SLCO1B3 screening in colorectal cancer patients using High-Resolution Melting Analysis method and immunohistochemistry

Abstract

Personalized medicine has made some major advances in colorectal cancer, but new biomarkers still remain a hot issue as an emerging tool with potential prognostic and therapeutic potential. We investigated for SLCO1B3 gene alterations and protein expression in colorectal cancer, using the novel high-resolution melting analysis technique and immunohistochemistry. Formalin-fixed paraffin-embedded tumor samples from 30 colorectal cancer patients were used. The screening for gene alterations was done by high-resolution melting analysis for all exons of SLCO1B3 gene. Organic anion-transporting polypeptide 1B3 protein expression was assessed by immunohistochemistry using the monoclonal mouse MDQ antibody. High level of polymorphism was observed in the SLCO1B3 gene. We identified three previously reported polymorphisms in exons 7, 12, and 14, 699G>A, 1557A>G, and 1833G>A, respectively. In the exon 5, one variant seems to correspond to an as yet unknown SLCO family member. The immunohistochemical study revealed that organic anion-transporting polypeptide 1B3 was expressed in 27/30 samples. Of great interest, the three samples, which were immunohistochemically negative, all appeared to accommodate mutations which lead to either early stop codons or other conformations of the tertiary protein structures affecting the antibody-epitope binding. The results of this study are of much interest as high-resolution melting analysis proved to be a reliable and rapid genotyping/scanning method for mutation detection of SLCO1B3 gene.

Keywords

Colon cancer HRMA SLCO1B3 mutation detection

Introduction

Colorectal cancer (CRC) is a major world health issue because of its high prevalence and mortality rates. It consists the second most common cancer diagnosed in Europe and the third most common cancer in the United States.^1–3 This cancer although highly curable by surgical resection at early stages, when it presents with metastases at diagnosis, which occurs in 25% of the patients, or when it becomes metastatic, which happens in 50% of the cases, it is considered largely incurable, despite the progress made in the development of novel therapeutics.³ Therefore, the discovery of novel molecular targets and targeted therapies is of major importance for an essential improvement of therapeutic outlook in this large group of patients.

SLCO1B3 gene belongs to solute carrier organic anion transporter family and encodes the human organic anion-transporting polypeptide (OATP) 1B3.⁴ It is located in chromosome 12 (2p12–31.7 to 12p12–37.2) (Figure 1(a).).⁵ This gene has six transcripts, two of which, SLCO1B3-001 and SLCO1B3-201, have a Consensus Coding Sequence (CDS) and comprise 16 and 15 exons, respectively,⁶ and their main difference is in the first and second exons of the 001 transcript (Figure 1(b)). OATP1B3 seems to be the only hepatic OATP transporting digoxin, amanitin, docetaxel, and paclitaxel. In addition, OATP1B3 expression has been suggested as a potential variable of tumor sensitivity to methotrexate treatment.^7–15 Moreover, besides the abundant expression in the periportal area in liver, OATP1B3 has been found to be expressed in several types of cancers. The expression of OATP1B3 in colon tumors and in the colon tumor cell lines was mainly cytoplasmic and clearly different from the membranous expression pattern in the normal liver. Cytoplasmic localization of OATP1B3 in tumor cells has also been found in primary breast cancer tissues. The aberrant expression of OATP1B3 in the cytoplasm of colon tumors was the impetus to investigate whether OATP1B3 functions differently in the setting of malignancy.^8,10

Figure 1.

(a) SLCO1B3 is located in chromosome 12 (2p12–31.7 to 12p12–37.2) (GeneCards. SLCO1B3. Genomic Location. 2012). (b1) The map of the transcript SLCO1B3-201 and (b2) the map of the transcript SLCO1B3-001.

The SLCO1B3 gene sequence (encoding OATP1B3) was shown to vary among different populations, as well as several single nucleotide polymorphisms (SNPs) were reported to date,^16–21 therefore the significance of SLCO1B3 polymorphisms on drug pharmacokinetics is becoming increasingly evident and the inter-ethnic differences exist in the allelic frequency of these variants suggests the need to characterize the full extent of SLCO1B3 genetic variability in any given population.¹⁷

The study of polymorphisms of SLCO1B3 gene presents a special interest as they seem to be implicated in the function of the protein and promises a possible discrimination among patients in order to achieve personalized treatment. In addition, high-resolution melting (HRM) analysis (HRMA) technique was chosen to serve as a powerful polymorphisms/mutation screening tool, based on the simplicity of the method, low cost, and high sensitivity and specificity.

