1.

INTRODUCTION

1.1 Cancer in India

With the establishment of the National Cancer Registry Programme,
cancer incidence data are available for five metropolitan areas and one rural area
in India since 1982. From 1990 through 1996, stomach remained as the leading
site of cancer in males in Chennai (AAR = 13.6/105) and Bangalore (9.5/105),
followed by Mumbai (6.4/105), Delhi (3.9/105), Bhopal (3.4/105) and Barshi
(1.2/105). Among women, stomach cancer is next to cancer of the cervix and
breast (AAR = 6.5/105) in Chennai. Stomach cancer has been reported as the
second most common malignancy in males in Chennai with a relative proportion
of 8 % in the Five year consolidated report of NCRP (NCRP, 2002).

1.2 Gastric Cancer

1.2.1 Epidemiology

Cancer of the stomach, or gastric cancer, is a disease in which stomach

cells become malignant (cancerous) and grow out of control, forming a tumor.
Almost all (95%) of stomach cancers start in the glandular tissue that lines the
stomach. The tumor may spread along the stomach wall or may grow directly
through the wall and shed cells into the bloodstream or lymphatic system. Once
beyond the stomach, cancer can spread to other organs.

Stomach cancers are classified according to the type of tissue where they
originate. The most common, adenocarcinomas, start in the glandular stomach
lining. Other forms include lymphomas, which involve the lymphatic system, and
sarcomas, which involve the connective tissue (muscle, fat, or blood vessels).

1.2.2 Stages of Cancer

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 There are different ways of staging stomach cancers. The two main ways are
the TNM system and the number system.

1.2.2.1 TNM System

TNM' stands for Tumour, Node, Metastasis. The system can describe the size of
a primary tumour, whether there are lymph nodes with cancer cells in them and
whether the cancer has spread to a different part of the body.

There are 4 stages of tumour size in stomach cancer

T1 means the tumour has grown no further than the inner layer of the
stomach
T2 means the tumour has grown into the muscle layer of the stomach
wall
T3 means the tumour has broken through the membrane covering the
outside of the stomach
T4 means the tumour has grown into other organs or body structures
nearby such as the liver, gullet or abdominal wall

There are 3 possible stages of lymph node involvement

N0 means there are no lymph nodes containing cancer cells

N1 means there are cancer cells in lymph nodes that are less than 3.0
centimetres away from the main tumour in the stomach
N2 means there are cancer cells in lymph nodes that are further than 3cm
away from the main tumour in the stomach OR in lymph nodes that are
connected to the main blood vessels around the stomach

There are two stages of metastasis

M0 means there is no cancer spread to other organs

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 M1 means the cancer has spread to other parts of the body

Number stages of stomach cancer

There are four main stages in this system - stage 1 to 4. Some doctors also refer
to stage 0.

Stage 0 or Carcinoma in situ

CIS or stage 0 stomach cancer refers early stage of stomach cancer.
cancer cells are contained within the lining of the stomach. So there is very little
risk of any cancer cells having spread. It is not usual for stomach cancer to be
diagnosed this early.

Stage one
This is the earliest stage of cancer. It is divided into 1a and 1b. Stage 1a
means the cancer has grown no further than through the inner lining of the
stomach, with no cancer in the lymph nodes (T1, N0, M0).

Stage 1b means either that

The cancer has still only just grown through the lining, but nearby lymph
nodes contain cancer cells (T1, N1, M0) OR
There are no cancer cells in the lymph nodes, but the cancer has grown
into the muscle of the stomach wall (T2, N0, M0).

Stage two
The cancer has grown into the muscle layer of the stomach wall and
nearby lymph nodes are affected (T2, N1, M0) OR
The cancer has grown right through the stomach wall (T3, N0, M0) OR
That the cancer has still only reached the lining of the stomach but lymph
nodes further than 3 cm away contain cancer cells (T1, N2, M0)

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Stage three
Stage 3 is also divided into a and b.

