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Abstract

We investigate the clinical and pathological features related to variations in colorectal tumour apoptosis, proliferation and angiogenesis and the influence of the latter in short-term mortality (2 years); 551 tumour samples from a prospective cohort of patients with colorectal cancer were examined and tumour biology markers were determined as follows: percentage of apoptotic cells, by the terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling technique; Ki-67 antigen, as a cell proliferation marker and density of microvessels (as a marker of angiogenesis). An increase in the percentage of cellular apoptosis is significantly related to the presence of poorly differentiated tumours, with vascular invasion (p < 0.001). The CD105 angiogenesis marker is not related to any clinical–pathological parameter except that of higher frequency in older patients (p = 0.03). Ki-67 is more frequently expressed in tumours with less nervous invasion (p = 0.05). Neither apoptosis nor angiogenesis present any significant association with short-term survival. The only marker clearly related to 2-year survival is Ki-67, which is shown to be a good prognostic factor in the multivariate analysis (hazard ratio = 0.49; 95% confidence interval = 0.27–0.90). Therefore, in a prospective cohort of colorectal cancer patients, only Ki-67 is a marker of good prognosis in short-term follow-up.

Keywords

Cell proliferation apoptosis angiogenesis colorectal neoplasms Ki-67 marker

Introduction

With an estimated incidence of 1.4 million cases and an overall mortality rate of 693,900 in 2012, colorectal cancer (CRC) is the third most common diagnosed malignancy and a major public health challenge worldwide.¹

Importantly, despite recent improvements in CRC management, approximately 40%–50% of early diagnosed CRC patients will relapse, which justifies the need to find new biomarkers with a prognostic value.²

In CRC patients, different histopathological features such as tumour grade, depth of invasion, lymph nodes and the presence of liver metastasis are prognostic factors.³ However, as these conventional prognostic parameters are not able to predict the outcome of every individual patient, there is still a need to find novel markers to help predict these patients’ outcome and guide therapy decisions more accurately.

Carcinogenesis is fundamentally characterized by alterations in cellular proliferation, apoptosis and formation of blood vessels. Given the evidence of apoptosis’ important role during CRC pathogenesis and progression, the significance of apoptotic rates as potential prognostic marker should be given serious consideration. Nevertheless, little research has been conducted into the prognostic significance of apoptosis in CRC. In this regard, a small number of studies have reported that in some distal colon carcinomas, reduced apoptosis is related to undesirable outcomes and metastasis.⁴ Others have suggested that there is an inverse relationship between apoptotic index (AI) and survival.^5–7 In a retrospective study presented by Alcaide et al.,⁸ it was reported for the first time the evidence that high AI is related to a significant decrease in CRC patients’ overall and disease-free survival.

Vascular–endothelial, hepatocyte and platelet-derived growth factors secretion by neoplasms stimulate endothelial migration and proliferation and promote angiogenesis.⁹ Neovascularization promotes tumour growth by supplying nutrients and oxygen.¹⁰ CD105 (endoglin), a co-receptor of the transforming growth factor-beta superfamily, has been proposed as a marker of neovascularization in solid malignancies. As sustained angiogenesis is one of the hallmarks of cancer, anti-angiogenic therapies are one of the most promising strategies for cancer treatment. Intratumoral microvessel density (MVD) is an independent prognostic indicator; thus, increased MVD correlates with shorter survival.^11–13 However, other investigators have failed to confirm these findings.¹⁴ Another important proliferation marker is Ki-67 antigen. Ki-67 (MKI67, KIA, MIB-1), a nuclear protein with a key role during cell division, can be detected during cell cycle active phases (G1, S, G2 and M) but not during the Gap0 phase. Although Ki-67 measurement has been widely used as a cell proliferation marker, few studies have considered the prognostic role of this protein, many of which report conflicting results.^15–19

In view of the dearth of prospective studies of biomarkers that might supply short-term prognostic information, this study was designed to evaluate the effects of angiogenesis (assessed by CD-105), apoptosis (by the terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) method) and proliferation (by the Ki-67 antibody) on short-term prognosis and the relations between these and diverse clinical–pathological variables in a large prospective cohort of CRC patients.

Materials and methods

Study design

A prospective, multicentre observational study was conducted, with the participation of seven Spanish national public health system hospitals from two different Autonomous Communities (Andalusia and the Basque Country). The following hospitals took part in the study: Antequera Hospital, Costa del Sol Hospital, University Hospital of Valme, University Hospital Virgen del Rocío, University Hospital Virgen de las Nieves, Galdakao-Usansolo Hospital and Basurto University Hospital.

Inclusion criteria for participation in this study were as follows: Patients diagnosed with new or incidental colon or rectum cancer, recruited during 2010–2012, stages I–IV and surgically treated either as an urgent case or according to schedule.

Data collection

Tissue specimens were obtained from curative surgery and processed by the corresponding Pathological Anatomy Unit. Prior to immunohistochemistry analyses, resected samples were formalin fixed for 48 h (formalin–tissue volume ratio 10:1), dehydrated with ethanol and embedded into paraffin blocks using an automated tissue processor.

A total of 551 tumour samples were analysed to detect markers of tumour biology (percentage of apoptotic cells by the TUNEL technique, Ki-67 antigen as a marker of cell proliferation and MVD as a marker of angiogenesis).

