Current possibilities of predicting the therapeutic response to neoadjuvant chemoradiotherapy in rectal cancer

Současné možnosti predikce léčebné odpovědi na neoadjuvantní chemoradioterapii u karcinomu konečníku

Standardem léčby pacientů s lokálně pokročilým karcinomem konečníku je neoadjuvantní chemoterapie v kombinaci s radiací. Hlavním cílem léčby je snížit riziko rekurence. S léčbou však mohou být spojeny také závažné nežádoucí účinky v důsledku postradiačního poškození v oblasti pánve. Mnoho skupin se snaží najít markery, které by umožňovaly předpovídat odpověď na léčbu. Tento přehledový článek přináší rešerši literatury provedenou za účelem identifikace hlavních studií zaměřených na použití klinických, radiologických, imunologických a molekulárních (proteinových, DNA a RNA) markerů. Pro každou skupinu předkládáme souhrn poznatků. Obecně lze shrnout, že interpretace v současné době získaných poznatků je spojena s určitou mírou nejednoznačnosti a nejednotnosti. Zdá se, že i v nejslibnější oblasti cirkulujících molekulárních biomarkerů je zapotřebí dalšího výzkumu, než bude možné stanovit jejich užitečnost v klinické praxi.

Klíčová slova:

karcinom konečníku – neoadjuvantní chemoradioterapie – cirkulující nádorová ctDNA – predikce – prognóza – odpověď – biomarker

Authors: Pazdírek F. ¹; Minárik M. ²; Benešová L. ³; Lohynská R. ⁴; Hoch J. ¹
Authors place of work: Department of Surgery, 2nd Medical Faculty, Charles University and University Hospital Motol, Prague 2 Elphogene, Prague 3 Center for Applied Genomics of Solid Tumors (CEGES), Genomac Research Institute, Prague 4 Department of Oncology, 1st Faculty of Med ¹
Published in the journal: Gastroent Hepatol 2020; 74(5): 393-403
Category:

Summary

Neoadjuvant chemotherapy in combination with radiation is currently the standard of care for patients with locally advanced rectal cancer. The main purpose of the treatment is to reduce the risk of recurrence, however at the same time it may be accompanied by severe adverse effects due to post-radiation pelvic damage. An effort towards finding markers allowing the prediction of the therapy response has been undertaken by many groups. In this review we have performed a literature search to identify the main studies directed at the use of clinical, radiological, immunological and molecular (protein, DNA and RNA) markers. We present a summary for each group with an overall conclusion that a certain level of ambiguity and disunity in interpretation of the results currently exists among the reported findings. Apparently, even in the most promising direction of circulating molecular biomarkers further work is needed before a clinical utility can be established.

Keywords:

rectal cancer – neoadjuvant chemoradiotherapy – circulating tumour ctDNA – prediction – prognosis – response – biomarker

Introduction

Current treatment of rectal cancer is based on a multimodal approach involving surgical, radiation and systemic therapies. Tumours in the early stage are being preferentially considered for direct surgical intervention. Patients with locally advanced rectal cancer (LARC), stages II and III are indicated for neoadjuvant treatment consisting of a combination of external irradiation and concomitant systemic chemotherapy (NCRT) administered prior to surgery. Following surgical treatment local recurrence is the principal risk factor. The main goal of neoadjuvant therapy is to reduce the risk of recurrence, as studies show that NCRT does not significantly improve the overall survival.

Neoadjuvant chemoradiotherapy can be applied in either short or long regimens. With interpatient variation a noticeable therapeutic response can be expected mainly after a long regimen. In 15–25% of the patients, NCRT leads to complete disappearance of the tumour, in others only a partial regression can be observed as shrinkage of the tumour, reduction in the number or a complete disappearance of positive nodes in the mesorectum. Patients with a complete response to NCRT have an excellent prognosis with almost 90% remaining in remission 5 years after the surgery [1]. Recent studies have shown that these patients may safely be deferred from surgical resection (non--operative management [NOM] or watch and wait approach) under strictly set criteria and the patients are followed up intensively [1–3].