Moreover, the expression and cellular localization of OATP1B3 was assessed by immunohistochemistry, using the monoclonal mouse MDQ anti-OATP1B3 antibody, which recognizes the N-terminal region of OATP1B3, for the same tissue samples.

HRMA is a polymerase chain reaction (PCR)-based method for detecting DNA sequence variation by measuring changes in the melting point of a DNA duplex. Until recently, there are not data as far as it concerns the use of HRM as a gene scanning method for the gene of solute carrier organic anion transporter 1B3 (SLCO1B3) in FFPE tissues from patients with CRC. Moreover, there are limited data about its corresponding protein organic anion-transporting polypeptide 1B3 (OATP1B3).

Materials and methods

We used 30 formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from patients diagnosed with CRC provided by the Department of Pathology of the General Hospital of Ioannina “G. Hatzikosta.” The institutional review board approved all of the protocols. Patient’s median age was 65 years: 13 were females and 17 were males. Histologically, 6 cases were diagnosed as poorly, 18 as moderate, and 6 as well-differentiated colon adenocarcinomas.

The genomic alterations of SLCO1B3 exons were assessed by HRM, while the expression and subcellular localization of OATP1B3 were carried out using immuno-histochemistry.

DNA extraction

Up to 5 of 10 µm FFPE tissue sections were gathered according to the standard method¹⁸ and were used for DNA extraction with “NucleoSpin^® FFPE DNA” kit (Macherey-Nagel cat number 740980) according to the manufacturer’s instructions. Extracted DNA was quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific).

HRMA

HRMA was performed on the LightCycler^® 480 Real-Time PCR System, using the LightCycler 480 High Resolution Melting Master Reaction Mix (Roche Diagnostics cat number 4909631001).

Specific pairs of oligonucleotide primers (forward and reverse) were designed using Primer3 (version 3.0) to bracket each exon (Table 1) of SLCO1B3. The reference sequence gene of SLCO1B3 is formatted and downloaded from the Ensemble (http://www.ensembl.org/Homo_sapiens/Transcript/Exons?db=core;g=ENSG00000111700;r=12:20963636-21243040;t=ENST00000261196).

Table 1.

Oligonucleotide primer sequences to carry out HRMA of the SLCO1B3 coding region.

Exon	Forward primer	Reverse primer	Amplicon size (bp)	Annealing temperature (°C)
1	5′-CATCATTGGACTCATAAAAACAAA-3′	5′-GAAATGTTTTAATTAGACAACCTCGAC-3′	151	45
2	5′-AACAGGTGATCATTTCAAACCA-3′	5′-AATCCATTGCAGCGTCTTGT-3′	150	57
3	5′-ATGTTCTTGGCAGCCCTGT-3′	5′-AAAGTTACCAATTTCAAAGCTTCC-3′	150	52
4	5′-TTCTAGGAAATTTGCTTGTGATTG-3′	5′-TGGCTCAGAGCTGTTTAACACTTA-3′	164
5	5′-CAAAATGTTCAATTTCATGTTGC-3′	5′-TTTTTCTACTATCTCAGGTGATGTTCC-3′	152	50
6	5′-AAAGTAAAACACTCTCTTGTCTCGAT-3′	5′-TCCAGGGAATGTAATCATGAAA-3′	219	57
7	5′-ACAGGTAGTTTGAATGCAATAGGA-3′	5′-TGCACTTAAAAGACACTATCATGG-3′	150	48
8	5′-CGATTTTTGACTGGCTTCTTTT-3′	5′-CAATTCCAAGTTGACAATGAATG-3′	315	55
9	5′-TCCAGTCTTTGAAAAGCATCC-3′	5′-ACCCAACAAAAAGTTAGCATGA-3′	161	55
10	5′-AGAATGGGTGAATTTGGTTGA-3′	5′-TGCACATAATCTTTTAATTTGATGG-3′	365	55
11	5′-CCCTCTCCTTATCCCCTTGT-3′	5′-GGAGAGGAGAAAAAGTGAAAACT-3′	287	55
12	5′-TTGGCAAATGTATTTGTTAATATTTC-3′	5′-CATGACAATGTTTTACAGGATCA-3′	256	55
13	5′-TGACATGTATTATTTCTTTGCCTTT-3′	5′-AGTGTTCTTATAACCATTGACTGGAA-3′	100	50
14	5′-TCTCATGTTGCAGGAGGAAT-3′	5′-TGTGTTTACGACAACTTACCCAAA-3′	150	50
15a	5′-TCGACTCTCTATTTTCTCTTTTCACA-3′	5′-TCCATTGGTCCACTGTAAGTAAA-3′	363
15b	5′-TCCAACATTCTTTACTTACAGTGGA-3′	5′-GGTTGGAAGAAGATCCATCG-3′	459
16	5′-GTGCATTTATCGCCACATTG-3′	5′-TTCATGCCTGAGGGAAACTT-3′	166	55

