Supplementary Material
Methodology
Procedure for grossing and study of pre-neoplastic lesions:
Formalin-fixed, paraffin-embedded gallbladder specimens were examined for the presence of
epithelial changes – normal mucosa, hyperplasia, metaplasia, dysplasia and carcinoma in situ.
The gallbladder specimen was collected from 350 patients. Each GB specimen was divided into
4 parts horizontally, i.e. fundus, body, tail and neck. Longitudinal strips cut from these areas
were embedded properly in separate blocks. Thus, for each gallbladder specimen, we had 4
blocks.for the purpose of grossing i.e. cutting sections to be able to examine histologically the
whole of the specimen for the presence of pre-neoplastic lesions.
Histological identifications of lesions:
From each block, one 5  thick section was cut and Hematoxylin-eosin (HE) staining was done.
All the parts of the gallbladder represented in the different blocks were screened for the
presence of any pre-neoplastic lesions by examining in detail the HE stained slides. In each
slide, the areas of interest were marked separately with coding.
Microscopic criteria as proposed by Albores-Saavedra et ali were employed for the diagnosis of
hyperplasia, metaplasia, dysplasia and carcinoma in situ as follows:
Normal gallbladder has variable branching fold mucosa. The surface epithelium is composed of
a single layer of uniform, tall columnar cells with basal nuclei. Lamina propria consists of loose
connective tissue with blood vessels.
Hyperplasia - It consists of pseudostratification of the epithelium, nuclear crowding, taller than
normal columnar cells with or without increased mucus production and occasional normal
mitotic features.
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Metaplasia – It appears as foci with gastric antral-type glands, located in any zone of the
gallbladder. The glands are branched, tortuous, which in some sections occupy large areas of
the lamina propria.
Dysplasia - In addition to the above mentioned changes, there is some loss of architecture and
disorganization of the epithelium as well as nuclear atypia. When atypical epithelial cells are
arranged in a single layer, they usually exhibit marked nuclear abnormalities and some
prominence of nucleoli. (Atypical hyperplasia and dysplasia are used interchangeably. However,
we have used the term dysplasia only).
A few goblet cells may be found both in hyperplasia and in atypical hyperplasia.
Carcinoma in-situ – This is the very initial stage when the abnormal cancer cells are confined
to the mucosal layer of the gallbladder.
Gallbladder Cancer- Histologically, the most common type of gallbladder cancer is
adenocarcinoma.
Genetic analysis in the pre-malignant histological lesions for Loss of Heterozygosity:
Multiple serial sections of 10 were cut from the block corresponding to the H&E stained slide,
in which pre-neoplastic lesions were found.
From the multiple 10  thick sections, micro-dissection of the pre-neoplastic lesions and the
corresponding adjoining normal area was done.ii Each type of lesion was micro-dissected and
collected in a separate vial for DNA extraction.
Each type of pre-neoplastic lesion i.e. hyperplasia, metaplasia and dysplasia was analyzed
separately for genetic studies. If there were multiple areas of one type of pre-neoplastic lesion in
one gallbladder specimen, all the areas were taken together for genetic studies. Also,
histologically normal areas were microdissected for genetic studies. Normal areas from normal
gallbladders were also microdissected for genetic studies.
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Study of Loss of Heterozygosity (LOH) through microsatellite markers:
The loss of heterozygosity was studied at microsatellite markers located intragenically or
adjacent to tumor suppressor genes of interest.iii,iv
In the present study, LOH was studied at 3p12, 3p14.2, 5q21, 9p21, 9q, 13q, 17p13, and 18q
locations using microsatellite markers. These microsatellite markers were chosen due to their
proximity to known tumor suppressor genes (TSG) as follows:
Location
microsatellite marker
size
Adjacent TSG