Data collection methods

In order to ensure consistency among centres and reviewers, clinical data from medical records and database were directly collected by qualified reviewers, following a data collection form and an instruction manual. Each patient was included in the study prospectively and consecutively from each of the participating hospitals. CRC diagnosis was based on anatomopathological findings after a biopsy by colonoscopy.

Inclusion criteria

Patients diagnosed with cancer of the colon (>15 cm above the anal margin) or of the rectum (≤15 cm above the anal margin), who underwent initial curative and/or palliative surgery for this condition.

Exclusion criteria

Patients diagnosed with colon or rectal cancer in situ. Patients not operable. Patients who had received prior radiotherapy or chemotherapy were excluded from the survival analysis.

Study variables

Explanatory (independent) variables

The following sociodemographic factors associated with the patient were evaluated: sex, age, family history and type of CRC symptom observed.

Clinical–pathological factors of the tumour

Location of the tumour (rectum or colon); degree of histological differentiation; tumour (T) or lymph node (N) staging, using the tumour–node–metastasis (TNM) clinical staging system; lymphovascular and perineural invasion; the presence of metastasis and state of the serum tumour markers CEA and CA19-9.

Characteristics of the initial treatment

Type of treatment applied is surgery, chemotherapy, radiotherapy, biological treatment or support treatment.

The main outcome variable is overall survival, defined as the time (in months) elapsed from the date of histological diagnosis until the date of death or last control. Events that occurred within 2 months of the first date were excluded from the analysis.

The molecular variables considered were the percentage of apoptotic cells (determined by the TUNEL technique), the presence of the Ki-67 antigen, as a marker of cell proliferation, and MVD, as a marker of angiogenesis.

Immunohistochemistry

Levels of Ki-67 and CD105 were determined by the streptavidin–biotin–peroxidase enzyme assay. In order to measure Ki-67, we employed MIB1 monoclonal antibody, which allows the detection of Ki-67 nuclear antigen in the formalin-fixed paraffin-embedded tissue sections and whose reactivity is not affected by any delay in fixation. Ki-67 expression was determined as the percentage of tumour cells positively stained with MIB1in each sample, being the nuclear staining the most common criterion of positivity. The median value was used to distinguish between high and low Ki-67 expression.

The results of each case were evaluated independently (blind study with anonymized samples) by two pathologists in a semi-quantitative analysis in which the intensity and percentage of staining were determined. Cases that produced disagreement were re-evaluated by a third pathologist.

Detection of cellular apoptosis using the TUNEL method

To detect apoptotic cells, the 3′-ends of the DNA fragments generated by apoptosis-associated endonucleases were labelled in situ using a commercial apoptosis detection kit (Roche Diagnostics GmbH, Mannheim, Germany). The sections were pre-treated with DNase as a positive control, and the absence of the enzyme was taken as a negative control. Cells are defined as apoptotic if the nuclear area is completely stained. Apoptotic bodies are defined as small globular bodies that are positively marked in the cytoplasm of the tumour cells, whether isolated or in groups; 1000 cells were counted for each sample. The cut-off point used was the median apoptosis value in each tumour series.

MVD (angiogenesis)

Endothelial cells were stained with CD105 to evaluate the density of intratumour microvessels. For each case, a histologic section was immunostained and the microvessels were counted by two investigators (who were blinded to diagnosis and to the immunohistochemical markers). Microvessels were only counted in intratumour ‘hotspots’ and were identified using high magnification (400×) for precise quantification. The median value observed was used to distinguish between high and low densities of intratumour microvessels.

Statistical analysis

A descriptive analysis was performed, using measures of central tendency, dispersion and position for the quantitative variables. A bivariate analysis was then performed to evaluate the differences in distribution of the independent variables with respect to dichotomized tumour markers according to the median values obtained. Student’s t test was used for the quantitative variables and the chi-square test for the qualitative ones. Kaplan–Meier survival analysis was performed to evaluate overall survival according to tumour markers, assessing mean survival rates with a 95% confidence interval (CI). Finally, Cox regression analysis was performed to evaluate markers previously found to be significant in the Kaplan–Meier analysis, adjusting for tumour staging, with the hazard ratio being described by the corresponding 95% CI. In each of these analyses, the level of statistical significance was p < 0.05. All analyses were performed with SPSS v15 statistical software.

Ethics

Patients were informed of the study objectives, invited to voluntarily participate and were included in the study sequentially. All of them signed a written informed consent to participate in the study. The Institutional Review Boards of the participating hospitals approved this project, in particular the Regional Committee of Clinical Trials of Andalucía (Sevilla, Spain)

Results

A total of 551 samples were evaluated. Valid samples for each of the studied markers were 457 for TUNEL, 487 for CD105 and 541 for Ki-67.

There were no differences in the two biological parameters – Ki-67 and the AI – concerning colon or rectal location, age, gender or past history of colorectal or other neoplasias (Table 1). However, CD105 was observed more frequently in older patients and in the colon, although in the last case without reaching statistical significance (p = 0.03 and p = 0.06, respectively). In addition, contrary to Ki-67 and CD105, increased apoptosis was significantly related to high-grade carcinomas and vascular invasion (p = 0.001 in both cases) and was related to the presence of lymph node metastasis, although the relation was not statistically significant (p = 0.06). Figure 1 shows three examples of tumours presenting positive expression for all the biological markers studied.

Table 1.

Distribution of sociodemographic and clinical characteristics according to the biological marker.