HRMA: high-resolution melting analysis.

Equal quantity of DNA template of each sample was used in each 20 µL of reaction volume with 0.2 µM and 2.5 mM final primer and MgCl₂ concentration, respectively. Cycling conditions were set as follows (Table 2): pre-incubation/denaturation of secondary structures 10 min at 95°C, followed by 55 amplification cycles comprising 1 min denaturation at 95°C, followed by 60-s primer annealing T (according to each primer), and a 60-s elongation period at 72°C. Final PCR product identification and genotypic differentiation were carried out by a single cycle of HRM curve (95°C for 60 s, 40°C for 60 s, and 65°C for 1 s). The annealing temperature is provided in Table 1.

Table 2.

The program of the LightCycler^® 480 Instrument.

Detection format	Block type	Reaction volume
SYBR Green I	96	20 µL
Programs
Program name	Cycles	Analysis mode
Pre-incubation	1	None
Amplification	55	Quantification
High-resolution melting	1	Melting curve
Cooling	1	None
Temperature targets
Target (°C)	Acquisition mode	Hold (hh:mm:ss)	Ramp rate (°C/s) (96 wells)	Acquisitions (per °C)
Pre-incubation
95	None	00:10:00	4.4
Amplification
95	None	00:01:00	4.4
Primer dependent^a	None	00:01:00	2.2
72	Single	00:01:00	4.4
High-resolution melting
95	None	00:01:00	4.4
40	None	00:01:00	2.2
65	None	00:00:01	1/1
95	Continuous	–	–	25
Cooling

Analysis of the experimental data with appropriate software

Analysis of results was achieved by the LightCycler 480 Gene Scanning Software using the “Second Derivative Maximum/2nd Derivative” analysis method.

The group with the most identical samples served as the “wild type” sequence for each exon. Thus, any sample with a different melting peak than that of the wild type was considered to carry some form of genetic mutation.

DNA sequencing

Sequencing analysis was performed on a representative sample of the “wild type” group and for each sample genotypic differences were demonstrated.

Sequence analysis

Purified samples were sent for sequencing to the VBC biotech Vienna. Results were assessed. The wild type of each exon was in accordance with the relatives from Ensembl genome browser (based on the site: Ensembl genome browser 67: Homo sapiens—Gene: SLCO1B3 ENSG00000111700. 2012). Then, each sequence was aligned with the corresponding wild type by using the multiple alignment algorithms in the Molecular Evolutionary Genetics Analysis 5.1 beta (MEGA 5.1 beta). Moreover, each nucleotide sequence was translated, using MEGA 5.1 beta.