Stage 3a means that

The cancer has grown into the muscle layer, but lymph nodes further than
3cm away contain cancer cells (T2, N2, M0) OR
The cancer has grown through the stomach wall, but only nearby lymph
nodes contain cancer cells (T3, N1, M0) OR
The cancer has grown right through the wall and into nearby tissues but
no lymph nodes contain cancer cells (T4, N0, M0)

In stage 3b, the lymph nodes are always affected. Either

The cancer has grown just through the stomach wall and lymph nodes
further than 3cm away contain cancer cells (T3, N2, M0) OR
The cancer has grown through the stomach wall into surrounding body
tissues and nearby lymph nodes contain cancer cells (T4, N1, M0)

Stage four
Stage four stomach cancer means cancer has either

Grown through the stomach wall into body tissues next to the stomach
and lymph nodes further than 3cm away contain cancer cells (T4, N2, M0) OR
Spread to other body organs through the lymphatic system or bloodstream
(any T or N, M1)

1.2.3 Risk Factors

The factors that can increase the risk of getting stomach cancer are as
follows :

Helicobacter pylori (H. pylori). Some studies suggest that a type of bacteria,
Helicobacter pylori, which lives in the stomach lining, is a major cause of

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stomach cancer. People with H. pylori infection have three to eight times greater
risk of developing gastric cancer than those not infected.

Diet. A diet high in preserved foods - such as those that are smoked, dried,
salted or pickled - that contain nitrates and nitrites are linked to stomach cancer.
These substances can be converted in stomach into compounds that increase
risk of stomach cancer. People who have diets rich in meat, cheese and whole
milk may be at increased risk of developing cancer in both the esophagus and
stomach.

Previous stomach surgery. Stomach surgery may result in higher levels of

nitrite-producing bacteria and bile in stomach, which increases the risk of
stomach cancer.

Stomach polyps. Polyps are small bumps or larger mushroom-like growths of

the lining of the stomach. Most types of polyps (such as hyperplastic polyps or
inflammatory polyps) do not increase a person's risk of stomach cancer, but
adenomatous, or benign polyps, sometimes develop into stomach cancers.

Pernicious anemia. Pernicious anemia is caused by a lack of a substance

needed to absorb this nutrient. Vitamin B12 helps form red blood cells. Anemia is
when red blood cells are not giving enough oxygen to body tissues. People with
this condtion may have gastric polyps and twice the rate of stomach cancer than
those without pernicious anemia. Older adults are more likely to have this
condition.

Genetics. Stomach cancers are two to four times more common for immediate
family members of those who have had the disease

Smoking and alcohol abuse. Both of these substances can irritate the lining of
the stomach, particularly the upper parts, and increase the risk of developing
cancer.

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Environmental exposure. Certain dusts and fumes in the workplace have been
linked to a higher-than-average risk of stomach cancer.

1.3 Cancer Genetics

Human cancers harbour numerous mutations and it has been proposed
that these result from some form of inherent genomic instability. Some cancers
have proven genomic instability or features that are indicative of this It is widely
accepted that carcinogenesis requires several genetic alterations that convey
selective growth advantages. Most cancers accumulate numerous genetic
changes at the level of the nucleotide, gene and/or chromosome, and many
cancers also acquire epigenetic changes that can have profound effects on gene
expression.
1.4 Cytogenetics

1.4.1 Human Chromosomes

Cytogenetics is the study of chromosomes and the related disease states
caused by abnormal chromosome number and/or structure. Chromosomes are
complex structures located in the cell nucleus, they are composed of DNA,
histone and non-histone proteins, RNA , and polysaccharides. They are basically
the "packages" that contain the DNA. Normally chromosomes can't be seen with
a light microscope but during cell division they become condensed enough to be
easily analyzed at 1000X. To collect cells with their chromosomes in this
condensed state they are exposed to a mitotic inhibitor which blocks formation of
the spindle and arrests cell division at the metaphase stage.

A variety of tissue types can be used to obtain chromosome preparations.

Some examples include peripheral blood, bone marrow, amniotic fluid, and
products of conception. Although specific techniques differ according to the type
of tissue used, the basic method for obtaining chromosome preparations is as
follows:

Sample log-in and initial setup.

