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Wydawca: Jolanta Jedlińska

Redaktor prowadzący: Beata Bednarczuk

Redaktor: Marek Darewski

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Warszawa 2020

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ISBN 978-83-200-6196-3

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About the Authors

María Dolores Ayllón
Unit of Hepatobiliary Surgery and Liver Transplantation. IMIBIC. CIBERehd, University Hospital Reina Sofía, University of Cordoba, Córdoba, Spain

Javier Briceño
Unit of Hepatobiliary Surgery and Liver Transplantation. IMIBIC. CIBERehd, University Hospital Reina Sofía, University of Cordoba, Córdoba, Spain

Ruben Ciria
Unit of Hepatobiliary Surgery and Liver Transplantation. IMIBIC. CIBERehd, University Hospital Reina Sofía, University of Cordoba, Córdoba, Spain

Michał Grąt
Department of General, Transplant and Liver Surgery
Medical University of Warsaw, Warsaw, Poland

Aleksandra Grela-Wojewoda
Deaprtment of Clinical Oncology, Maria Skłodowska-Curie National Research Institute of Oncology, Kraków Branch, Kraków, Poland

David A. Iannitti
Division of Hepato-Pancreato-Biliary Surgery, Department of Surgery, Carolinas Medical Center, Charlotte, NC, USA

Andrzej Jasiewicz Laboratory of Molecular Diagnostics, Cytogenetics and Flow Cytometry
Specialist Hospital in Brzozów, Brzozów, Poland

Piotr Kalinowski
Department of General, Transplant and Liver Surgery
Medical University of Warsaw, Warsaw, Poland

Andrzej L. Komorowski
Chair of General Surgery University of Rzeszów, College of Medical Science, Rzeszów, Poland

Artur Kowalik
Department of Molecular Diagnostics, Holycross Cancer Center, Kielce, Poland Division of Medical Biology, Institute of Biology, Jan Kochanowski University, Kielce, Poland

Maciej Krasnodębski
Department of General, Transplant and Liver Surgery Medical University of Warsaw, Warsaw, Poland

Maksymilian Kruczała
Department of Oncology University of Rzeszów, College of Medical Science, Rzeszów, Poland

Andrii Lukashenko
Department of Minimally Invasive and Endoscopic Surgery, Interventional Radiology National Cancer Institute, Kiev, Ukraine

Marco V. Marino
Department of General and Emergency Surgery, Azienda Ospedaliera, Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy

Łukasz Masior
Department of General, Vascular and Oncological Surgery Medical University of Warsaw, Warsaw, Poland

Barbara Niemiec
School of Medicine Collegium Medicum, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

Joanna Niemiec
Institute of Medical Sciences University of Rzeszów, College of Medical Science, Rzeszów, Poland Laboratory of Molecular Diagnostics, Cytogenetics and Flow Cytometry Specialist Hospital in Brzozów, Brzozów, Poland

Tomasz Pawlik
Department of Radiology Saint Raphael Hospital, Kraków, Poland

Olexii Potapov
Department of Minimally Invasive Surgery, Center for Innovative Medical Technologies of the NAS of Ukraine, Kiev, Ukraine

Mirosława Püsküllüoğlu
Department of Medical Education, Jagiellonian University Medical College, Kraków, Poland

Patrick Salibi
Division of Hepato-Pancreato-Biliary Surgery, Department of Surgery, Carolinas Medical Center, Charlotte, NC, USA

Jesse K. Sulzer, MD, PhD
Division of Hepato-Pancreato-Biliary Surgery, Department of Surgery, Carolinas Medical Center, Charlotte, NC, USA

Go Wakabayashi
Center for Advanced Treatment of Hepatobiliary and Pancreatic Diseases. Ageo Central General Hospital. Kashiwaza, Ageo City, Japan

Preface

Liver tumor management has evolved over the last 20 years. For both primary and metastatic liver malignancies, there is a large number of alternatives that can lead to improved outcomes. Considering that all patients need a multidisciplinary ap-proach, oncologists, physicians, surgeons, radiologists and pathologists really need to create multidisciplinary teams in which, on a case-by-case basis, optimal outcomes should be the main endpoint. The surgical approach has arisen as the cornerstone of all these advances as, at some point, full recovery requires the malignant areas to be fully cleared.

