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518
H-ras Gene Mutations in Salivary Gland
Mucoepidermoid Carcinomas
Jinyoung Yoo, M.D., Ph.D.1
Robert A. Robinson, M.D.,
BACKGROUND. The authors’ recent investigation of salivary gland tumors in ras
2
Ph.D.
1
Department of Pathology, Catholic University, St.
Vincent Hospital, Suwon, South Korea.
2
Department of Pathology, University of Iowa,
Iowa City, Iowa.
gene alteration has suggested that K-ras activation may not play a role in their
oncogenesis but H-ras may, especially in mucoepidermoid carcinomas. A study
was undertaken to assess the overall incidence of mutated H-ras genes in mucoepidermoid carcinomas and to discover its potential correlation with clinicopathologic parameters.
METHODS. Fifty samples from patients with salivary gland mucoepidermoid carcinoma were analyzed for point mutations at codons 12, 13, and 61 of the H-ras gene
using the polymerase chain reaction followed by automated direct sequencing
methodology.
RESULTS. Mutated H-ras genes were detected in 9 patients, for an overall incidence
of 18% (9 of 50 patients). All but 1 of the mutations occurred at codon 12: a
GGC-to-GTC transversion in 8 patients and a GGC-to-GAC transition in 1 patient,
resulting in the amino acid substitution of valine and aspartic acid, respectively, for
glycine. One of the samples showed concurrent mutations at codons 12 (GGC-toGTC) and 13 (GGT-to-GGA). None of the samples demonstrated mutations involving codon 61. The H-ras mutations were observed in 5% (1of 21), 17% (2 of 12), and
35% (6 of 17) of low, intermediate, and high grade lesions, respectively.
CONCLUSIONS. These data suggest involvement of H-ras activation in conjunction
with other yet-unknown events in the development and/or progression of mucoepidermoid carcinomas. It is noteworthy that a stepwise increase in the frequency
of H-ras mutations strongly correlates with tumor grade (P ⫽ 0.017). Molecular
analysis of this gene alteration may provide assistance in the determination of
tumor grade and differentiation. Cancer 2000;88:518 –23.
© 2000 American Cancer Society.
KEYWORDS: H-ras, mutation, mucoepidermoid carcinoma, salivary gland tumor,
sequencing.
as genes encode for a p21ras that is known to play an important
role in the regulation of normal signal transduction. Activation of
the ras genes, usually occurring by point mutation within specific
codons of the H-ras, K-ras, and N-ras genes, creates functional reduction of intrinsic GTPase activity of the proteins, leading to increased cell proliferation.1,2 Mutated ras genes have been identified in
a variety of human cancers,1– 4 although the frequency varies widely
depending on the tumor type. N-ras alteration is predominantly
found in hematopoietic malignancies5 and K-ras mutations are
largely detected in pancreatic, colorectal, and lung adenocarcinomas,6 –11 whereas H-ras gene mutations are rather rare but often
described in urothelial neoplasms.12
There has been little interest in the molecular biologic mechanisms underlying the oncogenesis of salivary gland tumors. The stud-
R
The authors are grateful to Christine Bromley,
Mary Maher-Sturm, and Janis Rodgers for their
technical assistance, and Sonya Park for her valuable help in the preparation of the manuscript.
Address for reprints: Robert A. Robinson, M.D.,
Ph.D., Department of Pathology, University of Iowa
Hospitals and Clinics, 200 Hawkins Drive 5232A
RCP, Iowa City, IA 52242-1009.
Received May 24, 1999; revision received September 20, 1999; accepted November 3, 1999.
© 2000 American Cancer Society
H-ras in Mucoepidermoid Carcinomas/Yoo et al.
ies to date are quite limited, and only a few reports of
isolated cases of the alterations in ras, RB1, and p53
genes in some salivary gland tumors have been published.13,14 Of all malignant salivary gland tumors, mucoepidermoid carcinoma is the most common, accounting for up to 9% of these lesions.15 A previous
investigation (submitted for publication) of ras activation in salivary gland tumors demonstrated the relevance of H-ras mutation to mucoepidermoid carcinoma, which prompted us to investigate the
mutational status of this gene in a large series of the
tumors to determine whether mutational activation of
H-ras is an important molecular event in the development and progression of mucoepidermoid carcinomas.
