Liquid Biopsy, author: doctor of medical sciences Jakub Dobruch, Department of Urology Medical Centre of Postgraduate Education Department of Urology, the European Health Centre in Otwock.

 

Introduction

According to the World Health Organization (www.globocan.iarc.fr) in 2012, the cancer was detected in 7.410.376 men. At the same time because of them died 4.653.385. It is estimated that over the next two decades, the number of patients with cancer will increase to almost double and cancer will become the primary and fundamental problem of civilization.

Cancer “Urology” represent almost one-quarter of the malignant tumors observed in men. The most common of these is the cancer of the glandular prostate (PCa-prostate cancer). In 2012, it has been detected in 1.094.873 men, and there has been only 307481 deaths because of it. PCA ranks second in terms of incidence and the fifth in terms of mortality among all malignant tumors. It has been calculated that over the last 5 years diagnosed with PCa live 3.857.500 men. This phenomenon is explained by, inter alia, the slow development of the PCa. It is estimated that the tumour doubles its weight on average after 4 years.  Introduction General the determination of prostate-specific antigen (PSA) has been a recognition of the PCa. The increase has number of patients for whom treatment with radical, the number of patients with advanced PCa has been reduced. In the United States the mortality rate specific for PCa is systematically reduced since 1995. Unfortunately, it was found that with the spread of the determination of PSA and earlier detection of advanced form of cancer is observed an unexpectedly large nadrozpoznawalność PCa, i.e. the detection of cancers that were it not for the DOG would not be detected. About men with such diagnosis says that they will die of cancer and not because of it. Not surprisingly a very large number of patients, who for many years living with the PCa and often die from other than cancer. Nadrozpoznawalność PCa was the primary argument used by the American institution of the USPSTF (US Preventive Services Task Force) resurgent PSA.

Recently, the “medical market” there are medicines that affect the survival of patients with the advanced form of the PCa. The mechanism of action of individual drugs, however, is different, and the order of their use is unknown. Abiraterone and enzalutamide are hormone treatment. Cabazitaxel is a cystostatycznym and the isotope Radium has an effect on the cells of the PCa via Alpha radiation. Sipuleucel is a form of treatment immunomodulującego. It is not known which of them will bring the most beneficial in the Group of men suffering from the PCa in a similar State. Molecular studies, the results of which have been published recently (The evolutionary history of lethal metastatic prostate cancer. Gundem G et al. Nature 2015; 520:353-357.) yielded the basis for considerations on the alleged effectiveness of drugs with different mechanism of action in individual cases. From deceased persons due to the PCa downloaded portions of each outbreak of cancer and genetic profile building them cells. It was noted that the cells that make up the various outbreaks have specific mutations not present within other metastases. It was called “leaf mutations” (LF). Mutations that are common to all metastases, that is to say, their presence has been detected in the cells that build all identified outbreak was named “trunc mutations” (TM). And those that occurred in at least two areas called “branch mutations (BM). It is understood that the drugs, which point corresponds to the changes caused by the TM will be effective with respect to each outbreak of PCa. On the contrary, those which will relate only to LF or BM will be characterised by less effective. Are many cases of progression of PCa happening that in “background” supposedly man’s hormonally treated effectively. About its effectiveness provide has little PSA. Meanwhile, imaging studies document the growing number of metastases or increasing their size. The results of that study explain this phenomenon. In clinical practice, we use tools whose purpose is to detect macro-consequences of biological process ongoing for quite some time. A good example is the dog. It is known that the re-activation of a biochemical After Prostatectomy radical ahead of clinical progression of a few years. Unfortunately, with the development of the PCa decreases the importance of PSA as a marker of cancer. Little PSA levels after radical prostatectomy does not preclude a future recurrence.

These observations are the challenges with which the measured modern uro-Oncology. In the case of the PCa, PSA level does not mean that there is a need for a biopsy of the prostate. The correct result of the first biopsy does not mean that you do not need to perform the next. Excision or destruction of radical prostate does not necessarily indicate a doszczętności treatment despite typeable values. Similarly, low levels of PSA don’t always suggests the lack of progression of the PCa in the course of hormone treatment. Cancer patients resistant to eradicate (CRPC) the beneficial effect of treatment immunomodulującego or treatment involving the administration of the radioisotope of Radium does not correlate with the concentration of PSA. The individual has metastasized in the CRPC may respond differently to treatment. Download snippets from each of them and the assessment of the genetic profile of cells building them is not justified. One of the methods subject to intensive research, which could resolve some of the dilemmas.

LIQUID BIOPSY

The term biopsy was made as a result of two words – bios meaning life, and opsis-watching, looking. It was introduced in the mid-19th century to describe the microscopic examination of tissue taken from a living individual in contrast to an autopsy or post-mortem examinations. Fluid biopsy (LB) is the equivalent of the classic biopsy but the source of the test cells are the blood. In fact, it is assumed that the LB means the examination of the genetic material isolated from blood in search of a specific disease/phenomenon. Its source can be cancer cells circulating in the blood (CTC-circulating tumor cells), while LB also refers to a free analysis, current outside the cells genetic material (cfDNA-circulating free DNA). In healthy subjects, the number of cfDNA is very small. Significantly increased in patients with cancer. Cancer cells release your genetic material during apoptosis or necrosis but also as a result of the secretion of the small air sacs called egzosomami DNA from cancer cells is called ctDNA (circulating tumor DNA). It is believed that the analysis of ctDNA reveals genetic disorders present in each cancer outbreaks, although the contribution of individual changes to the total pool of ctDNA is certainly different. In addition to the cfDNA genetic material of blood is RNA, including microRNAs. Study of ctDNA can therefore provide reliable information about the State in which the cancer regardless of the nature and type of treatment.