3p14.2
D3S1766
225bp
FHIT gene

3p12
D3S1274
128bp
DUTT1 gene

5q21
D5S409
146 bp
APC gene

17p13
D17S786
148 bp
p53 gene

9p21
IFNA
147 bp
p16 gene

9q
D9S127
154 bp
FCMD gene

18q
D18S34
110 bp
DCC gene

13q
D13S153
160 bp
RB1 gene
DNA extraction from the tissue: DNA extraction of the tissue was done using QIAamp DNA
FFPE tissue DNA extraction kit (Qiagen, Maryland, USA). The extracted DNA (quality and
quantity) was tested using picodrop spectrophotometer (Biotron Healthcare, Cambridge, UK).
The yield of the extracted DNA was 10.2 to 64.5 ng/ul. Genomic DNA was also extracted from
whole blood samples using QIAamp DNA blood kit (Qiagen, Maryland, USA).
Standardization of polymerase chain reaction (PCR): The markers were selected for each
microsatellite marker from the Genome Database (http://www.gdb.org/). PCR was standardized
for each microsatellite marker. The amplification was checked on 2% agarose gel. The details of
the markers, sequence, their primers and product size are as follows:
i.
D3S1766
Forward Primer:
tetranucleotide repeat
ACCACATGAGCCAATTCTGT
3
tcta
Reverse Primer:
ACCCAATTATGGTGTTGTTACC
Expected Product size: 225
ii.
D3S1274
dinucleotide repeat
Forward Primer:
TTATACATCAGTCTCTGGGAAACAC
Reverse Primer:
TACTGTGCATATAGGTTCCTGTGA
ca
Expected Product size: 134
iii.
D5S409
dinucleotide repeat
Forward Primer:
GGGATGAAGTGTGGATAAAC
Reverse Primer:
TAGGATGGCAGTGCTCTTAG
ca
Expected Product size: 146
iv.
D17S786
dinucleotide repeat
Forward Primer:
TACAGGGATAGGTAGCCGAG
Reverse Primer:
GGATTTGGGCTCTTTTGTAA
ca
Expected Product size: 149
v.
IFNA
dinucleotide repeat
Forward Primer:
TGCGCGTTAAGTTAATTGGTT
Reverse Primer:
GTAAGGTGGAAACCCCCACT
ca
Expected Product size: 147
vi.
D9S127
dinucleotide repeat
Forward Primer:
CCCTCAAAATTGCTGTCTAT
Reverse Primer:
AGATTGATTGATACAAGGATTTG
ca
Expected Product size: 154
vii.
D18S34
dinucleotide repeat
Forward Primer:
CAGAAAATTCTCTCTGGCTA
Reverse Primer:
CTCATGTTCCTGGCAAGAAT
Expected Product size: 110
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ca
viii. D13S153
dinucleotide repeat
ca
Forward Primer:
GTT TCA TGT TGG TGT ACG TC
Reverse Primer:
TTG TGG AAA GGA GCG TAT CT
Expected Product size: 160
PCR for Fragment Analysis: The 5’ end of forward primers was labeled with either FAM (blue),
NED (yellow but black on screen), VIC (green) or PET (red) (fluorescent dyes). Touchdown
PCR conditions were used for all the microsatellite markers. Genomic DNA was amplified by a
touchdown PCR with 25 μl reaction mixture. Initial denaturation at 940 C for 5 minutes, 11 cycles
of 950 C for 20 sec, 650 C to 560 C for 55 sec and 720 C for 20 seconds, then 30 cycles at 900 C
for 20 sec, 550 C for 20 sec and 720 C for 20 seconds and final extension at 720 C for 35
minutesv. A few modifications were done e.g. the final extension time at 720 C was modified to
35 minutes to reduce stutter bands and the number of cycles was increased from 24 to 30 in the
PCR to increase the yield. Since degradation of DNA by formalin fixation limits reproducible
amplification of DNA to fragments smaller than 250 base pairs, the primers used were designed
to bracket relatively short DNA fragments between 100 and 200 base pairs. Simplex PCR was
done for each primer separately as all the PCR products were of approximately similar fragment
size.
The PCR products were then checked on 2% agarose gel by staining with ethidium bromide.
The PCR products from 5 primers were mixed in a particular ratio for a sample and then
denatured at 950 C for 3 minutes. So, multiplexing of the samples was done before analysis on
a sequencer.
Study of informative polymorphic markers: DNA from corresponding blood and/ or normal
gallbladder tissue was first analyzed to find out which markers were heterozygous in each
patient. These heterozygous microsatellite markers were called informative markers.
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Fragment Analysis: The amplified PCR products were analyzed using the automated
fluorescent fragment analysis (ABI sequencer, Applied Biosystems, USA) which is considered
better than conventional radiolabeling methods.vi,vii Alleles of different lengths give different peak
heights which are used to define the presence or absence of allelic imbalance. Liz-600 was
used as a size standard. Stutter bands were formed during the amplification of microsatellite
markers as a consequence of polymerase slippage. Different four color fluorescent dyes were
used to resolve PCR products of comparable size from different loci. Binning for each
