Circulating tumor cells are an important link between primary tumors and metastases. A longitudinal monitoring of their numbers and properties can provide valuable information on therapy response and disease progression for patients with colorectal cancer. As several techniques for the detection of circulating tumor cells are notorious for yielding low detection rates in patients with non-metastatic colorectal cancer, the present study aimed to perform a proof-of-principle study using the Maintrac® approach for an assessment of circulating epithelial tumor cells (CETCs) in patients with colorectal cancer receiving neoadjuvant and/or adjuvant radio/chemotherapy (R/CT). CETCs in the peripheral blood of 22 patients with colorectal cancer were quantified by fluorescence image analysis (Maintrac®) before and after the first cycle of a neoadjuvant and/or adjuvant R/CT, as well as before and after surgical resection of the primary tumor. To determine that blood-borne CETCs originate from tumor tissues, spheres were cultured from CETCs as well as from primary tumor tissue and compared with the expression of tumor-specific antigens. Within the scope of this study, it was demonstrated that the Maintrac® method allows for the precise detection and characterization of CETCs in the blood of patients with colorectal cancer independent of tumor stage. Furthermore, correlations between CETC parameters and patients' response to neoadjuvant and/or adjuvant R/CT that have been described in previous literature could be reproduced. Whether the observed trends are of a general nature and suitable as an auxiliary criterion for prognosis and treatment decisions remains to be shown. Patients with rectal cancer may benefit from CETC monitoring as a method to select suitable patients for adjuvant therapy.
For both sexes, colorectal cancer is the second leading cause of cancer-related death, globally (9.2%) (
In the last 30 years, the survival of patients suffering from colorectal cancer has increased markedly, owing mainly to the introduction of screening programs and of new therapeutic agents (
Circulating tumor cells, readily accessible from blood samples of patients with solid tumors, are an important link between primary tumors and metastases. A longitudinal monitoring of their numbers and properties can provide valuable information on therapy response and disease progression. Various studies demonstrated a correlation between circulating tumor cells and metastases, survival and therapy response for patients with different types of cancer (
Ki-67 is a non-histone nuclear protein, which is expressed in actively proliferating cells throughout the cell cycle, but not in quiescent (G0) cells (
While circulating tumor cells were shown to have prognostic potential for tumors of different entities (
A total of 22 patients, diagnosed with colorectal cancer, were enrolled in this study between October 2018 und August 2020. Before treatment, all patients passed a complete clinical evaluation including clinical history, physical examination, rectoscopy/colonoscopy, relevant blood examination and chest/abdominal computed tomography. Local stage was determined according to the TNM classification of the UICC (
The study was based on the Ethics Declaration of Helsinki and was approved by the Ethics Committee of the University of Bayreuth. Participants provided their written informed consent to participate in this study.
For all rectal cancer patients, treatment responses were assessed according to the pathological results after surgery, and graded by histological evaluation of the surgical specimens according to the criteria described by Dworak
For a proper assessment of therapy response, we additionally compared the tumor size and lymph node status as assessed by computed tomography and/or endosonography of each patient before and after neoadjuvant R/CT. According to Dworak regression grade, as well as TNM re-staging, each patient was individually assigned either to the group of good or poor responders to neoadjuvant R/CT (
Peripheral blood (7.5 ml) from 22 patients with colorectal cancer at different stages of disease was drawn into blood count tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant and processed 24 h after collection.
