Patients
During a period of 1 year between December 2003
and December 2004 metastatic CRC patients who were
admitted to the Akdeniz University Medical Faculty
Department of Medical Oncology were recruited to the
study. The eligibility criteria were: histologically proven
metastatic adenocarcinoma of the colon or rectum with
measurable disease, no prior chemotherapy for metastatic
disease, age ≥18 and ≤76 years, Eastern Cooperative Oncology
Group (ECOG) performance status ≤2, life expectancy
>3 months, adequate hematological and biochemical
parameters (hemoglobin ≥10 g/dl, neutrophils ≥2x10
9/l,
platelets ≥100x10
9/l, bilirubin ≤1.25x institutional upper
limit of normal range (ULN), aspartate aminotransferase
and alanine aminotransferase ≤5 times ULN, serum creatinine
≤1.25 ULN or creatinine clearance >50 ml/min). All
patients gave written consent before the treatment they
received. Main exclusion criteria were: prior exposure
to irinotecan, severe concomitant conditions, inflammatory
bowel disease, malabsorption [
15]. Prior 5-FU based
adjuvant chemotherapy was not accepted as an exclusion
criteria. Hypertension, diabetes mellitus and coronary
heart disease were scored as positive for comorbidities.
Treatment
We conducted a non-randomised prospective, observational
study recruiting consecutive patients. Five patients
were treated with the IFL (irinotecan 125 mg/m2 IV,
5-FU 500 mg/m2 IV bolus, leucovorin 20 mg/m2 IV bolus
weekly for four weeks every six weeks) regimen and the
other twenty-five patients were treated with XELIRI (irinotecan
250 mg/m2 IV day 1 and capecitabine 2000 mg/
m2/day days 1-14, every 3 weeks) regimen as the first-line
treatment by the decision of their own treating physicians
[15],[16]. Treatment was continued till disease progression
and unacceptable toxicity. Patients were followed up every
3 months after completion of treatment to evaluate
overall survival. Response was evaluated by RECIST criteria
at every 2-3 cycles during treatment. Toxicity was
evaluated during treatment according to the National Cancer
Institute Common Toxicity Criteria (version 3, 2003).
Protocol specified dose reductions and delays were based
on previous cycle toxicity utilizing both hematological
nadirs and hematological/biochemical parameters on the
day of next treatment.
Tumor specimen collection
Detailed descriptions of the specific characteristics of
the specimen collection have been published previously
[17]. Tissue samples from 30 patients with metastatic
CRC were studied. One section from the tumor together
with a section from the adjacent normal tissue, if available,
were blocked. These sections were cut at 5 μm thickness
and stained with hematoxylin-eosin (H&E;). The sections
stained with H&E; were reviewed and re-examined
by the same pathologist. DNA was isolated from the tissue specimens of the tumor, adjacent normal tissue and
also from the blood samples. Serial 5 μm thick sections of
selected tissue blocks were obtained on glass slides, and
the areas of interest were microdissected after matching
with an adjacent section stained with H&E.; To eliminate
cross-contamination, disposable microtome blades were
used [18].
PCR and MSI analysis
The method utilized for the characterization of microsatellite
alterations was based on PCR amplification [5].
Samples of genomic DNA were used to amplify sequences
from 5 of the following mononucleotide and dinucleotide
microsatellite loci: BAT-25, BAT-26, D5S346, D2S123,
D17S250. These specific microsatellite loci were derived
from the National Cancer Institute (NCI) reference [19].
Using this reference panel, microsatellite instability-high
(MSI-H) tumors were defined as having instability in two
or more markers, whereas microsatellite instability-low
(MSI-L) tumors were defined as having instability in one
marker. Lack of instability in any marker described the
microsatellite stable (MSS) group [20]. Genomic DNA
was amplified by PCR in a total of 25 μl of reaction mixture,
which included 100 nm primers, 100 μM each of
dNTP, 1xPCR buffer, 1.5 mM magnesium chloride and
2 units of Tag DNA polymerase (Fermentase, USA) [21].
Aliquots (5 μl) of the PCR products were added to 5 μl
of loading buffer (95% formamide, 20 mM EDTA, 0.05%
bromophenol blue and 0.05% xylene cyanol) and the entire
was denatured at 94oC for 2 minutes and placed on
ice. Aliquots (8 μl) were electrophoresed on 8% polyacrylamide
gels (PAGE) and silver nitrate was used for
the detection of bands [22]. MSI was identified as either
a deletion or a band shift. A band shift was defined as
an abnormal and reproducible pattern which revealed expansion,
contraction or rearranged bands [23]. All altered
cases were analyzed at least twice by an additional PCR
and an electrophoretic run to confirm the results.
Statistical analysis
The association of clinical and MSI related factors
with the best objective response was analyzed with the
univariate logistic regression analysis. Survival curves for
the progression-free survival (PFS) and the overall survival
(OAS) were drawn according to the method of Kaplan
and Meier [23]; Cox regression analysis was used to
test associations with survival. A multivariate regression
analysis was planned if more than two clinic factors had p
values <0.10. P values of less than 0.05 were considered
to indicate statistical significance. All statistical calculations
were performed by using SPSS for Windows version
11.0 (SPSS Inc. Chicago, IL).
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