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 Tissue culture (feeding and maintaining cell cultures).
Addition of a mitotic inhibitor to arrest cells at metaphase.
Harvest cells.It involves exposing the cells to a hypotonic solution followed
by a series of fixative solutions. This causes the cells to expand so the
chromosomes will spread out and can be individually examined.
Stain chromosome preparations to detect possible numerical and
structural changes.

1.4. 2 Chromosome Morphology

Under the microscope chromosomes appear as thin, thread-like
structures. They all have a short arm and long arm separated by a primary
constriction called the centromere. The short arm is designated as p and the
long arm as q. The centromere is the location of spindle attachment and is an
integral part of the chromosome. It is essential for the normal movement and
segregation of chromosomes during cell division. Human metaphase
chromosomes come in three basic shapes and can be categorized according to
the length of the short and long arms and also the centromere location.
Metacentric chromosomes have short and long arms of roughly equal length
with the centromere in the middle. Submetacentric chromosomes have short
and long arms of unequal length with the centromere more towards one end.
Acrocentric chromosomes have a centromere very near to one end and have
very small short arms. They frequently have secondary constrictions on the short
arms that connect very small pieces of DNA, called stalks and satellites, to the
centromere. The stalks contain genes which code for ribosomal RNA.

The ideogram is basically a "chromosome map" showing the relationship

between the short and long arms, centromere (cen), and in the case of
acrocentric chromosomes the stalks (st) and satellites (sa). The specific banding
patterns are also illustrated. Each band is numbered to aid in describing
rearrangements.

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 Metacentric Submetacentric Acrocentric
(Chromosome 14) (Chromosome 9) (Chromosome 1)
Fig. 1 : Three types of human chromosomes based on centromere location. 1(a): metacentric,
1(b) submetacentric, 1(c) Acrocentric
1.4.3 Chromosome Analysis

All clinical Cytogenetic analyses are done on chromosome preparations

that have been treated and stained to produce a banding pattern specific to each
chromosome. This allows for the detection of subtle changes in chromosome
structure. The most common staining treatment is called G-banding. A variety of
other staining techniques are available to help identify specific abnormalities.
Once stained metaphase chromosome preparations have been obtained they
can be examined under the microscope. Typically 15-20 cells are scanned and
counted with at least 5 cells being fully analyzed. During a full analysis each
chromosome is critically compared band-for-band with it's homolog. It is
necessary to examine this many cells in order to detect clinically significant
mosaicism .

Following microscopic analysis, either photographic or computerized

digital images of the best quality metaphase cells are made. Each chromosome
can then be arranged in pairs according to size and banding pattern into a
karyotype. The karyotype allows to closely examine each chromosome for

8
structural changes. A written description of the karyotype which defines the
chromosome analysis is then made.

Normal Chromosomes
Normal human somatic cells have 46 chromosomes: 22 pairs, or
homologs, of autosomes (chromosomes 1-22) and two sex chromosomes. This
is called the diploid number. Germ cells (egg and sperm) have 23
chromosomes: one copy of each autosome plus a single sex chromosome. This
is referred to as the haploid number. One chromosome from each autosomal
pair plus one sex chromosome is inherited from each parent.

Fig. 2 : Picture of a typical human karyotype - Preparing a karyotype

1. 4. 4 Chromosome Abnormalities
Although chromosome abnormalities can be very complex there are two
basic types: numerical and structural. Both types can occur simultaneously.

Numerical abnormalities involve the loss and/or gain of a whole chromosome or

chromosomes and can include both autosomes and sex chromosomes.
Structural abnormalities involve changes in the structure of one or more
chromosomes. They can be incredibly complex but for the purposes of this
discussion we will focus on the three of the more common types:
Deletions involve loss of material from a single chromosome. The effects
are typically severe since there is a loss of genetic material.

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 Inversions occur when there are two breaks within a single chromosome
and the broken segment flips 180 (inverts) and reattaches to form a
chromosome that is structurally out-of-sequence.
Translocations involve exchange of material between two or more
chromosomes. If a translocation is reciprocal (balanced) the risk for
problems to an individual is similar to that with inversions: usually none if
familial and slightly increased if de novo.