Variables	TUNEL (%)					CD105 (%)					Ki-67 (%)
	≤1.4		>1.4		p	≤10.66		>10.66		p	≤29		>29		p
	N	%	N	%	p	N	%	N	%	p	N	%	N	%	p
Sex
Male	158	50.8	153	49.2	0.087	159	49.1	165	50.9	0.291	182	50.0	182	50.0	0.478
Female	97	59.5	66	40.5	0.087	89	54.6	74	45.4	0.291	95	53.7	82	46.3	0.478
Age
Mean – SD	69.5	10.8	68.1	11.5	0.192	68.1	11.6	70.1	10.2	0 . 038	68.6	10.9	69.3	11.1	0.491
Delay patients
≤180 days	150	51.0	144	49.0	0.729	154	51.3	146	48.7	1	157	46.2	183	53.8	0.601
>180 days	29	54.7	24	45.3	0.729	28	51.9	26	48.1	1	30	50.8	29	49.2	0.601
Signs/symptoms of presence of disease
Asymptomatics	21	51.2	20	48.8	0.855	20	48.8	21	51.2	0.902	24	55.8	19	44.2	0.637
Signs/symptoms moderate or severe	234	54.0	199	46.0	0.855	228	51.1	218	48.9	0.902	253	50.8	245	49.2	0.637
Altered bowel rhythm
No	139	54.5	116	45.5	0.808	120	48.0	130	52.0	0.217	142	50.5	139	49.5	0.813
Yes	116	53.0	103	47.0	0.808	128	54.0	109	46.0	0.217	135	51.9	125	48.1	0.813
Tumour location
Colon	206	53.0	183	47.0	0.506	196	48.9	205	51.1	0.067	223	50.7	217	49.3	0.693
Rectal	49	57.6	36	42.4	0.506	52	60.5	34	39.5	0.067	54	53.5	47	46.5	0.693
Size of tumour
Small local extension (T0–T1–T2)	67	56.8	51	43.2	0.479	64	53.3	56	46.7	0.544	77	56.6	59	43.4	0.124
Large local extension (T3–T4)	182	52.4	165	47.6	0.479	179	49.6	182	50.4	0.544	192	48.5	204	51.5	0.124
Presence of lymph node metastasis
Absence	153	56.9	116	43.1	0.063	147	51.8	137	48.2	0.43	160	50.0	160	50.0	0.728
Presence	90	47.6	99	52.4	0.063	90	47.6	99	52.4	0.43	106	52.0	98	48.0	0.728
Histology
Adenocarcinoma	234	53.4	204	46.6	0.508	228	50.6	223	49.4	1	255	50.8	247	49.2	0.691
Mucinous adenocarcinoma	19	61.3	12	38.7	0.508	16	51.6	15	48.4	1	19	55.9	15	44.1	0.691
Staging (Dukes)
A	50	54.3	42	45.7	0.25	50	52.6	45	47.4	0.452	61	56.5	47	43.5	0.176
B	104	59.4	71	40.6		94	50.3	93	49.7		97	46.2	113	53.8
C	75	45.7	89	54.3		80	49.1	83	50.9		94	53.4	82	46.6
Metastasis	17	58.6	12	41.4		14	45.2	17	54.8		13	40.6	19	59.4
Differentiation
Low degree	189	56.8	144	43.2	0 . 001	171	49.4	175	50.6	0.728	193	50.1	192	49.9	1
High degree	22	32.8	45	67.2	0 . 001	30	46.2	35	53.8	0.728	36	50.7	35	49.3	1
Vascular invasion
Absence	188	59.1	130	40.9	0 . 001	179	52.2	164	47.8	0.342	189	50.1	188	49.9	0.648
Presence	17	33.3	34	66.7	0 . 001	28	60.9	18	39.1	0.342	31	54.4	26	45.6	0.648
Perineural invasion
Absence	186	56.9	141	43.1	0.744	182	51.7	170	48.3	0.539	185	48.2	199	51.8	0.056
Presence	20	52.6	18	47.4	0.744	20	58.8	14	41.2	0.539	29	64.4	16	35.6	0.056
Carcinoembryonic antigen (CEA)
Normal (0–5)	145	56.9	110	43.1	0.411	135	51.1	129	48.9	0.783	148	49.8	149	50.2	0.876
Anormal (<5)	61	51.7	57	48.3	0.411	66	53.2	58	46.8	0.783	63	48.5	67	51.5	0.876
CA 19-9
Normal (1–37)	88	56.8	67	43.2	0.667	88	52.1	81	47.9	0.991	81	45.0	99	55.0	0.096
Anormal (>37)	13	50.0	13	50.0	0.667	15	50.0	15	50.0	0.991	19	63.3	11	36.7	0.096
Screening test
No	210	54.5	175	45.5	0.456	204	51.6	191	48.4	0.585	227	51.1	217	48.9	1
Yes	43	49.4	44	50.6	0.456	43	47.8	47	52.2	0.585	48	50.5	47	49.5	1
Tunnel (%)
Cut-off value≤1.4						122	54.5	102	45.5	0.218	129	51.8	120	48.2	0.552
>1.4						97	48.0	105	52.0	0.218	105	48.6	111	51.4	0.552
CD105 (%)
Cut-off value≤10.66											127	52.0	117	48.0	0.097
>10.66											104	44.1	132	55.9	0.097

SD: standard deviation; N: number of valid observations; TUNEL: terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling.