In the current study, 50 DNA samples of mucoepidermoid carcinomas in the salivary glands were
analyzed to evaluate the frequency and pattern of
alterations of the H-ras gene and to discover a possible
correlation between the mutation and clinical or morphologic parameters. DNA was isolated from archival
paraffin-embedded tissues. After amplification of the
first and second exons of the H-ras gene, containing
codons 12/13 and 61, respectively, by using the polymerase chain reaction (PCR), mutational activation
was investigated with automated direct sequencing
analysis.
MATERIALS AND METHODS
Patients and Tissue Specimens
Fifty cases of mucoepidermoid carcinoma were collected; these included 11 mucoepidermoid carcinomas described in a previous investigation (submitted
for publication), all of which were also investigated for
the presence of H-ras mutation at codon 61 in the
current study. All specimens were obtained from patients undergoing either excisions or biopsies for their
tumor at the University of Iowa Hospitals and Clinics.
None of them had received therapy before surgery.
Clinical and follow-up information was obtained from
the patients’ charts and supplemented with data from
the tumor registry. The important clinicopathologic
characteristics of the patients are summarized in Table 1.
There were 18 female and 32 male patients; their
ages at the time of diagnosis were between 10 and 85
years. The various sites included parotid glands in 19
cases; palate in 7 cases; submandibular glands in 6
cases; maxillary sinus in 5 cases; floor of the mouth in
4 cases; retromolar trigone in 2 cases; buccal mucosa
in 2 cases; and tongue, inferior temporal fossa, lingual
plate, and parapharyngeal and pterygomandibular areas in 1 case each. The tumor sizes ranged from 0.6 to
8.0 cm in greatest dimension.
519
The tissue samples were fixed in 10% buffered
formalin. After routine embedding, light microscopy
led to the final diagnosis. Histologic review was performed by one of the authors (J.Y.) to confirm the
diagnosis. Low grade tumors showed abundant cystic
components, very rare mitoses, frequent mucin-containing cells, and minimal atypia in the squamous
cells. Intermediate grade tumors had a nearly solid
growth pattern, occasional mitoses, and occasional
mucin-containing cells. No keratinization was seen.
High grade tumors had marked nuclear atypia of the
squamous cells, but mucin-containing cells could also
be found. The tumors consisted of 21 low grade, 12
intermediate grade, and 17 high grade lesions. Only
sections containing more than 50% tumor tissue without necrosis and hemorrhage were selected for study.
DNA Extraction
Microdissection and DNA extraction were performed
as previously described, with minor modifications.16
Briefly, 5 sections (10 ␮ thick) were cut from the selected paraffin blocks and stained with hematoxylin
and eosin. All of the slides (lacking coverslips) were
used for microdissection. Precisely identified tumor
tissue was obtained with care by use of a needle to
assure that more than 75% of the recovered cells were
tumor, as opposed to unremarkable connective tissue
elements, necrotic debris, or inflammatory or hemorrhagic cell populations. No attempt was made to separate different components of the tumor. The tissue
was transferred to a sterile Eppendorf tube, suspended
in 100 ␮1 of lysis buffer (50 mM Tris, l mM ethylenediaminetetraacetic acid, 0.5% Tween 20, containing proteinase K), and incubated at 55 °C overnight.
Nucleic acid was purified by phenolchloroform extraction and precipitated with ethanol. Quantitation was
carried out by ultraviolet (UV) absorption.