Numerical and structural abnormalities can be further divided into two main
categories: constitutional, that are inborn and acquired, those that arise as
secondary changes to other diseases such as cancer.

1.5 REVIEW OF LITERATURE

Chromosomal Aberrations in Lymphocytes of Healthy Subjects and Risk of

Cancer
Pavel Rossner, Paolo Boffetta, Marcello Ceppi, Stefano Bonassi, Zdenek
Smerhovsky, Karel Landa, Dagmar Juzova, and Radim J. Srm

Increased frequency of chromosomal aberration (CA) in peripheral blood

lymphocytes is a predictor of cancer, but further data are needed to better
characterize CA as marker of cancer risk. Cytogenetic records of 11,834 subjects
who were free of cancer at the moment of blood drawing and who underwent
cytogenetic examination for preventive purposes were gathered and linked to the
national cancer registry, revealing a total of 485 cancer cases. Subjects were
classified according to the percentiles of CA distribution within each laboratory as
low (033rd percentile), medium (3466th percentile), and high (66100th
percentile). Subjects were further classified by occupational exposure and by
subclass of CA. We found a significant association between the overall cancer
incidence and the presence of chromosome-type aberrations [relative risk (RR)
for high vs. low CA level = 1.24; 95% confidence interval (CI), 1.031.50] but not
chromatid-type aberrations. Stomach cancer showed a strong association with

10
frequency of total CA (RR = 7.79; 95% CI, 1.0160.0). The predictivity of CA
observed in subjects exposed to various classes of carcinogens did not
significantly differ from the group of nonexposed subjects. This study contributes
to validation of CA as a predictive marker of cancer risk, in particular, of stomach
cancer; the association between CA frequency and cancer risk might be limited
to chromosome type aberrations.

Impact of Types of Lymphocyte Chromosomal Aberrations on Human

Cancer Risk:Results from Nordic and Italian Cohorts
Lars Hagmar, Ulf Stromberg, Stefano Bonassi, Inger-Lise Hansteen, Lisbeth
Ehlert Knudsen, Carita Lindholm, and Hannu Norppa

The frequency of cells with structural chromosomal aberrations (CAs) in

peripheral blood lymphocytes is the first genotoxicity biomarker that has shown
an association with cancer risk. CAs are usually divided into chromosome-type
(CSAs) and chromatid-type aberrations (CTAs), with different mechanisms of
formation. From a mechanistic point of view, it is of interest to clarify whether the
cancer predictivity of CAs is different with respect to CSAs and CTAs. We report
here cancer risk for cytogenetically tested, healthy subjects with respect to
frequency of CAs, CSAs, and CTAs in peripheral blood lymphocytes, using
Nordic (1981 subjects with CA data, 1871 subjects with CSA/CTA data) and
Italian (1573 subjects with CA data, 877 subjects with CTA/CSA data) cohorts,
with a median follow-up of 17 years. High levels of CAs at test were clearly
associated with increased total cancer incidence in the Nordic cohorts and
increased total cancer mortality in the Italian cohort. In the Nordic cohorts,
significantly elevated cancer risks were observed for subjects with both high
CSAs and high CTAs at test, and these variables showed equally strong cancer
predictivity. The results of the Italian cohort did not indicate any clear-cut
difference in cancer predictivity between the CSA and CTA biomarkers. There
was no significant effect modification by age at test, gender, country, or time
since test. The results suggest that both DNA double-strand breaks and other

11
initial DNA lesions responsible for CSAs and CTAs are associated with cancer
risk.