The Maintrac® approach was used for identification, quantification and further characterization of CETCs (
Only a small subpopulation of CETCs possessing additional stem cell properties is able to grow into metastases. By enumeration of CETCs able to clonally grow into CETC microspheres under specific conditions, we specified and quantified this subpopulation. Therefore, CETCs and leukocytes were isolated from peripheral blood as described earlier, plated at a density of 2x105 cells/ml in RPMI-1640 supplemented with l-glutamine, HEPES, penicillin/streptomycin and growth factors such as EGF, insulin and hydrocortisone, and incubated under standard cell culture conditions (37˚C, 5% CO2) in a sterile incubator. Every five days, the cultures were inspected under an inverted light microscope (PrimoVert) and fresh culture medium was added. Between days 21 and 28 of incubation, spheres were collected from the culture flasks, pelleted (250 x g, 7 min), and resuspended in 500 µl PBS. Immunostaining of spheres was performed using FITC-conjugated mouse anti-human EpCAM-antibody (clone HEA-125; Miltenyi Biotec GmbH), PE-conjugated mouse anti-human CD44-antibody (BD Biosciences) or mouse anti-human CD133-antibody (clone 7; BioLegend) for 20 min at 4˚C in the dark. The samples were then diluted in PBS/EDTA and transferred into the wells of a 96-well microtiter plate (Greiner Bio-one). Analysis of fluorescence was performed using a fluorescence scanning microscope (ScanR; Olympus). To verify vitality, PI staining of spheres was performed before the analysis. Finally, only vital CTC spheres with intact morphology and without PI staining were counted.
In case of surgery of the primary tumor, a small piece of tissue from the middle of the tumor (ø depending on the size of the tumor) was obtained in a sterile falcon in 10 ml transportation medium (RPMI-1640, 5% FBS, 5 µg/ml insulin, 2.75 µg/ml transferrin, 20 mM sodium selenite, 55 µg/ml sodium pyruvate, 1 µM hydrocortisone, 1,000 U/ml penicillin, 1,000 µg/ml streptomycin, 250 mg/ml amphotericin B, 15 mM HEPES, 100 µg/ml gentamycin, 5 µg/ml metronidazole) directly from the operating theater and kept at 4˚C for transportation. All samples were processed within 24 h after withdrawal. For further processing, the tumor tissue was washed 3-5 times in PBS by extensive shaking and put into a sterile petri dish. Before the tissue was chopped into small pieces of about 1 mm in diameter by anti-parallel movement of two scalpels, it was covered with a small amount of sphere culture medium (RPMI-1640 supplemented with l-glutamine, HEPES, penicillin/streptomycin and growth factors such as EGF, insulin and hydrocortisone). After one more washing step with PBS, the tissue was enzymatically homogenized with Accumax™ solution (Sigma-Aldrich; Merck KGaA) for 45 min under continuous mixing at rt. Then the cell suspension was filtered using a cell strainer (mesh size 0.44 µm; Greiner Bio-one) to eliminate bigger cell clumps and centrifuged at 240 x g for 10 min at rt. The resulting cell pellet was resuspended in 1 ml of culture medium and the number of vital cells was determined by bromophenol blue staining. Finally, the cells were plated in a concentration of approximately 0.6x106 vital cells/ml in culture medium in 6-well plates and incubated at standard cell culture conditions (37˚C, 5% CO2) for several weeks. All cultures were checked for bacterial infections daily and in the case of a minor infection isolated and treated with additional antibiotics, or in case of a major infection, discarded. If primary tumor spheres were detectable after a few weeks, a small amount of the culture was harvested and immunostained for further characterization and documentation.
Statistical analysis was performed using SigmaPlot (version 14.0; Systat Software Inc.) for Windows. Comparisons between variables were performed using ANOVA (analysis of variance) followed by a post hoc test for parametric data, or Kruskal-Wallis test followed by Dunn's test for nonparametric data. The significance level was set at P<0.05.
A total of 22 patients with histologically confirmed colorectal cancer (16 patients with rectal cancer, 6 patients with colon cancer) were enrolled in this study. Patients' characteristics are given in
Considering all patients (ICD10: C18 and C20), 1 patient was at stage I (4%), 7 patients were at stage II (32%), 12 patients were at stage III (55%), and 2 patients were at stage IV (9%). Six patients (27%) suffered from colon cancer and 16 (73%) from rectal cancer. The age of the patients ranged from 51 to 80 years (median 65.5 years). The median number of CETCs of all 22 patients with colorectal cancer was 55 CETCs per 100 µl cell suspension (ranging from 0 to 640) from which colon cancer patients (ICD10: C18) had a median CETC number per 100 µl of 45 (ranging from 0 to 145), and rectal cancer patients (ICD10: C20) of 65 (range from 0 to 640). No statistically significant differences in CETC numbers were observed in correlation to tumor size, lymph node status or distant metastasis (data not shown).