On the other hand, in up to 40% of the patients, the tumour extent remains unaffected by the treatment. Patients undergoing NCRT are exposed to a number of adverse side effects due to post-radiation pelvic damage, which can result in fibrosis, anal sphincter dysfunction, incontinence and sexual dysfunction. The neoadjuvant treatment also increases the risk of postoperative complications and aggravates the symptoms of LARS (Low Anterior Resection Syndrome). The aim is to choose the appropriate group of patients profiting the most from the individualised treatment. For these so-called nonresponders, it would be more appropriate to modify (intensify regimen with total neoadjuvant approach) or in some cases omit preoperative treatment and thus eliminate its side effects [4]. Over the past years a number of markers have been proposed to assess the likelihood of response in order to avoid unnecessary damage in LARC patients and to save uneconomical consumption of financial resources. Here we present a brief overview or a full spectrum of these markers ranging from clinical, radiological and immunological parameters to molecular biomarkers (including proteins and RNA/DNA) to more recently explored plasma-circulating tumour-derived elements (circulating tumour cells, CTC and circulating tumour DNA, ctDNA).

Database search

Using Reference Manager software (Thomson ISI, Philadelphia, PA) we have performed MEDLINE® database search in article titles and abstracts for keywords ([prediction] OR [predictive]) AND (response) AND (neoadjuvant) AND ([therapy] OR [treatment]) AND ([rectal cancer] OR [colorectal cancer]). Only English-language literature was selected. By entering these keywords, we have obtained a total of 366 relevant literature refe- rences. We have subsequently excluded 110 references not directly related to the topic of NCRT in LARC patients. In addition we have found 25 summary studies, of which 5 were meta-analyses or systematic reviews. The total numbers of studies according to the studied biomarker are given in Fig. 1. The number of studies in each year is also given in detail in Graph 1. Based on the search, markers can be divided into clinical, radiological, protein--based biomarkers, genetic, immunological and circulating tumour elements.

Graph 1. The number of studies in each year.<br>
Graf 1. Počty studií v jednotlivých letech. — Graph 1. The number of studies in each year.
Graf 1. Počty studií v jednotlivých letech.

A. Clinical markers

Tumour size, tumour location – distance from the anal margin, tumour differentiation

A number of studies has been performed to investigate whether tumour size, degree of differentiation and T and N stage can be a prognostic marker of NCRT in rectal tumours. Some work has shown that pathological complete response (pCR) was more often associated with small tumour size, good tumour differentiation and early T and N [5,6]. These results have not been confirmed in all studies [7]. In terms of localisation, tumours of the middle and distal third of the rectum have a better response to NCRT than tumours of the proximal third [8,9]. Well-differentiated tumours also have a better clinical response to preoperative treatment [10]. After preoperative chemoradiotherapy it is common to detect lower numbers of lymph nodes and studies have been published on a significant association of pCR and the number of detected lymph nodes, patients with <12 nodes have a significantly higher proportion of pCR (p = 0.004) [11]. In an extensive retrospective work, Lorimer et al analysed a cohort of 27,532 patients from 1,179 hospitals [12]. He found that the likelihood of achieving pCR was independently associated with earlier diagnosis, female gender, lower T and N stages, a prolonged interval between the termination of chemoradiotherapy and surgery, as well as treatment in an institution with a higher patient volume.

Anaemia

Tumour hypoxia is associated with resistance to radiotherapy [13]. Oxygenation of the tumour depends on its vascular supply, microcirculation and on the amount of haemoglobin in the blood. A large proportion of cancer patients suffer from anaemia. Anaemia is caused by the tumour or the treatment itself. Anaemia has been shown to be a negative prognostic factor in head and neck, lung, cervical and oesophageal tumours. Similarly, in rectal tumours, anaemia can have a negative effect on response, local recurrence and OS [14]. The work of Lee et al showed that anaemic patients (haemoglobin lower than 90 mg/l) with locally advanced rectal cancer undergoing NCRT achieved a lower percentage of pCR than patients without anaemia (p = 0.012). Multivariate analysis showed that anaemia before treatment (p = 0.035), tumour stage T and nodal involvement (p = 0.020 and 0.032) were independent prognostic factors for local disease control [15]. On the other hand, T. Clarke et al did not demonstrate an association between pretreatment haemoglobin levels and pCR [16].

