OVIDIU MIRCEA ZLATIAN of tumor heterogeneity in... · 2010. 11. 22. · OVIDIU MIRCEA ZLATIAN...
Transcript of OVIDIU MIRCEA ZLATIAN of tumor heterogeneity in... · 2010. 11. 22. · OVIDIU MIRCEA ZLATIAN...
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STUDY OF TUMOR HETEROGENEITY IN COLORECTAL
CANCER BASED ON PROTEIC EXPRESSION
AND GENE MUTATION
ŞI MUTAŢIILOR GENICE
THESIS ADVISOR
PROFESSOR, PhD MIHAI CRUCE
OVIDIU MIRCEA ZLATIAN
CRAIOVA
2010
PhD Student
OVIDIU MIRCEA ZLATIAN
UNIVERSITY OF MEDICINE AND PHARMACY
OF CRAIOVA
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INTRODUCTION
Colorectal cancer (CRC) is the second cause of mortality by cancer in Europe and has a
high incidence, concordant with prolonging the life expectancy.
A major challenge in understanding the colorectal cancer is the characterization of
phenotypic and genotypic changes of the neoplastic cells. The main obstacle is the haotic
evolution of the neoplastic process, which is also the main cause of the failure of therapy, due to
the fact that sooner or later cellular chemoresistant clones are selected. One of the consequences
of this type of evolution is the divergence of daughter cells characteristics which induce the
tumor heterogeneity phenomen.
By definition the intratumor heterogeneity supposes the onset of differences between
neoplastic cells during tumor development. It was proved the existence of cell subpopulations for
every histological type and location of the cancer, both in humans and in experimental disease in
animals.
The tumor heterogeneity has a theoretical importance in understanding the global
evolution of neoplastic process, but also in establishing the histological and molecular
phenotypes and further in assignment and conduction of therapy. Cell subpopulations
therapeutically targeted are contigous both spatially and as origin and act as ecosystems which
manifest characteristics transcending those of individual cells.
In order to understand the development and evolution of such systems a fusion of
methods of population biology and those of cell biology is needed, for the purpose of
deciphering the fundamental causes which underlie the onset of heterogeneity and the common
denominator of these populations. The study of the general phenomen of tumor heterogeneity
suppose the investigation of the causal relations between phenotype and genotype.
In order to experimentally study the tumor heterogeneity I proposed first to examine the
epidemiology of colorectal cancer and the risk factors involved in colorectal oncogenesis, the
morphopathology of colorectal cancer and the molecular mechanisms involved in oncogenesis
and tumor progression.
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I established as first experimental objective the statistical evaluation of prevalence of
colorectal cancer in the Oltenia area and determination of the possible correlations between
clinical and pathological parameters of the patients with colorectal cancer.
A second objective is the examination of the phenotypic expression of tumor
heterogeneity by an immunohistochemical study of the proteins expressed by neoplastic cells
from the colorectal tumors.
The correlation of observed phenotypic heterogeneity with the genotypic heterogeneity
which determines it was the third objective of the present thesis which proposes the detection of
some mutations of the Kras and p53 genes by molecular methods, due to their important role in
tumor initiation and progression.
Keywords: colorectal cancer, immunohistochemistry, tumor heterogeneity, Kras and
p53 mutations.
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I. STATE OF KNOWLEDGE
In Chapter I – The epidemiology and risk factors of the colorectal cancer I presented
the geographical distribution of CRC I presented the geographical distribution of the CRC
prevalence, which is raised in developed countries, and also that of CRC mortality, increased in
countries with low or medium economical level, due to the poor access to performant diagnosis
and therapy services (Coleman, Quaresma, 2008). I showed the time variations of these
variables, reffering to Romania (Zavoral, Suchanek et al, 2009). Then I described the risk factors
for colorectal cancer, both those which belong to the patient (age, intestinal inflammatory
diseases, race, ethnicity, genetic disorders), as well as belonging to environment (alimentation,
obesity, smoking status, alcohol consumption, diabetes, infectious factors).
In Chapter 2 – Morphopathology of colorectal cancer I described the anatomy and
normal structure of the colon and rectum, from the macroscopic and microscopic perspective. I
presented the main premalignant lesions of the colorectal mucosa and I showed the
morphological steps of the transition from the normal mucosa to colorectal benign tumors. I
summarized the main colorectal malignant tumors, with a short macroscopic and microscopic
description. Then I presented the main immunohistochemical markers used to phenotype and
evaluate the prognosis of colorectal tumors. Finally, I analyzed the natural evolution of
colorectal cancer, the local (invasion) and at dinstance (metastazing) extension. .
În Chapter 3 – The colorectal cancer genetics I described the most important genetic
phenomena which determine the initiation and progression of CRC, by presenting the main
mechanisms which underlie the genomic instability, which induce the evolution of colorectal cancer
by facilitating the acquisition of mutations associated with malignant phenotype. I presented the main
tumor suppressing genes affected by mutations, with the observation of the high number of genes
mutated in CRC, but for most of them the mutations frequency is small, which implies an enormous
colorectal cancer heterogeneity, which refelects the heterogeneity of clinical aspects. Then I showed
some modifications of tumor cell biology, like activation of growth factors pathways or alterations in
regulatory mechanisms of the stem cells. Finally I discussed the applications of colorectal cancer‟s
molecular biology data in detection, prognosis and therapy.
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II. OWN CONTRIBUTIONS
CHAPTER 4. CLINICAL AND STATISTICAL STUDY OF THE CASES
WITH COLORECTAL MALIGNANT AND BENIGN TUMORS
I performed a statistical anlysis for the purpose of evaluating the prevalence of CRC in
the Oltenia area and correlation with clinical and pathological parameters of the patients.
BIOLOGICAL MATERIAL
I studied 788 patients with malignant and benign colorectal tumors, from whom were
preleved biopsies in clinical or internal medicine clinics of the Regional Clinical Emergency
Hospital of Craiova, Romania, between the years 2003-2009. By removing the patients with
incomplete date, I selecten for the study 650 patients, from whom 606 had malignant tumors
(334 men and 272 women).