Valid/missing cases: 474/77 (TUNEL), 487/64 (CD105) and 541/10 (Ki-67).

Cut-off values: 1.4 (TUNEL), 10.66 (CD105) and 29 (Ki-67).

Figure 1.

Three examples of tumours presenting positive expression of the biological markers used in this study. (a) Numerous nuclei stained for Ki-67 (400×). (b) Intraneoplastic vascular proliferation. Staining with CD105 (200×). (c) Fragmented nuclei positive for the TUNEL technique are observed in a poorly differentiated carcinoma (200×).

With respect to the clinical serum tumour marker CEA, no relationship was found. However, Ki-67-positive tumours were less frequent with increased levels of CA 19-9 (p = 0.09), while Ki-67 was less frequent when neural invasion took place (p = 0.056) although in both cases without reaching statistical significance (Table 1).

To our knowledge, this study is the first to analyse the possible association between the presence of symptoms and alterations in proliferation, apoptosis or angiogenesis. In fact, no differences were found in this respect between asymptomatic and symptomatic tumours (Table 1). Neither was any relationship observed when each symptom was considered separately (data not shown). Furthermore, no clear relationship was observed between the different biological factors investigated, except that more new vessels were formed in tumours with a high expression of Ki-67 (p = 0.09).

Table 2 shows survival segmented by biological marker and by tumour staging. Overall survival at 2 years of follow-up was significantly decreased in patients with a low number of Ki-67-positive cells (Figure 2). This prognostic information persisted in the multivariate analysis (Table 3). However, there were no differences in survival concerning the percentages of CD105 and TUNEL positive cells.

Table 2.

Survival segmented by levels of markers and tumour staging.

Markers		N	Events	Survival			p
				Mean	95% CI
				Mean	Lower	Higher
TUNEL (%)	≤1.4	213	18	40.2	39.2	41.3	0.106
TUNEL (%)	>1.4	189	26	38.5	36.4	40.7	0.106
CD105 (%)	≤10.66	204	18	39.4	38.4	40.5	0.304
CD105 (%)	>10.66	192	24	38.5	36.4	40.6	0.304
Ki-67 (%)	≤29	230	32	38.4	36.2	40.6	0 . 039
Ki-67 (%)	>29	214	16	40.5	39.5	41.4	0 . 039
Stages (Dukes)	A	96	5	41.8	40.5	43.0	<0.001
	B	179	14	40.3	39.1	41.5
	C	147	21	35.8	32.6	39.0
	Metastasis	16	7	24.1	19.6	28.5

CI: confidence interval; N: number of valid observations; TUNEL: terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling.

Total valid cases: 402 (TUNEL), 396 (CD105) and 444 (Ki-67).

Cut-off values: 1.4 (TUNEL), 10.66 (CD105) and 29 (Ki-67).

Bold values represent p < 0.05.

Figure 2.

Survival of patients grouped according to Ki-67 expression. Survival of patients with primary tumours lacking Ki-67 expression (thick line) is significantly shorter than that of patients with tumours presenting Ki-67 expression (thin line).

Table 3.

Survival assessed through the multivariate Cox regression model.

Variables		β	p	Hazard ratio	95% CI
		β	p	Hazard ratio	Inferior	Superior
Ki-67	≤29		0.02	1.00
Ki-67	>29	−0.71		0.49	0.27	0.90
Stages (Dukes)	A		<0.001	1.00
	B	0.48		1.61	0.57	4.53
	C	1.18		3.24	1.22	8.61
	Metastasis	2.46		11.76	3.82	36.25

CI: confidence interval.

n evaluated = 432.

Bold values represent p < 0.05.

We also considered the question of prognostic information from these three markers in patients treated with chemotherapy after surgery. However, no such information was obtained (Figure 3).

Figure 3.

Survival of patients undergoing chemotherapy after surgical resection grouped according to Ki-67 expression. No differences were found between Ki-67-positive tumours (thick line) and those negatives (thin line).

Discussion

This article considers the biological aspects of CRC, seeking to establish novel markers providing short-term prognostic information. To do so, we analyse the fundamental processes of proliferation, apoptosis and angiogenesis in the evolution of tumours in a prospective cohort of CRC patients.²⁰

Defects in the response to apoptosis are implicated not only in pathological aspects of CRC but also in its resistance to ionizing radiation and chemotherapeutic drugs. It has been demonstrated in a previous retrospective series with long follow-up that apoptotic cells, evaluated by the TUNEL assay, are more frequently present in samples from CRC patients with a worse prognosis than in those with a good one.⁸ Other studies have also reported this inverse relationship between AI and survival.^5–7 The varying or conflicting results reported by such studies, as in the case of the prognostic significance of cleaved caspase-3,^21–23 has led to the investigation of other markers to clarify the relationship between tumoral cell apoptosis and patients’ survival.^21–23 In this regard, it is worth to highlight the inverse relationship reported between AI and survival, when apoptosis is measured by the M30 antibody,^24,25 as AI may reveal a physiological effort to eliminate neoplastic cells with genetic alterations such as DNA mutations or mitotic errors. In other words, if mutated neoplastic cells that can inhibit programmed cell death are the only ones to survive and continue proliferating, then apoptosis would allow the survival of the most aggressive cancer cells for tumour formation by selection. However, it is interesting to examine whether this prognostic information is valid for the first 2 years of follow-up, which is when the majority of events occur. In our series, although tumours with elevated AI were more frequent in patients with a short survival, the association did not reach statistical significance (p = 0.1). Therefore, apoptosis does not represent a clear marker with prognostic information, at least in short-term follow-up.

