Original Articles
ABSENT RAS GENE MUTATIONS IN HUMAN ADRENAL CORTICAL NEOPLASMS AND PHEOCHROMOCYTOMAS
JUDD W. MOUL,* JAY T. BISHOFF, SHEILA M. THEUNE AND ESTHER H. CHANG
From the Departments of Surgery and Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, and Urology Service and Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, D.C.
ABSTRACT
A variety of human tumors have been studied for ras mutations to date. However, little is known about the prevalence and significance of ras gene activation in adrenal neoplasms. Recently, a study of 10 primary human pheochromocytomas found no evidence for ras mutations. To our knowledge no survey of ras mutations in adrenocortical neoplasms has been reported. Therefore, we analyzed deoxyribonucleic acid (DNA) from 17 archival tumors (8 adrenocortical carcinomas, 6 pheochrom- ocytomas, 2 adrenal adenomas, 1 aldosteronoma, 2 fresh pheochromocytomas and 1 fresh benign adrenal gland) for activating mutations at the 12, 13 and 61 codons of N-ras, H-ras and K-ras. DNA was extracted from archival tissues using 3 different methods: a simplified boiling method, a proteinase-K-phenol chloroform extraction and a novel heat-stable protease Thermus rt41A tech- nique. The boiling and heat-stable protease methods provided for more consistent polymerase chain reaction amplifications than the more laborious phenol chloroform method. This heat-stable protease Thermus rt41A method had not been reported previously for use in archival DNA extraction. Polymerase chain reaction amplified the ras gene regions of interest, and mutations were screened by mutation-specific oligonucleotide probe hybridization of Southern and slot blots. Polymerase chain reaction-generated mutation-specific positive and negative controls were used in the hybridization protocol. With these controlled conditions no definite mutations were detected at codons 12, 13 or 61 of N, H or K-ras. Ras activation via point mutations at these codons rarely, if ever, occurs in adrenal neoplasms.
KEY WORDS: adrenal neoplasms; pheochromocytoma; genes, ras; polymerase chain reaction; carcinoma
Mammalian ras genes code for high related proteins of 189 amino acids, which are known as p21 proteins. The p21 proteins bind to the inner cell membrane and because of their strong resemblance to G-proteins they are believed to be involved in signal transduction.1 The 3 most widely studied ras genes, K- ras (Kirsten), H-ras (Harvey) and N-ras are most commonly converted from the normal proto-oncogene by single point mutations occurring in codons 12, 13 and 61. Ras proteins bind guanine nucleotides, guanosine triphosphate and guanosine diphosphate, and dephosphorylation from guanosine triphos- phate to guanosine diphosphate inactivates the signal pathway function of the proteins. It is postulated that these point mutations stabilize ras proteins in their active state, thereby continuing signal transduction and contributing to unregulated cellular growth.1
During the last few years, because of advances in molecular biology techniques the incidence of ras mutations in various animal and human neoplasms has been elucidated.1-3 Mutations at the 12 and 61 codons have been seen most frequently in human tumors but the incidence of these mutations varies
Accepted for publication October 9, 1992.
Supported by Grants G190AZ01, R090BJ-01 and 090BR-01, Uni- formed Services University and the Henry M. Jackson Foundation, and Grants 2860 and 2862, Department of Clinical Investigation, Walter Reed Army Medical Center.
The opinions and assertions contained herein are the private views of the authors and are not to be construed as reflecting the views of the United States Army or the Department of Defense.
* Requests for reprints: Department of Surgery, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, Maryland 20814-4799.
widely depending on the type of neoplasia.1-3 Recently, K-ras mutations were found to represent an unfavorable prognostic factor for adenocarcinoma of the lung4 but similar associations have not been found for other tumors to date.1-3 The exact role of the ras oncogenes in carcinogenesis remains to be deter- mined, although clinical work with colon tumors and recent animal work suggest that these mutations may be an early event and may proceed frank neoplasia.5,6 It is generally agreed, however, that ras mutations alone are not sufficient to induce human cancers but that other oncogenes and/or tumor sup- pressor genes or other promoting agents interact in a multistep phenomenon.6
Little is known about the molecular pathogenesis of most adrenal neoplasms. Pediatric neuroblastomas, most of which can be considered of adrenal origin, have been studied exten- sively regarding N-myc amplification,7-10 ras expression11 and rare ras mutations.12-14 Pheochromocytomas, also generally of adrenal location, express transcripts of c-myc, c-fos and ret proto-oncogenes but the significance of this is unknown.15-18 Only 1 previous study has analyzed pheochromocytomas for activated ras, finding no mutations in 10 tumors.14 Aside from these studies of adrenal medullary neoplasms of neural crest origin, no analysis of the prevalence of ras mutations in adre- nocortical tumors has been done.