Using the Maintrac® method we detected CETCs in 100% of colorectal cancer patients included in this study. CETC numbers of all patients during the course of therapy are specified in
CETCs from patient #1 were investigated for their proliferative activity by growing non-adhesive suspension cultures. Formation of EpCAM-positive spheres was observed after the first cycle of neoadjuvant R/CT (5 spheres/100 µl blood). Interestingly, CETCs from all other samples did not show any sphere formation. During surgery of patient 1, a small piece of tumor tissue was set aside and stored on ice until further processing. After separation and washing, primary tumor cells were cultured under the same conditions as CETCs, also resulting in the formation of spherical structures. Both, spheres from the primary tumor, as well as spheres from the peripheral blood of patient #1 were further characterized by immunostaining (
14 patients with rectal cancer received neoadjuvant R/CT, 7 (50%) of whom showed a good response (
9 patients (41%; 6 patients with colon cancer and 3 patients with rectal cancer) received adjuvant chemotherapy and CETCs were quantified before surgery, before the beginning of chemotherapy and after the first cycle of adjuvant therapy (
Interestingly, all patients showed decreasing CETC numbers under adjuvant chemotherapy.
Ki-67-positive CETCs were detected in 20 patients (91%) and the percentage ranged from 0-100 (median: 25 Ki-67-positive CETCs/100 µl cell suspension). The median of Ki-67-positive CETCs/100 µl cell suspension in colon cancer patients was 18 (ranging from 0 to 170), and in rectal cancer patients 25 (ranging from 0 to 169). Although the differences in the Ki-67-positive CETCs at the three time points were not statistically significant neither for patients with neoadjuvant R/CT (P=0.202), nor in the group of patients with adjuvant CT (P=0.151), there was a trend in the number of Ki-67-positive CETCs to decrease under adjuvant CT, and to increase in patients receiving neoadjuvant R/CT (
Although disseminated tumor cells play a major role in the metastatic process of tumors, their detection and monitoring does not play a decisive role in standard clinical procedures. Monitoring of circulating tumor cells in the blood of cancer patients during therapy has already been shown to be a powerful prognostic tool for tumors of different entities including colorectal tumors (
In recent years, different techniques have been described for the detection of circulating tumor cells (
Up until now, only a few studies investigated the role of circulating tumor cells for evaluating the response to neoadjuvant R/CT for patients with rectal cancer. In a study by Zitt
As discussed above, we observed a heterogenic reaction of the CETC profile for patients receiving neoadjuvant R/CT. In contrast, a constant decrease in CETC numbers during adjuvant therapy was found. A potential explanation for this discrepancy may be the tumor burden in the adjuvant versus the neoadjuvant situation. While the neoadjuvant therapy targets the whole, intact tumor, in the adjuvant situation the tumor burden is low, because only microscopic tumor residues remain in the patient after surgery which may be more sensitive to chemotherapy and radiation. In addition, because of the reduced number of tumor cells, the development of resistance is less likely when compared to the neoadjuvant situation (
The proliferation marker Ki-67 is widely utilized in routine clinical diagnostic of breast cancer patients (
Finally, the general trends reported in this study could be exemplified by a case report of a rectal cancer patient (#1) receiving neoadjuvant R/CT, as well as adjuvant CT. This patient seemed to benefit from the surgery and from the additional adjuvant CT as CETC numbers decreased continuously after surgery to reach zero level on the last day of adjuvant therapy. This patient has remained free of relapse until nine months after the completion of therapy.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
RS, AK, KP and MG contributed to the design of the present study and developed the methodology. MG collected the bioinformatics data, performed the experiments, analyzed the results and wrote the manuscript. AK contributed to the collection of patient data. RS, AK and KP critically revised the manuscript and approved the final version to be published. All authors agreed to be accountable for all aspects of the study. All authors have read and approved the final manuscript. MG and RS confirm the authenticity of all the raw data.