Liver metastases have traditionally been considered as the last stage of onco-logical disease. Patients would be referred for palliative chemotherapy and just a few would have had “miraculous” responses due to unknown circumstances. Since the be-ginning of the first decade of the 21st century, the scenario for the management of these patients has dramatically changed. The early series of Rene Adam, which demonstrated improved outcomes after surgical resection for colorectal liver metasta-ses, have now become standard practice. The surgical approach has demonstrated that for some patients it turns into a feasible and safe alternative.

In the modern management of liver tumors, oncologic management has switched from the maximum tolerable strategy to the minimum effective one. In other words, less is more in the oncological global approach to our patients. Nowadays, the approach to liver malignancies, including metastatic tumors, has a new ally, which is the minimally invasive approach. Several laparoscopic and robotic series have demon-strated that a less aggressive approach than the classic open one may offer patients improved outcomes with reduced complications. Performing an optimal resection with no complications and adjusting adequate timing, type and dosage of chemotherapy, leads to improved outcomes. Multidisciplinary teams nowadays have to strike a bal-ance between individualized biological-based chemotherapy, percutaneous strategies, surgical approaches (from minimally invasive procedures to radical extreme resec-tions or two-stage surgeries), and even liver transplantation in very carefully selected cases.

This book offers a whole range of modern strategies for managing secondary liver malignancies. From diagnosis to non-operative strategies, with the emphasis on operative procedures, there are many possibilities for the patient in terms of treat-ment pathways. All of them are important and necessary. For the benefit of our pa-tients, a deep knowledge of current evidence may lead to improved outcomes, a better quality of life, optimization of resources and a proper application of evidence-based medicine.

Go Wakabayashi. MD, PhD. FACS

Director, Center for Advanced Treatment of Hepatobiliary and Pancreatic Diseases, Chief, Surgical Services. Ageo Central General Hospital. 1-10-10 Kashiwaza. Ageo City. Japan.

Sample

About the Authors

Preface

1. Genetic profile of colorectal cancer liver metastases
– Joanna Niemiec, Andrzej Jasiewicz, Barbara Niemiec, Artur Kowalik

2. Liver imaging techniques
– Tomasz Pawlik

3. Strategies for colorectal cancer metastasis in the liver
– Andrzej L. Komorowski

4. Strategies for non-colorectal cancer metastasis in the liver
– Piotr Kalinowski

5. Metastatic colorectal cancer (mCRC) systemic therapy and management strategy
– Aleksandra Grela-Wojewoda, Mirosława Püsküllüoğlu, Maksymilian Kruczała, Barbara Niemiec

6. Open approach to liver tumours
– Piotr Kalinowski

7. ALPPS technique for the treatment of liver metastasis
– Łukasz Masior, Michał Grąt

8. Liver transplantation in the treatment of metastatic liver disease
– Maciej Krasnodębski, Michał Grąt

9. Laparoscopic approach to liver metastases
– Ruben Ciria, María Dolores Ayllón, Javier Briceño

10. Thermal Ablation for Metastatic Liver Tumors
– Jesse K. Sulzer, Patrick Salibi, David A. Iannitti

11. Robotic approach to liver lesions
– Marco Vito Marino

12. Novel training approaches to the surgical management of liver lesions
– Olexii Potapov, Andrzej L. Komorowski

13. Major complications in the management of liver lesions
– Andrii Lukashenko

Footnotes

1.
Genetic profile of colorectal cancer liver metastases
Joanna Niemiec, Andrzej Jasiewicz, Barbara Niemiec, Artur Kowalik