B. Radiological markers

MRI is routinely used for preoperative staging of rectal tumours. Determining the T and N stages can be used for prediction as described above. Measurement of the tumour volume did not show an association with pCR [17]. On the contrary, a comparison of tumour signal intensity yielded better results. Patients with TRG 1 had a higher preoperative tumour signal intensity than patients with TRG 2, 3. However, there was no difference in signal between the TRG 1 and 4 group of patients [18]. Contrast-enhanced MRI and MRI (DWI) better reflect tumour perfusion and cell density, better reflect tumour biology, and could potentially be used for prediction. In particular, tumour areas with poor perfusion and signs of hypoxia may be resistant to the effects of NCRT [13]. In MRI using a contrast agent, it is possible to determine the perfusion index (PI), taking into account the microcirculation in the tumour. A high perfusion index (PI) indicates hypoxia in the tumour and may predict a poor response to NCRT. DeVries showed that the PI value was significantly increased in the group of responders compared to the group of nonresponders [19]. Unfortunately, low PI has not been shown to predict pCR [19–21]. Another important parameter determined in MRI (DWI) is the ADC (apparent diffusion coefficient), which describes the degree of proton mobility of free water molecules. In necrotic tissue we find a rapid diffusion of protons of water molecules due to impaired membrane integrity and ADC is high. By contrast, tissues with a high cell density show low ADC.

DWI has been studied by a number of authors with conflicting results. In the previously mentioned work of DeVries, it was shown that in the group of nonresponders there was a higher level of ADC (p < 0.001). Lambrecht et al showed promising data in 22 patients. Low pretreatment ADC was associated with a higher number of pCR (p 0.003) [22]. By contrast, Barbaro et al found that low pretreatment ADC was associated with a poor response to NCRT [23]. Elmi et al found that higher pretreatment ADCs corresponded to a response to treatment with a sensitivity of 75% and a specificity of 48% [24]. The results are therefore contradictory. The position of MRI after the end of chemoradiotherapy and evaluation of possible tumour regression is important. The best results are obtained when MRI is done at least 6 weeks after the end of NCRT. In the work of Maas et al 50 patients were examined 6–8 weeks after the end of NCRT. Clinical examination (DRE), endoscopic examination and MRI (T2W, DWI) were performed. 34% of the patients achieved pCR. MRI alone had a success rate of 79% in predicting pCR, in combination with clinical examination and endoscopy this success rate increased to 98% [25]. In the following work, the success rate of MRI prediction alone exceeded 90% [26]. Using MRI, we are also able to determine the degree of tumour regression by the relative proportion of fibrosis and viable tumour tissue. These results correlate with histopathologically determined regression (TRG) [27]. FDG PET/CT is not used as a standard method to determine preoperative staging in rectal tumours. Pretreatment use of PET CT to predict NCRT has yielded conflicting results. The standardised uptake value (SUV) or SUVmax is used to determine the degree of FDG (fluoro-D-glucose) tumour saturation in PET/CT. There is work focused on monitoring the dependence of SUV and pCR, unfortunately without statistical significance. Goldberg et al monitored whether SUV values before treatment and after the first week of NCRT could predict pCR. She found that the decrease in SUVmax was more pronounced among responders [28]. The work of Kim et al was similarly designed, except that a control PET/CT was performed 5–6 weeks after the end of the NCRT. The assessed SUVmax found at the second examination was lower in the group of responders [29]. Martoni et al demonstrated that SUV values below 27 before treatment predict pCR with high sensitivity but very low specificity (10.6%) [30]. Neither of the imaging methods is reliable to determine prediction before NCRT starts. The use of MRI in the early stages of NCRT seems promising.