The present study was approved by the Ethics Committee of the University of Bayreuth (approval no. O 1305/1-GB; Bayreuth, Germany). Written informed consent was obtained from all patients.
Not applicable.
Katharina Pachmann holds a patent protecting the Maintrac® method used in the present study (patent no. EP 3128325 B1; dated February 8th, 2017). The other authors declare that they have no competing interests.
3-Dimensional spheres cultured from peripheral blood or primary tumor tissue from patient #1. Immunostaining of spheres cultured from (A) primary tumor tissue or (B) CETCs from peripheral blood from patient #1. The epithelial origin of the spheres was identified by staining with anti-EpCAM antibody. PI was used as a vitality marker. Additionally, EpCAM-positive spheres were investigated for the expression of specific stem cell markers (CD44, CD133) and PD-L1. EpCAM, epithelial cell adhesion molecule; PI, propidium iodide; PD-L1, programmed death-ligand 1; CETC, circulating epithelial tumor cell.
Number of CETCs in the blood of patients with rectal cancer with good response to neoadj. R/CT. Blood samples were drawn before R/CT, after the first cycle of R/CT and after completion of R/CT immediately before surgery. Left, boxplot with median CETC values, quartiles and variability at each time point; right, individual CETC numbers at all time points, each line represents one patient. Patient #4 (360/10/60 CETC/100 µl), patient #7 (246/200/125 CETC/100 µl), patient #8 (105/64/65 CETC/100 µl), patient #5 (54/30/n.d. CETC/100 µl), patient #6 (40/105/70 CETC/100 µl), patient #3 (0/30/15 CETC/100 µl), patient #2 (n.d./10/0 CETC/100 µl). Assignment of patients in
Number of CETCs in the blood of patients with rectal cancer with poor response to neoadj. R/CT. Blood samples were drawn before R/CT, after the first cycle of R/CT and after completion of R/CT immediately before surgery. Left, boxplot with median CETC values, quartiles and variability at each time point; right, individual CETC numbers at all time points, each line represents one patient. Patient #1 (40/73/225 CETC/100 µl), patient #9 (10/0/0 CETC/100 µl), patient #10 (205/50/125 CETC/100 µl), patient #11 (0/390/n.d. CETC/100 µl), patient #12 (225/75/640 CETC/100 µl), patient #13 (155/189/210 CETC/100 µl), patient #14 (10/0/n.d. CETC/100 µl). Assignment of patients in
Number of CETCs in the blood of patients with colorectal cancer with adj. CT. Blood samples were drawn directly before surgery, 6-8 weeks after surgery and after the first cycle of adj. CT. Left, boxplot with median CETC values, quartiles and variability at each time point; right, individual CETC numbers at all time points, each line represents one patient. Patient #1 (C20; 225/65/20 CETC/100 µl), patient #15 (C20; 55/270/225 CETC/100 µl), patient #16 (C20; n.d./215/110 CETC/100 µl), patient #17 (C18; 45/145/80 CETC/100 µl), patient #18 (C18; n.d./60/35 CETC/100 µl), patient #19 (C18; 35/40/20 CETC/100 µl), patient #20 (C18; n.d./45/0 CETC/100 µl), patient #21 (C18; n.d./45/20 CETC/100 µl), patient #22 (C18; 110/105/20 CETC/100 µl). Assignment of patients in
Boxplots with median values, quartiles and variabilities of Ki-67-positive CETCs in the blood of patients with colorectal cancer. Left, patients with neoadj. R/CT; blood samples were drawn before the beginning of the neoadj. R/CT, after the first cycle of R/CT and after completion of R/CT (before surgery). Right, patients with adj. CT; blood samples were drawn directly before surgery, 6-8 weeks after surgery and after the first cycle of CT. CETC, circulating epithelial tumor cell; neoadj., neoadjuvant; adj., adjuvant; CT, chemotherapy; R/CT, radio/chemotherapy.