INTRODUCTION

During progression of colorectal cancer, the acquisition of metastatic potential and the cancer cell transformation progress in parallel and influence genetic profile of future metastases of colorectal cancer (CRC) to liver. The first event in cancer progression (initiation) is primary mutation (or mutations), which lead to a cellular proliferation and impaired program of cell differentiation. During the first step of cancerogenesis (stage 0 colorectal carcinoma or carcinoma in situ) abnormal cells are found only in the innermost layer (mucosa) of colon and/or rectal wall. The next steps of cancer progression are a consequence of new genetic and epigenetic events, acquired by cancer cell, leading to changes in their morphology and function and to switch-on the programme of cancer stroma remodelling. The above-mentioned processes and a plethora of interactions, could be divided into the following steps: disaggregation of malignant cells (detachment from the primary tumour, migration and spread into nearby normal tissue and disruption of tissue architecture), intravasation (penetration of the wall of blood or lymphatic vessel and entrance into bloodstream), evasion of the immune system (cell survival in the hostile environment of the systemic circulation). The process of CRC cells extravasation and liver parenchyma infiltration is divided into four interrelated phases: (1) microvascular phase with arrest of circulating malignant cells infiltrating the liver within the hepatic sinusoids and with their adhesion to the endothelium, which is followed by extravasation, (2) interlobular micrometastasis phase with evasion of host defences; (3) angiogenic micrometastasis phase; and (4) established hepatic metastasis (colonisation of target organ) [1]. At each step of cancerogenesis and metastatic cascade selection of cancer cell clones takes pace. This process shapes the genetic makeup of secondary tumours, and is responsible for its clinical appearance. The disease-free interval between primary tumour and relapse, which is frequently observed in the clinic, results from presence of dormant micrometastases in multiple tissues. The length of the aforementioned interval depends on the process of transformation from micrometastases into detectable macrometastases [1–19].

1.1. ACQUISITION OF METASTATIC CAPACITY – GENETIC PATHWAYS OF COLORECTAL CANCER TUMORGENESIS

The knowledge about driver[1]/hereditary mutations and further mutational cascade is very important for genetic counselling, especially in case of families with high-risk of CRC, because it helps to plan prophylactic strategies and predict clinical course of the disease (Table 1.1) [20–30].

1.1.1. CANCEROGENESIS MODELS AND CLASSIFICATIONS OF COLORECTAL CANCER

The ideal model of cancer progression should explain both dynamic of cancer growth or metastases formation and bring some predictive information. In case of CRC such model has not been proposed. Although molecular markers (testing KRAS, NRAS, BRAF gene status) are the most important in selection of molecularly targeted therapy for metastatic CRC patients (see below).

These pathways of CRC development may be determined on the basis of its molecular features: (1) the pathway activated by mutations in APC (Adenomatous polyposis coli), characterized by chromosomal instable (CIN)[2] phenotype, (2) the pathway initiated by mutations in DNA mismatch repair genes (MMR)[3], leading to a DNA microsatellite instability (MSI) [4] phenotype, (ii) the pathway initiated by global genome hypermethylation, resulting in switch-off of the tumour suppressor genes, indicated as CpG island methylator phenotype (CIMP)[5] [21, 25].

According to CRC sequence model, proposed by Fearon and Vogelstein, certain mutations are directly related to different stages of tumour development. The best example of this model is cancerogenesis on the basis pathway activated by mutations in APC[6] mutation which is depicted on Figure 1.1 [20–24]. Hereditary or somatic APC mutation is an initial/driver mutagenic event (ie. tumour initiation), leading to differentiation defects and other gene mutations (TP53, KRAS, TP53, DCC: Deleted in Colorectal Cancer, BRAF, TGF-b: transforming growth factor β, PI3KCA: Phosphatidylinositol 3-kinase, GNAS: Guanine Nucleotide Binding Protein: G Protein, AKT1, ARID1A and SOX9, etc.). The above-mentioned additional mutagenic and/or epigenetic events, collectively known as tumour promotion, are responsible for the proliferation of mutated cell clone or clones, growth[7] and progression of colorectal cancer. CRC developed based on APC mutation are characterised by chromosomal instability (CIN)[8] and as a consequence of CIN, by is the loss of tumour suppressor genes[9] (Figure 1.1). Hereditary APC mutation is responsible for Familial Adenomatous Polyposis [21]. In the Table 1.1 other hereditary mutations related to CRC risk are presented [20–24]. The most frequently CRC develops on the basis of hereditary or somatic APC mutations (60–56%). In case of hereditary APC mutation the risk of CRC is 2–4% (Table 1.1).