C. Protein-based biomarkers

Carcinoembryogenic antigen (CEA)

Molecular prediction is based on the search for specific markers present in blood plasma or directly in tumour tissue. These markers are associated with cellular regulation, DNA repair, the onset and progression of cancer and the molecular mechanisms responsible for tumour chemoradiosensitivity.

The CEA (carcinoembryogenic antigen) oncomarker is a membrane glycoprotein that is formed in epithelial cells during foetal development. It affects cell adhesion, but its complete function is unknown. It also has an indirect immunosuppressive effect on T-lymphocytes. The half-life in the body is 7 to 14 days, the physiological serum level is up to 3 µg/ml, in smokers it is slightly increased to 5 µg/ml. It may be increased in liver cirrhosis and gastrointestinal inflammation. Its sensitivity in colorectal cancer reaches up to 71%, but this only applies to advanced and metastatic tumours, in the early stages the sensitivity is less than 25%. Thus, CEA cannot be used as a diagnostic marker, but CEA levels adjusted by tumour size could reflect the malignancy of rectal cancer. Moreover, CEA levels could be used as a marker for monitoring the progression of cancer [31].

A number of studies has been performed looking for a relationship between pretreatment CEA levels and the outcome of preoperative chemoradiotherapy.

A large retrospective study analysed a group of 530 patients with rectal cancer undergoing NCRT. The patients were irradiated with a total dose of 50.4 Gy and given 5-FU at the same time, followed by surgery. Overall, 20% of the patients achieved pCR. The mean pretreatment CEA level in non-smokers with cPR was 2.9 ng/ml, in those who did not achieve cPR the pretreatment level was 8.3 ng/ml. Interestingly, in this work, only 57% of the patients included in the analysis had CEA levels available [32]. Similarly, an extensive study from the MD Anderson Cancer Center has shown that pretreatment levels of CEA > 2.5 ng/ml are associated with significantly lower numbers of pCRs [6]. A cohort of 323 patients from China showed that pretreatment CEA levels ≤ 5 ng/ml were an independent predictor of increasing the chance of achieving pCR [33]. The works of other authors ended with a similar conclusion [34–36]. The change in CEA levels before and after NCRT were also been investigated. In particular, a low level of CEA, or a decrease after the end of NCRT, could indicate a good response to NCRT [37].

According to recent work analysing a group of 354 patients, the group of patients who achieved pCR had significantly lower CEA levels at baseline compared to the group that did not achieve pCR [10].

These encouraging results are counteracted by the work of Kalady et al who failed to demonstrate an association between pretreatment CEA and pCR in a cohort of 242 patients. The only endpoint that had a positive effect on achieving pCR was a time of longer than 8 weeks following surgery [7]. Also, Clarke et al did not show a correlation between pretreatment levels of CEA and pCR [16].

Nevertheless, it appears that elevated pretreatment levels of CEA may adversely affect the response to chemoradiotherapy in rectal tumours.

Thymidylate synthase (TS)

TS is an enzyme involved in the metabolism of thymidine. During its inhibition, certain metabolic products accumulate and thus DNA is damaged. The effect of 5-FU is mediated by binding to TS. 5-FU acts as an antimetabolite and irreversibly inhibits TS. High TS expression in CRC is associated with 5-FU resistance. TS can be examined at the protein level by IMC or at the RNA level by reverse transcriptase. At the same time, the TS gene polymorphism is investigated. In the work of Jakob et al low pretreatment expression of the TS gene in tumour tissue led to a good therapeutic response; in addition, together with Ki-67, it had a positive prediction in achieving pCR [38]. The work of Negri et al did not confirm this [39]. Research on TS polymorphism has not yielded better results either [40].