Immunohistochemistry

Tissue sections of 3–4 µm width were cut, applied on microscope slides, and incubated overnight at 65°C for optimum tissue–glass adhesion. Deparaffinization, rehydration, and heat-induced epitope-retrieval (HIER) were achieved by incubating the slides in preheated (65°C) pH 9 target retrieval solution (cat number S2375)for 20 min at 93°C. DAKO’s Autostainer/PT link system was used for the immunostaining process. A 30-min blocking step with 5% dry milk preceded the Ab application. Monoclonal antibody MDQ (mMDQ) (PROGEN Biotechnik, cat number 651140) was used for the immunolocalization of the N-terminal 24 amino acid epitope of OATP1B3, at a final concentration 1:10. Primary antibody was diluted with Dako REAL™ Antibody Diluent (DAKO, Code S2022) in 1% blocking reagent and applied on slides for 30 min. Endogenous peroxidase was blocked using Dako REAL peroxidase blocking solution (Code S2023) for 10 min. DAKO’s special engineered Dextran backbone enriched with peroxidase molecules and goat secondary antibody molecules against rabbit and mouse immunoglobulins (Dako REAL EnVision™/HRP, Rabbit/Mouse ENV, Code Code K5007) was applied on slides for 20 min followed by a 5-min Dako REAL DAB+ Chromogen (DAKO, Code K5007) detection system and a 2-min hematoxylene (QS H-3404 Vector Laboratories) exposure. All incubation steps were performed at room temperature unless stated otherwise. Finally, sections were dehydrated with alcohol/xylene baths and stabilized with mounting medium. Immunostaining intensity was assessed by two experienced pathologists, who graded the results, using endothelial cells and tissue macrophages as internal controls.

Results

HRMA results with sequence confirmation

Based on our results, we observed high polymorphism rate in the SLCO1B3 gene (Table 3). Three previously reported polymorphisms are identified, in exons 7, 12 and 14, which correspond to 699G>A with amino acid variation Met233Ile, encoding the OTP1B3/OATP-8-M233I (Rs7311358),^11,19,20 1557A>G with amino acid variation (Ala519Ala) encoding the OTP1B3/OATP-8-A519A (Rs2053098), and 1833G>A with amino acid variation Gly611Gly (Rs3764006), respectively.

Table 3.

Immunohistochemical expression of OATP1B3 (score scale (0, 1+, 2+, 3+) reflecting antibody product expression intensity) and the type of mutation identified by HRMA in each case.

Patient number	Sex	Age	Differentiation	mMDQ anti-OATP1B3 antibody	Polyclonal anti-OATP1B3 antibody	Mutated exons	Type of mutation	Protein alteration	Hetero-/homozygote
1	F	70	Well	+	−
2	F	50	Poor	++	−	3	Substitution of A to C	Substitution of I to S	Homozygote
3	M	75	Intermediate	++	−
4	F	64	Intermediate	+	−	5	Deletion of A		Heterozygote
5	F	49	Intermediate	++	−	13	Deletion of A	Stop codon	Homozygote
6	M	61	Poor	++	−	3	Substitution of A to C	Substitution of I to S	Homozygote
						14	Deletion of A and substitution of G to A	Stop codon	Heterozygote
7	F	69	Intermediate	−	−	2^a	Deletion of A and substitution of C to T		Homozygote
8	F	58	Intermediate	+	−
9	F	72	Well	−	−	2	Insertions		Homozygote
10	M	74	Well	++	−	1	Deletion of A in intron area		Heterozygote
11	M	70	Intermediate	+++	−
12	M	45	Poor	++	−	11	Substitution of T to A	Substitution of S to I	Homozygote
13	M	57	Well	++	−	13	Substitution of C to A and A to C	Substitution of QP to HH	Homozygote
14	M	66	Intermediate	++	−
15	M	42	Intermediate	++	−	6,	Substitution of TA to AG	Stop codon	Homozygote
						7	Substitution of A to G	Substitution of M to I	Homozygote
16	M	74	Intermediate	++	−
17	M	68	Poor	+	−
18	M	51	Poor	++	−
19	F	47	Well	++	−	11	Deletion of C in intron area		Homozygote
20	M	69	Intermediate	++	−	2^a	Insertion of A and deletion A		Homozygote
21	F	61	Intermediate	+	−
22	F	63	Intermediate	−	−	10,	Deletion of two As in intron area		Heterozygote
						2^a	Deletions of A and substitution of C to T		Homozygote
23	M	79	Intermediate	+	−	6,	Substitution of A to T and G to T	Stop codon	Homozygote
						7,	Substitution of A to G	Substitution of M to I	Homozygote
						13	Substitution of C to A, A to C, and insertion of A	Substitution of QPELKALAM to HHRIESTCN	Homozygote
24	M	77	Intermediate	++	−	14	Deletion of A and substitution of G to A	Stop codon	Heterozygote
25	M	66	Intermediate	++	−
26	F	48	Intermediate	++	−
27	F	68	Poor	+++	−	5	Substitution of G to A and deletion of GA	Stop codon	Homozygote
28	F	58	Well	++	−	12	Insertion of A	Stop codon	Heterozygote
29	M	59	Intermediate	++	−
30	M	75	Intermediate	++	−