Polymerase Chain Reaction
A 121-base pair (bp) DNA fragment of the first exon and
a 219-bp DNA fragment of the second exon of the H-ras
gene, containing codons 12/13 and 61, respectively, were
amplified by using as primers 5⬘-CTG-AGG-AGC-GATGAC-GGA-ATA-TAA-GC-3⬘ (sense) (Glen Research, Sterling, VA) and 5⬘-CTC-TAT-AGT-GGG-GTC-GTA-TTCGTC-CA-3⬘ (antisense), which flank codons 12 and 13,
and 5⬘-TGA-GCC-CTG-TCC-TCC-TGC-AGG-ATT-C-3⬘
(sense) and 5⬘-GCC-AGC-CTC-ACG GGG-TTC-ACCTGT-A-3⬘ (antisense), which flank codon 61. The composition of the PCR reaction mixture (100⬃1) was as
follows: 0.5 ␮g of genomic DNA in 20 mM TrisHCl (pH
8.4), 50 mM KCl, 2.0 mM MgCl2, 0.2 mM each of deoxyribonucleoside triphosphate, 0.5 ␮M each primer, and 2.5
units Platinum Taq DNA polymerase (Life Technologies,
520
CANCER February 1, 2000 / Volume 88 / Number 3
TABLE 1
List of Patients and H-ras Status
Age/gender
Site/size (cm)
Gradea
H-ras
Codon
Nucleotide
Amino acid
54/F
71/M
Lt. parapharyngeal/3.1
Lt. maxillary sinus/5.4
55/M
52/M
73/M
64/M
42/M
44/F
45/M
51/M
72/M
81/M
84/M
39/M
81/M
80/F
64/M
11/M
74/M
15/M
37/M
52/M
40/F
48/M
10/F
84/F
43/M
34/M
62/M
35/F
34/F
10/F
34/M
78/F
39/M
25/F
59/F
45/F
60/M
85/M
62/F
69/F
78/M
49/M
33/F
51/F
84/M
43/M
74/F
48/M
Lt. floor of mouth/4.0
Rt. maxillary sinus/3.3
Rt. parotid/5.0
Lt. parotid/3.0
Rt. submandibular gland/4.5
Lt. parotid/3.0
Lt. parotid/2.8
Rt. retromolar trigone/1.5
Lt. parotid/5.0
Rt. parotid/2.2
Rt parotid/6.0
Tongue/1.0
Lt. submandibular gland/3.9
Rt. palate/4.0
Rt. parotid/1.8
Rt. palate/1.5
Rt. parotid/4.0
Lt. palate/2.0
Lt. parotid/8.0
Lt. parotid/1.0
Rt. retromolar trigone/0.9
Rt. buccal mucosa/1.0
Lt. palate/2.0
Rt. maxillary sinus/1.5
Rt. lingual plate/3.0
Rt. parotid/1.2
Lt. submandibular gland/3.0
Lt. parotid/4.5
Lt. parotid/0.6
Lt. palate/2.9
Rt. maxillary sinus/3.9
Rt. parotid/3.1
Palate/2.5
Lt. pterigomandibular/2.7
Rt. floor of mouth/2.0
Lt. parotid/0.6
Inf. temporal fossa/2.5
Lt. palate/0.8
Lt. submandibular gland/2.0
Rt. parotid/2.0
Lt. maxillary sinus/2.1
Floor of mouth/3.0
Submandibular gland/2.5
Lt. parotid/0.9
Floor of mouth/5.0
Rt. submandibular gland/2.9
Lt. parotid/4.0
Buccal mucosa/2.2
3
3
3
3
3
3
2
2
1
3
1
3
3
3
1
3
1
1
1
3
1
2
1
1
1
1
2
2
1
3
1
1
2
2
1
1
2
3
1
3
1
2
1
2
3
1
1
3
2
2
3
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Mutant
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
12.2
12.2
13.3
12.2
12.2
12.2
12.2
12.2
12.2
12.2
GGC 3 GTC
GGC 3 GTC
GGT 3 GGA
GGC 3 GTC
GGC 3 GTC
GGC 3 GTC
GGC 3 GTC
GGC 3 GAC
GGC 3 GTC
GGC 3 GTC
Gly 3 Val
Gly 3 Val
Gly 3 Gly
Gly 3 Val
Gly 3 Val
Gly 3 Val
Gly 3 Val
Gly 3 Asp
Gly 3 Val
Gly 3 Val
b
a
b
1: low; 2: intermediate; 3: high grade.