Modern molecular biology techniques allow relatively quickly isolate and examine the genetic material of the cancer cells. The first reports were, however, not Oncology but prenatal diagnosis [Lun, Tsui NB, Chan KC, Leung YOU, Lau TK, Charoenkwan P, et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relativemutation dosage on DNA in maternal plasma. Proc Natl Acad Sci U S A 2008; 105:19920-5]. It turned out that the LB you can detect chromosomal trisomie 21, 18 and 13 in fetuses, and thus replace invasive amniopunkcję biopsy. In Oncology, in 2014, the first evidence for clinical utility determination of “free” of genetic material (cfDNA) in the blood, the relative amount of correlated with survival of patients in a two-year follow-up [Bettegowda, c., Sausen, m., Leary, R.J., Kinde, i., et al., Detection of circulating tumor DNA in early-and late-stage human malignancies. SCI Transl. Med 20.

Diagram of a biopsy of the smooth images figure 1. cfDNA isolates of plasma formed after centrifuging, 10 ml of blood that you previously downloaded from the test. After extracting the genetic material followed by its sequencing and comparison with normal human genetic material. Thanks to know which part of the genome is inadequately represented in the test. Analysis of cfDNA can be carried out at least bi-directional searching for known, previously a specific mutation (qualitative study) or by analyzing the total amount of all genetic disorders relative to a healthy state (Figure 2). In the latter case, the result of LB is presented through a pointer CNI (copy number instability). CNI reflects the number of times a given DNA region compared to the sample taken earlier from the same patient or sample taken from a person healthy, which the result was assembled in the manufacturer’s test. The result of the comparison for the enumeration of CNI can be represented graphically (circos plot-circus scheme.). Figure 3 provides an example of the result of LB in 5 healthy people (3A).

Apply a liquid biopsy in urology in the first paragraphs of this study mentioned the diagnosis of prostate cancer. Eventually, the PCa will detect based on biopsy of the prostate. Indications for its implementation, regardless of the number of previously travelled biopsy, are still the subject of numerous works. Per year in the United States carried out more than a million biopsies, the PCa is diagnosed in approximately 200 thousand. men. The majority of men after the biopsy merely observed severe bleeding from the urinary tract, or from the road. What twenty, however, requires hospitalization and its the reason most of them are septic complications. It is understood that looking for methods that would reduce the number of unnecessary biopsy performed. Recently published results of a study which evaluated the importance of LB in the Group of patients with PCa and the men healthy. It was noted that a factor of CNI was significantly higher in 204 patients with PCa (44; 95% CI: 30-67) than in the rest of the 207 men (9; 95% CI: 5-1.12) not affected by this cancer. After the analysis of the results of this study have been developed commercially available test (Second OpinionTM Prostate Cancer Evaluation) that can be used in determining indication for prostatic biopsy. The accuracy of this test in the diagnosis of the PCa exceeds 90%. This means that the Second OpinionTM may be helpful in those who want to make sure the need to repeat the biopsy in a situation in which there are still classic to it.

Figure 1. Diagram of a liquid biopsy (by CHRONIX BIOMEDICAL; www.chronixbiomedical.com/connect/technology)

aaaaaaaaaaaaaasssssssssssssssssssss

 

Figure 2. Diagram that shows the types of cfDNA analysis and its interpretations (by CHRONIX BIOMEDICAL; www.chronixbiomedical.com/connect/technology)

pu-2

Figure 3. Diagram showing the result of LB in the five “healthy” people (A) and in 5 patients with PCa (B). Areas of the genome that vary from a healthy state is marked with dots green (more repetitions of a given piece of DNA than in normally-gains) or red (less repetitions of a given piece of DNA than in normally-losses).

2016-11-09-09_23_51-clinichal-chemistry_prostate-cancer_2015-pdf

Plans for further development

Setting indications for biopsy is only one of many possible uses of LB in relation to patients with PCa. Prostate cancer is one example out of many cancers for which the liquid biopsy may be useful. Table 1 includes the potential use of LB in the daily practice of uro-Oncology. I’m convinced that soon we will begin to discuss about them.

pu3

A summary
Liquid Biopsy is an example of a new kind of research that could revolutionize cancer detection and treatment of patients affected. Is it the blood test, which can be helpful in determining the indication for prostatic biopsy. It is possible that in the near future, serially repeated biopsy fluid will be used for the election of a new treatment and monitor its effectiveness.

Dr hab. n. med. Jakub Dobruch, FEBU. Urological Review, The Polish Association of Urology bimonthly magazine. No. 5/2016