HRMA: high-resolution melting analysis; mMDQ: monoclonal antibody MDQ; F: female; M: male; OATP: organic anion transporting polypeptide; A: adenine; T: thymine; G: guanine; C: cytosine; I: isoleucine (nonpolar, neutral); S: serine (polar, neutral); M: methionine (nonpolar neutral); Q: glutamine (polar, neutral); P: proline (nonpolar, neutral); H: histidine (polar, 10% positive).

Moreover, in exon 5, one variant seems to correspond to an as yet unknown SLCO family member (Homo sapiens cDNA FLJ31312 fis, clone LIVER1000224, highly similar to solute carrier organic anion transporter family member 1B3, dbj|AK055874.1|). Some other nucleotide substitutions which have not been reported yet were found.

Furthermore, the observed deletion and/or insertion of adenine was evaluated with caution as it might be caused by the use of formaldeyde during tissue fixation. Changes were also observed in the nucleotide sequence in the area of introns of exons 1 and 11.

Immunohistochemistry

The immunohistochemical study revealed that OATP1B3 was expressed in 27/30 studied cases and was located in the cytoplasm (Figure 2). Interestingly in positive cases, almost all cancer cells were stained positive.

Figure 2.

Immunohistochemical staining of colon adenocarcinoma tissue sample. There is cytoplasmic expression of the antibody in neoplastic and not in normal epithelial tissue. (a) x20, (b) x40.

Furthermore, protein expression was intense in 40% of the cases studied while weak was the dominant pattern in 63.3% of studied cases.

HRMA of the three OATP1B3 negative samples confirmed the presence of polymorphisms in exons 2 and 3 of the transcript SLCO1B3-001 which either led to early stop codons or to other conformations of the tertiary protein structures prohibiting properly the antibody-epitope binding, thus inability in protein detection.

One of them had an insertion of 22 nucleotides in the area of exon 2 of the transcript SLCO1B3-201, which causes deletion of the start codon. As a consequence, the protein OATP1B3 is not produced at all, making impossible for the mMDQ to detect. The other two cases have the same mutation, a deletion of adenine and a substitution of cytosine to thymine in the second exon of the transcript SLCO1B3-00. This exon belongs to the untranslated area of the gene SLCO1B3. We suggest that these mutations may change the ribosomal binding site (RBS), which is a sequence on messenger RNA (mRNA) that is bound by the ribosome when initiating protein translation.

Discussion

The great interest for SLCO1B3, as far as it concerns cancer, is that its protein OATP1B3, although it is expressed in the human liver under normal conditions, can also be expressed in various human cancer tissues that have been associated with prognosis and clinical outcomes. The expression of OATP1B3 in colon tumors and in the colon tumor cell lines was mainly cytoplasmic and clearly different from the membranous expression pattern in the normal liver. This is in accordance with our immunohistochemical results (Figure 2). The aberrant expression of OATP1B3 in the cytoplasm of colon tumors was the impetus to investigate whether OATP1B3 functions differently in the setting of malignancy.¹⁰ The molecular entity of cancer-associated OATP1B remains undetermined. Only Nagai et al.²¹ reported the identification of a new OATP1B3 mRNA isoform expressed in human colon and lung cancer tissues, which they named cancer-type OATP1B3 (Ct-OATP1B3). Such data raised our interest to investigate this transporter, applying HRMA in FFPE samples derived from patients with CRC. The upper goal was to scan possible polymorphisms/mutations of the transporter SLCO1B3 in this group of patient, with possible implications in the pathogenesis and treatment of CRC.