Number of CETCs and Ki-67-positive CETCs in the blood of patient #1 with rectal cancer receiving neoadj. R/CT, as well as adj. CT after surgical removal of the primary tumor (R0-resection). Blood samples were drawn prior to the neoadj. R/CT (40 CETCs/100 µl), after the first cycle of R/CT (73 CETCs/100 µl), immediately before surgery (225 CETCs/100 µl), 6-8 weeks after surgery/before the beginning of adj. CT (65 CETCs/100 µl), after the first cycle of adj. CT (20 CETCs/100 µl) and on the last day of adj. CT (0 CETCs/100 µl). CETC, circulating epithelial tumor cell; neoadj., neoadjuvant; adj., adjuvant; CT, chemotherapy; R/CT, radio/chemotherapy.
Clinicopathological characteristics of patients with colon (C18) and rectal (C20) cancer included in this study.
Clinicopathological characteristics | Number of patients with colon cancer, n (%) | Number of patients with rectal cancer, n (%) |
---|---|---|
Total | 6(27) | 16(73) |
Age, years | ||
>60 | 5(23) | 11(50) |
≤60 | 1(4) | 5(23) |
Sex | ||
Female | 3(14) | 6(27) |
Male | 3(14) | 10(45) |
Tumor size |
||
T1 | 0 (0) | 0 (0) |
T2 | 1(4) | 3(14) |
T3 | 4(18) | 11(50) |
T4 | 1(4) | 2(9) |
Lymph node status |
||
Positive | 6(27) | 4(18) |
Negative | 0 (0) | 12(54) |
Distant metastasis | ||
Positive | 1(4) | 1(4) |
Negative | 5(23) | 15(68) |
Neoadjuvant therapy | 1(17) |
14(64) |
Adjuvant therapy | 6(27) | 3(14) |
aObtained by histopathological examination of a surgical specimen;
bone patient with carcinoma of the colon ascendens also had rectal cancer, for which neoadjuvant radiochemotherapy had been performed prior to study entry.
Responses of patients with rectal cancer (C20) after receiving neoadjuvant R/CT.
TNM | ||||
---|---|---|---|---|
Patient number | Before R/CT | After R/CT | Regression grade | Response category |
1 | µ, T2; µ, N0 | yp, T3b; yp, N1 | 1 | Poor |
2 | µ, T2; c, T3; µ, N1 | yp, T2; yp, N0 | 3 | Good |
3 | µ, T3; µ, N+ | yp, T2; yp, N0 | 2 | Good |
4 | µ, T3; µ, N0 | yp, T2; yp, N0 | 3 | Good |
5 | µ, T3; µ, N1; c, M1HEP | yp, T3; yp, N0 | 3 | Good |
6 | c, T4; c, N2b | yp, T3b; yp, N0 | 3 | Good |
7 | c, T3; c, N+ | yp, T3a; yp, N1b | 3 | Good |
8 | µ, T3; µ, N+ | yp, T3a; yp, N0 | 3 | Good |
9 | µ, T3; µ, N0 | yp, T3b; yp, N0 | 1 | Poor |
10 | µ, T2; µ, N+ | yp, T3; yp, N0 | 2 | Poor |
11 | c, T3; c, N1 | yp, T4a; yp, N1b | 3 | Poor |
12 | µ, T2; µ, N+ | yp, T3a; p, N0 | 1 | Poor |
13 | µ, T3; µ, N1 | yp, T3b; yp, N0 | 1 | Poor |
14 | µ, T3; µ, N1; c, M1aPUL | yp, T4a; yp, N0; c, M1a | 1 | Poor |
Patients with rectal cancer (C20) were assigned to either the group of good or poor responders according to Dworak regression grade and TNM re-staging. µ, stage determined by ultrasonography; c, stage determined by clinical examination; y, stage assessed after R/CT; p, stage given by histopathological examination of a surgical specimen; TNM, tumor node metastasis; R/CT, radio/chemotherapy.