Detection of codon 12 mutation in the k-ras oncogene in pancreatic tumors

Detecção de mutação no códon 12 do oncogene K-ras em tumores pancreáticos

DETECTION OF CODON 12 MUTATION IN THE K-ras ONCOGENE IN PANCREATIC TUMORS

Márcia Saldanha Kubrusly, Euclides Matheucci Junior^*, Kátia Ramos Moreira Leite, Ana Maria de Mendonça Coelho, Osmar Monte^*, Marcel Cerqueira Cesar Machado and Henrique Walter Pinotti

RHCFAP/2954

KUBRUSLY, M.S. et al. - Detection of codon 12 mutation in the K-ras oncogene in pancreatic tumors. Rev. Hosp. Clin. Fac. Med. S. Paulo 54 (1): 17- 20,1999.

SUMMARY: Mutations at codons 12, 13, or 61 of the H-ras, K-ras, and N-ras have been detected in human neoplasias by a variety of techniques. Some of these techniques are very sensitive and can detect K-ras mutation in 90% of the cases of pancreatic adenocarcinomas. We analyzed 11 samples of pancreatic adenocarcinoma, three samples of pancreatic mucinous cystadenoma, and two samples without tumors in formalin-fixed paraffin embedded tissue sections. K-ras mutations at codon 12 were detected by a two-step PCR-enriched technique in all the samples of pancreatic adenocarcinoma, but not in cystadenoma or control samples. This technique may be useful for early detection of pancreatic cancer.

DESCRIPTORS: Point mutation. Oncogene K-ras. Pancreatic adenocarcinoma. Polymerase chain reaction (PCR).

Through improved molecular biology techniques, it has been possible to detect genetic mutations that cause several types of tumors and thus to gain a better understanding of the tumorigenesis process at both cellular and molecular levels. In general, these mutations involve genes that are engaged in the control of cellular growth-the so-called protooncogenes and suppressor tumor genes. Mutations of these genes, now called oncogenes, can cause irregularities in the control of cellular proliferation, leading to the development of cancer. The list of activated oncogenes associated with different types of neoplasia is increasing every day. A better understanding of the molecular basis of transformation of cells into malignant tumors could contribute to the establishment of new criteria for prevention, diagnosis, prognosis, and treatment of human neoplasms.^{1, 2, 11, 12, 13, 14, 22,26,27}

We have directed our attention to pancreatic tumors, for which even with the achievements of imaging techniques and cytopathologic analysis, early diagnosis of the disease is rarely achieved, and the cure rate is less than 10%.^{1, 11, 12, 13, 14, 22,31}

The ras genes serve as a model showing that small genetic changes may result in transformed or neoplasic cells. All three ras genes (N-ras, Harvey-ras and Kirsten-ras) are able to code for proteins of homologous structure containing 188 or 189 amino acids, even though they have different sequences and sizes. These proteins are commonly referred to as p21 ras. They have GTP-ase activity and function as molecular switches in signal transduction.

The ras genes have been related to oncogenesis of many tumors for some years, and they seem to be activated by point mutations (that is, mutation in just one base of DNA). This type of mutation can occur in all three genes of the ras family at the codons 12, 13, and ⁶¹, with substitutions of corresponding amino acids in the ras proteins. The mutations result in the expression of altered protein products that are capable of transforming cells into a malignant phenotype.

More than 90% of pancreatic adenocarcinomas contain mutated ras genes with the site of mutation restricted to codon 12. The substitution of a nucleotide in codon 12 is the most frequent mutation identified in human tumors. This substitution at the first or second base of codon 12, which results in a change from GGT (glycine) to GTT (valine), AGT (serine), GAT (aspartic acid), or CGT (arginine), may precede the development of malignancy. The high prevalence of mutation in these tumors suggests that point mutations in the K-ras oncogenes may form the basis for new molecular tests that can be used to detect early stages of pancreatic tumors ^1-23.

Among the various existing methods for detection of point mutations in ras genes, the simplest and most easily reproducible one is the polymerase chain reaction (PCR). Some protocols for PCR increase the sensitivity of the reaction by amplifying the target (mutated oncogenes) in vitro, highlighting their presence relative to normal genes ^2-14. Consequently, it is possible to detect one cell heterozygous for the K-ras codon 12 mutation in the presence of more than 500 normal cells, an equivalent sensitivity of one mutant allele to 1000 normal alleles. Therefore, this method plays an important role in the detection of mutations in neoplasias, as well as in pre-malignant stages.

MATERIALS AND METHODS

Formalin-fixed and paraffin-embedded tissue block material from the pathological archives of Sirian-Lebanese Hospital, São Paulo, Brazil, obtained at surgery from sixteen patients (Table 1), was used for the analysis.