In the pathway initiated by mutations in DNA MMR, related to MSI phenotype, called the Lynch syndrome (hereditary non-polyposis coli), initial/driver mutagenic events are mutations in MMR, i.e..: MLH1, MSH2, PMS2, MSH6 (Table1.1). In MMR-related CRC, about 20% of cases results from germline mutations, while about 80% of cases from epigenetic silencing. Lynch syndrome is less frequent (Table 1.1) than Familial Adenomatous Polyposis, Patients with CRC characterised by MSI have a better prognosis than microsatellite stable patients. Moreover it was shown that MMR status could predict response to immune check point inhibitors[10] in metastatic CRC patients [21, 25].

It was shown that CRC developed on the basis of the pathway initiated by global genome hypermethylation (CIMP) present characteristic clinico-pathological features, i.e.: the high rate of mutations (KRAS or BRAF), wild type TP53, proximal colon location, mucinous histological type, higher age at diagnosis, poor differentiation, and higher occurrence in female gender and older patients. Several of the above-mentioned are also related to MSI. There are conflicting data concerning prognostic and predictive value of CIMP [21, 25]

An alternative to the above-mentioned hypotheses of cancer development and progression, is the “Big Bang” hypothesis of colon cancer evolution. This model was proposed after experiments with extensive multi-regional tumour sampling and reconstruction of topographic distributions of public (acquired before growth) and private (acquired during growth) mutations. “Big Bang” model is based on the concept that the majority of genomic alterations accumulate during the early stages of carcinogenesis, before the development of a big tumoural mass. It was shown, and that even small polyps have multiple pathogenic mutations in crucial driver genes (APC, KRAS/NRAS, BRAF, FBXW7 and TP53). As a consequence, human colorectal tumours grow as single co-clonal expansions, where most of the mutational intratumour heterogeneity (ITH), commonly observed in human tumours, originates from the first few divisions of growth. In this model, tumour growth is an evolutionary process, while many “detectable” private mutations in the final tumour originate from the first few divisions. It was calculated, for a simple exponential expansion at a diploid locus, that frequencies in the final tumour are 50% for public mutations, 25% when a private mutation occurs during the first division, 12.5% during the second division and 6.25% during the third division. [20–24].

Based on the gene expression studies 4 molecular subtypes (CMS) of colorectal cancer has been identified [21]:

1. CMS1 (hypermutated): driver mutation: BRAF; characterized by: strong immune activation, high PD1 activation, sensitivity to immune check inhibitors, intermediate prognosis (worse after relapse), diagnosed in 14% of cases, at less advanced stages (I–II).

2. CMS2 (canonical): driver mutation: APC; characterized by: high expression of WNT and MYC targets; epithelial differentiation, very low immune infiltration and activation, sensitivity to anti-EGFR MAbs, good prognosis (superior survival rates also after relapse), diagnosed in 40% of cases.

3. CMS3 (catabolic): driver mutation: KRAS; characterized by: upregulation of multiple metabolic signatures (sugar, amino acids, fatty acids, nitrogen), epithelial differentiation, low immune infiltration and activation, intermediate prognosis, diagnosed in 10% of cases.