Epidermal growth factor receptor (EGFR)

EGFR is a transmembrane protein belonging to the growth factor receptor tyrosine kinase (HER) family. Upon binding of the ligand to the extracellular domain of the receptor, intracellular tyrosine kinase residues are autophosphorylated, leading to activation of a signalling cascade involving the RAS / RAF / MAPK, PI3K / AKT and STAT / AKT pathways, important for cancer development and progression. The RAS (KRAS) pathway to MAPK is involved in cell cycle regulation, gene transcription and cell division. The PI3K / AKT pathway regulates signals affecting cell survival and signals preventing apoptosis. The EGFR receptor also activates the STAT / AKT pathway, which acts to transcribe genes involved in the cell survival process. Autoantibodies targeting the EGFR receptor play an important role in the treatment of metastatic colorectal cancer. The effectiveness of this treatment depends on the status of KRAS. In patients in whom KRAS is mutated, treatment is ineffective. Some studies have shown that low EGFR expression leads to a good therapeutic response, others have shown the opposite. Research into the EGFR polymorphism has also yielded conflicting results [40–42]. The determination of the KRAS mutation and its use to predict pCR has also not been confirmed [43,44].

Survivin

Survivin is a small molecule involved in cell cycle regulation and inhibition of apoptosis, expressed during embryogenesis as well as numerous cancers. Its expression in the tumour correlates with a more aggressive tumour phenotype and chemotherapeutic resistance [45]. In rectal tumours, the effect of survivin expression on chemoradiotherapy was investigated and controversial results were found. Terzi et al found no correlation between survivin expression and response to preoperative chemoradiotherapy and prognosis [46]. Kim et al, by contrast, found that high immunohistochemical expression of survivin in a pretreatment tumour biopsy was associated with a lower response to preoperative chemoradiotherapy in locally advanced rectal tumours [47].

Cyclooxygenases

Cyclooxygenase is represented in two isoforms (COX1,2) involved in the metabolic conversion of arachidonic acid to prostaglandins, including prostaglandin E2, which is one of the major mediators of inflammation and angiogenesis. COX2 can inhibit apoptosis, promote angiogenesis and modulation of cell differentiation, and increase tumour aggressiveness and metastatic potential [48].

Unfortunately, COX research in predicting the response to NCRT in locally advanced rectal cancer has yielded conflicting results [49,50]. A number of other proteins has been investigated. The results are contradictory. Increased activity of 86 kinases can with high success predict pCR [51]. The dependence of p21, Bax, Bcl-2, Ki-67 expression on pCR was different in different work [52–57].

p53 protein

The p53 protein is a transcription factor that, among other functions, prevents the formation of tumours, hence it is a tumour suppressor. The p53 protein regulates the expression of many other factors that can control cell growth, apoptosis (programmed cell death), DNA repair, ageing of cell populations and angiogenesis. P53 looks for damaged sites on DNA and, if it finds such sites, starts transcription of the p21 gene, which stops cell division until the damaged site is repaired. If this is not possible, the cell will trigger programmed cell death. The function of the p53 protein is regulated by a number of mechanisms, including oxidative stress, osmotic shock, fever, cell membrane damage, DNA damage and more. Inactivation of p53 is important in the development of colorectal cancer. P53 also plays an important role in the effect of NCRT on the tumour, so its role has been investigated extensively. Immunohistochemical detection of p53 protein in tumour tissue revealed that cells expressing a wild type p53 protein were chemoradiosensitive, while those expressing the mutated p53 protein were resistant. Some work confirmed that wild type p53 (unmutated gene or decreased expression of p53 protein) is associated with a good response to NCRT [58–60]. In other works, this was not confirmed [41,52].

D. Genetic DNA/RNA markers

Chromosomal aberrations

Acquisition or loss of a portion of a chromosome can lead to alterations in oncogenes and tumour suppressor genes that are important for the development and progression of colorectal cancer. When Chen et al examined chromosomal aberrations, he found fewer regions obtained, and a specific loss of the 12p13.31 region in patients with pCR. Analysis of this region revealed 8 genes related to the tumour‘s response to treatment [61].