DNA extraction from paraffin-embedded pancreatic tissue: Each section (5-10 µm) cut from the block was extracted twice with xylene to remove the paraffin. This organic extraction was followed by two washes with 100% ethanol to remove the solvent. The ethanol was removed by drying the samples in a dry bath.

One ml of xylene was added to each tube, and the tubes were closed and vortexed at room temperature for about 30 minutes. The samples were then centrifuged at 13000 rpm for 5 minutes. The xylene was removed from each sample with Pasteur pipette. These three steps were repeated. Next, 0.5 ml of 100% ethanol was added to each tube and centrifuged immediately for 5 minutes. The ethanol was removed with Pasteur pipette, and the samples were dried at room temperature.

To the dry samples were added 100 µl of digestion buffer (Tris 50 mM pH 8.5/ EDTA 1 mM/ Tween 20 0.5 % ) containing 200 µg/ml of proteinase K. The samples were incubated overnight at 37°C. Proteinase K was inactivated by heating at 95°C for 10 minutes. Then the samples were centrifuged briefly, and an aliquot (50 to 100 µl) of the supernatant was used for the amplification¹⁷.

Polymerase chain reaction (PCR): Amplification with Taq polymerase was performed in 100 µl of the reaction mixture containing 2U of polymerase, 50 pmol of each primer (Table 2), 50 µM of each deoxyribonucleoside triphosphate (dATP, dCTP, dGTP, dTTP), 2.0 mM Mg2+, 60 mM KCl, and 10 mM Tris-HCl pH 8.8.

Each cycle of PCR was conducted at 96°C for one minute for denaturation, 55°C for one minute for annealing, and 73°C for 30 seconds for extension. The first PCR was comprised of 12 cycles, followed by digestion with MvaI, and then a second amplification of 24 cycles. The product of this second PCR was again digested with the enzyme MvaI.

The samples then underwent electrophoresis on a 2.5 % agarose gel containing 1µg/ml ethidium bromide. This step was completed to check for the presence of amplified DNA, comparing with known molecular weight standard (ladder 123 GIBCO), and was subsequently photo-documented.

RESULTS

The technique utilized is a two-step amplification. In the first PCR using oligonucleotides A and B, a fragment of 157 base pairs is amplified, which contains two sites of restriction for the enzyme MvaI (CCA/TGG) if codon 12 is normal (glycine), or one site if codon 12 contains a mutation in either of its first two bases. Therefore, wild-type fragments cleave to yield products of 29, 114, and 14 base pairs, whereas mutant fragments cleave to yield products of 143 and 14 base pairs. When a second PCR is performed with oligonucleotides A and C, only fragments of 143 base pairs are amplified. The second amplification produces a product of 135 base pairs, which cleaves with MvaI at one site if the codon 12 is normal, but fails to cleave if a mutation exists in the first two bases of codon 12. The wild-type shows a 106 base pairs product.

The results after separation of the digested polymerase chain reaction products on agarose gel were as follows:

1. In none of the three cases of mucinous cystadenoma (patients1, 9, and 14) was a mutation detected; only a wild-type allele band of 106 base pairs was seen.

2. There was no mutation detected in non-neoplastic tissue (patients 2 and 3); only a wild-type allele band of 106 base pairs was seen.

3. In all cases of pancreatic tumors with metastases (patients 4, 7, 8, 10, 11, 12, 15, and 16), mutation was detected; a band of 135 base pairs indicative of a mutated gene was seen.

In cases 5 and 6, there was a technical problem during the amplification process, making the analysis difficult.

The lane M refers to the molecular weight marker ( ladder 123 Gibco) utilized and the other lanes represent the patient numbers related in table 1.

DISCUSSION

The survival rate over 5 years in pancreatic cancer patients in the United States was not greater than 3-5% ²⁵ in the last decade. However, early detection of pancreatic cancer could help radical curative treatment achieve reduced rates of mortality caused by this disease. For patients with pancreatic tumors of less than 2 cm in diameter, the survival rate over 5 years can reach 41% after a radical duodenopancreatectomy. Therefore, if we could accomplish early detection of pancreatic cancer (detection of tumors with less than 2 cm of size and confined to the pancreas) the survival after a radical resection would certainly increase.

The main problem in early diagnosis of any malignant tumor is the definition of the population at risk of developing the lesion. Accordingly, the population at risk for developing pancreatic cancer has not been not clearly defined. Chronic pancreatitis has been considered a risk factor for pancreatic cancer ¹³. Another risk factor is premalignant lesions characterized by mucinous ductal dilatation (intraductal tumor). Carcinomatous transformation is present at the time of surgical treatment in more than 50% of these patients with these lesions ³⁰.

