An open-label comparative cohort trial has been conducted to determine if the addition of MSC to surgery and radio- and/or chemotherapy adds any therapeutic benefit compared to surgery and radio- and/or chemotherapy alone.
To be eligible, patients had to have cytologically or histologically documented colorectal cancer (Dukes A–D); a WHO performance status of 0, 1 or 2; adequate organ functions; life expectation of at least 6 months; and minimal age of 18 years. No restrictions were made regarding the date of the diagnosis. Similarly, no restrictions were made for prior radio- and/or chemotherapy, but all the patients had to undergo curative surgery at the time of diagnosis of their disease. This included complete removal of the primary tumour with adequate lymphadenectomy. In cases of existing distant metastatic lesions, indicated by preoperative investigations, hepatic and/or lung resection had also been carried out. At the time of the enrolment into this study, however, several patients had already had measurable and inoperable metastatic disease. Exclusion criteria were severe parenchymatous liver disease not connected to the metastatic cancer; severe kidney failure; respiratory insufficiency; history of severe cardiovascular disease; history of other type of cancer; pregnancy; lactation. The Regional Helsinki Committees approved the protocol, and all patients gave written informed consent before entering into the study.
The two cohorts of patients (MSC and ‘control’) were formed according to the patients' preference, since MSC was freely available as an over-the-counter product. Patients were asked if they prefer to take MSC, and thus enrolled in the MSC cohort. Those patients who refused to take the preparation formed the control cohort. The decision of treatment group assignment according to patients' preference was carried out on ethical basis, that is, those patients who otherwise would take MSC, got it free, and those who otherwise would not wish to take, served as control. By this procedure, no direct randomisation or stratification was performed. At the time of check-ups, control patients were always routinely asked if they take any dietary supplement or medical nutriment including MSC and, randomly chosen sera of these patients were analysed for 2-methoxy-benzoquinone (MBQ) and 2,6-dimethoxy-benzoquinone (DMBQ) contents, which are specific indicators of MSC administration. Patients answering ‘yes’ to the question of taking MSC, or having benzoquinone-positive sera, were excluded from the control cohort. The measurement of absorption of methoxy-substituted benzoquinones (MBQ and DMBQ), whose markers were in the form of glycosides in crude wheat germ, and were liberated as aglycones by the process of fermentation, served as a specific monitoring technique of MSC administration, and thus also of compliance. Sample preparation was carried out by solid-phase extraction of the sera, followed by reversed-phase high-performance liquid chromatography. Markers MBQ and DMBQ were detected by mass spectrometry using single-ion recording. The compounds were analysed by atmospheric pressure chemical ionisation. The detection limit was 2
ng per 10
ml serum. Besides this analytical monitoring, patients of the MSC cohort had to account for the empty containers of the nutriment before having received the new supply.
The survival analysis was chosen with power 1–β=0.8=80% and with a significance level α=0.05=5%; for these options, using the Freedman's procedure, the minimal required sample size was 62 patients per group, assuming a disease-free survival probability in the control group to be roughly equal to 50% at average follow-up, and an estimated disease-free probability increased by 25% in the MSC group, as deducible from previous animal studies, as well as from early clinical experiences.
Beyond the anticancer therapy for the group of patients of the MSC cohort, 9
g of MSC, dissolved in 150
ml of water, were taken orally once daily uninterruptedly and continuously throughout the study but, at least for more than 6 months. The treatment time was measured as the interval between the first and the last visits completed. Patients of the control cohort received the anticancer treatments alone. The term ‘anticancer treatments’ stands for the 5-FU-based standard Mayo Clinic chemotherapy regimen and/or radiation therapy following surgery.
All patients were evaluated at baseline, at the end of the first month, and every 12 weeks afterwards. Evaluation included assessment of all measurable lesions by imaging (radiographic, ultrasonic or magnetic resonance) techniques, laboratory tests (haematology, chemistry and urinalysis), physical examination and data collection of treatment-related toxicities. Tumour progression was defined as an increase of at least 25% in the overall area of the tumour size or the appearance of any new lesions. Deaths were also reckoned in progression. Time-related events were measured from the date of diagnosis.
The primary end point of this study was to compare progression-free survivals of the two cohorts. For this case, the two-tailed, unstratified log-rank test (Kaplan–Meier method), where P<0.05 indicates statistical significance, was used. For other comparisons, the z-test, Mann–Whitney's z-test, Fisher's exact test and Student's t-test were applied.
For the analysis of the effects of different variables (age, sex, disease staging, chemotherapy, radiotherapy, MSC administration) on survival, Cox's proportional hazards (PH) model was used. The Cox PH assumptions for this analysis have been evaluated for the six independent variables taken into account (see also ) by using the Schoenfeld residuals (Schoenfeld, 1982
) of the general form
Multivariate analysis of survival of colorectal patients (Cox-regression), proportional hazards model: χ2=22.756; P=0.0009
for each covariate xu
, such that ruj
is the difference between the covariate value for the failed j
th observation and the average of the covariate values, weighted on the basis of estimated hazard from the Cox model; the residual analysis carried out by means of the generalised method, developed by Grambsch and Therneau (1994)
, has shown no evidence of violations of Cox's PH assumptions.