Concerning measurement methods, although apoptosis can be detected by other methods, TUNEL is the approach most commonly adopted and is considered the reference standard.²⁶ Moreover, it also presents a very good correlation with the M30 antibody.²⁷

Although a previous study by Kim et al.²⁸ in rectal cancer has found that apoptosis is associated with lymphatic invasion, other studies show opposite results.²⁹ In this regard, even though we did not reach statistical significance (p = 0.06) in our prospective series, we found a statistically significant association between apoptosis and poorly differentiated tumours and tumours with vascular invasion, which is in line with a previous study evidencing that apoptosis is greater in advanced than in earlier stages.⁸

In our series, proliferation, measured by the Ki-67 antigen, was independently related to survival. In this respect, Anjomshoaa et al.³⁰ also reported an inverse association between colon cancer progression and proliferative tumour activity. Moreover, Adams et al.¹⁷ found Ki-67 to be predictive of improved survival during the first 18 months following diagnosis, while Salminen et al.³¹ observed that proliferative activity in rectal cancer, as measured by Ki-67, was associated with increased survival compared to patients with low Ki-67. Similar results were obtained recently by Melling et al.¹⁹ It is very important to understand the mechanisms underlying this inverse relationship, as proliferation could play a more significant role at the beginning than at the end of the neoplastic process.

Although Fodor et al.¹⁸ reported that Ki-67 proliferation index was not a useful prognostic factor for CRC patients, they also highlighted the need to continue with a further evaluation of this marker as a predictive factor for the additional benefits derived from adjuvant chemotherapy. In this respect, however, our own results do not show that the expression of Ki-67 is related to survival in patients with colorectal carcinomas who have undergone chemotherapy. Finally, Peng et al.¹⁵ in a study of stage II tumours found an association between Ki-67 expression and a worse prognosis. Despite these two studies with different results to ours, the observations made in this work will make a significant contribution to the knowledge on this topic.

Our findings also show that an abnormal result for the CA 19-9 serum marker tends to be associated with lower Ki-67 although without reaching statistical significance (p = 0.09), which might indicate that in more advanced stages of the disease, with a lower proportion of cells positive for Ki-67, there would be a greater presence of the clinical marker CA 19-9.

Our series revealed a positive association between the neoformation of vessels with Ki-67 expression (i.e. the greater the proliferation, the greater the formation of vessels) although without reaching statistical significance (p = 0.09). A similar relationship was found by Zlobec et al.¹³

Several studies show contradictory results about the role of angiogenesis in colorectal tumours. Thus, although Bossi et al.³² and Pietra et al.³³ reported that an increased vascularization was not a valuable prognostic factor for CRC patients, in their study with factor VIII, Saclarides et al.³⁴ and Frank et al.,³⁵ reported a significant correlation between angiogenesis, recurrence, metastasis and survival. Moreover, Zlobec et al.¹³ and Li et al.³⁶ reported that CD105 was a marker of disease progression. However, our study did not find angiogenesis to be a prognostic indicator in short-term follow-up. Nevertheless, it could be a crucial factor in late phases of the disease, with the progression to a metastatic state.³⁷

In our study, we targeted cancer vasculature via CD105 instead of other commonly used pan-endothelial markers such as CD31, CD34 or Factor VII as the endoglin antibody particularly binds to the activated endothelial cells implicated in tumour angiogenesis but not to the vascular endothelium in normal tissues.³⁸

This study demonstrates a very limited value for TUNEL assay and CD-105 as short survival biomarkers for CRC. A wide range of other markers that influence these phenomena in the human body should also be assessed in CRC. In fact, there are numerous factors that can influence these parameters (e.g. cell cycle phase, circadian rhythms and cell types unrelated to the tissue of interest) which have not been considered in this study.

In conclusion, our study demonstrates that Ki-67 labelling is an independent prognostic parameter for survival at short-term follow-up. Nevertheless, further pre-clinical and clinical investigation is required.

Footnotes

Appendix 1

The REDISSEC CARESS-CCR (Results and Health Services Research in Colorectal Cancer) group