microsatellite marker was done. Analysis was done using Genemapper version 4.0 software.
LOH was calculated as:
(peak height of normal allele 2)/ (peak height of normal allele 1)
LOH
=
(peak height of tumor allele 2)/ (peak height of tumor allele 1)
LOH is strongly indicated by ratios less than 0.5 or higher than 2.0.
Fractional allelic Loss (FAL) index was defined as the total number of microsatellite markers
with LOH in an epithelial sample divided by the total number of informative markers examined. It
was calculated as an expression of the amount of allelic loss for all chromosomal loci.viii
Immunohistochemistry for p53, p16 and p27:
The 5μ tissue section was cut on poly-l-lysine coated slide.
Deparaffinization and hydration: The slides were heated at 650 C for 3 minutes. Then the
slides were incubated in xylene at room temperature for 10 minutes followed by a series of
decreasing ethanol concentrations for 5 minutes each. Finally, the slides were rinsed in running
water for 3 minutes.
Antigen Retrieval:
The slides were then kept in citrate buffer for 30 minutes at 1000C
(microwave treatment), cooled at room temperature and then washed in running water.
Blocking: The slides were kept in 4% H2O2 in Methanol for 30 minutes for endogenous
peroxidase blocking.
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The slides were then washed in running tap water for 10 minutes and rinsed with RO water and
finally washed with Tris buffer.
Primary Antibody: The p53 primary mouse monoclonal antibody clone DO7 (Bio SB, Santa
Barbara, CA, USA; Catalogue no. BSB 5846) was put in the desired dilution (1:1600) and
incubated overnight at 40 C. For p16 immunohistochemistry, p16 mouse monoclonal antibody;
Santa
Cruz Biotechnology;
catalogue
no.
SC-1661
at
dilution of
1:100,
for
p27
immunohistochemistry and for p27 rabbit anti-human polyclonal antibody; Spring Bioscience,
Pleasantan, CA; catalogue no: E2604 at dilution of 1:1000 were used. Slides were brought at
room temperature the following day and then washed with Tris buffer.
Secondary antibody and Horseshoe-radish-peroxidase (HRP): MACH 2 Universal HRPPolymer Detection kit (Biocare Medical, Concord, CA, USA) was used and after adding the
secondary antibody, the slides were kept at room temperature for half an hour and then washed
with Tris buffer. Finally, di-amino-benzidine (DAB) was added and observed for the reaction
colour development.
Counterstaining with hematoxylin was done. Finally, the slides were washed in water, dried at
room temperature, mounted and labeled.
Positive control sections were cut and prepared in the same manner and were put with every
batch of samples.
Grading of IHC: Grading of IHC was done on the basis of two features: intensity of the stain
and the proportion of stained area.
The intensity in the best stained positive control slide was taken as grade 3 positivity. The stain
intensity in the patient slides were compared with the control and was graded accordingly: grade
3 (intensity equal to the positive control slide); grade 0 (negative); grade 1 (indefinite or very
light and grade 2 (stain intensity intermediate to grade 1 and 3. The proportion of positive areas
was graded as follows: 0 (negative), 1 (less than 30% area of the area),2 (30 – 60% area), 3 (60
– 90% area) and 4 (more than 90% area).
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References:
i
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ii
Tadokoro H, Shigihara T, Ikeda T, Takase M, Suyama M. Two distinct pathways of p16 gene
inactivation in gallbladder cancer. World J Gastroenterol 2007; 13(47):6396-6403.
iii
Wistuba II, Behrens C, Virmani AK, Mele G, Milchgrub S, Girald L, et al. High resolution chromosome
3p allelotyping of human lung cancer and preneoplastic /preinvasive bronchial epithelium reveals multiple,
discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res 2000;
60:1949-60.
iv
Boldog F, Gemmill RM, West J, Robinson M, Robinson L, Li E, et al. Chromosome 3p14 homozygous
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v
Wistuba I, Behrens C, Milchgrub S, Virmani AK, Jagirdar J, Thomas B, et al. Comparison of molecular
changes in Lung cancers in HIV-positive and HIV-Indeterminate subjects. JAMA 1998;279(19):15541560.
vi
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2688-91.
vii
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viii
Maitra A, Wistuba II, Washington C, Virmani AK, Ashfaq R, Milchgrub S, et al. High-Resolution
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