METHODS
By descriptive statistics I discovered correlations between patient age, tumor location,
macroscopic aspect, grade and histological type, which were verified by correlative statistic, and
those with a high semnification were more deeply analyzed. As statistical methods I used the
simple Pearson correlation and that based on rank (Spearman). In order to detect differences
between groups of patients I used the Student t-test and for nominal data I utilized non-
parametric tests (CHI squared and Fischer‟s exact test for small groups).
The statistical calculations and graphics were executed using the software packages SPSS
17 (SPSS Inc., Chicago, IL, USA) and Microsoft Excel 2010.
RESULTS AND DISCUSSION
Regarding the location of tumors, CRC had a high incidence in sigmoid colon (217 cases
– 35,81%) and rectum (268 cases – 44,22%). I observed an increased incidence in advanced
ages, as the mean age was 64,77 years, without significant sex differences (p=0,619).
I also noticed thatfor the first colic segments (from cec to descendent colon) the incidence
of CRC is higher in women, and the situation is inverse for sigmoid and rect, the report
women/men is 0,85 for cec and 1,72 for rect (Figure 1), the association between patients sex and
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rectal location is statistically significant. The tumor location was also associated with the
differentiation grade (p=0,0650).
Figure 1. The distribution of the studied cases by tumor location and patients’s sex
The patients‟s age significantly differ according to localization of tumors (ANOVA test‟s
p=0,190), the difference between colonic and rectal tumors is significant.
Vârsta pacienţilor diferă semnificativ în funcţie de localizarea tumorilor (test ANOVA,
p=0,190), diferenţa între tumorile cu localizare colonică şi rectală fiind semnificativă (p = 0,015).
Most colorectal carcinoma (68,48%) had a moderate grade of differentiation. I
demonstrated that the histopathological type of papillary carcinoma is strongly associated with
well-differentiated grade (CHI square‟s p<0,0001), and that the infiltrative and stenosant forms
are almost always associated with intestinal wall invasion (invaded in 132 from the 138 cases
with these macroscopic forms, CHI square‟s p<0,0001) and of organs in the neighbourhood (28
from 138 cases, CHI square‟s p = 0,0256 <0,05). Also the invasion of the organs was present in
23,08% of the poorly differentiated carcinoma.
The logistic regression showed a significant influence (p<0,05) of tumor location (B=-
0,233) and grade (B=+0,302) on the presence of lymphnodes invasion, results similar with other
studies (Chou, Row et al, 2010). In our study ande statistic modeling had a rate of correct
prediction of 65%, not very high but which justify using the statistical model for estimating
lymphnodes invasion.
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CHAPTER 5. HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL
UNVEILING OF TUMOR HETEROGENEITY IN COLORECTAL CANCER
Immunohistochemistry plays an important role in differentiating tumor types, assessment
of agresiveness and recognizing of metastasis origin. Although the molecular analysis are
increasingly used, many therapeutical protocols are still based on histological types and
immunohistochemical phenotypes. From these reasons I performed a histological study, a
necessary step in every research regarding the characterization of tumor heterogeneity.
BIOLOGICAL MATERIAL
The patients were randomly selected from our cases with a histopathological diagnosis of
colorectal cancer, namely 50 cases: 27 men (54%) and 23 women (46%), with a mean age of
59,7 years (41-79 years). For each case clinical and pathological informations were recorded:
age, sex, tumor location, histological type and tumor grade.
METHODS
The samples were obtained from surgery, were formalin-fixed and included in paraffin
using the classic protocol. Afterwards, the blocks were cutted at the microtome in sections 3-5
µm thick.
The histochemical and immunohistochemical study was performed in Pathological
Anatomy laboratory from the national “Victor Babes: Institute from Bucharest, Romania.
Histochemical methods. The sections were stained with haematoxiline-eosine and were
examined using the Zeiss Axiostar Plus microscope and the Olympus camera, resolution 5 MP.
Immunohistochemical methods (IHC). For the IHC study we used the sections which
were sheeted on glass slides coveredwith polilysine. The slides were processed using the three
stages method Avidin - Biotin Peroxidase (ABC) (HSU 1981). We used as IHC markers:
cytokeratins (CK7, CK20), mucins (MUC1, MUC2), Ki-67, PCNA (Proliferaating Cells Nuclear
Antigen), P53, KRAS (Kirsten-RAS), BCL2, PTEN (Phosphatase and Tensine homologue) ,
EGFR (Epidermal growth factor receptor.
RESULTS AND DISCUSSION
Hematoxiline-eosine stain permitted the assessment of microscopical grade of the
studied adenocarcinoma: well (figure 2A), moderated and poorly differentiated (figure 2B), the
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most frequent grade was G2. Histologically, most adenocarcinoma haven‟t special
characteristics, 9 were mucinous, 8 coloid and 1 with signet-ring cells.
A B Figure 2. A. Well differentiated adenocarcinoma with infiltration in submucosa,
adiacent to normal mucosa. HE stain, 100x. B Poorly differentiated colonic adenocarcinoma –
solid area of tumor cells without glandular differentiation. HE stain, 100x.
IHC detection of cytokeratins detected the expression of cytokeratin 20 in most cases
(80%) with a focal pattern in tumoral cells, and of CK7 in 8 cases with diffuse pattern (figure
3A). 78% of adenocarcinoma presented the CK20+/CK7- phenotype, other studies identified this
phenotype in 65,85%. The phenotype CK20-/CK7+ was identified in 5 cases (10%) on the
proximal colon, in the literature being reported the absence of this phenotype in distal colon.
Also in my study I found 3 cases (15,78%) with expression of both cytokeratins (CK20+/CK7+),
distally located, other studies of adenocarcinomas reporting his presence in 22% of the cases.
Expression of mucins (MUC 1 and (MUC2) in normal colorectal mucosa has the
pattern MUC2+/MUC1-, phenotype present in only 18%of the cases, which shows the frequency
of alterations of mucin expression in adenocarcinoma. We also identify the following patterns:
MUC1+/MUC2- in 38% cases, MUC1+/MUC2+ in 36% cases and MUC1-/MUC2- in 8% cases
(figure 3B). Procentul ridicat de expresie al proteinei MUC1 (74%) este frecvent întâlnit în
carcinoamele colorectale.