Jose María Quintana Lopez¹, Marisa Baré Mañas², Maximino Redondo Bautista³, Eduardo Briones Pérez de la Blanca⁴, Nerea Fernández de Larrea Baz⁵, Cristina Sarasqueta Eizaguirre⁶, Antonio Escobar Martínez⁷, Francisco Rivas Ruiz⁸, Maria M. Morales-Suárez-Varela⁹, Juan Antonio Blasco Amaro¹⁰, Isabel del Cura González¹¹, Inmaculada Arostegui Madariaga¹², Amaia Bilbao González⁷, Nerea González Hernández¹, Susana García-Gutiérrez¹, Iratxe Lafuente Guerrero¹, Urko Aguirre Larracoechea¹, Miren Orive Calzada¹, Josune Martin Corral¹, Ane Antón-Ladislao¹, Núria Torá¹³, Marina Pont¹³, María Purificación Martínez del Prado¹⁴, Alberto Loizate Totorikaguena¹⁵, Ignacio Zabalza Estévez¹⁶, José Errasti Alustiza¹⁷, Antonio Z Gimeno García¹⁸, Santiago Lázaro Aramburu¹⁹, Mercé Comas Serrano²⁰, Jose María Enríquez Navascues²¹, Carlos Placer Galán²¹, Amaia Perales²², Iñaki Urkidi Valmaña²³, Jose María Erro Azkárate²⁴, Enrique Cormenzana Lizarribar²⁵, Adelaida Lacasta Muñoa²⁶, Pep Piera Pibernat²⁶, Elena Campano Cuevas²⁷, Ana Isabel Sotelo Gómez²⁸, Segundo Gómez-Abril²⁹, F. Medina-Cano³⁰, Julia Alcaide³¹, Arturo Del Rey-Moreno³², Manuel Jesús Alcántara³³, Rafael Campo³⁴, Alex Casalots³⁵, Carles Pericay³⁶, Maria José Gil³⁷, Miquel Pera³⁷, Pablo Collera³⁸, Josep Alfons Espinàs³⁹, Mercedes Martínez⁴⁰, Mireia Espallargues⁴¹, Caridad Almazán⁴², Paula Dujovne Lindenbaum⁴³, José María Fernández-Cebrián⁴³, Rocío Anula Fernández⁴⁴, Julio Ángel Mayol Martínez⁴⁴, Ramón Cantero Cid⁴⁵, Héctor Guadalajara Labajo⁴⁶, María Heras Garceau⁴⁶, Damián García Olmo⁴⁶, Mariel Morey Montalvo⁴⁷, Javier Mar⁴⁸.

¹Unidad de Investigación. Hospital Galdakao-Usansolo, Galdakao-Bizkaia/Red de Investigación en Servicios de Salud en Enfermedades Crónicas–REDISSEC.

²Epidemiologia Clínica y Cribado de Cáncer, Corporació Sanitaria Parc Taulí, Sabadell/Red de Investigación en Servicios de Salud en Enfermedades Crónicas–REDISSEC.

³Servicio de Laboratorio. Hospital Costa del Sol, Málaga/REDISSEC.

⁴Unidad de Epidemiología. Distrito Sevilla, Servicio Andaluz de Salud.

⁵Centro Nacional de Epidemiología, Instituto de Salud Carlos III, Madrid/REDISSEC.

⁶Unidad de Investigación. Hospital Universitario Donostia/Instituto de Investigación Sanitaria Biodonostia, Donostia -REDISSEC.

⁷Unidad de Investigación. Hospital Universitario Basurto, Bilbao/REDISSEC.

⁸Servicio de Epidemiología. Hospital Costa del Sol, Málaga -REDISSEC.

⁹Department of Preventive Medicine and Public Health, University of Valencia/CIBER de Epidemiología y Salud Pública (CIBERESP)/CSISP-FISABIO, Valencia.

¹⁰Unidad de Evaluación de Tecnologías Sanitarias, Agencia Laín Entralgo, Madrid.

¹¹Unidad Apoyo a Docencia-Investigación. Dirección Técnica Docencia e Investigación. Gerencia Adjunta Planificación. Gerencia de Atención Primaria de la Consejería de Sanidad de la Comunidad Autónoma de Madrid.

¹²Departamento de Matemática Aplicada, Estadística e Investigación Operativa, UPV–REDISSEC.

¹³Epidemiologia Clínica y Cribado de Cancer, Corporació Sanitaria Parc Taulí, Sabadell/REDISSEC.

¹⁴Servicio de Oncología. Hospital Universitario Basurto, Bilbao.

¹⁵Servicio de Cirugía General. Hospital Universitario Basurto, Bilbao.

¹⁶Servicio de Anatomía Patológica. Hospital Galdakao-Usansolo, Galdakao.

¹⁷Servicio de Cirugía General. Hospital Universitario Araba, Vitoria-Gasteiz.

¹⁸Servicio de Gastroenterología. Hospital Universitario de Canarias, La Laguna.

¹⁹Servicio de Cirugía General. Hospital Galdakao-Usansolo, Galdakao.

²⁰IMAS-Hospital del Mar, Barcelona.

²¹Servicio de Cirugía General y Digestiva. Hospital Universitario Donostia.

²²Instituto de Investigación Sanitaria Biodonostia, Donostia.

²³Servicio de Cirugía General y Digestiva. Hospital de Mendaro.

²⁴Servicio de Cirugía General y Digestiva. Hospital de Zumarraga.

²⁵Servicio de Cirugía General y Digestiva. Hospital del Bidasoa.

²⁶Servicio de Oncología Médica. Hospital Universitario Donostia.

²⁷Instituto de Biomedicina de Sevilla. Hospital Universitario Virgen del Rocío, Sevilla.

²⁸Servicio de Cirugía. Hospital Universitario Virgen de Valme, Sevilla.

²⁹Servicio de Cirugía General y Aparato Digestivo. Hospital Dr. Pesset, Valencia.

³⁰Servicio de Cirugía General y Aparato Digestivo. Agencia Sanitaria Costa del Sol, Marbella.

³¹Servicio de Oncología M_edica. Agencia Sanitaria Costa del Sol, Marbella.

³²Servicio de Cirugía. Hospital de Antequera.

³³Coloproctology Unit, General and Digestive Surgery Service, Corporació Sanitaria Parc Taulí, Sabadell.

³⁴Digestive Diseases Department, Corporació Sanitaria Parc Taulí, Sabadell.