4. CMS4 (mesenchymal): genetic/molecular driver: miR-200 (downregulation) TGF-b pathway; characterized by: upregulation of genes implicated in EMT, activation of TGF-b signalling and VEGF/VEGFR and integrin pathways, angiogenesis, matrix remodelling pathways and the complement-mediated inflammatory system, high stromal infiltration, postulated sensitivity to PDGFRA, KIT, HSP90, poor prognosis (worse overall survival and relapse-free survival), tend to be diagnosed at more advanced stages (III–IV).

The above-mentioned classification could be helpful in treatment selection and improvement of overall survival of CRC patients.

Figure 1.1.

The genetical mechanism of APC gene inactivation. Germinal (hereditary) APC mutation is present in each somatic cell in one of homologous chromosomes. In the second homologous chromosome, APC copy works well and keeps cells healthy. Before the tumour formation, The loss of heterozygosity (LOH) takes place. In cells with a mutated APC gene (in LOH mechanism), there is no functional APC protein, and therefore b-catenin, which is released from complex with E-cadherin and catenins, could not undergo proteasomal degradation (in healthy cell APC before degradation form complex with b-catenin). Then b-catenin is translocated to the nucleus and, as a co-activator of T-cell factor (TCF)-lymphocyte enhancer factor (LEF), activates a key cell-cycle regulatory genes, i.e.: LGR5, c-Myc, Axin2 and cyclin D1, deleted in colorectal cancer (DCC) and TP53.

1.1.2. MOLECULAR MARKERS WITH PREDICTIVE ROLE FOR COLORECTAL CANCER

Currently decision about adjuvant systemic therapy is made upon colorectal cancer staging called Duke’s classification. Only in case of metastatic CRC molecular testing is applied for prediction of sensitivity to monoclonal anti-EGFR antibodies: cetuximab and panitumumab. The above-mentioned treatment have been available for about a decade and currently patients whose tumours do not show mutations in the KRAS, NRAS and BRAF gene are eligible for it [31].

The stratification of patients for treatment with Cetuximab and Panitumumab is based on the analysis of exons 2–4 in both KRAS, NRAS and BRAF[11]. The presence of mutations in one of these genes is found in about 65% of patients (KRAS in 50%, NRAS in 5% and BRAF in 10% of patients respectively) and it indicates a lack of sensitivity to anti-EGFR antibodies (Figure 1.2). This is because protein products of mutated KRAS, NRAS and BRAF, independently of EGFR (which is upfront regulator of EGFR/RAS/MAPK cascade), cause continuous activation of the MAPK pathway (and therefore cell proliferation) (Figure 1.2) [31].

For KRAS or NRAS or BRAF testing, very sensitive detection methods (e.g. qPCR[12], NGS[13] or ddPCR[14]) should be applied, because it is essential to detect a therapy-resistant clones (with a mutation in the KRAS or NRAS or BRAF genes), which may be in the minority in the tumour mass. The testing of these mutations is performed on postoperative material (primary tumour as well as material from metastases to distant organs). However, it should be kept in mind that the concordance of the detected mutations between the primary tumour and metastasis reaches 90% (see next chapter) [31].

There are also emerging biomarkers which are not recommended for routine patient management outside of a clinical trial. Amplification of HER2 and MET drive primary (de novo) resistance to anti-EGFR treatment but still there is clear evidence. The prognostic role of PIK3CA mutation is unknown and needs extensive research. However, the exon 20 PIK3CA mutation can predict resistance to anti-EGFR therapy. PIK3CA and PTEN alternations co-occur with KRAS or BRAF mutations. Then PTEN loss of expression is intensively studied by IHC as a valuable predictive biomarker for CRC treatment [31].

Figure 1.2.

Mechanism of anti-EGFR monoclonal antibodies action and explanation why in tumours with mutation in KRAS or NRAS or BRAF this therapy is ineffective.