A number of other studies have suggested that ras oncogenes might be important in adrenocortical tumors. The cellular oncogene c-ki-ras is amplified 30 to 60-fold in the Y1 cell line established from the mouse adrenocortical tumor LAF 1, which is homologous to the human c-ki-ras-2 gene.19-21 A more recent
study implicated mutational activation of a G-protein, similar to ras in human adrenocortical tumors.22 A mutation in the & coding gene of G-protein, Gi2 (resulting in an oncogene GIP- 2), was detected in 3 of 11 adrenocortical tumors (27%). Because of the similarities of G-proteins to ras, it is important to determine if ras mutations also occur in human adrenocortical neoplasms.
To address the question of whether ras mutations occur in adrenocortical tumors and to confirm the findings for pheo- chromocytomas,14 deoxyribonucleic acid (DNA) from 17 archi- val neoplasms (8 adrenocortical carcinomas, 6 pheochromocy- tomas, 2 adrenal adenomas and 1 aldosteronoma) and 3 fresh specimens (2 pheochromocytomas and 1 normal adrenal gland) were studied by polymerase chain reaction and oligonucleotide hybridization for codon 12, 13 and 61 mutations of N, H and K-ras. In addition to the ras mutation data, we studied various methods of obtaining DNA suitable for polymerase chain re- action from archival pathological material. A simple technique using a heat-stable protease, Thermus rt41A, provided DNA that was suitable for this polymerase chain reaction application.
MATERIALS AND METHODS
Clinical material. Paraffin archival pathology blocks from surgical adrenalectomy cases were obtained from the pathology departments at Walter Reed Army Medical Center, National Naval Medical Center, Armed Forces Institute of Pathology and Wilford Hall Medical Center. There were 8 adrenocortical carcinomas, 6 pheochromocytomas, 2 adrenal adenomas and 1 aldosteronoma available for study. Pathology was reviewed at the Armed Forces Institute to confirm histology. An operation had been done in these cases and the blocks were processed between 1985 and 1990. In addition, 3 specimens were obtained directly at operation via snap-freezing in liquid nitrogen in the operative suite at Walter Reed Army Medical Center (2 pheo- chromocytomas and 1 benign adrenal gland). These fresh spec- imens were frozen by 1 of us (J. W. M.) within 5 minutes of operative removal.
Extraction of DNA from archival material. The diagnosis and accuracy of the chosen blocks were confirmed by light micros- copy of standard hematoxylin and eosin stained sections. One paraffin block chosen as having the most concentrated area of neoplasm was used for each of the 17 archival cases. Excess paraffin surrounding the embedded tumor tissue was shaved away with a scalpel blade followed by microtome cutting of 3, 10 um. sections into sterile 500 ul. Eppendorf tubes. Special care was taken to clean the microtome blade with 95% ethanol and a Bunsen burner between each case to avoid inadvertent cross-contamination.
Deparaffinization was achieved by adding 400 ul. xylene to each tube, incubation at 37C for 15 minutes and centrifugation at 13,000 times gravity for 5 minutes. Xylene was removed and 400 ul. 100% ethanol were added. Then, the samples were incubated at room temperature for 5 minutes and centrifuged for 5 minutes. Excess ethanol was removed and the remaining tissue pellet was desiccated. DNA was extracted from the deparaffinized tissue by 3 methods: 1) a simplified boiling technique similar to that of Shibata et al with slight modifica- tion,23,24 2) a proteinase-k/phenol/chloroform method25,26 and 3) a method using a heat-stable protease, Thermus rt41A, referred to as “Pre-Taq.”27
For the boiling method the pellet was resuspended in 150 ul. double distilled autoclaved water and heated to 100C for 10 minutes. The tissue and water were mixed well with the pipette tip and a 10 to 20 ul. aliquot was placed in a new 500 ul. Eppendorf tube for subsequent polymerase chain reactions. The tissue suspensions were stored at 4C between uses. For the proteinase-K/phenol/chloroform extraction the deparaffinized tissue pellet obtained from the same blocks was added to 100 ul. of a proteinase-K/digestion buffer solution (200 µg./ml.