³⁵Pathology Service, Corporació Sanitaria Parc Taulí, Sabadell.

³⁶Medical Oncology Department, Corporació Sanitaria Parc Taulí, Sabadell/REDISSEC.

³⁷General and Digestive Surgery Service, Parc de Salut Mar, Barcelona.

³⁸General and Digestive Surgery Service, Althaia- Xarxa Assistencial Universitaria, Manresa.

³⁹Catalonian Cancer Strategy Unit, Department of Health, Institut Catalá d’Oncología.

⁴⁰Medical Oncology Department, Institut Catal_a d’Oncología.

⁴¹Agency for Health Quality and Assessment of Catalonia eAquAS- and REDISSEC.

⁴²Agency for Health Quality and Assessment of Catalonia eAQuAS-, CIBER de Epidemiología y Salud Pública eCIBERESP.

⁴³Servicio de Cirugía General y del Aparato Digestivo, Hospital Universitario Fundación Alcorcón, Madrid.

⁴⁴Servicio de Cirugía General y Aparato Digestivo, Hospital Universitario Clínico San Carlos, Madrid.

⁴⁵Servicio Cirugía General y del Aparato Digestivo, Hospital Universitario Infanta Sofía, San Sebastián de los Reyes, Madrid.

⁴⁶Servicio de Cirugía General y del Aparato Digestivo, Hospital Universitario La Paz, Madrid.

⁴⁷REDISSEC. Unidad de Apoyo a la Investigaci_on, Gerencia Asistencial de Atención Primaria de la Comunidad de Madrid, Madrid.

⁴⁸REDISSEC. Unidad de Gesti_on Sanitaria. Hospital del Alto Deba. Mondragon-Arrasate. Spain.

Acknowledgements

We thank Maria del Carmen Delgado for her excellent technical assistance. M.R. and J.M.Q. contributed to experimental design and conception. M.R., C.A., T.T., M.G.A. and F.R.R. contributed to writing of the manuscript. C.A., T.T., M.M.M.S.-V., A.R., A.E. and J.M.Q. contributed to data collection. R.C.M.G., I.Z., M.S.C., J.J.B.M. and R.G.M. contributed to histopathological evaluation. C.A. and T.P. contributed to evaluation of microvessel density. R.F. and A.M.B. contributed to evaluation of cellular apoptosis. C.A., I.R. and A.M.B. contributed to evaluation of proliferation. F.R.R. contributed to statistical analysis. M.R. supervised the study and revised the manuscript. All authors read and approved the final manuscript. The members in the REDISSEC CARESS-CCR (Results and Health Services Research in Colorectal Cancer) group are listed in .

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was partially supported by grants from REDISSEC (RD12/0001/0010 and RD16/0001/0006), Instituto de Salud Carlos III (PI 09/00910, PI13/00013, PI 18/01181) and the European Regional Development Fund.

Informed consent

Informed consent was obtained from all patients and the study was approved by the ethics committee at our institution.

ORCID iD

Maximino Redondo

References

Torre

Bray

Siegel

et al . Global cancer statistics, 2012. CA Cancer J Clin 2015; 65(2): 87–108.

Das

Kalita

Pal

Predictive and prognostic biomarkers in colorectal cancer: a systematic review of recent advances and challenges. Biomed Pharmacother 2017; 87: 8–19.

Lindmark

Gerdin

Påhlman

et al . Prognostic predictors in colorectal cancer. Dis Colon Rectum 1994; 37: 1219–1227.

Sinicrope

Hart

Hsu

et al . Apoptotic and mitotic indices predict survival rates in lymph node-negative colon carcinomas. Clin Cancer Res 1999; 5(7): 1793–1804.

Bendardaf

Ristamaki

Kujari

et al . Apoptotic index and bcl-2 expression as prognostic factors in colorectal carcinoma. Oncology 2003; 64(4): 435–442.

Hilska

Collan

YUO

O’Laine

et al . The significance of tumor markers for proliferation and apoptosis in predicting survival in colorectal cancer. Dis Colon Rectum 2005; 48(12): 2197–2208.

Garrity

Burgart

Mahoney

et al . Prognostic value of proliferation, apoptosis, defective DNA mismatch repair, and p53 overexpression in patients with resected Dukes’ B2 or C colon cancer: a north central cancer treatment group study. J Clin Oncol 2004; 22(9): 1572–1582.

Alcaide

Funez

Rueda

et al . The role and prognostic value of apoptosis in colorectal carcinoma. BMC Clin Pathol 2013; 13: 24.

Weidner

Intratumor microvessel density as a prognostic factor in cancer. Am J Pathol 1995; 147(1): 9–19.

10.

Folkman

Angiogenesis and breast cancer. J Clin Oncol 1994; 12: 441–443.

11.

Peng

Liu

X-L

Liu

et al . Co-evolution of cancer microenvironment reveals distinctive patterns of gastric cancer invasion: laboratory evidence and clinical significance. J Transl Med 2010; 8: 101.

12.

Uzzan

Nicolas

Cucherat

et al . Microvessel density as a prognostic factor in women with breast cancer: a systematic review of the literature and meta-analysis. Cancer Res 2004; 64(9): 2941–2955.

13.

Zlobec

Holler

Tornillo

et al . Combined histomorphologic and immunohistochemical phenotype to predict the presence of vascular invasion in colon cancer. Dis Colon Rectum 2009; 52(6): 1114–1121.