1.1.3. CLONE SELECTION IN CRC LIVER METASTASES – IMPLICATION FOR CONCORDANCE BETWEEN PRIMARY TUMOUR AND METASTASIS

Metastatic CRC disease at presentation includes lymph nodes (35–40%), liver (50–60%), lung (10–30%), and peritoneum (5–20%). Mutational heterogeneity could be explored by mutational discordance between primary tumour and metastases. In case CRC the reported median concordance was 93.7% (range 67–100) for KRAS, 99.4% (range 80–100) for BRAF, 93% (range 42–100) for PIK3CA, 92.9% (range 73–100) for TP53, and 100% (range 90–100) for NRAS. In case of CRC metastases to the liver the level of concordance is above 90%. The results from the published studies point that the greater the number of genes tested, the lower the rate of absolute concordance. KRAS and BRAF was concordant at more than 90% but extending the tested panel to few genes lowers the concordance below 90% (50–80) and finally sequencing of 1000 genes in a separate cohort caused concordance fall to only about 5% between primary tumour and metastases.

The lower reported concordance could be attributable to intra-tumour heterogeneity and resistance of metastases to the cytotoxic and biologic treatment. It was additionally shown that, metastases possess greater number of mutations then primary tumour. On the other hand, applying sensitive methods could improve concordance. The concordance strongly depends on adequate sample used for testing as well. The liver metastases could be easily biopsied to collect enough material for molecular diagnostics comparing to with metastases to the lung which are more challenging to sample.

In conclusion there is high concordance across tested biomarkers between primary tumours and their liver metastases, especially in case of markers with predictive value (KRAS, NRAS, BRAF). The above result suggests that molecular testing could be done by using one of the two above-mentioned localizations [32].

1.2. DISAGGREGATION, INVASION AND INTRAVASATION OF MALIGNANT CELLS

Dissociation or disaggregation is the process, in which tumour cells acquire diverse alterations in gene expression and therefore cellular functions, such as a decrease in epithelial markers (E-cadherin) and an increase in mesenchymal markers (N-cadherin, vimentin, fibronectin etc.), which is called epithelial to mesenchymal transition (EMT). Moreover, in this process the cancer cell, expand migratory and invasive capacities and therefore detaches from the primary tumour and invade surrounding tissues [34, 35, 36].

During this phase, cancer cells interact with neighbouring stromal cells (fibroblasts, macrophages[15], lymphocytes, neutrophils, endothelial cells, dendritic cells, platelets), extracellular matrix (ECM) components (collagen, elastin, laminin, etc.), leading to: (1) neoangiogenesis[16], (2) activation and proliferation of cancer associated fibroblasts[17] (desmoplasia), (3) proteolytic degradation and extracellular matrix remodelling (enrichment in fibrin and collagen-1 stimulating motility of cancer cells), (3) stimulation or blockade of lymphangiogenesis, (4) macrophage recruitment, (5) production and secretion of cytokines and growth factors, (6) lymphatic infiltration (which is favourable prognostic factor both in primary CRC and in liver metastases), etc. [2–19]. Moreover, the plethora of stroma-coupled cytokines, growth factors, proteolytic enzymes and chemokines mediate influx of bone marrow stem cells and progenitor cells. The above-mentioned cells penetrate to the surroundings of the primary tumour and acquire the ability to survive detached from extracellular matrix components (after proteolytic degradation of ECM)[18]. The last ability constitutes a crucial property of metastatic cells [1].

Finally, tumour cells invade the basement membrane and endothelium of local blood and/or lymphatic vessels and enter the vasculature [1, 34, 35, 36]. The last process is called intravasation and precedes hematogenous or lymphagenous dissemination of CRC cells to distant anatomical sites [36]. Cancer cells may enter both blood and lymphatic vessels. Intravasation is facilitated by impaired structure of blood vessels formed during cancer neoangiogenesis. It is postulated that entry of cancer cells into the lymphatic vasculature might be easier, because of their permeability and absence of a regular basement membrane. However not all tumours present lymphatic vasculature [35]. It is worth noting that the lymphatic system does eventually drain into the systemic venous system, and therefore, metastatic cells finally spread through the hematogenous route [35].