Patient had two mutations.
H-ras in Mucoepidermoid Carcinomas/Yoo et al.
Gaithersburg, MD). Amplification was then performed
on an MJ Research PTC-100 thermal cycler (Watertown,
MA) for 35 cycles, which consisted of an initial denaturation at 94 °C for 1 minute; a cycle of 94 °C, 55 °C, and 72
°C for 1 minute each; and 72 °C for 5 minutes on the last
cycle. Negative controls without DNA template were run
in parallel to exclude contamination of reagents. As a
positive control, we used T24 bladder carcinoma cell
lines, which are known to have H-ras codon 12 mutation. The PCR reactions were routinely checked for the
appropriately amplified DNA on an ethidium bromide–
stained agarose gel under UV light transillumination.
Automated Direct Sequencing
The PCR products of 50 samples were purified with a
QIAquick PCR purification kit (Qiagen, Valencia, CA)
and then sequenced with a 373A DNA sequencer (Applied Biosystems, Foster City, CA) by using dye-primer
conditions recommended by Applied Biosystems.
Both strands were sequenced for each DNA analyzed,
and genomic DNA from control samples was sequenced in parallel to confirm the mutations.
Statistical Methods
We used the Mantel–Haenszel chi-square test to establish correlation between the presence of H-ras mutation and tumor grade (Mantel, 1966).
RESULTS
The H-ras mutations at codons 12 and 13 as well as at
codon 61 were evaluated in 50 DNA samples of mucoepidermoid carcinomas. The prevalence of mutation was 18% (in 9 of 50 samples). Single mutation was
detected in eight tumors and double mutations in one.
All but one of the mutations were in the second base
of codon 12. Eight samples had a G-to-T transversion,
substituting valine for glycine (Fig. 1), whereas one
showed a G-to-A transition, replacing glycine with
aspartate. One patient (Case 4) exhibited concurrent
mutations involving codons 12 and 13: a GGC-to-GTC
transversion at codon 12 and a GGT-to-GGA heterozygous transversion (no change in amino acid) at codon
13. None of the patients had H-ras mutations at codon
61.
There was no association of the mutations with
the patients’ age, gender, tumor size, or survival. However, the number of cases with mutations increased
with increasing severity of histologic change. Of the
H-ras mutant carcinomas, 1 was low grade (1 of 21,
5%), 2 intermediate (2 of 12; 17%), and 6 high (6 of 17;
35%) (Fig. 2). The low grade tumors had a 50:50 distribution of squamous and nonsquamous cells (mucin-producing and intermediate cells). The intermediate and high grade tumors had few (⬍10%) mucin-
521
containing cells. The correlation between the
incidence of H-ras mutation and tumor grade was
found to be statistically significant (P ⫽ 0.017). Clinicopathologic characteristics of the tumors are given in
Table 1, along with mutation status.
DISCUSSION
Alterations of oncogenes and/or tumor suppressor
genes have frequently been implicated in the oncogenesis of various human malignancies. However, little is known about the molecular event in salivary
gland tumors. Studies of these tumors in ras gene
alteration have only included scattered small series of
isolated cases. No K-ras mutations were demonstrated
in cases with adenoid cystic carcinoma, pleomorphic
adenoma, and pleomorphic carcinoma,14,17 whereas
mutated H-ras genes were present in 35% of the pleomorphic adenomas.13 In a previous study (submitted
for publication), salivary gland carcinomas were investigated for the presence of point mutations in H-ras
and K-ras genes, and the frequencies of the H-ras and
K-ras activation were 21% and 8%, respectively. Of the
11 mucoepidermoid carcinomas included, there were
detectable ras mutations in 5 tumors (45%) and K-ras
activation in 1 (9%). The differing results of previously
reported studies are probably due to the wide range of
investigated cases, the selection of lesion types, and
technical variations.