Chromosomal Aberrations in Lymphocytes Predict Human Cancer

Independently of Exposure to Carcinogens

Stefano Bonassi, Lars Hagmar, Ulf Stromberg, Alicia Huici Montagud, Hkan
Tinnerberg, Alessandra Forni, Pirjo Heikkila, Saskia Wanders, Peter Wilhardt,
Inger-Lise Hansteen, Lisbeth E. Knudsen, and Hannu Norppa,

An increased risk of cancer in healthy individuals with high levels of

chromosomal aberrations (CAs) in peripheral blood lymphocytes has been
described in recent epidemiological studies. This association did not appear to be
modified by sex, age, country, or time since CA test, whereas the role played by
exposure to carcinogens is still uncertain because of the requisite information
concerning occupation and lifestyle was lacking. It has been evaluated whether
CAs predicted cancer because they were the result of past exposure to
carcinogens or because they were an intermediate end point in the pathway
leading to disease. A nested casecontrol study was performed on 93 incident
cancer cases and 62 deceased cancer cases coming from two prospective
cohort studies performed in Nordic countries (Denmark, Finland, Norway, and
Sweden) and Italy. For each case, four controls matched by country, sex, year of
birth, and year of CA test were randomly selected. Occupational exposure and
smoking habit were assessed by a collaborative group of occupational hygienists
Logistic regression models indicated a statistically significant increase in risk for
subjects with a high level of CAs compared to those with a low level in the Nordic
cohort (odds ratio, 2.35; 95% confidence interval, 1.31 4.23) and in the Italian
cohort (odds ratio, 2.66; 95% confidence interval, 1.26 5.62). These estimates
were not affected by the inclusion of occupational exposure level and smoking
habit in the regression model. The risk for high versus low levels of CAs was
similar in subjects heavily exposed to carcinogens and in those who had never,

12
to their knowledge, been exposed to any major carcinogenic agent during their
lifetime, supporting the idea that chromosome damage itself is involved in the
pathway to cancer. The results have important ramifications for the
understanding of the role played by sporadic chromosome damage for the origin
of neoplasia-associated CAs.

Non-random structural chromosomal changes in primary gastric cancer

Anna D. Panani , and Charis Roussos

15 cases of primary gastric cancer were analysed cytogenetically by

direct culture of tumors cells and G banding technique. Nonrandom structural
aberrations presenting common chromosomal breakpoints. Chromosomes most
commonly involved were according to frequency 1,11,14,7,17,6,8 and 13.
Chromosome 11 was involved as add(11)(p15), while the pericentromeric area of
chromosome 14 was constantly participated in aberrations. Isochromosomes
i(1q), i(8q), i(13q), i(14q) and i(17q) were constantly found. Furthermore
translocations t(1;7), t(7;14), t(6;17) and t(5;14) were identified.

Cytogenetic studies in primary gastric cancer.

Ochi H, Douglass HO Jr, Sandberg AA.

Five primary gastric cancers were analysed with a G-banding technique.

All tumors had clonal chromosome abnormalities; a total of 67 numerical and 83
structural karyotypic anomalies were identified as clonal. The most prominent
and recurring numerical abnormality was a missing Y chromosome (three of four
tumors in the male patients). No recurrent structural abnormalities were found.
However, the breakpoints at bands 1p22, 3p21, and 19p13 were frequently
involved.

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Alterations of tumor suppressor and tumor-related genes in the
development and progression of gastric cancer
Gen Tamura

The development and progression of gastric cancer involves a number of

genetic and epigenetic alterations of tumor suppressor and tumor-related genes.
The majority of differentiated carcinomas arise from intestinal metaplastic
mucosa and exhibit structurally altered tumor suppressor genes, typified by p53,
which is inactivated via the classic two-hit mechanism, i.e. loss of heterozygosity
(LOH) and mutation of the remaining allele. LOH at certain chromosomal loci
accumulates during tumor progression. Approximately 20% of differentiated
carcinomas show evidence of mutator pathway tumorigenesis due to hMLH1
inactivation via hypermethylation of promoter CpG islands, and exhibit high-
frequency microsatellite instability. In contrast, undifferentiated carcinomas rarely
exhibit structurally altered tumor suppressor genes. Thus, while the molecular
pathways of gastric carcinogenesis are dependent on histologicalbackground,
specific genetic alterations can still be used for risk assessment, diagnosis, and
prognosis.