proteinase-K, 1 mM. ethylenediaminetetraacetic acid [EDTA], 50 mM. TRIS-hydrochloric acid, pH 8.5 and 0.5% Tween-20) and incubated at 55C for 3 hours. The tubes were centrifuged to remove any excess moisture from the top and sides before being incubated at 95C for 8 to 10 minutes to inactivate the proteinase. A volume of phenol equal to the volume of the sample was added to each tube, mixed for 10 minutes by inversion and centrifuged for 10 minutes at 13,000 times grav- ity. The top layer was removed and placed in a new 500 ul. Eppendorf tube to which an equal volume of 24 parts chloro- form to 1 part isoamyl alcohol solution was added. The tubes were mixed by inversion and centrifuged as described previ- ously. The top layer was removed and placed in a new Eppendorf tube. The volume in the tubes was estimated and 1/ 15 of the volume of 3.0 M. sodium acetate was added. The DNA was precipitated by adding 2.5 times estimated volume of -20C 100% ethanol. The tubes were then mixed well by inversion and placed at -20C overnight. The tubes were centrifuged (10 minutes at 13,000 times gravity) and the supernatant was removed. The DNA pellet was resuspended in 100 ul. digestion buffer (50 mM. TRIS-hydrochloric acid, pH 8.5, 0.1 mM. EDTA and 0.5% Tween-20). The concentration of DNA in the samples was estimated by comparison to X-Hind III control in ethidium bromide stained gel electrophoresis. Depending on concentra- tion, 10 to 30 ul. of the DNA solution were used to achieve an ideal polymerase chain reaction substrate of 0.1 ug. genomic DNA.28
The DNA extraction using the heat-stable protease, Thermus rt41A or Pre-Taq,27 starts with the gray-white tissue pellet obtained from deparaffinization. Then, 40 ul. double distilled autoclaved water were added to each dried sample and mixed thoroughly with a sterile pipette. Assuring that particulate solution was obtained, 1 ul. of each sample was removed and suspended in 30 ul. polymerase chain reaction buffer (50 mM. potassium chloride, 10 mM. TRIS, pH 8.3, 2 mM. magnesium chloride and 0.01% gelatin). To this mixture was added 0.3 units of Pre-Taq and the samples were incubated at 94C for 30 minutes under mineral oil in the thermal cycler. The samples were then centrifuged for 2 minutes at 13,000 times gravity and 5 ul. were added to the polymerase chain reaction mixture.
Extraction of DNA from freshly collected samples. Adrenal specimens that had been snap-frozen in liquid nitrogen were stored at -70C until use. A portion of the tumor was removed from storage and resuspended in liquid nitrogen for several seconds followed by pulverizing the tissue with a mortar and pestle. This finely ground frozen tissue was then subjected to standard DNA extraction, dialysis and quantitation.29
Polymerase chain reactions. Polymerase chain reaction was used to amplify selectively the regions of the human H, K and N-ras genes containing codons 12, 13 and 61.30 Primer se- quences used have been published previously31 or are available upon request. The polymerase chain reaction mixture contained 5 ul. polymerase chain reaction buffer (100 mM. TRIS-hydro- chloric acid, pH 8.3, 500 mM. potassium chloride, 15 mM. magnesium chloride and 0.1% gelatin), 1 ul. nucleotide mixture (10 mM. each of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate and deoxythymi- dine triphosphate), 1.5 ul. each (20 mM.) of the sense and antisense primers, DNA suspension and double distilled auto- claved water to a volume of 45 ul. This mixture was heated to 95C for 5 minutes in a Cetus/Perkin-Elmer DNA Thermal Cycler followed by ice cooling. Five ul. of a Taq mixture containing 2 units of thermostable Taq polymerase were added and the mixture was overlaid with 1 drop (approximately 50 ul.) of mineral oil. The specimens were subjected to 40 cycles of polymerase chain reaction on the aforementioned cycler as follows: 96C for 30 seconds (denaturation), 56C for 1 minute (annealing) and 74C for 1 minute (extension) for Clontech
RIGHTS LINKY
primers* or 94C, 55C and 72C, respectively, for 1 minute each for the Dupont primers.t Mineral oil was removed via chloro- form extraction and the success of the polymerase chain reac- tion was assessed by running 8 ul. of the final product plus 2 ul. of blue running buffer (0.25% bromophenol blue, 0.25% xylene cyanol and 15% Ficoll in water) on a 2% (weight-in- volume) agarose gel. After standard electrophoresis (100 volts for 1.5 hours) in 45 mM. tris-borate-EDTA the polymerase chain reaction product was visualized as approximately 80 to 120 base pair bands via ethidium bromide staining using a 123 base ladder for molecular size comparison (fig. 1). The DNA
* Clontech Laboratories, Inc., Palo Alto, California.