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A B Figure 3. A. Expression of citokeratin 20 in a well differentiated colorectal adenocarcinoma
(IHC stain, 100X). Focal positivity in tumor cells and difuse in normal epithelial cells. B. Expression
of MUC2 mucin in a case of moderated differentiated adenocarcinoma (IHC stain, 200X). The
positivity is present in cytoplasm and membrane of the tumor cells.
Ki-67 expression was detected in all the studied cases with indexes between 10 and 95%
(figure 4A), with increased intratumoral heterogeneity, with great differences between indexes on the
same section. I observed a big number of Ki-67 positive cells in the tumoral glands. It couldn‟t be
established a statistical significant relationship between positivity index for Ki-67 and histological
grade (ANOVA analysis for groups with differentiation G1, G2 and G3, p=0,014, < 0,05).
PCNA expression was also present in all cases with various degrees of intratumoral
heterogeneity, both at the level of whole tumor as well as in the malignant glands (figure 4B). the mean
index of PCNA was 15-30% in well differentiated adenocarcinoma, 25-60% in moderated
differentiated and 50-90% in poorly differentiated. It was observed a significant correlation between
PCNA index and differentiation grade (ANOVA analysis for the three grades, p=0,085, >0,05).
A B Figure 4. A. Expression of Ki-67 in a case of moderated differentiated adenocarcinoma
(IHC stain, 100X). Positivity index:70% of the tumor cells. B. Expression of PCNA in a moderated fifferentiated adenocarcinoma, positivity index: 70%.
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P53 expression was observed in 43 cases (86%), the positivity index varied between 5
and 80%, previous studies (Bousserouel , Kauntz et al, 2010) reported between 30% and 60,6%
positive cases. P53 supraexpression wasn‟t identified in normal tissue adiacent to the tumor. The
positivity was relative homogenous in tumoral mass (figure 5), in sections at different levels the
indexes were similar (low intratumoral heterogeneity).
In the interpretation of these results we must account for that immunohistochemically
detected supraexpression of P53 protein means an accumulation in the cells. But not all mutations of
p53 gene lead to his accumulation, so it is possible the existence of false negative results.
Absence of tumor positivity for P53 was correlated with poor differentiation (p<0,001), as
other studies found (Carneiro, Ramalho et al, 2006). The supraexpression of P53 was associated with
raised mean indexes of PCNA and Ki-67 (Student t-test, p=0,0042 and p=0,0025, respectively),
suggested that the P53 protein accumulated in cells which already had a growth advantage by
mutation which increase the proliferation rate.
By correlating these results with those of molecular analysis, we observed that the
positivity for P53 wss present in all cases in which were investigated the p53 gene‟s
polymorphisms. 5 of the 6 samples with positivity index above 50% had the Pro/Pro allele
(present in 50% of the studied cases, Fisher‟s p = 0,017). We consider that this associations is
due mainly to high frequency of Pro/Pro allele in colorectal cancer patients, rather than a link of
this polymorphism with the immunohistochemical expression of P53.
A B
Figure 5. Colon adenocarcinomas marked for P53 (IHC stain, 100X). A. Well
differentiated adenocarcinoma, P53 index: 80% of tumor cells, with adiacent negative normal
tissue. B. Moderately differentiated adenocarcinoma, P53 index:70% of tumor cells.
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The KRAS protein has a positivity index between 5 and 95%. The immune rewaction
was considered positive when at least 30% of the tumor cells were marked. The expression of
KRAS protein was identified to 26 (52%) of the cases (figure 6A), most indexes were over 50%.
These percents are close to those reported by similar studies (Cejas, Lopez-Gomez et al, 2009).
The Kras expression was signicantly raised in poorly differentiated adenocarcinoma (6 cases of
9, p=0,037), possibly due to their rapid replication and increased chance to accumulate
oncogenic mutations.
By correlating Kras mutations detected by molecular methods (33,33% cases) with
KRAS expression (52 % cases) it can be observed that in 3 (75%) of the 4 cases with KRAS
mutations, the expression was increased with a positivity index over 70%. Other studies of this
relation showed a significant increase of Kras expression in the cells with KRAS mutations
(p=0,010).
The high index of positivity for KRAS and P53 in these patients suggests that the
mutations of these 2 genes are present in the majority of tumor cells (low intratumoral
heterogeneity), meaning that a single cell clone is responsible for tumor development. In order to
confirm this hypothesis are needed studies with both markers and analysis of double-marked
cells.
A B Figure 6. A. Well differentiated adenocarcinoma with positivity for KRAS protein (IHC
stain, 100X). KRAS positivity index: over 80% of the tumor cells. Next to the tumor it can be
observed a normal tissue negative for KRAS. B. Colon adenocarcinoma marked for BCL2 (IHC
stain, 100X). Positivity for KRAS in the cytoplasm of tumor cells and also in lymphocytes.
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The BCL2 expression was identified in a small number of cases (12%) and varied in
intensity. The positivity pattern was predominantly cytoplasmatic, more pregnant in the apical
region, although occasionally was identified perinuclear positivity (figure 6B). the marked tumor
cells were mostly located within the tumoral glands from the parabasal and superficial area of
tumors. Although the tumoral positivity was heterogenous, the lymphocytes were positive in all
sections examined (internal positive control).
Expression of nuclear protein PTEN was investigated in 12 cases. The 8 positive cases
(66,67%) were well differentiated adenocarcinomas and the 4 negative cases were 3 moderately
differentiated and 1 well differentiated adenocarcinomas. The normal colorectal epitelia adiacent
to tumor areas has a nuclear positivity for PTEN (figure 7A).
It was showed that PTEN supression is induced by RAS/ERK pathway, which explains
why from the 4 negative cases 3 were positive for KRAS.
S-a arătat că supresia PTEN este indusă prin calea RAS/ERK, astfel putându-se explica
faptul că dintre cele 4 cazuri negative pentru PTEN, 3 au fost pozitive pentru KRAS.