Footnotes

[1] A driver mutation is an alteration that gives a cancer cell a fundamental growth advantage for its neoplastic transformation. It differs from passenger mutations, which do not determine the development of the cancer.

[2] Chromosomal instability (CIN) is characterized by a widespread numerical chromosomal aberrations, subchromosomal aberrations, amplifications and loss of heterozigosity. One of the major negative results of CIN is the loss of tumour suppressor genes.

[3] DNA mismatch repair (MMR) genes recognize and repair erroneous insertions, deletions, and mis-incorporations of nucleotides that can arise during DNA replication.

[4] Microsatellite instability results from germline mutations or epigenetic silencing in the mismatch repair (MMR) genes (MLH1, MSH2, PMS2, MSH6) and is characterized by alterations in the length of specific areas of the genome containing microsatellites (short sequences of nucleotide bases, repeated multiple times).

[5] DNA methylation is an enzymatic process in which a methyl group is added to the 5’-position of cytosine by DNA methyltransferases (DNMT) to produce 5-methylcytosine. Usually, the favourite substrate for DNMT are CpG (cytosine preceding guanine) islands. This regions are common in promoter sites. In cancer cells, CpG islands may be aberrantly hypermethylated, causing inappropriate silencing suppressors gene or might be aberrantly unmethylated – activating oncogenes [25].

[6] APC mutation is responsible for lack of functional APC protein, while aberrant APC protein, is not able to form complex with β-catenin and direct it (β-catenin) to proteasomal degradation. Then free β-catenin is translocated to the nucleus and, as a co-activator of T-cell factor (TCF)-lymphocyte enhancer factor (LEF), activates a key cell-cycle regulatory genes, ie.: LGR5, c-Myc, Axin2 and cyclin D1, deleted in colorectal cancer (DCC) and TP53.

[7] Recent studies carried out that APC loss induced intestinal differentiation defects, whereas proliferation defects and the nuclear accumulation of b-catenin required the additional activating mutations in KRAS gene. On the other hand APC restoration in developed tumours, induced cell differentiation and tumour regression, without tumour relapse. Moreover, TP53 loss alone, was insufficient to cause colon carcinogenesis initiation, but markedly increased carcinogen-induced tumour incidence and determined the development of aggressiveness and metastagenicity of invasive cancer. It was additionally shown that number of genetic changes in CRC, correlated with the degree of dysplasia and invasiveness. [21]

[8] Chromosomal instability (CIN) is characterized by a widespread numerical chromosomal aberrations, subchromosomal aberrations, amplifications and loss of heterozygosity. One of the major negative results of CIN is the loss of tumour suppressor genes.

[9] A tumour suppressor gene, control a progression of the cell through the cell cycle and are responsible for DNA repair. The lack of their function is responsible for cancerogenesis.

[10] Immune checkpoints are molecules on certain immune cells that need to be activated (or inactivated) to start an immune response. For example, PD-1 is a checkpoint protein on cytotoxic T lymphocytes (Tc). When PD-1 attaches to PD-L1 it prevents the attack of Tc on the cells presenting PD-L1. During cancer progression cancer cells might use these checkpoints to avoid being attacked by the immune system. Drugs that target these checkpoints, and unlock cytotoxicity of Tc, are called checkpoint inhibitors.