Chromosomal biomarkers of genomic instability relevant to cancer

Michael Fenech

It is generally acknowledged that a crucial event in the initiation and

evolution of cancer is the acquisition of a genomic instability phenotype. This
review focuses on mechanisms of chromosomal instability including aneuploidy,
chromosome rearrangement and breakage-fusion-bridge cycles. The role of
micronutrient deficiency, such as folate deficiency, in the causation of

14
chromosomal instability is briefly reviewed and the concept of recommended
dietary allowances for genomic stability is introduced. In addition, the techniques
for measuring the various chromosomal instability events are discussed with a
focus on the cytokinesis-block micronucleus assay as an almost complete
system for measuring these various genetic mishaps.

Interphase cytogenetics of gastric and esophageal adenocarcinomas.

Rao PH, Mathew S, Lauwers G, Rodriguez E, Kelsen DP, Chaganti RS.

Numerical changes affecting chromosomes 1, 2, 3, 4, 6, 7, 8, 10, 11, 12,

16, 17, 18, and the X and Y chromosomes have been analyzed using
chromosome-specific centromeric alpha-satellite repeat DNA probes in a panel of
biopsies of six gastric and three esophageal adenocarcinoma and one
epidermoid carcinoma of esophagus obtained at surgery. For each case, with
each probe, the number of hybridization signals were determined in 200 nuclei.
Hybridization of each probe to phytohemagglutinin-stimulated normal peripheral
blood lymphocytes served as controls. Monosomy was defined by loss of one
signal in 15% or more cells and trisomy or tetrasomy was defined by the
presence of 3 or 4 signals in 7% or more cells, respectively. The Y chromosome
was lost in 6 of 8 cases and monosomy 10 was seen in 5 of 10 cases. Trisomy
for chromosomes 17, 8, 7, 12, 11, and 1 was seen in 4 of 10, 4 of 10, 4 of 10, 2
of 10, 2 of 8, and 2 of 10 cases, respectively, and tetrasomy for chromosome 7
was seen in 1 of 10 cases. These data show that the Y chromosome and
chromosomes 10, 8, 7, 17, and 12 are most frequently involved in
nondisjunctional changes in these tumors. They also document the feasibility and
utility of interphase cytogenetics of gastric adenocarcinomas

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2. OBJECTIVE

To study the chromosomal aberrations in gastric cancer patients

using cytogenetics

3. MATERIALS & METHODS

3.1 PREPARATION OF REAGENTS

3.1.1 RPMI 1640

RoseWell Park Memorial Institute 1640 (RPMI - 1640) medium stimulates
a large proportion of lymphocytes to grow. Adequate cells are therefore available
for further preparation of intact chromosomes for karyotyping. Consequently,
clear karyograms to detect chromosome abnormalities can be drawn. It is a
ready-to-use medium suitable for heparinised blood and lymphocyte enriched
blood.
10.5g of RPMI-1640 medium (Hi Media Laboratories) was added to 750
ml distilled water and mixed with a magnetic stirrer. To the mixed solution, 2g of
sodium bicarbonate (NaHCO3) was added and the pH was adjusted to 7.2 using
0.1N HCl. The solution was made to 1000ml using distilled water and was then
filtered using Millipore filter (0.45 m). Antibiotics penicillin (100 IU/ml) and
streptomycin (0.1 mg /ml) were added to the filtrate and the medium was stored
at 8 C for further use.

3.1.2 Foetal Bovine Serum (FBS)

Foetal Bovine Serum (FBS), as a source of specific growth factors, is

necessary for cell cultivation. Gamma () irradiated Foetal Bovine Serum (Hi
media Laboratories) was stored at -20C.

3.1.3 Phytohaemaglutinin (PHA)

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 Peripheral blood lymphocytes do not normally divide. For mitosis to take
place, they need to be stimulated by adding the mitogen phytohaemagglutinin
(PHA). PHA is made from beans. PHA activity is the determining factor when
stimulating lymphocyte growth, because: too much PHA has a toxic effect and
not enough PHA results in poor mitosis

3. 1.4 Giemsa Staining Solution

1g of Giemsa powder was mixed with 54ml of glycerol with the help of
magnetic stirrer for 2 hours at 56C and was shifted to room temperature after
which 84 ml of methanol was added and mixed well for 1 hour. The solution was
filtered using Millipore filter (0.45 m ). The filtrate was stored at 4C.
Working standard was prepared by mixing 2ml of stock solution with 2ml
of 10% Sodium dihydrogen phosphate buffer and 46ml of sterile distilled water.