EGFR was identified in 21 cases (42%). The EGFR was also positive in blood vessels
(figure 7B) which demonstrates microangiogenesis in invasion front and tumoral glands.
A B Figure 7. A. Expression of PTEN in a moderately differentiated adenocarcinoma (IHC
stain, 100X), nuclear positivity both in tumor cells as well as in the stromal and inflammatory
cells(internal positive control). B. Expression of EGFR in a colorectal adenocarcinoma (IHC
stain, 100X). Staining is negative in tumor cells and positive in blood vessels.
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CHAPTER 6. DETECTION OF ACTIVATING MUTATIONS IN CODONS
12 AND 13 OF THE KRAS GENE INVOLVED IN COLORECTAL
PATOLOGY
Kras mutations occur in 30-45% of CRC patients and are restricted to certain codons. The
most frequent mutations are in the 12 and 13 codons from exon 2, rarely in codons 59 and 61
from exon 3 (Bousserouel, Kauntz et al, 2008; Bukhari, Niazi, 2010). We proposed to elucidate
the correlation between Kras mutations in 12 and 13 codons in patients with colorectal cancer
and some clinical and pathological characteristics.
The molecular analysis of Kras and p53 mutations by PCR-RFLP method was performed
in the Molecular Genetics Laboratory from “Victor Babeş” National Institute, Bucharest,
Romania.
BIOLOGICAL MATERIAL
The molecular tests were performed on bioptic samples obtained by colonoscopy from 12
patients with colorectal adenocarcinoma (test lot) and 12 patients with other inflammatory colic
disorders (control lot), formalin fixed and included in paraffin blocks. We tried to match the age
and sex structure of the control lot with that of the test lot (p coefficients > 0,05) in order to
extract valid conclusions from small lots.
METHODS
Genomic DNA extraction used the QIAamp DNA Mini Kit (QIAGEN). For every
sample we used 10-20 sections 5 μm thick. The evaluation of DNA quantity and purity was
performed by ultraviolet spectrophotometry (figure 8).
The PCR-RFLP assay was performed according to literature recommendations. PCR
reactions were performed in a final volume of 25 μL, containing 1X reaction buffer,
deoxinucleotide triphosphate (dATP, dCTP, dGTP, dTTP) 0,2 mM, magnesium chloride 1,5
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mM, primers 1 μM and 1,5 units “DNA Taq polymerase” (Promega). All PCR reactions were
performed using the thermocycler Gene Amp PCR System 9700 (ABI).
Enzymatic restriction reaction can detect the point mutations which create or destroy a
recognition situs for restriction enzymes. The result is a restriction fragment lengh polymorphism
(RFLP).
In order to analyze Kras mutations in codons 12 and 13 we performed enzymatic
restriction with the enzymes Mva I (Microccocus varians) and Hae III (Haemophilus
aegypticus).
Separation of restriction products was performed by submerse electrophoresis in agar
gel 2-4% i TBE at/cm, during 30-60 minutes.The migrated nucleic acids were visualized in UV
light after staining with ethidium bromide.
RESULTS AND DISCUSSION
Spectrophotometry showed DNA genomic concentrations above 1200 ng/ µl in all
cases, and values of the fraction Abs. 260 / Abs. 280 between 1,70 and 2,06 (figure 8).
Figure 8. Spectrofotometric evaluation of purity and concentration of DNA extracts
obtained with QIAamp Tissue Kit (Qiagen) from formalin fixed and paraffin included tumoral
tissues. Sample 1 (patient D.M , male 65 years, diagnosed with colonic adenocarcinoma).
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The PCR-RFLP method for Kras’s codon 12 mutations showed: a DNA fragment of
106 bp corresponding to the normal allele, without mutations in codon 12 („wild type”) and a
135 bb fragment which corresponds to mutant allele, which couldn‟t be cleaved by the restriction
enzyme because codon 12 mutation modified the recognition situs for MVA I enzyme (figure 9).
Figure 9. Agar gel electrophoresis in TBE 1X on agar gel of products obtained by PCR-
RFLP analysis for detecting mutations in Kras gene. 1 and 3: samples with mutation in codon
12; 2: sample without mutation in codon 12; 4: k- Kras 12 negative control sample; 5: k+ Kras
12 positive control sample; M: 100 bp DNA Ladder (Fermentas); N: unrestricted PCR
amplicon; R: PCR amplicon after enzymatic restriction with Mva I enzyme (Fermentas).
By PCR-RFLP analysis 4 (33,33%) of the 12 colorectal adenomas analyzed presenting an
activating mutation in the codon 12 of the Kras gene, compared with one sample (8,33%) in the
control lot, percentages which enframes between those from literature (35-55%).
The Kras mutations were more frequent in proximal colon (33,33% compared with
16,67%), but due to the small number of cases we can‟t prove the significance. The wild
phenotype was more frequent in females compared with males.
After performing the PCR-RFLP analysis for detecting mutations in codon 13 of the
Kras gene, for all analyzed samples we obtained 3 DNA fragments of 85, 48 and 29 bp
respectively, corresponding to the wild-type allele, without mutation. If it would be present a
mutation in codon 13 we obtained just two DNA fragments, one of 74bp and the other of
85 bp (figure 10).
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Figure 10. 1, 2, 3: samples without mutation in codon 13 of the Kras gene; 4: k- Kras 13
negative control sample; 5,6: k+ Kras 13 positive control sample; M1: 25 bp DNA Step Ladder
(Promega); M2: GeneRuler 50 bp DNA Ladder (Fermentas); N: unrestricted PCR amplicon; R:
PCR amplicon after enzymatic restriction with Hae III enzyme (Fermentas).
In our study none of the 12 analyzed samples presented mutation in codon 13 of the
Kras gene. Taking into account the small number of analyzed samples and the frequency
between 7 and 10% of these mutations in CRC patients (Andreyev, Norman et al, 2001), our
result is plausible.
Regarding the prognostic importance of Kras mutations in CRC patients, it has been
shown that the glycin-valin mutation in codon 12 significantly decreases the surviving rate,
regardless of the therapy. There are controversies about association between Kras mutations
and prognosis in patients with metastases, which haven‟t been treated with anti -EGFR
antibodies, as some studies reports a significant correlation with unfavourable prognosis
(Cejas, Lopez-Gomez et al, 2006).