In this study, although high levels of polymorphism were observed in the exons of the SLCO1B3 gene, the results could not confirm that any of these polymorphisms presented with greater frequency in patients with CRC. Actually, three previously reported polymorphisms in exons 7, 12 and 14 were identified. In exon 5, one variant seems to correspond to an as yet unknown SLCO family member (Homo sapiens cDNA FLJ31312 fis, clone LIVER1000224, highly similar to solute carrier organic anion transporter family member 1B3, dbj|AK055874.1|). In addition, many other novel variants were identified, which were not reported yet.

As it is already known, in the general population, more than 50 SLCO1B3 polymorphisms, most of which are located in regulatory regions, have been identified along with a variety of haplotypes. Two clusters of deletions at −7 to −4 and −28 to −1 in the 5′-untranslated region (UTR) that may result in reduced mRNA stability or problems with initiation of translation have also been reported.^17,20 Among all these polymorphisms, functional analysis indicated that the variant 699G>A, which was also detected in our study, alters transport levels and kinetics of OATP1B3 and the change appears to be substrate-specific, while 699G>A expression was not different from that of wild type. Moreover, 699G>A is the most common OATP1B3 polymorphism in individual of all racial backgrounds (Hispanic Americans = 0.833, Chinese American = 0.795, Caucasian Americans = 0.779, African Americans = 0.478, and Caucasian Europeans 0.71).^11,19,20 This commonly inherited polymorphism M233I (as well as S112A) influences the degree to which testosterone is imported into cells through SLCO1B3 playing a possible important role in men with prostate cancer.²²

Immunohistochemically, it was revealed that three cases were completely negative for the mMDQ protein. A possible hypothesis is that these mutations may not permit the protein of OATP1B3 to be produced throughout. The mMDQ antibody was expressed immunohistochemically in the rest twenty seven (27) cases because the epitope of the antibody is found in the area of protein which is encoded by the second exon of the transcript SLCO1B3-201 or the third exon of the transcript SLCO1B3-001. Thus, mutations after this exon, deletions, insertions, or premature stop codons, might produce conformational changes in the OATP1B3 polypeptide, but they do not interfere with recognition of this epitope. However, nobody can exclude that other mutations could also exist in the positive immunohistochemical speciments.

Neither this study can elucidate the precise role of OATP1B3 in CRC. Because of the limited number of samples (only 30), the interpretation of the results was quite difficult. The use of more samples could possibly show some polymorphisms/mutations to be presented more often in this group of patients. Our results could not confirm that a polymorphism or mutation was more often in the patients with CRC, suggesting a possible pathophysiological correlation with CRC. The three already reported variants that have been found may represent just polymorphisms of the population and they are not connected to the disease.

It is noteworthy the fact that there are relatively often deletion and insertions of the base adenine. A possible explanation may be that the DNA was isolated from FFPE tissue. Analysis of nucleic acids from FFPE samples is crucial in today’s clinical research. It is known that the formalin fixation procedure lowers the success of PCR amplification because of cross-linking between protein and DNA. In addition, standard embedding protocols require heating the formaldehyde-soaked samples. The temperatures used for embedding in paraffin cause further molecular reactions, some irreversible.^23–28 Artifacts could be the consequence of formalin damaging or cross-linking cytosine nucleotides. Thus, the Taq DNA polymerase would not recognize them and incorporate an adenosine instead of a guanosine (because of the so-called A-rule). In this study, some of the detected mutations are because of formalin damaging. (Table 3).

Several experiments took place in order conditions of HRMA and concentrations of the reagents to be optimized. It has been proven that a careful optimization of HRMA is rewarded by high-throughput, economy, and simple and accurate performance of HRMA. Various techniques were employed to reduce non-specific amplification, such as increasing the annealing/extension temperature, reducing the primer concentration, optimizing the MgCl₂ concentration. In this study, the concentration of the salt (MgCl₂) proved to be a very determinant factor of the method, as it may increase resolution and improve clustering.²⁹ Moreover, high concentration of DNA template was prohibitive in HRMA during the first applications of the method. When a few simple rules are taken into account (i.e. avoid long fragments and multiple melt domains), designing a HRMA screen for a gene is rather straightforward.^28–31 Moreover, HRMA seems to represent a more sensitive approach to detect a minimal fraction of mutated cells in neoplastic tissue that, in many cases, is not achievable by direct sequencing.³² Among the many applications of HRMA method, HRMA provides a convenient way to detect SNP mutations in a targeted gene without performing full fragment sequencing. Although HRMA cannot define mutations by their melting transitions, it can significantly reduce the DNA sequencing burden, but positive results still require sequencing for diagnostic confirmation²⁷ Its ease of use, simplicity, flexibility, low cost, nondestructive nature, superb sensitivity, and specificity make HRMA the method of choice to screen patients for pathogenic variants. Because HRMA is still a rather young technology, one can only expect exciting further developments.