t Dupont NEN, Boston, Massachusetts.
A.
15 12 11 10 9 8 6 4 2 1 (123)
B.
15 12 11 10 9 8 6 4 2 1
C.
1
2
4
6
8
9
10
11
12
15
N.C.
from these gels was subsequently transferred to a nylon mem- brane via Southern transfer and hybridized with wild-type probes to confirm the success of the polymerase chain reaction. A visible electrophoresis gel band and confirmatory hybridiza- tion signal were the criteria to signify successful polymerase chain reaction amplification. Only these successful amplifica- tion samples were chosen for slot blotting. A no template DNA sample was used in each polymerase chain reaction experiment to assess for possible contamination.
Slot blotting and hybridization to oligonucleotide probes. The remaining polymerase chain reaction product for each specimen (approximately 42 ul.) was mixed with 200 ul. 0.4 M. sodium hydroxide plus 25 mM. EDTA and heated to 95C for 2 minutes to denature the DNA. The tubes were subsequently placed on ice and 200 ul. 1 M. TRIS-hydrochloric acid, pH 7.4, were added to neutralize the DNA. A volume of 55 ul. of this solution was then placed on 8 identical nylon membranes via a Schleicher and Schuell Minifold II slot blot system (No. SRCO7210). Nylon membranes were air dried, ultraviolet cross-linked for 2 minutes with a DNA Transfer Lamp* and vacuum baked for 2 hours. Nylon membranes were prehybridized for 1 hour at 37C in a shaking water bath and hybridized in fresh solution for 4 to 14 hours at 37C (fig. 1). Hybridization solution consisted of 5 × sodium chloride, sodium phosphate, EDTA buffer, 5 × Denhardt’s solution, 0.5% sodium dodecyl sulfate and 100 mM. sodium pyrophosphate, pH 7.5.29 The oligonucleotide probe protocol was essentially done as described by Verlaan-de Vries et al.32 The Clontech oligonucleotide probes consisted of 63, 20 mer sequences corresponding to the wild-type and known mu- tations at the 12, 13 and 61 codons for H, K and N-ras genes. The exact sequences of these probes are available upon request. To make radioactive labeled probe, an aliquot of 15 ul. (1 pmol.) of each oligonucleotide 20 mer sequence was added to 27 ul. of autoclaved double distilled water and heated to 65C for 5 minutes. After this procedure the mixture was placed on ice and 5 ul. 10 x T4 kinase buffer (0.5 M. TRIS-hydrochloric acid, pH 7.5, 0.1 M. magnesium chloride, 50 mM. dithiothreitol, 1 mM. spermidine and 1 mM. EDTA), 2 ul. T4-kinase enzyme and 1 ul. y-32phosphorus (y-32P) deoxyadenosine triphosphate were added. This entire mixture was then incubated at 37C for 20 minutes. Following hybridization, the nylon filters were initially washed twice with 6 X standard sodium citrate for 20 and 10 minutes, respectively, at room temperature. A tetrame- thylammonium chloride wash solution (3 M. tetramethylam- monium chloride, 50 mM. TRIS-hydrochloric acid, pH 8.0, 2 mM. EDTA and 0.1% sodium dodecyl sulfate) was then used for a 10-minute wash at room temperature followed by 2 washes at 59 to 71C for 20 minutes depending on the probe.30 Nylon membranes were then exposed to radiographic film using inten- sifying screens at -70℃ for 1 to 24 hours. Membranes were stripped for reuse using 0.2 M. sodium hydroxide at 65℃ wash for 20 to 25 minutes depending on radioactive counts. Neutral- ization of these blots was then performed with sequential washes using distilled water, 0.5 M. tris-hydrochloric acid, pH 7.5, and 2 × standard sodium citrate, respectively, for 10 minutes each at room temperature. All nylon membranes were probed with a single mutation-specific oligonucleotide (no mix- ing of multiple probes in the same reaction) and all hybridiza- tion experiments were repeated by separate investigators to confirm results.
