Clinical Influence of Ploidy and Cancer Stem Cells and Other Parameters in Stage IV Colorectal Cancer

Materials and Methods

Patients. The medical records of 100 patients with histologically-proven CRC, International Union of Cancer Control stage IV (8) between 2000 and 2009 were retrospectively reviewed and their paraffin-embedded tissue samples from the primary site of CRC were collected. All were consecutive non-elected cases from a single center and all patients were treated outside of clinical trials. No patients were candidates for surgical treatment (either curative or palliative); however, all received palliative chemotherapy according to established protocols.

Chemotherapy regimens were based on single agent leucovorin modulated 5-fluorouracil (5-FU) (Mayo clinic) or combination treatments of 5-FU (De Gramont or simple infusion and leucovorin) with either oxaliplatin or irinotecan, or capecitabine with or without bevacizumab or cetuximab.

Records with complete data (for the parameters used as prognostic factors) were included in the analysis. Follow-up was continued until death from CRC or from any other cause, and patients who remained alive were censored as of January 1, 2015. Overall survival was the primary endpoint. This protocol was approved by the Ethics Committee of the National and Kapodistrian University of Athens on the 5th of April 2013 (approval number: 7315).

Prognostic variables. Twenty-four potential prognostic variables were selected. These included patient-related variables such as age, body surface area (BSA), gender, performance status (PS) according to the Karnofsky performance status scale index (before chemotherapy initiation, and after receiving it) response to therapy, location of distant metastases (lymph nodes, liver, lung, abdominal, pelvic, locoregional disease, bone, skin, adrenal glands) symptoms of fever, side-effects from therapy such as neutropenia, anemia, thrombocytopenia, nausea and vomiting, diarrhea, anorexia, weight loss, mucositis, fatigue; anemia with need for administration of erythropoietin and red blood cells transfusion, and laboratory parameters such as albumin, C-reactive protein (CRP), carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA 19.9) values.

DNA measurements (ploidy). For DNA measurements, the Feulgen staining technique was applied as previously described (9). The nuclei of Feulgen-stained cells from the histology of the primary colorectal tissue biopsy were evaluated for DNA ploidy using a Nikon eclipse microscope (Nikon, Tokyo, Japan) connected to a Nikon CCD video camera and an IBM Pentium 4/PC cell measurement software (Image Pro Plus v. 5.1; Media Cybernetics Inc., Silver Springs, MD, USA). Areas of the Feulgen-stained sections containing pathological lesions, identified in adjacent stained slides, were selected for DNA content analysis.

A total of 200-300 nuclei with clear boundaries appearing to have no loss of membrane integrity were analyzed in each tissue sample. Cytometric measurements were performed at a magnification of ×200 and calculated automatically according to the algorithms described previously by measuring the nuclear integrated optical density (IOD), representing the cytometrical equivalent of DNA content (10).

The procedure was performed for all nuclei, and the overall mean represents the DNA content or DNA index (DI). The mean IOD of human lymphocytes (control cells) was used as the diploid standard (2c) and reference for DI calculation for targeted cells. DNA histograms were generated and a tumor was classified as diploid when the DI ranged from 0.9 to 1.1 and the relevant DNA histogram revealed only one peak at 2c, and aneuploid when either of these two criteria was absent.

Stem cell measurements. The immunohistochemical score (IHS) for immunostaining of CD133, as a marker of stem cells, was calculated for each case. The scoring system used was similar to previously published methods (4). CD133 immunostaining was measured in the cytoplasm of isolated cancerous cells and in luminal secretions of malignant gland and towards the luminal surface of malignant glandular structures. The extent of positively stained epithelial cells was estimated and classified on a four-point scale as follows: no staining=0%, 1=1-10%, 2=11-25%, 3=26-50%, and 4=51-100%.

Statistical analysis. Summary statistics of continuous variables were based on the mean followed by the corresponding 95% confidence interval, or standard deviation and range. In the case of severely dispersed variables values were transformed into the natural logarithm; in such cases the reported statistics were based on the geometric mean. Categorical variables are summarized by absolute and relative frequencies.

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Figure 1.

Survival according to ploidy.

One-way analysis of variance (ANOVA) or Chi-squared statistic were applied in order to investigate the association of patient responses with clinical characteristics.

Time-to-event analysis with regards to overall survival was investigated by log-rank statistics and illustrated by Kaplan–Meier curves. Variables with level of significance of α=1% at the univariate analysis as well as demographics and stem cells were inserted into the Cox proportional hazard regression model in order to identify potential prognostic factors for survival.

All tests were two-sided and the acceptable level of statistical significance was set at 5% and were performed with SPSS 12.0 statistical package (SPSS Inc., Chicago, IL, USA).