Mutations and methylations in cancerous tissue

Early works on detecting somatic mutations in known tumour supressor genes and oncogenes suggested that evaluation of the cancerous tissue for mutation status of the TP53 gene could be used in prediction of the response to NCRT treatment. A meta-analysis published by Chen et al in 2012 confirmed that a wild type TP53 is associated with a better response, however this hypothesis has not been confirmed by others [60].

Sun et al have examined the presence of the KRAS mutation and MGMT (methylation promoter) in patients with CRC. The KRAS mutation decreased significantly after NCRT in the nonresponder group. By contrast, a higher pretreatment level of MGMT was found in the group of responders [62].

Inherited single nucleotide DNA polymorphisms (SNPs)

SNPs are inherited genetic variants that indicate the general predisposition of the organism to the tumour processes and also the response to the treatment – e. g. the ability to repair DNA in connection with radiation-chemotherapeutic action. Spindler et al investigated SNPs in TS, EGFR, Sp1-216 and its association with pCR. The TS 2/2 genotype and heterozygote of the EGFR A61G gene predicted cPR with high success [63]. Other authors have not been as successful [64].

RNA expression profiling

The microarray technique is already used in the prediction and individualisation of cancer treatment for breast cancer (Oncotype DX, MammaPrint). Similarly, genes are being sought to predict the effect of NCRT in rectal tumours. These groups of genes include DNA repair genes, genes regulating apoptosis, and signalling pathways for cell growth. Ghadimi et al demonstrated significantly different expression in 54 genes in the group of responders and nonresponders. The predictive value of this test was 84%, sensitivity 78% and specificity 86% [65]. Similarly, Rimkus et al identified a set of 42 genes associated with pCR prediction with a sensitivity of 71% and a specificity of 86% [66].

The Korean group identified 95 genes predicting pCR in a cohort of 31 patients. The accuracy of this prediction was 84%. Brettingham-Moore et al performed a microarray analysis on a group of 51 patients. She tried to verify a panel of genes used for prediction by Ghadimi, Rimkus and Kim. It has failed to demonstrate correlation in the expression of these genes and prediction of the therapeutic response [67]. Similar to DNA analysis, microRNA gene expression (miRNA) analysis was performed. Differential expression of 53 miRNAs was found in responders versus nonresponders. The largest difference was found for 14 miRNAs. MiRNA-622 and miRNA--630 showed 100% sensitivity and specificity in pCR prediction [68]. The authors believe that miRNA affects genes involved in the repair of cells damaged by radiotherapy. MiRNA-630 has previously been shown to adversely affect the DNA repair capacity of oxaliplatin-damaged cells, specifically NSCLC. Kheirelseid et al extracted miRNA from 12 paraffin blocks in rectal tumours. He found that miRNA-16, 590, 153 predicted pCRs with 100% accuracy. This is a very promising technique due to the good availability of paraffin blocks, unfortunately it has not yet been tested on a wider cohort [69]. Lopes-Ramos et al identified 27 differentially expressed genes between patients with pCR and patients with incomplete responses to NCRT. Predictive gene signatures using subsets of these 27 differentially expressed genes peaked at 81.8% accuracy. However, signatures with the highest sensitivity showed poor specificity and vice versa, when applied in an independent set of patients. These results indicate that currently available predictive signatures are highly dependent on the sample set from which they are derived, and their accuracy is not superior to current imaging and clinical parameters used to assess response to NCRT [70].

Spontaneous apoptosis

The degree of spontaneous apoptosis was examined as a marker of radiosensitivity. Several studies have shown an association between the presence of spontaneous apoptosis in pretreatment tumour biopsies and the subsequent complete response to NCRT. By contrast, McDowell and Tannapfel et al have not shown any such association [71,72].