Diabetic patients represent one risk group that would seem to be a particularly good candidate for screening for the early diagnosis of pancreatic tumor, especially in patients 60 or more years old in the early stages of diabetes with no family history of diabetes¹⁰. Diabetes is present in about 60-80% of patients with pancreatic cancer. Therefore, patients who develop diabetes as described above must be considered as a population at risk of developing pancreatic cancer. ^{20, 21}

Despite the many sophisticated diagnostic methods for detecting pancreatic cancer that have been introduced in the last decade, the prognosis remains unchanged. Among the imaging diagnostic tests, some authors have find that endoscopic ultrasound is the most sensitive one; however, this technique is not able to show any difference between tumor and inflammatory process, more effective diagnostic techniques are needed for early detection of pancreatic cancer.

Methods capable of detecting genetic mutation can be used to diagnose early pancreatic cancer. The most common mutation is related to the K-ras oncogene. This mutation appeared in 100% of our studied cases, although, in the literature, it was detected in about 90% of the cases ^{1, 10, 13}. In the present study, the use of the two-step PCR amplification and two-step restriction enzyme digestion may have resulted in the 100% sensitivity rate. Similar results, reaching 100% of sensitivity, have been accomplished by Banerjee et al. ⁵ using a similar two-step method, .

Analysis for the presence of the K-ras mutation could be one of the most sensitive diagnostic test for pancreatic cancer now available. It is recognized that mutation related to the K-ras oncogene is, definitively, more common than the p53 tumor suppressor gene ¹⁹.

Since the mutation of K-ras gene is not present in samples of pancreatic duct of patients with chronic pancreatitis with or without hyperplasty ²², the mutation of K-ras gene detected in pancreas secretions must be related with the development of malignancy.

The high prevalence of K-ras gene mutation in pancreatic cancer, as verified in this study and others ^1,5,11,24,27 suggests the possibility for the detection of the mutation in pancreatic juice¹², collected by endoscopy after pancreatic duct brushing or cholangiopancreatography ^3,28,29, in duodenal juice ²³, and even in stools ⁶. The possibility of detecting the K-ras gene mutation in these samples increases the plausibility of early diagnosis of pancreatic cancer, the incidence of which has been increasing in many countries around the world, promoting it to an important public health care issue. However, additional studies are required to evaluate the utility of this technique for the early diagnosis of pancreatic cancer.

RESUMO

RHCFAP/2954

KUBRUSLY, M. S. e col. - Detecção de mutação no códon 12 do oncogene K-ras em tumores pancreáticos. Rev. Hosp. Clín. Fac. Med. S. Paulo 53 (1): 17 - 20,1999.

Muitos dos oncogenes detectados em neoplasias malignas humanas pertencem à família do gene ras. Mutações nos códons 12, 13 ou 61 em um dos tres genes ras; H-ras, K-ras e N-ras, convertem esses genes em oncogenes ativos.

Ensaios rápidos para detecção dessas mutações pontuais, tais como a reação em cadeia de polimertização têm sido desenvolvidos nas últimas décadas e usados para investigar o papel dos genes ras mutados na patogênese de tumores humanos. As mutações no gene ras podem ser encontradas numa variedade de tipos de tumores. Incidências mais altas aparecem em adenocarcinomas do pâncreas (90%) e cólon (50%).

Analisamos 11 amostras de tumores primários de pâncreas com diferentes metástases, três amostras de cistadenoma mucinoso e dois casos de ausência de tumor de material incluído em parafina, de onde extraímos o DNA para realização das amplificações.

Os resultados mostraram que todos os casos de tumores apresentaram a banda de 135 pares de bases correspondente ao gene mutado e para os normais, a banda característica de 106 pares de bases. Nos três casos de cistadenoma mucinosos, não detectamos a banda de 135 pares de bases , apenas a banda de 106 pares de bases.

DESCRITORES: Mutação pontual. Oncogene K-ras. Adenocarcinoma pancreático. Reação de polimerização em Cadeia.

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Received for publication on the 18/09/98

*

From Medical Investigation Laboratory (LIM 35) of the Gastroenterology Surgery Department of the Faculty of Medicine - São Paulo University and the Molecular Biology Laboratory

of Physiology Department of Santa Casa Hospital. Supported by FAPESP nº 97/03669-4

Publication Dates

• Publication in this collection
  12 Sept 2000
• Date of issue
  Feb 1999

History

• Received
  18 Sept 1998