In spite of the problems of the quality of DNA in this study, because it was derived from FFPE tissues and the several tests which were needed in order to standardize the method for each exon, HRMA remains a sensitive scanning method that allows rapid detection of DNA sequence variations without cumbersome post-PCR methods. The HRMA method gave us the possibility to screen most of the exons of SLCO1B3, sending for sequencing just a part of the samples. To our knowledge, this is the first study of SLCO1B3 gene using HRMA in FFPE tissue from patients with CRC and in comparison with immunohistochemistry. The interpretation of the results was difficult because of the limited number of samples (30). Accurate determination of specificity requires analysis of large numbers of known wild type samples. The “gold standard” of the HRMA method could be achieved if PCR products for each exon were sequenced for polymorphisms variations and subsequently HRMA should be evaluated for every exon. This would help us to interpret better our experimental data of HRMA

Archival tissue specimens, such as FFPE tissues, represent a vast source of tissue genomic DNA, easily obtained from clinical archives. Nowadays, the medicine lives the time of “targeted therapies” and needs to make use of all this archival tissue. In the fight against cancer, the request for rapid and reliable mutation screening in human cancers is continuously increasing for the definition of clinical samples and orienting targeted therapies. SLCO1B3 seems to be a quite good target as it seems to play a role in the delivery of anticancer drugs. Its pathobiological significance in CRC may not have been elucidated yet, neither to our study. However, it seems to be an interesting molecule. The development of targeted therapies needs attractive techniques for mutation scanning with ease of use, high sensitivity and specificity, low cost, and rapid sample turnaround. These requirements make HRMA ideal for use in routine clinical diagnostic settings. In addition, HRMA is an interesting method which combines biology (PCR amplification), physics (the phenomenon of fluorescence), and mathematics (the use of the software)—three sciences in one.

Conclusion

The results of this study are of much interest as HRMA proved to be a reliable and rapid genotyping/scanning method for mutation detection of SLCO1B3. Moreover, it is important that the finding of immunohistochemical negative samples proved to have accommodate mutations which lead either to early stop codons or to other conformations of the tertiary protein structures affecting the antibody-epitope binding. In this study, HRMA shed light on SLCO1B3 gene, promising to reveal more in the near future.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Ferlay

Steliarova-Foucher

Lortet-Tieulent

. Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer 2013; 49(6): 1374–1403.

Siegel

Miller

Jemal

. Cancer statistics, 2016. CA Cancer J Clin 2016; 66(1): 7–30.

Fakih

. Metastatic colorectal cancer: current state and future directions. J Clin Oncol 2015; 33(6): 1809–1824.

Konig

Cui

Nies

. Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J Biol Chem 2000; 275(30): 23161–23168.

Genatlas. Gene database, SLCO1B3, http://genatlas.medecine.univ-paris5.fr/fiche.php?n=10757 (2008, accessed 15 May 2012).

Ensembl genome browser 67. Homo sapiens. Gene: SLCO1B3 ENSG00000111700, http://www.ensembl.org/Homo_sapiens/Transcript/Summary?db=core;g=ENSG00000111700;r=12:20963636-21243040;t=ENST00000545880 (2012, accessed 15 May 2012).

Abe

Unno

Onogawa

. LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers. Gastroenterology 2001; 120(7): 1689–1699.

Kounnis

Ioachim

Svoboda

. Expression of organic anion-transporting polypeptides 1B3, 1B1, and 1A2 in human pancreatic cancer reveals a new class of potential therapeutic targets. Onco Targets Ther 2011; 4: 27–32.

Smith

Marsh

Scott-Horton

. Variants in the SLCO1B3 gene: interethnic distribution and association with paclitaxel pharmacokinetics. Clin Pharmacol Ther 2007; 81(1): 76–82.