14.

Lindmark

Gerdin

Sundberg

et al . Prognostic significance of the microvascular count in colorectal cancer. J Clin Oncol 1996; 14(2): 461–466.

15.

Peng

Wang

Elevated preoperative carcinoembryonic antigen (CEA) and Ki67 is predictor of decreased survival in IIA stage colon cancer. World J Surg 2013; 37(1): 208–213.

16.

Allegra

Paik

Colangelo

et al . Prognostic value of thymidylate synthase, Ki-67, and p53 in patients with Dukes’ B and C colon cancer: a National Cancer Institute-National Surgical Adjuvant Breast and Bowel Project collaborative study. J Clin Oncol 2003; 21(2): 241–250.

17.

Adams

Tzankov

Lugli

et al . New time-dependent approach to analyse the prognostic significance of immunohistochemical biomarkers in colon cancer and diffuse large B-cell lymphoma. J Clin Pathol 2009; 62(11): 986–997.

18.

Fodor

Hutchins

Espiritu

et al . Prognostic and predictive significance of proliferation in 867 colorectal cancers. J Clin Pathol 2012; 65(11): 989–995.

19.

Melling

Kowitz

Simon

et al . High Ki67 expression is an independent good prognostic marker in colorectal cancer. J Clin Pathol 2016; 69(3): 209–214.

20.

Quintana

Gonzalez

Anton-Ladislao

et al . Colorectal cancer health services research study protocol: the CCR-CARESS observational prospective cohort project. BMC Cancer 2016; 16: 435.

21.

Noble

Vyas

Al-Attar

et al . High levels of cleaved caspase-3 in colorectal tumour stroma predict good survival. Br J Cancer 2013; 108(10): 2097–2105.

22.

Leonardos

Butler

Hewett

et al . The activity of caspase-3-like proteases is elevated during the development of colorectal carcinoma. Cancer Lett 1999; 143(1): 29–35.

23.

Jonges

Nagelkerke

Ensink

et al . Caspase-3 activity as a prognostic factor in colorectal carcinoma. Lab Invest 2001; 81(5): 681–688.

24.

Evans

Morrison

Heriot

et al . The correlation between colorectal cancer rates of proliferation and apoptosis and systemic cytokine levels; plus their influence upon survival. Br J Cancer 2006; 94(10): 1412–1419.

25.

Rupa

deBruine

Gerbers

et al . Simultaneous detection of apoptosis and proliferation in colorectal carcinoma by multiparameter flow cytometry allows separation of high and low-turnover tumors with distinct clinical outcome. Cancer 2003; 97(10): 2404–2411.

26.

Koornstra

Rijcken

DeJong

et al . Assessment of apoptosis by M30 immunoreactivity and the correlation with morphological criteria in normal colorectal mucosa, adenomas and carcinomas. Histopathology 2004; 44(1): 9–17.

27.

Koornstra

deJong

Hollema

et al . Changes in apoptosis during the development of colorectal cancer: a systematic review of the literature. Crit Rev Oncol Hematol 2003; 45(1): 37–53.

28.

Kim

Lee

Chun

et al . Apoptosis and its correlation with proliferative activity in rectal cancer. J Surg Oncol 2002; 79(4): 236–242.

29.

Ikenaga

Takano

Saegusa

et al . Apoptosis of colon cancers assessed by in situ DNA nick end-labeling method. Pathol Int 1996; 46(1): 33–37.

30.

Anjomshoaa

Nasri

Humar

et al . Slow proliferation as a biological feature of colorectal cancer metastasis. Br J Cancer 2009; 101(5): 822–828.

31.

Salminen

Palmu

Vahlberg

et al . Increased proliferation activity measured by immunoreactive Ki67 is associated with survival improvement in rectal/recto sigmoid cancer. World J Gastroenterol 2005; 11(21): 3245–3249.

32.

Bossi

Viale

Lee

et al . Angiogenesis in colorectal tumors: microvessel quantitation in adenomas and carcinomas with clinicopathological correlations. Cancer Res 1995; 55(21): 5049–5053.

33.

Pietra

Sarli

Caruana

et al . Is tumour angiogenesis a prognostic factor in patients with colorectal cancer and no involved nodes. Eur J Surg 2000; 166(7): 552–556.

34.

Saclarides

Speziale

Drab

et al . Tumor angiogenesis and rectal carcinoma. Dis Colon Rectum 1994; 37: 921–926.

35.

Frank

Saclarides

Leurgans

et al . Tumor angiogenesis as a predictor of recurrence and survival in patients with node-negative colon cancer. Ann Surg 1995; 222(6): 695–699.

36.

Gardy

Seon

et al . Both high intratumoral microvessel density determined using CD105 antibody and elevated plasma levels of CD105 in colorectal cancer patients correlate with poor prognosis. Br J Cancer 2003; 88(9): 1424–1431.

37.

Vermeulen

Van den Eynden

Huget

et al . Prospective study of intratumoral microvessel density, p53 expression and survival in colorectal cancer. Br J Cancer 1999; 79: 316–322.

38.

Saad

Jasnosz

Tung

et al . Endoglin (CD105) expression in endometrial carcinoma. Int J Gynecol Pathol 2003; 22(3): 248–253.

Clinical–pathological characteristics and short-term follow-up associated with proliferation,apoptosis and angiogenesis in a prospective cohort of patients with colorectal tumours