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CHAPTER 7. EVALUATION OF SOME POLYMORPHIC VARIANTS OF
THE P53 GENE IN COLORECTAL PATHOLOGY
The tumor suppressor p53 gene, located on 17p13 chromosome, is one of the most
frequently mutated genes in all human cancers. A lot of studies reported the presence of somatic
or ereditary mutations of p53 gene in CRC, their frequency reaching 45%. It is largely believed
that in half of human cancers the p53 gene is affected by somatic mutations (Markowitz,
Bertagnolli et al, 2009).
The genetic polymorphisms, especially uninucleotidic (Single Base Polymorphisms -
SNP) were associated with predisposition to several cancers, but our understanding of their
implication in pathogeny of colorectal cancer is still at the beginning. In p53 gene were
identified several such polymorphisms, both in coding and non-coding regions. We proposed to
study the distribution of some of these polymorphic variants in patients with colorectal cancer.
7.1 EVALUATION OF CODON 72 POLYMORPHISMS OF THE P53
GENE
SNP de la codonul 72 (Arg72Pro) este un polimorfism cunoscut de mult şi frecvent
întâlnit în cancerul colorectal. Alela Arg72 inhibă apoptoza, probabil datorită localizării
mitocondriale şi a fost asociată cu creşterea riscului de dezvoltare a tumorilor (Katkoori, Jia et al,
2007).
În acest studiu ne-am propus să studiem polimorfismele la nivelul codonului 72 al genei
p53 prin RFLP-PCR la lotul de pacienţi cu CRC comparativ cu lotul martor, pentru a studia
posibila asociere dintre aceste polimorfisme şi riscul de dezvoltare a CRC şi de asemenea pentru
a evalua corelaţia lor cu alţi factori clinico-demografici.
BIOLOGICAL MATERIAL
Am luat în studiu aceleaşi blocuri de parafină folosite pentru evaluarea mutaţiilor Kras,
provenite de la două loturi de pacienţi: 12 pacienţi cu CRC (lot test) şi 12 pacienţi cu afecţiuni
colice inflamatorii (lot martor).
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METHODS
Din fiecare bloc de parafină s-au realizat secţiuni de 10 μm, care au fost colectate în
tuburi de 1,5 ml. Dintre acestea am folosit două secţiuni pentru extracţia ADN.
Extracţia ADN genomic din ţesuturile înglobate în blocuri de parafină s-a efectuat după
un protocol preluat din literatură (Shi, Cote et al, 2009), constând în deparafinare, tratarea în
două bai de xilen şi două de alcool absolut, proteoliză şi separarea ADN prin precipitare.
Cantitatea şi calitatea ADN-ului rezultat au fost analizate spectrofotometric.
Reacţia PCR a folosit primeri cu specificitate pentru codonul 72 din exonul 4. Reacţia a
fost realizată într-un volum final de 25 μL care conţinea 50 ng ADN genomic, 1X PCR buffer
(Biotools) cu 2 mM MgCl2, 0,4 μM din fiecare primer (Genescript), 50 μM dNTP (Biotools) şi
0,5 U ADN polimerază.
Reacţia de restricţie enzimatică pentru analiza mutaţiilor din codonul 72 al genei p53,
produsele de amplificare PCR de 279 pb a fost restricţionat cu enzima BstUI (1 U la 37°C timp
de 16 ore)
RESULTS AND DISCUSSION
În cazul polimorfismului p53 Arg72Pro, alela sălbatică Arg /Arg a produs două benzi
(160 şi 119 pb), varianta Pro / Pro a fost identificată printr-o bandă unică (279 bp) şi varianta
heterozigotă Pro/Arg a produs trei benzi (279, 160, şi 119 pb) (figura 11).
Figura 11. Analiza RFLP a polimorfismului Arg72Pro al p53, prin folosirea enzimei
BstUI. Legenda: M: ladder alelic 100 pb, 1-6: produşi de digestie; Arg/Arg sălbatic este clivat de
BstUI rezultând două fragmente de 119 şi 160 pb, alela mutantă Pro/Pro produce un fragment
de 279 pb. 1 şi 6: forma heterozigotă Arg/Pro. 2 şi 3: forma mutantă. 4 şi 5: forma sălbatică.
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From our study resulted that the frequency of wild-type allele Arg/Arg for SNP Arg72Pro
was 41,66% in test lot and 33,33% in control lot, and the mutant Pro/Pro allele had a double
frequency in test lot. It can be seen a correlation (risk ratio: 2, p=0,249), sustained by previous
studies which reported a significant association of the mutant Pro/Pro phenotype with risk for
colorectal cancer. By correlating the p53 polymorphisms with clinical and pathological
characteristics, we observed that the Pro/Pro allele is associated with tumor location but not with
other variables.
Another result which can be of great importance is significantly increased frequency of
the Arg/Arg allele (risk ratio: 2,5, p=0,084) compared with Pro/Pro. This phenomenon was
associated with exposure to hypoxia or ultraviolet radiation. Another possible cause is the diet
and therefore, indirectly, the lifestyle.
We also detected a possible association between the frequency of Pro/Pro mutation and
distal location (p=0,242), but not with other variables (age, sex, habitate), results similar with
other studies (Bojesen şi Nordestgaard, 2008).
These correlations should be verified in the future on bigger lots, in the aim of obtaining
results usefull for determining the agresiveness of colorectal cancer.
The causal semnification of associations between any genetic polymorphisms with
colorectal cancer remains unveiled, especially for some SNP located in uncoding or unknown
regions of the genome.
The mutations of Kras and p53 genes haven‟t necessarily impact on their function and
for this reason we tried to correlate the presence of detected Kras mutations in codon 12 with
p53 polymorphisms in codon 72. from the 4 cases with mutations in codon 12 of the 12 of Kras
gene, two (50%) presented the Arg/Pro phenotype in codon 72 of p53, one case Arg/Arg and
one case Pro/Pro phenotype. Both cases with Kras muations associated with Arg/Pro phenotype
were males over 72 years of age. If we account for the increased frequency of Kras mutations
observed and of the phenotype Arg/Pro in advanced ages, is little probable an association
between age and mutational status of the two genes (Fisher‟s p=0,595).