3.2 STUDY SUBJECTS

The patients admitted in Medical and Surgical Gastroenterology wards
(Unit I & II ) of Government General Hospital, Chennai were selected for the
study. 10 early gastric cancer patients (7 male & 3 female) were taken for
cytogenentic analysis and their case history including histopathology records
were collected from the Medical records section of the hospital. All patients
underwent Upper Gastro endoscopy to examine the stage of cancer before
cytogenetic analysis. Two gender-matched controls were included in the study.

3.3 CYTOGENETIC ANALYSIS

Culturing of Peripheral Blood Lymphocytes (PBLs) was performed using
standard technique (Hungerford et al. 1965).

3.3.1 Blood Collection:

1.8 ml of peripheral blood was collected from each patient by
venopuncture into tubes containing 0.2 ml Sodium heparin. Heparinized blood

17
was maintained at 48C and transported to the cytogenetics laboratory within
24 hours .

3.3.2 Culturing of PBLs :

The blood samples were transferred to culture vials and cultures were set
up in RPMI 1640 medium. RPMI 1640 medium was supplemented with 20%
Foetal Bovine Serum (Hi Media Laboratories) and 4 % PHA (Gibco Laboratories).
After 69 hours of incubation at 37C, colchicine was added and harvesting was
done.

3.3.3 Harvesting & Slide Preparation :

After 20 minutes incubation at 37C (REMI YORK Incubator ), cells were
collected by centrifugation (REMI Centrifuge) at 1200 rpm for 10 minutes. The
pellet was resuspended in 7ml of prewarmed hypotonic solution (0.075 M KCl)
and incubated for 7 min at 37C . The suspension was centrifuged and the
pellet was resuspended in 7 ml of fixative solution - acetic acid/methanol (1:3,
vol/vol). After incubation for 1 hr at room temperature, the mixture was pelletted
and resuspended in 7 ml of fixative solution. After 30 minutes of incubation at
room temperature the mixture was centrifuged and pellet was resuspended in
7ml fixative solution and left at room temperature for 15 minutes. The mixture
was finally centrifuged and the pellet resuspended with fixative solution was used
to prepare slides. These slides were air dried and stained with 5% Giemsa
solution (pH 6.8). Slides from each culture were numbered and scored.

3.3.4 Scoring :
100 well-spread plates with 46 1 centromeres were examined (Rossner
et al. 1998) in each slide using Fluorescent microscope (Olympus BH 20i). The
vertical/horizontal (V/H) position of the plates were noted to avoid repetition of
plate identification. Immersion oil was used to view the plates under 100X. Plates
with well banded chromosomes were photographed using a CCD camera

18
attached to the microscope and the metaphase plates were analysed for CAs
including CSAs (chromosome- type breaks, ring chromosomes, marker
chromosomes, and dicentrics) and chromatid type aberrations (CTAs; including
chromatid type breaks and chromatid exchanges) (Rossner et al. 2005).

4 . RESULTS

The major type of chromosomal aberrations observed in the study group is

tabulated in Table 1.

VARIABLES Case1 Case2 Case3 Case4 Case5 Case6 Case7 Case8 Case9 Case10
Age 50 45 54 34 62 64 79 75 60 58
Sex M F F F M M M M M M
Clinical Ant Sto Fun Sto Sto Fun Ant Sto Sto Fun
Examination
- Growth
No. of cells 62 38 45 52 56 39 47 52 66 53
scored
CSA * 14 14 *
CTA 16 17 17 16 17 16

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20
 Karyotype showing CTA in chromosome 16

5. DISCUSSION
Human chromosome 16 and 17 show chromatid type aberrations which is
substantiating the earlier literature findings. Prospective studies in gastric cancer
will reveal the biomarkers in human genome responsible for gastric cancer.

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 6. CONCLUSION

Thus the cytogenetic studies were carried out in gastric cancer

patients and the human chromosomes 16 and 17 were found to be the biomarkers of
this study.

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