Clinical Frontline
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