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7.2 EVALUATION OF VAL217MET-RS35163653 AND GLY120ALA-
RS35993958 POLYMORPHISMS OF THE P53 GENE
In this study I demonstrated the utility of qRT-PCR method for detection of p53
mutations in DNA from paired samples: feces and biopsy. We used the TaqMan (Applied
Biosystems) probes for detecting of two p53 polymorphisms (Val217Met - rs35163653,
Gly120Ala - rs35993958), with little known clinical significance and unknown role in colorectal
cancer. The investigations of these polymorphisms were performed in Molecular Genetics
Laboratory from university of medicine and pharmacy of Craiova.
BIOLOGICAL MATERIAL
The studied patients had ages between 32 and 84 years with a mean of 68,17; the report
between females and males was 19:34 (42,19% women and 57,81% males). From the 53
patients, 5 were diagnosed with benigne tumors, one with Crohn disease, two with malignant
suspicion and the rest with colorectal neoplasms.
All samples were evaluated by a pathologist in order to establish the histological type,
differentiation grade and tumoral stage. From all patients biopsies were gathered from tumor, in
49 cases were also sampled feces for analyzing the fecal DNA.
METHODS
Isolation of genomic DNA from the tissue samples we used the Wizard® Genomic DNA
Purification Kit (Promega, Madison, WI), and for isolation of total DNA from feces the
QIAamp® Stool Mini Kit (Qiagen).
Quantification and purity determination of the isolated DNA was performed by
spectrophotometric method, with Eppendorf Biophotometer.
PCR reaction was performed in a final volume of 25 μL. We used TaqMan® Gene
Expression Master Mix and TaqMan® SNP Genotyping Assays (Applied Biosystems, Foster
City, CA), specific for every polymorphism studied. TaqMan Gene Expression Master Mix
contains AmpliTaq Gold UP (Ultra Pure) DNA polymerase, deoxiribonucleotide triphosphates
(dNTPs), ROX™ passive reference, as well as buffer components optimized for sensibility,
precision, specificity and reproductibility.
The cycling was performed with the system Real-Time Corbet RotorGene 6200 HRM.
The TaqMan Gene Expression Master Mix protocol indicates the execution of just 40 cycles in
21
the 1 minute attachement/extension, after that the amplification curves haven‟t entered in the flat
phase. Accordingly, we gradually increased the number of amplification cycles and the time of
the attachment/extension step, and we found optimal values of 50 cycles and 90 seconds.
The qRT-PCR was realized with TaqMan (Applied Biosystems) probes marked with
specific fluorescent dyes.
RESULTS AND DISCUSSION
After spectrophotometric check of extracted DNA purity we performed the qRT-PCR
reaction. The obtained data were processed (figure 12) using Rotor-Gene 6000 Series Software
1.7 (Build 75), copyright 2000-2006 Corbett Research. In table 1 it is presented the correlation
between observed fluorescent signals and allele sequency.
Table 1. Correlation between observed fluorescent signals and allele sequency
Fluorescence Result
Only VIC dye fluorescence Homozigosity for allele 1
Only 6FAM dye fluorescence Homozigosity for allele 2
Fluorescent signals for both dyes Heterozigosity for alleles 1 and 2
Figure 12. Data obtained by qRT-PCR and processed with specialized software.
For SNP rs 35993958, in 5 samples of fecal DNA the fluorescent signal was absent on
both alleles and for another 5 samples the signal was present only on allele 1 (homozigosity). For
22
all other paired samples the fluorescent signal was present on both alleles (heterozigosity). From
the unpaired ones, 6 were heterozigous and one homozigous.
For SNP rs 5163653, 4 samples of fecal DNA the fluorescent signal was absent on both
alleles, with the observation that in one samples the signal was absent on both probes, which
suggests the poor quality of this type of DNA.
For the other samples of genomic DNA the fluorescent signal was present only on the
allele 1 (homozigosity).
Our study detected a mutant phenotype of p53 gene in SNP Gly120Ala in 5 cases
(9,43%). The patients were mostly (80%) aged over 55 years, 60% were females and 40% had
tumors located on nthe right colon. All analyzed cases were homozygous for SNP Val217Met
phenotype.
We can conclude that the p53 gene has an important role in development of the colorectal
cancer due to its expression on both allelic variants for SNP rs35993958 and expression only on
allele 1 for SNP rs35163653.
Because the fluorescent signal was present for a significantly high number of probes for
genomic DNA isolated from feces, it can be sustained the use of this testing in screening
programs of colorectal cancer (Itzkowitz, Jandorf et al, 2007; Tomlinson, Webb et al, 2008).
This test is accessible for patients due to high compliance.
23
FINAL CONCLUSIONS
The clinical and statistical study showed a correlation between patient‟s sex and tumor
location on the right or left colon, and also between local invasion and well differentiated
adenocarcinoma (p=0,0426). By statistical regression and modeling was demonstrated a
high influence of location (Odds Ratio = 0,792) and tumor grade (Odds Ratio = 1,353) on
invasion of the lymph nodes, fact with practical importance in diagnosis and therapy.
The expression profiles of cytokeratins and mucins are modified in colorectal cancer,
compared with those of the normal mucosa, fact which demonstrate their usefulness in
defining tumoral phenotypes which can be used in prognosis and therapy. The cell
proliferation markers, Ki-67 and PCNA, were positive with an index between 10 and
95% in all cases. The expression of tumor suppressing protein P53 was identified in 86%
of cases, and that of KRAS oncoprotein in 52%.
The intratumoral fenotipic heterogeneity was raised for proliferation markers with high
index differences, as oncoproteins showed an homogenous expression in tumor mass, the
heterogeneity was expressed only intertumorally. That‟s why we recommend the use of
proliferation markers in order to asses the intratumor heterogeneity and of tumor
suppressor or oncogenic proteins for the intertumor heterogeneity.
We ascertained a positive correlation with statistical significance (p=0,037) between
expression of KRAS protein and activating mutations of Kras gene.