10.

Lee

Belkhiri

Lockhart

. Overexpression of OATP1B3 confers apoptotic resistance in colon cancer. Cancer Res 2008; 68(24): 10315–10323.

11.

Letschert

Keppler

Konig

. Mutations in the SLCO1B3 gene affecting the substrate specificity of the hepatocellular uptake transporter OATP1B3 (OATP8). Pharmacogenetics 2004; 14(7): 441–452.

12.

Mahagita

Grassl

Piyachaturawat

. Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport. Am J Physiol Gastrointest Liver Physiol 2007; 293(1): G271–G278.

13.

Kalliokoski

Niemi

. Impact of OATP transporters on pharmacokinetics. Br J Pharmacol 2009; 158(3): 693–705.

14.

Kim

. Organic anion-transporting polypeptide (OATP) transporter family and drug disposition. Eur J Clin Invest 2003; 33(Suppl. 2): 1–5.

15.

Lockhart

Harris

Lafleur

. Organic anion transporting polypeptide 1B3 (OATP1B3) is overexpressed in colorectal tumors and is a predictor of clinical outcome. Clin Exp Gastroenterol 2008; 2008(1): 1–7.

16.

Tsujimoto

Hirata

Dan

. Polymorphisms and linkage disequilibrium of the OATP8 (OATP1B3) gene in Japanese subjects. Drug Metab Pharmacokinet 2006; 21(2): 165–169.

17.

Boivin

Cardinal

Barama

. Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3: genetic variability and haplotype analysis in white Canadians. Drug Metab Pharmacokinet 2010; 25(5): 508–515.

18.

Galani

Alexiou

Miliaras

. Expression of stem cell marker nestin and microRNA-21 in meningiomas. Turk Neurosurg 2015; 25(4): 574–577.

19.

Schwarz

Meyer

Schwabedissen

Tirona

. Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity. Pharmacogenet Genomics 2011; 21(3): 103–114.

20.

Sissung

Troutman

Campbell

. Transporter pharmacogenetics: transporter polymorphisms affect normal physiology, diseases, and pharmacotherapy. Discov Med 2012; 13(68): 19–34.

21.

Nagai

Furihata

Matsumoto

. Identification of a new organic anion transporting polypeptide 1B3 mRNA isoform primarily expressed in human cancerous tissues and cells. Biochem Biophys Res Commun 2012; 418(4): 818–823.

22.

Hamada

Sissung

Price

. Effect of SLCO1B3 haplotype on testosterone transport and clinical outcome in Caucasian patients with androgen-independent prostatic cancer. Clin Cancer Res 2008; 14(11): 3312–3318.

23.

Dobrovic

. Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies. Mol Cancer 2009; 8: 82.

24.

Williams

Ponten

Moberg

. A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am J Pathol 1999; 155(5): 1467–1471.

25.

Krypuy

Mitchell

. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies. BMC Cancer 2008; 8(142): 142.

26.

Vorkas

Poumpouridou

Agelaki

. PIK3CA hotspot mutation scanning by a novel and highly sensitive high-resolution small amplicon melting analysis method. J Mol Diagn 2010; 12(5): 697–704.

27.

Wang

. Is high resolution melting analysis (HRMA) accurate for detection of human disease-associated mutations? A meta-analysis. PLoS ONE 2011; 6(12): E28078.

28.

Erali

Voelkerding

Wittwer

. High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 2008; 85(1): 50–58.

29.

Kapabiosystems. Introduction to high resolution melt analysis, http://www.kapabiosystems.com/public/pdfs/kapa-hrm-fast-pcr-kits/Introduction_to_High_Resolution_Melt_Analysis_Guide.pdf (n.d., accessed 21 October 2011).

30.

Ruskova

Raclavsky

. The potential of high resolution melting analysis (HRMA) to streamline, facilitate and enrich routine diagnostics in medical microbiology. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2011; 155(3): 239–252.

31.

Vossen

Aten

Roos

. High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum Mutat 2009; 30(6): 860–866.

32.

Taylor

. Mutation scanning using high-resolution melting. Biochem Soc Trans 2009; 37(2): 433–437.