Polymerase chain reaction-generated mutation-specific posi- tive and negative control. Polymerase chain reaction-generated mutation-specific positive controls were synthesized for all pertinent base substitutions at the 12, 13 and 61 codons of K, H and N-ras genes similar to the methods of Rochlitz et al.33 Using the concept that single base mismatches in the interior of the 5’-primer have minimal effect on the polymerase chain
* Photodyne, New Berlin, Wisconsin.
reaction amplification,34 the 20 mer Clontech oligonucleotide probes corresponding to all known 12, 13 and 61 codon muta- tions in the 3 ras genes were used as the 5’-primer along with the standard wild-type 3’-primer used in the tumor amplifica- tions. The polymerase chain reaction amplification thus incor- porated the single base mutation present in the 5’-primer into the product DNA and mutation-specific positive control ma- terial was created. Human placental DNA (Clontech) was used as a template for these polymerase chain reactions with cycle parameters of 96C for 30 seconds, 56C for 15 seconds and 74C for 30 seconds times 25 cycles. The quality of the polymerase chain reaction was determined by 2% agarose gel electropho- resis and the quantity of DNA product was assessed using - Hind-III marker. These positive controls were then slot-blotted and the quantity of polymerase chain reaction product used was adjusted to match approximately the quantity of polymer- ase chain reaction product obtained for the tumor samples. Escherichia coli transfer ribonucleic acid was used as a carrier during slot blotting for uniform distribution of the DNA. Nine types of control slots were created containing wild-type (no mutation) and mutation-specific material: K12, K13, K61, N12, N13, N61, H12, H13 and H61. The codons 12 and 13 control blots contained 7 samples corresponding to wild-type (negative con- trol) and 6 mutations, and the codon 61 blots had 8 samples representing wild-type and 7 mutation possibilities. For each of these samples 8 identical blots were made. A positive-nega- tive control blot was always hybridized/washed autoradi- ographed with corresponding tumor blots. During the high stringency tetramethylammonium chloride wash the autoradi- ography signal for known negatives (wild-type) on the control blots had to disappear. Autoradiographic signals that persisted on the known negative control samples after the aforemen- tioned protocol washing indicated residual nonspecific binding of the probe, which required 1 or more extra tetramethylam- monium chloride washes at prescribed temperature.30 The hy- bridization signal of the known positive control remained on the control blot (example for K-ras 61 controls shown in figure 2) indicating single base-specific hybridization.
RESULTS
Ras mutational analysis. For the cohort of 20 human adrenal specimens (8 adrenocortical carcinomas, 8 pheochromocyto- mas, 2 adrenal adenomas, 1 aldosteronoma and 1 normal ad- renal gland) no K, H or N-ras 12, 13 or 61 codon mutations were detected. Potential false-positive results due to nonspecific binding of mutation-specific probe were controlled by using blots containing known negative (wild-type) samples. To con-
-
-
-
GAA (GLU)
AAA CGA CTA (PRO) (HIS #1) (HIS #2)
CCA CAT
(LYS)
(LEU)
firm that the mutation-specific probe was detecting a single base mutation, if present, a polymerase chain reaction-gener- ated positive control was used for each experiment.
