PATHOLOGICAL SOCIETY OF GREAT BRITAIN AND IRELAND INTERNATIONAL COLLABORATIVE
AWARD (01 October 2012 Grant Reference No: ICA 2012/01) REPORT
Title of Project: Molecular alterations in gastric cancer from Nigerian patients
Introduction:
About 190 formalin-fixed, paraffin-embedded (FFPE) gastric cancer samples obtained from five Nigerian
institutions were included in the project. The research activities were carried out by Dr Henry O. Ebili at the
Molecular Pathology and Cell Biology Laboratory of the Division of Pathology, University Of Nottingham, Queen’s
Medical Centre campus, Nottingham, United Kingdom, under the supervision of Prof Mohammad Ilyas, between
July 8, 2013 and May 30, 2014.
Materials and Methods
Following months of optimization to achieve adequate amplification for old degenerate formalin-fixed, paraffinembedded (FFPE) DNA samples, a modification of the Quick Multiplex Consensus PCR (QMC-PCR) protocol
(Fadhil et al. J Clin Pathol, 2010;63:134-140) was found to adequately amplify our sample set. This protocol
consists of 2 amplification stages: an initial multiplex PCR of gene regions of interest, and a subsequent
singleplex PCR using 1:100 dilution of the amplicons from the multiplex PCR, the same cycling durations and
primer concentrations as the multiplex PCR, but specific annealing temperature.
We also optimized the pre-melting steps for use in the LightScanner HRM analysis and derived a pre-melting
protocol which includes the simple steps of adding 15-20µl of mineral oil overlay onto each sample in the wells
and centrifuging the plate at 3000 rpm for 2 minutes before melting on the LightScanner.
High Resolution Melt Analysis was utilized to screen for point mutations in KRAS, BRAF and PIK3CA, on 2
different HRM platforms: the ABI 7500 Fast Real Time HRM and the LightScanner HRM platforms.
Results
1. The modified QMC-PCR protocol enhanced the amplification of FFPE DNA samples for downstream
applications such as HRM (Figure1A&B).
2. Correct use of the LightScanner pre-melting protocol gave highly reproducible results
3. HRM of the amplification products is highly reproducible across the 2 different HRM platforms (Figure 1C,
D, E & F)
4. HRM results are unaffected by the starting DNA quantity, as long as the samples achieve adequate
amplification during PCR (an adequate amplification is regarded as a CT value of 30 or less and sigmoid
or near-sigmoid amplification curve with an amplitude)
5. Experimental data was obtained for 189 gastric cancer samples thus: KRAS (exons 2, 3 and 4), BRAF
(exons 11 and 15), and PIK3CA (exons 1, 9 and 20)
A total of 108 gastric cancer cases showed aberrant melting in at least one of the gene regions tested,
giving a 57% combined point mutation rate for KRAS, BRAF and PIK3CA in gastric carcinoma from
Nigerian patients.
KRAS
A total of 40/189 cases (about 21%) of cases showed aberrant melting in at least one of the 3 KRAS
exons tested (exon 2: 10 cases, exon3: 24, and exon4: 8). Furthermore, 2 samples showed aberrant
melting in more than one KRAS exon (exons 2/3: 1case; exons 3/4: 1 case). These samples will be
subjected to Sanger Sequencing for confirmation.
BRAF
Twenty-eight (28/189; about 14.8%) cases showed abnormal melting for exon 11(8 cases) and exon 15
(23 cases) of BRAF. Three (3) cases were aberrant at both exons 11 and 15. These 3 cases were also
aberrant at KRAS exon 2, and will be subjected to confirmation by Sequencing.
PIK3CA
A total of 40 samples (about 21% of cases) were abnormally melting in at least one of exons 1 (31), 9 (8),
and 20 (14) of PIK3CA. Three cases were aberrant at more than one exon (exon1/9: 2 cases, exon 1/20:
1 case). Interestingly, more samples were aberrantly melting at exon 1 of PIK3CA than at exons 9 and 20
combined; considering that mutations at exon 1 of PIK3CA are reported to be rare in gastric cancer from
other populations (Sukawa et al. Digestion, 2014; 89:12-17). These abnormally melting samples form 4
well-defined clusters (Figure 1 G & H). If these aberrant melting patterns are confirmed to be due to
mutations, this will add evidence to the growing notion that the genetic alterations which give rise to
cancers in indigenous African populations may be qualitatively and quantitatively different from those
which cause cancer in other populations (Fackenthal et al. Int J Cancer, 2012; 131:1114-23). On the
other hand, these aberrant melting patterns observed may simply be innocuous, synonymous SNPs
which may not have been reported in other studies.
A total of 9 samples showed aberrant melting patterns in both KRAS and BRAF. Considering the mutual
exclusivity of KRAS and BRAF mutations in cancers (Maughan et al. Lancet, 2011; 377:2103-14), these 9
samples will be subjected to further confirmation by Sequencing.
A
C
B
D
E
F
G
Figure 1A: PCR of KRAS exon 3 from using conventional protocol showing inadequate amplification of most samples. B: PCR of same
sample set as in A, using the modified QMC protocol showing significantly improved amplification of all the samples. C, D, E & F: HRM
Analysis of exon 15 of BRAF using the ABI 7500 Fast Real Time and the LightScanner platforms showing highly reproducible results
across both platforms seen here as identical Difference Plots, or Curves (C & E), and Melting Curves (D & F). G & H: Normalized Melt
Curves and Difference Plots from the HRM Analysis of PIK3CA exon 1 using the ABI platform showing four distinct clusters of gastric
cancer samples having aberrant melting patterns.