By PCR-RFLP method we demonstrated the higher prevalence of activating mutations in
codon 12 of the Kras gene in colorectal cancer patients (33,33%) compared with control
lot (8,33%), with a high frequency for tumors on proximal colon. We didn‟t detect
mutations in codon 13 of the Kras gene in patients from the test and control lots.
We used the qRT-PCR to identify two polymorphic variants of the p53 gene (SNP
rs35993958 and rs35163653) in genomic DNA extracted from feces, and we detected a
mutant phenotype in SNP Gly120Ala in 9,43% of the cases studied, but all cases were
were homozygote for SNP Val217Met.
The statistical correlation of the results of immunohistochemical study with the gene
mutations detected by molecular methods permitted us to establish a link between
mutation-dependent genotipic heterogeneity and phenotypic heterogeneity which depends
on extratumoral factors.
24
SELECTIVE BIBLIOGRAPHY
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mutations in patients with colorectal cancer: the „RASCAL II‟ study. Br J Cancer 2001;85: 692–
696.
Bojesen S.E., Nordestgaard B.G. The common germline Arg72Pro polymorphism of p53
and increased longevity. Genetics and Molecular Research 2008;9:2:651-660.
Bousserouel S., Kauntz H., Gosse F., et al. Identification of gene expression profiles
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Bukhari M.H., Niazi S., Ghani R. et al. Detection of DNA by scraping the formalin-fixed
paraffin-embedded tissue. Proc Inst Mech Eng H 2008;222:999-1003.
Carneiro F.P., Ramalho L.N., Britto-Garcia S., Ribeiro-Silva A., Zucoloto S. Immunohistochemical expression of p16, p53, and p63 in colorectal adenomas and
adenocarcinomas. Dis Colon Rectum 2006;49:588-94.
Cejas P., Lopez-Gomez M., Aguayo C., Madero R., de Castro Carpeno J. et al. KRAS
Mutations in Primary Colorectal Cancer Tumors and Related Metastases: A Potential Role in
Prediction of Lung Metastasis. PLoS ONE 2009;4(12):e8199. doi:
10.1371/journal.pone.0008199.
Chou J.F., Row D., Gonen M., Liu Y.H., Schrag D., Weiser M.R. Clinical and pathologic
factors that predict lymph node yield from surgical specimens in colorectal cancer: a population-
based study. Cancer 2010;116:2560-70.
Coleman M.P., Quaresma M, Berrino F. et al. Cancer survival in five continents: a
worldwide population-based study (CONCORD). Lancet Oncol 2008;9(8):730-756.
Itzkowitz S.H., Jandorf L., Brand R., Rabeneck L., Schroy P.C., III, Sontag, S., Johnson
D., Skoletsky J., Durkee K., Markowitz S., Shuber A. Improved fecal DNA test for colorectal
cancer screening. Clin Gastroenterol Hepatol 2007;5(1):111-117 available from: PM:17161655.
Katkoori V.R., Jia X., Shanmugam C., Wan W. et al. Prognostic significance of p53 codon
72 polymorphism differs with race in colorectal adenocarcinoma. Clin. Cancer Res.
2009;15:2406-2416.
Markowitz S.D., Bertagnolli M.M. Molecular origins of cancer: Molecular basis of
colorectal cancer. N Engl J Med 2009;361:2449-60.
Shi S.R., Cote R.J., Wu L. et al. DNA extraction from archival formalin-fixed, paraffin-
embedded tissue sections based on the antigen retrieval principle: heating under the influence of
pH. J Histochem Cytochem 2002;50:1005-11.
Su J., Zheng J. Use of tumor proliferation marker ki-67 and PCNA in surgical pathology.
Zhonghua Bing Li Xue Za Zhi 2009;38:568-71.
Tomlinson I.P., Webb E., Carvajal-Carmona L., et al. A genome-wide association study
identifies colorectal cancer susceptibility loci on chromosomes 10p14 and 8q23.3. Nat Genet
2008;40:623-30.
Zavoral M., Suchanek S., Zavada F., Dusek L., Muzik J., Seifert B., Fric P. Colorectal
cancer screening in Europe. World J Gastroenterol 2009;15(47):5907-5915.
25
CURRICULUM VITAE
Name: ZLATIAN
Surname: OVIDIU
Date and place of birth: 24.05.1976, Craiova, Dolj
Citizenship: Romanian
Marital status: married
Actual workplace and position: Assistant, Department of Bacteriology-Virology-Parasitology,
Faculty of General Medicine, University of Medicine and Pharmacy of Craiova.
Scientific title: Assistant Professor
Education:
Institution Period Grade or diplomas
School no. 9, Craiova 1982-1990
„Carol I”, Highschool
Craiova, profile chemistry-
biology
1990-1994
Faculty of General Medicine,
University of Medicine and
Pharmacy of Craiova,
promotion 2000
1994-2000 General medicine
physician
Science Project: “Molecular mechanisms of mitochondrial maladies with neurological
manifestations”, coordinated by Professor PhD Mihai Cruce.
Medical professional experience:
Period: Place: Institution: Position:
1.01.2001 - 31.12.2001 Craiova Spitalul Clinic Nr. 1 Medic stagiar
1.01.2002 - 31.12.2006 Craiova
Laborator clinic şi
microbiologie,
Spitalul Clinic Nr. 1
Medic rezident,
Medicină de laborator
01.03.2007 până în
prezent Craiova
SC Omnimed
Laborator SRL
Medic specialist,
Medicină de laborator
Didactic professional experience:
Perioada: Localitatea: Instituţia: Funcţia:
1.03.2007 till
present Craiova
Faculty of General Medicine,
University of Medicine and
Pharmacy of Craiova, promotion
2000
Assistant
Professor
26
PUBLICATIONS
Articles in extenso, in national CNCSIS journals
Zlatian O., Jalbă C.S., Roşu Lucica, Cruce M. Tumor heterogeneity in colorectal cancer:
immunohistochemical study. Annals of the Romanian Society for Cell Biology, 2011;XV:1
(in press).