DNA extraction from archival adrenal tissue. Three methods were investigated that would reliably provide DNA extraction suitable for polymerase chain reaction amplification of these 70 to 120 base pair ras gene segments. The proteinase/phenol/ chloroform extraction method25,26 was initially used but only 15 of 80 polymerase chain reactions (19%) resulted in visible bands on electrophoresis confirmatory gels. These bands were con- firmed by Southern transfer and blot hybridization with wild- type probes. The more simplified boiling method23,24 was suc- cessful for 78 of 137 polymerase chain reactions (57%). The heat-stable protease, Thermus rt41A, method was successful for 35 of 39 reactions (90%).
DISCUSSION
We found no H, K or N-ras codon 12, 13 or 61 mutations in a cohort of human adrenocortical and medullary neoplasms, and benign adrenal tissue using the polymerase chain reaction/ oligonucleotide mismatch hybridization assay. With regard to pheochromocytoma, our results are in agreement with those of Moley et al, who found no ras mutations in a group of 10 primary human pheochromocytomas.14 By combining our re- sults with those previously reported,14 no mutations have been detected in 18 pheochromocytomas, which virtually rules out an important role for ras mutational activation in the genesis of this neoplasm. Pheochromocytoma would appear to be sim- ilar to neuroblastoma in the respect that ras mutational acti- vation is rare.12-14 Our findings for adrenocortical neoplasms, which to our knowledge have not been studied previously, suggest that ras mutations in these tumors are also rarely, if ever, present.
That ras mutations are absent in this cohort of adrenocortical neoplasms is of particular interest in comparison to the finding of G-protein gene mutations in a group of similar tumors.22 Lyons et al screened a large number of human tumors for activating mutations in the a-polypeptide chains of certain G- proteins. These G-proteins are similar to ras in that specific site mutations inhibit the guanosine triphosphate activity and alter signal pathways, thereby contributing to transformation. The a chain gene of a particular G-protein, Gi2, was mutated in codon 179 from arginine to cysteine or histidine in 3 of 11 adrenocortical neoplasms (27%). This mutation in the @-gene converted the Gi2 proto-oncogene to an oncogene termed GIP- 2. The ras p21 proteins resemble G-proteins in sequence ho- mology and function, and have been considered as potential intermediaries in the signal transduction pathways.35 Although the exact mechanism is unknown, ras activation has been found in a variety of tumors and may have a more general role in the signaling pathway.22 G-proteins usually mediate hormonal reg- ulation of cell function other than proliferation.22 Lyons et al postulate that the mutations found in Gi2 in adrenal neoplasms implicate this G-protein to be important in a signaling pathway that promotes proliferation. Since Gi2 may be the primary proliferative pathway for adrenal tumors, ras may be less im- portant for this particular neoplasm. It is unknown if this finding of activated GIP-2 precludes or lessens the likelihood of activated ras. The relationship, if any, between our findings of absent ras mutations and those of Lyons et al of GIP-2 activation in adrenocortical tumors remains to be determined.
Aside from oncogenes, recent work has focused on the pos- sible role of tumor suppressor genes in adrenal neoplasms. Tumors of the endocrine system show frequent familial inher- itance patterns and, consequently, have been an area of active investigation, looking for tumor suppressor genes as an etiology for tumorigenesis. Pheochromocytomas, along with medullary thyroid carcinoma, are part of the dominantly inherited cancer syndrome multiple endocrine neoplasia type 2A. The multiple endocrine neoplasia type 2A gene has recently been mapped to
the centromeric region of chromosome 10.36-38 However, unlike the prototype model for tumor suppressor genes, allelic loss on chromosome 10 near the multiple endocrine neoplasia type 2A locus has been an uncommon event in pheochromocytomas.39 Pheochromocytomas have been reported to have allelic dele- tions in a hypervariable region of DNA on chromosomes lp and 22q.36-40 One of the larger series evaluated 41 pheochromocy- tomas for loss of heterozygosity and found that 42% of the tumors had allele loss on chromosome lp, 31% on chromosome 22q, 24% on chromosome 17p and 16% on chromosome 3p.41 They also showed no significant loss of heterozygosity on chromosome 10 near the multiple endocrine neoplasia type 2A locus. Of further importance was the finding that loss of het- erozygosity on chromosome lp was associated with the clinically important tumor marker-urinary metanephrine excretion.41 Regarding adrenocortical tumors, Koufos et al suggested that a recessive oncogene located on chromosome 11p confers pre- disposition to adrenocortical tumors.42 Support for their inves- tigation is provided by the demonstration that loss of hetero- zygosity at loci on chromosome 11p was observed in a family with 2 children who had adrenocortical carcinoma.43 Yano et al reported a series of 17 benign and malignant adrenocortical tumors and found that all patients with adrenocortical carci- noma whose normal somatic tissues were heterozygous for a specific locus on chromosome 17p had lost 1 allele in the tumor sample.44 Four patients had also lost an allele on chromosome 11p and 3 had lost an allele on chromosome 13q. Conversely, there was no evidence for loss of heterozygosity at chromosomes 17p, 11p or 13q among the patients with adrenal adenomas or hyperplastic lesions. Further study is necessary, however, since in many other human malignancies tumor suppressor gene(s) may be important in the pathogenesis of adrenal neoplasms.