E. Immunological markers

Lymphocytes play an important role in the body‘s immune response to the tumour and also mediate the effect of NCRT. Since the decrease in T lymphocytes in the experiment worsened the effect of radiation in mice, both the numbers of lymphocytes in the peripheral blood and in the tumour are examined [73]. A higher proportion of CD8 + lymphocytes in the tumour have been shown to be an independent prognostic factor for achieving pCR after NCRT [74]. Peripheral blood lymphocytes are higher in patients with pCR [75]. Tada et al confirmed that pretreatment circulating lymphocyte counts are higher in patients with a good NCRT response than in the minimal response group. Furthermore, in the proportion of these lymphocytes, T and Th lymphocytes predominate at the expense of B lymphocytes. T and B cell levels decline during chemoradiotherapy [76]. Similar results have been shown in tumours of the uterus, cervix, breast and nasopharynx. Higher pretreatment number of lymphocytes had a beneficial effect on the prognosis of patients. Not all authors have succeeded in confirming this [16]. The relationship between circulating and tumour infiltrating lymphocytes (TIL) is not clear yet. Milne et al showed that the level of circulating lymphocytes does not correlate with the number of TILs in ovarian cancer. Circulating lymphocytes and TIL correlate favourably with the patients’ prognosis, but independently of each other [77].

F. Circulating tumour elements including circulating tumour cells (CTC) and circulating tumour DNA (ctDNA)

CTC

Cells are released from the tumour into the bloodstream. Tumour cells can then be detected in the peripheral blood, but also in the bone marrow. The presence of CTC is thought to be associated with the ability of the tumour to establish metastases and thus a worse prognosis. In non-metastatic breast cancer, CTC has a prognostic value. Patients with CTC before treatment have a worse prognosis, which is also negatively associated with the number of circulating tumour cells [78]. The inclusion of neoadjuvant chemotherapy in the treatment of patients with non-metastatic HER2-negative but CTC-positive breast tumours did not lead to the elimination of circulating tumour cells [79].

In prostate cancer, the disappearance of CTC after 13 weeks of treatment is associated with a good prognosis and has a better predictive value than a decrease in PSA.

Approximately 20–30% of patients with advanced colorectal cancer have more than one CTC in 7.5 ml of peripheral blood [80]. The presence of CTC in the blood is then associated with worse OS in these patients. Magni et al monitored CTC levels at baseline and during multimodal treatment of locally advanced rectal cancer. She found that 18.9% of the patients had at least one CTC at the start of treatment. CTC decreased during NCRT. It did not show a relationship between the presence of CTC and the T, N stage. She found that patients with a good response to CRT had a decrease in CTC after stopping neoadjuvant treatment. The presence of CTC alone at the beginning of treatment was not a statistical prognostic factor [81]. Similarly, Sun et al investigated the amount of CTC in locally advanced rectal cancer. In patients with a good response to NCRT, the amount of CTC decreased significantly after CRT was stopped. Responders had higher amounts of CTC in the peripheral blood before treatment and less CTC after the end of CRT than non-responders [82]. In Troncarelli Flores et al, CTCs were also examined for kinetics and expression of TS and RAD23B protein. The cohort consisted of 30 patients. CTC levels were determined before and after NCRT. In most patients, CTC levels decreased during treatment. None of the patients who achieved pCR showed TS and RAD23B protein expression in CTC [57].

Cell-free DNA (cfDNA) and circulating tumour DNA (ctDNA)