In our analysis of 50 mucoepidermoid carcinomas, we found H-ras gene mutations in 9 (18%). This
observation suggests that H-ras alterations may play
some role in pathways leading to the development of
such lesions. H-ras alterations were restricted to
codons 12 and 13. All but one of the mutations were
present in the second base of codon 12. With one
exception (Case 34), the identical mutations were
noted, resulting in the same amino acid change (glycine to valine). This preferential member of the ras
gene family and the predominant type of nucleotide
transition at a specific site might indicate an unusual
sensitivity of this codon in mucoepidermoid carcinoma cells for tumorigenesis. The precise mechanism(s) leading to this disclosed genetic alteration,
however, remain(s) to be disclosed.
The incidence of H-ras gene activation in mucoepidermoid carcinomas was significantly lower in this
study (18%) than in a previous series (45%) the authors performed. This discrepancy could be the result
of the number of cases analyzed in the two studies and
also possibly due to the larger proportion of nonmutated low grade tumors analyzed in this study. The
current investigation includes more samples of mucoepidermoid carcinomas (11 vs. 50) and might by
chance have detected more mutation negative tu-
522
CANCER February 1, 2000 / Volume 88 / Number 3
mors. Furthermore, more patients with lower grade
tumor were included (1 vs. 21 low grade, 1 vs. 12
intermediate grade, and 9 vs. 17 high grade lesions). It
was not possible to establish any correlation between
the mutation status and tumor grade because of the
limited sample size of our earlier study. In this extended analysis, tumors with H-ras mutations had
poorer differentiation; six were high grade lesions, two
intermediate, and one low. These results suggest a
strong correlation between the presence of H-ras gene
mutation and tumor grade in mucoepidermoid carcinomas (P ⫽ 0.017). Molecular analysis of this gene
alteration may find application as a modifying role in
staging.
It has been reported that certain amino acid substitutions indicate a poorer prognosis, whereas hydrophobic amino acid replacements are associated with a better
prognosis.18 In patients with colorectal carcinoma, K-ras
12 mutation, particularly G-to-T change (valine), was
FIGURE 1. H-ras mutation using automated DNA sequencing is demonstrated. This figure, representing Patient 10, represents sequencing of the
reverse strand: (A) wild-type sequence, and (B) a point mutation in the second
base of codon 12 (GGC-to-GTC) of the forward strand as indicated by a C-to-A
transversion (arrow) in the reverse strand.
FIGURE 2. The incidence of H-ras mutations in mucoepidermoid carcinomas
of different tumor grade is shown.
reported to be associated with increased risk of relapse
and death.19 In this study, however, the presence of
H-ras 12 mutation resulting in substitution of valine did
not increase the risk of recurrence. Of interest, one sample carried the sequence GGA in place of the wild-type
GGC at codon 13, in addition to the H-ras 12 mutation.
Even though concurrent mutations might increase the
potential for progression, no difference was observed in
the prognosis of patients with a single mutation versus
the one whose tumor contained double mutations. The
significance of concurrent mutations thus cannot be
established at present.
Mucoepidermoid carcinoma is composed of a
dual population of cells, that is, the mucous cell and
the squamous cell. In lesions of low grade, the majority of the cell population is mucous cells, whereas a
predominant squamous element characterizes the
high grade form. It may be that the molecular pathobiology differs between these two cell populations.
However, no attempt was made in our studies to separate these two components of the tumor. It will be of
interest to investigate the DNA samples from each
population of lesions of different tumor grade to determine which cell component is responsible for the
ras gene alteration.
In summary, H-ras mutations, predominantly at
codon 12, were detected in 9 tumors (18%), 8 of which
had a single mutation and 1 of which contained double mutations. Mutations were observed more often in
higher grade lesions. Based on these and our previous
results, we conclude that mutational activation of the
H-ras gene may contribute to the development of this
particular salivary gland tumor. It is also noteworthy
that the presence of H-ras mutations strongly correlates with tumor grade. Mutation detection may be of
help in the determination of tumor grade and differentiation.
H-ras in Mucoepidermoid Carcinomas/Yoo et al.
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