An equally important aspect of our study was the documen- tation of the success rate of polymerase chain reaction ampli- fication for archival adrenal tissues. A number of different techniques for extracting the DNA from paraffin samples have been reported. Before the advent of polymerase chain reaction, Goelz25 and Dubeau26 et al introduced DNA extraction from paraffin blocks using proteinase K digestion followed by phenol-chloroform extraction for purposes of Southern blot analysis. These procedures are laborious and provide ample opportunity for cross-contamination between samples. Impraim et al first reported that DNA extracted by the afore- mentioned method was suitable for polymerase chain reaction.45 However, Shibata et al initially reported a simplified technique in which a single 5 to 10 u. paraffin-block slice was deparaffin- ized and used directly for polymerase chain reaction.23,24 The simplified technique certainly is faster and less steps decrease the risk of contamination. However, it is limited by the product length that can be amplified. More recently, Wright and Manos reported that deparaffinization followed by proteinase K diges- tion without phenol-chloroform extraction works well to polym- erase chain reaction amplify products of more than 800 base pairs.46 None of these investigators, however, reported the success rate of polymerase chain reaction amplifications for archival tissues using these methods. Unlike DNA extracted from fresh tissue, polymerase chain reaction amplifications, even of relatively short sequences (80 to 130 base pairs) such as used in ras studies, are inconsistent. Type of fixative used, interval between removal of tissue and fixation, duration of fixation, nuclease levels in tissues, size of product to be ampli- fied and duration of storage have all been implicated to con- tribute to variable results.46,47 Our results using the proteinase K/phenol/chloroform and boiling technique do provide suitable template DNA for polymerase chain reaction but success is inconsistent. It is interesting that the simplified boiling pro- vided more consistent polymerase chain reaction results than the proteinase K/phenol/chloroform extraction, which is sim- ilar to the recent results of other investigators.48,49 Chen et al found that a simplified boiling technique using water containing
Chelex-100 was superior to proteinase-K/phenol/chloroform extraction for archival liver specimens.48 Park et al also found that phenol-chloroform extraction of archival genital tract tis- sues already subjected to deparaffinization and proteinase-K digestion did not improve polymerase chain reaction amplifi- cation.49 Certainly, for widespread use of archival material for polymerase chain reaction the most simplified methods are desirable. Perhaps if Chelex-100 was added to our boiling extraction or if proteinase-K digestion was performed without phenol-chloroform extraction our success rate with these meth- ods may have been greater.
It has been reported recently that a nonspecific protease purified from a highly thermophilic bacteria, Thermus rt41A, can be used to prepare cervical tissue and blood samples rapidly and consistently for amplification of specific DNA sequences using polymerase chain reaction.27 McHale et al used this method to amplify by polymerase chain reaction 93 base pairs and 450 base pair fragments, and reported a 95% reliability with fresh blood and tissue.27 With archival tissue we modified the technique of McHale et al and achieved a 90% polymerase chain reaction success rate for these adrenal samples amplifying 80 to 120 base pair fragments. Further work is obviously nec- essary to determine if this simple method can provide suitable DNA substrate from archival tissues for longer gene segment polymerase chain reaction amplifications.
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