Zlatian O., Jalbă C.S., Ioana M., Cimpoeru Alina, Yousaf Sajjad, Cioboata Elena, Pintilie B,
Amelia Bârcă, Cruce M, Roşu Lucica. Evaluation of activating mutations in codons 12 and
13 from exon 2 of kras oncogene in patients with colorectal adenocarcinoma. Annals of the
Romanian Society for Cell Biology, 2010;XIV:2:114-120.
Jalbă C.S. , Jalbă B.A., Vladoi Anca-Daniela, Ioana M., Zlatian O., Cruce M. Correlations
between vascular endothelial growth factor expression and colorectal cancer. Annals of the
Romanian Society for Cell Biology, 2010;XIV:2:99-105.
Roşu Lucica, Puiu Ileana, Zlatian O., Bejenaru L., Călina Daniela, Katramados M.
Imunodeficienţa – Factor favorizant al parazitozelor intestinale la copii cu infecţie gripală.
Craiova Medicală, 2005;VII:2:146-150.
Roşu Lucica, Zamfirescu Luiza, Zlatian O., Puiu Ileana, Ştefan Lorena, Boroghină Adela.
Aspecte etiologice in otitele medii supurate. Factori de risc asociaţi. Medicina Modernă,
2005; XII:3:138-140.
Manolescu Mirela, Mixich Rodica, Ungureanu Anca, Zlatian O. Incidenţa infecţiilor cu
Mycobacterium Tuberculosis în Spitalul Clinic de Urgenţă Craiova în perioada 1990-2004.
Medicina Modernă, 2005;XII:5:229-231.
Cruce M., Ardelean A., Zlatian O. Remodelarea matricei extracelulare şi inducţia
fenotipului celular. Analele Societăţii Române de Biologie Celulară, 2004;IX:2:59-67.
Cruce M., Stănoiu B., Ardelean A., Zlatian O. Semnalizarea prin receptorii serin/treonin
kinazici ai TGF- β. În: Căi si reţele de semnalizare celulară, Editura Aius, Craiova,
2004;vol3: pag.24-31.
Cruce M., Stănoiu B., Pirici D., Ardelean A., Zlatian O. Semnalizarea dependentă de
proteoliza reglată. În: Căi si reţele de semnalizare celulară, Editura Aius, Craiova, 2004, 93-
97.
Roşu Lucica, Petrică Cristina, Stoicescu Irina, Zlatian O., Lazaroniu G. Chlamidia,
Mycoplasma şi Ureaplasma, musafiri nepoftiţi în sfera urogenitală. Medicina Modernă, 2004;
11;1:20-24.
Ardelean A., Cruce M., Stănoiu B., Buteică Elena, Zlatian O. Remodelarea matricei
extracelulare si migraţia celulelor crestelor neurale. Analele Societăţii Române de Biologie
Celulară, 2003;VIII:2:51-58.
Stănoiu B., Ardelean A., Buteică Elena, Pisoschi Cătălina, Baniţă Monica, Zlatian O., Cruce
M. Angiogeneza si remodelarea matricei extracelulare. Analele Societăţii Române de
Biologie Celulară, 2003;VIII:1:62-75.
Cruce M., Stănoiu B., Ardelean A., Baniţă Monica, Buteică Elena, Pirici D., Zlatian O.
Remodelarea mucoasei colorectale si cancerul colorectal. Analele Societăţii Române de
Biologie Celulară, 2003;VIII:1:82-88.
27
Studies published in abstract books at national or international conferences:
Zlatian O., Pleşea E., Roşu Lucica, Pirici D., Cruce M. Modificări ale profilului
imunohistochimic în neoplasmul de colon. A XXXIX-a Conferinţă Naţională de Imunologie,
Călimăneşti-Căciulata, Craiova, Romania, 24-26 septembrie 2009.
Roşu Lucica, Siloşi Isabela, Rogoz Suzana, Petrescu F., Rogoveanu I., Biciuşcă V., Zlatian
O., Ilie Andreea. TNF-alpha în hepatitele virale B şi C. . A XXXIX-a Conferinţă Naţională
de Imunologie, Călimăneşti-Căciulata, Craiova, Romania , 24-26 septembrie 2009.
Zlatian O., Cruce M., Pirici D., Ioana M. Detecţia deleţiilor/mutaţiilor genei p53 în
adenoamele colorectale prin FISH. Al IX-lea Congres National al Societăţii Române de
Morfologie, Craiova, Romania , 28-31 mai 2008.
Roşu Lucica, Zlatian O., Rosu Alexandra, Godeanu Lucia Statie, Comişel Sorina, Cara Lia,
Nania Daniela, Picu Mihaela. Rolul markerilor imunologici în managementul bolnavilor cu
infecţie HIV..A XII-a Conferinţă Naţională de Microbiologie cu participare internaţională,
Sibiu, România, 30.10-1.11 2008.
Zlatian O., Roşu Lucica, Manolescu Mirela, Mixich Rodica, Ilie Andreea, Porumbiţă
Cătălina. Aportul investigaţiilor bacteriologice în confirmarea diagnosticului clinic de
tuberculoză renală. A 3-a Conferinţă a ALMR cu participare internaţională, Iasi, Romania
27-30 iunie 2007.
Ioana M., Zlatian O., Stănoiu B., Buteică Elena, Ciobanu Felicia, Cruce M., Mixich F.
Diagnosticul molecular al pacienţilor cu cancer colorectal ereditar non-polipozic (HNPCC).
Zilele UMF din Craiova, Romania, ed. 36, 9-11 iunie 2006.
Zlatian O., Ioana M., Buteică Elena, Hertzog Zorica, Stănoiu B., Cruce M. Evaluarea prin
PCR a potenţialului de transformare malignă a zonelor displazice ale mucoasei colorectale.
Zilele UMF din Craiova, Romania, ed. 36, 9-11 iunie 2006.
Dena Bianca, Manolescu Mirela, Ungureanu Anca, Roşu Lucica, Zlatian O., Puiu
Alexandra. Enterococcus antibiotics resistance – present and perspective. 29th Balkan
Medical Week, Golden Sands, Varna, Bulgaria, 28-30 september, 2006.