Cell-free DNA (cfDNA) is characterised as short fragments (150–200 bp in length) released into the bloodstream from cells undergoing necrosis or apoptosis or generated by a mechanism of an active release [83]. It consists of two fractions, one originating from normal (non-cancerous) cells and another from the cancerous cells. The latter is referred to as circulating tumour DNA (ctDNA). CfDNA and, later, ctDNA have been studied in association with prediction of response to NCRT in LARC patients. Zitta et al determined cell-free DNA levels before treatment, after cessation of CHRT and after surgery. The group was divided into nonresponders and responders. The median pre-treatment cfDNA level was 4.2 ng/ml, 1 ng/ml after CRT completion and 4.1 ng/ml after surgery. He found that pre-treatment cfDNA levels of nonresponders and responders did not differ. At the end of treatment, however, the cfDNA levels were higher in the nonresponder group. In virtually all patients, the cfDNA levels decreased after NCRT. The limit of this work was the small number of patients [84]. In 2011, Agostini published a cohort of 67 patients with LARC. He measured cfDNA levels before and after NCRT. It determined the total concentration of cfDNA, the proportion of long and short DNA fragments, the so-called DNA integrity index. Like Zitt, he did not detect a correlation between pre-treatment cfDNA levels and responses to NCRT. He was able to show that in patients with a good response to treatment, the index of responders was significantly lower in responders than in nonresponders after the end of NCRT DNA integrity [85]. In the work of Sun et al not only was total free DNA sought, but specific tumour mutations in blood plasma were sought as well. Sun has verified that the amount of cfDNA in patients with CRC is significantly higher than in healthy individuals. He also determined the concentration of two DNA fragments (100 bp and 400 bp) in the plasma before and after NCRT. He found that the fragment concentration of 400 bp was significantly lower after NCRT termination in the responder group [62]. In the following works, specific circulating tumour DNA was already sought. Carpinetti et al identified specific DNA fragments in each of the 4 patients by harvesting tumour tissue and then whole-gen sequencing of tumour DNA [86]. He then looked for these fragments in blood plasma and found that patients with a good response to treatment had a decrease in ctDNA during NCRT. Recurrence of ctDNA levels was associated with disease progression and preceded an increase in CEA and the manifestation of recurrence on imaging. Unfortunately, the study was only done on a very small number of patients, hence further work is needed.

The main challenge of ctDNA detection is its differentiation from a high background of cfDNA naturally occurring in plasma. In our recent work, we have demonstrated a two-level approach to ctDNA testing using a rapid method with lower sensitivity followed by examining the negative samples using a more costly, high-sensitive approach. We have only confirmed the negative prognostic value of baseline ctDNA positivity, while no predictive value was found for ctDNA dynamics during the first week of the treatment [86].

As we mentioned, neoadjuvant chemoradiotherapy can cause significant side effects, especially adverse effects and in particular post-radiation pelvic damage, which can result in fibrosis, anal sphincter dysfunction, incontinence and sexual dysfunction. The neoadjuvant treatment also increases the risk of postoperative complications. Those patients who have minimal or no response to NRCT do not benefit from this treatment and are burdened by its adverse effects. Treatment administered in this way then delays surgery, prolongs incapacity and increases the funds spent on treatment. It is also evident from the research that the number of studies has been growing steadily in recent years and their field focus has expanded. Initially, clinical and molecular markers were investigated, now the research focuses on genetic markers, the use of imaging methods for prediction and most recently, the prediction of therapeutic response based on the determination and analysis of ctDNA. It is clear that none of the markers examined is sufficiently reliable to predict the therapeutic response to NCRT in rectal tumours. Another problem is that the determination of some promising markers is time- and money-consuming and is not suitable for routine clinical practice.

Conclusion

In summary, currently no clinical or laboratory marker is sufficiently reliable to predict therapeutic response to preoperative chemoradiotherapy in rectal cancer. Some are promising, however. The localisation of the tumour itself is important in relation to the therapeutic response. Many studies have shown that a low pretreatment CEA level is more often associated with a good response to NCRT in rectal tumours. Among radiological markers, the use of MRI early during NCRT seems to be promising. Recently, the use of ctDNA to predict treatment response has been investigated. Studies suggest that declining ctDNA levels during treatment may be a predictor of a successful response to NCRT, and in particular the dynamics of early changes in ctDNA levels at the onset of NCRT. Further research is still to be done in this area.

Submitted/Doručeno: 20. 9. 2020

Accepted/Přijato: 1. 10. 2020

Filip Pazdírek, MD

Department of Surgery

2^nd Medical Faculty

Charles University and University Hospital Motol, Prague

V Úvalu 84/1

150 06 Praha 5

filip.pazdirek@gmail.com

Conflict of Interest: The authors declare that the article/ manuscript complies with ethical standards, patient anonymity has been respected, and they state that they have no
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Redakční rada potvrzuje, že rukopis práce splnil ICMJE kritéria pro publikace zasílané do bio medicínských časopisů.

Zdroje

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