[11] Initially, the application of the drug was based on the results of immunohistochemical testing of the expression of EGFR protein on the surface of cancer cells combined with the lack of mutation in exon 2 of the KRAS gene. However, after retrospective analysis of data from clinical trials it turned out patients with EGFR immunonegativity responded to the treatment, while patients with mutation in the exon 2 of KRAS gene did not benefit from treatment with anti-EGFR therapy. As a result, the requirement for targeted therapy in colorectal cancer was the lack of mutation in the exon 2 of KRAS gene. However, the results of the above-mentioned molecularly targeted therapy in combination with chemotherapy were not satisfactory as compared to the application of chemotherapy alone. In order to improve efficacy, retrospective analysis of predictive value of another exons of the KRAS, NRAS and BRAF was performed on tissue samples from patients participating in clinical trials. It was found that only patients without mutations in mutations in exons 3 and 4 of the KRAS gene and 2-4 of the NRAS exon benefited from anti-EGFR treatment. Another important information obtained from this retrospective analysis was that in patients without mutation in KRAS exon 2, who presented mutations in other exons of KRAS or NRAS, anti-EGFR treatment accelerated the progression of disease.

[12] In probe-based qPCR (quantitative polymerase chain reaction), many mutations can be detected simultaneously in each sample by use of pre-designed target-specific fluorescent-lebelled probes or primers attaching to defined DNA regions. Emission of fluorescence, from probes or primers, is present only if particular mutation (or lack of mutation) is present in the probe. During each cycle, the fluorescence is measured and fluorescence signal increases proportionally to the amount of replicated DNA (or fluorescent-lebelled probe attached to DNA) and hence the DNA is quantified in “real time”.

[13] NGS (next-generation sequencing) is a high-throughput methodology that enables rapid sequencing of the base pairs in DNA or RNA samples.

[14] During droplet digital PCR (ddPCR) very low concentration of DNA with particular mutation, can be detected in a background of high numbers of non-target nucleic acids. In this method, each PCR is performed in thousands nanoliter droplets. The absolute number of target molecules initially present in the original sample can be determined by the ratio of positive to total number of droplets.

[15] M1 macrophages (CD11b+, CD80+, CD86+, CD204+, CD284+, CD14+, CD68+) present anti-tumour activity, while M2 macrophages (TAM: tumour-associated macrophages; CD23+, CD169+, CD206+, CD163+, CD14+, CD68+) are tumour growth favouring once, and are responsible for: immunosuppression, inactivation of T lymphocytes, tumour angiogenesis, production of proteolytic enzymes remodelling extracellular matrix, stimulating tomor motility and production of growth factors.

[16] Neoangiogenesis is switched on when oxygen diffusion from the normal capillary network is unable to supply tumour larger than 1-2 mm, this process is related to the balance between proangiogenic factors: VEGF (Vascular endothelial growth factor), PDGR (Platelet-derived growth factor), FGF (Fibroblast growth factor), angiopoietin, etc; and anti-angiogenic factors: endostatin, angiostatin, and TSP (Thrombospondins), etc.

[17] Early in tumourogenesis fibroblasts inhibit early stages of tumour progression [13, 14]. during cancer growth, as a result of mechanical tension, fibroblasts acquire the miyofibroblast phenotyoe, characterised (among others) by the expression of αSMA. Therefore they became CAFs and they are similar to myofibroblasts, which are active during wound healing [14]. The tumours CAFs are involved in many aspects of tumour progression: (1) secretion of variety of factors promoting carcinogenesis (angiogenesis, invasion and metastasis) (2) mechanical mdification of tumour stroma – they generate force- and protease-mediated tracks into ECM and cancer cells follow these leading fibroblasts along these tracks, (3) convection of signals via mechanotransduction (conversion of phisical stimulation into chemical signals active in cancer progression), (4) immuno-supression mediated by CAFs derived factors (imminoediting: elimination, equilibrium, and escape), (5) resistance to anti-cancer therapies, (6) reprogramming of metabolism in tumour microenviranment, (6) provide an early growth advantage to metastatic cancer cells: it was shown that metastatic cells can bring their own soil, i.e. stromal components including activated cafs, from primary sites to secondary tumour sites. [13-19].

[18] Anoikis is programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extracellular matrix. The aforementioned ability is related to overexpression of focal adhesion kinase by CRC cells, which on the other hand contributes to conferring survival by activating certain molecular pathways, including ERK and AKT [1].