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Regular Articles |
From the Department of Pathology,*
Brigham and
Women's Hospital and Harvard Medical School,
Boston, Massachusetts; the Department of
Pathology,
Universidade Federal do
Paraná, Curitiba, Brazil; and the Departments of
Pathology§
and Obstetrics and
Gynecology,¶
University of Washington,
Seattle, Washington
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pathological diagnosis of uterine smooth muscle tumors requires evaluation of mitotic activity, nuclear atypia, tumor necrosis, and perhaps to a lesser extent, cellularity and circumscription.4,5 Uterine smooth muscle tumors may have any one of these features and still be considered benign variants of leiomyoma. When several of these features are present, histological distinction of uterine leiomyosarcomas from leiomyomas is not always possible. The diagnostic difficulty posed by this apparent overlap in morphological phenotype is reflected in diagnostic terms such as "smooth muscle tumor of uncertain malignant potential" and "atypical leiomyoma with recurring potential." Whether smooth muscle tumors of uncertain malignant potential or atypical leiomyomas represent true biological intermediates, such as the relationship between colonic adenomas and carcinomas, is unknown.
One approach taken to understand the differences between benign and malignant uterine smooth tumors has been investigation of their respective cytogenetics. Leiomyosarcomas typically have complex karyotypic abnormalities.6-12 Their karyotypes show both numerical and structural aberrations, which often preclude identification of some derivative chromosomes in given metaphases. These aberrations are often unstable, resulting in significant variation from metaphase to metaphase within a tumor. In contrast to leiomyosarcomas, benign leiomyomas have normal karyotypes or simple cytogenetic abnormalities in approximately 40% of tumors.13-15 The most common aberrations in leiomyomas include a translocation between chromosomes 12 and 14 and deletions of the long arm of chromosome 7.16,17 The t(12;14) results in aberrant expression of a member of the high mobility group (HMG) family of architectural factors, HMGIC, from chromosome 12.18,19 This translocation breakpoint maps near the estrogen receptor ß (ESR2) on chromosome 14, but does not significantly alter this gene's expression.20 Other chromosomal changes found in uterine leiomyomas include trisomy 12, and rearrangements involving chromosomes 3, 6, 10, or 13.21-25 Of note, rearrangements involving chromosome 6 band p21 in uterine leiomyomas involve another high mobility group family member, HMGIY.26 The cytogenetics of benign variants and smooth muscle tumors of uncertain malignant potential have yet to be studied in detail.27-31
To understand better the molecular pathogenetic distinction(s) between these tumors, we analyzed uterine leiomyosarcomas and leiomyomas for two other potential markers of genomic instability, loss of heterozygosity (LOH) and microsatellite instability (MI).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our study materials consisted of 29 archival hysterectomy
and myomectomy specimens from the Division of Women's and Perinatal
Pathology, Brigham and Women's Hospital, Boston, MA and from a
previously described collection of uterine smooth muscle tumors (D.R.H.
and W.A.P.).32,33
Three pathologists (B.J.Q., A.P.P., and
C.P.C.) reviewed hematoxylin and eosin stained histological sections to
either confirm or reclassify the diagnosis of leiomyoma or benign
variant, and leiomyosarcoma based on current criteria. Benign vari-ants
of leiomyomas included mitotically active, cellular, and
symplastic (atypical) types, as well as a lipoleiomyoma and an
epithelioid leiomyoma. Leiomyosarcomas in our panel included spindle
cell tumors with varying pleomorphism and tumors with epithelioid
differentiation (Table 1)
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Five-micrometer sections were prepared from archival paraffin
blocks. One section was stained with hematoxylin and eosin and used as
a reference for dissection. Tumor or normal tissue was then collected
by excising the paraffin embedded tissue from two additional sections
with sterile surgical blades. DNA was extracted from paraffin fragments
by incubation at 62°C in 300 µl of extraction buffer (50 mmol/L
Tris-HCl, pH 8.5; 1 mmol/L EDTA; 0.5% Tween-20; and 0.2 µg/µl
proteinase K) for 36 hours. The paraffin emulsion was microfuged
briefly and 2 µl of clarified aqueous phase was removed for use as
template for polymerase chain reaction (PCR). Primer pairs were
selected primarily from the Cooperative Human Linkage Center's Human
Screening Set (Weber version 8, Research Genetics,
Huntsville, AL) and analyzed the following microsatellite markers:
D7S1824, D10S1435, D10S2325, D10S1432, D10S1418, D10S218, D10S188,
D10S2327, D10S541, D10S1765, D10S1239, D10S1213, D11S2000, D12S375,
D14S606, D14S1426, D15S643, D16S521, D16S291, D18S843, D18S464,
D18S542, D21S1446, D21S2052, D21S2055, DXS6810. PCR amplification was
carried out in 25 µl reaction volumes containing 2 µl of DNA
solution, 50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3, 1.5 mmol/L
MgCl2, 200 µmol of each dNTP (supplemented with 50 µmol
of 32P-
-dCTP), 50 nmol of each forward and reverse
microsatellite primer, and 1 U of AmpliTaq DNA polymerase (Perkin
Elmer, Norwalk, CT). Twenty-nine cycles of amplification were performed
in a thermocycler (DNA Thermocycler 480, Perkin-Elmer) with the
following profile: denaturation at 95°C for 30 seconds, annealing at
55°C for 45 seconds, extension at 72°C for 1.5 minutes. The first
three cycles were preceded by a 4-minute denaturation step at 95°C.
The last cycle was followed by an extended incubation for 7 minutes at
72°C. PCR products were separated on 7% polyacrylamide gels and
visualized by autoradiography.
Loss of heterozygosity at a microsatellite locus is manifested by partial or total absence of either one of the two PCR products in tumor compared to some normal tissue. Samples were scored positive for LOH if the ratio of autoradiogram intensity for the alleles changed by a factor of 2 or more between tumor and normal tissues.34 LOH was obvious by visual inspection (without scanning densitometry) in most cases. MI was scored positive if novel bands were present in tumor samples compared to normal tissues.35
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined archival material from 16 leiomyosarcomas and 13 leiomyomas by polymerase chain reaction and analyzed 26 microsatellites on 11 chromosomes. Markers surveying the 11 chromosomes (including 7, 9, 10, 11, 12, 14, 15, 16, 18, 21, and X) were selected for evaluation based on previous reports of cytogenetic or molecular genetic abnormalities in either leiomyosarcomas or leiomyomas.10,11,16,17,36-38
We found LOH involving microsatellite markers on chromosomes 10, 15,
18, and 21 in uterine leiomyosarcomas. Interestingly, 57.2% (8 of 14)
informative leiomyosarcomas had LOH for at least one marker on
chromosome 10. In contrast, LOH at markers on chromosomes 15, 18, 21,
and X was infrequent, involving 16.5% (1 of 6) of informative tumors
tested for each of the microsatellite markers. The genomic region
subject to LOH on chromosome 10 was large, involving markers on both p
and q arms (viz., in cases 2, 3, 6, 8, and 12 in
Figure 1
) in 45.5% (5 of 11) of
informative tumors. LOH was observed on the short arm of chromosome 10
in 54.5% (6 of 11) of informative tumors. The long arm also had LOH in
50% (7 of 14) of informative cases. In cases 9 and 10, the contiguous
region of LOH included at least the 10q21–23 region. Multiple
interstitial deletions were present in case 3.
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. One of two
alleles for two different markers (eg, D10S218 and D10S1213) was absent
in one portion of the tumor whereas the other allele was lost in a
sample from another portion (lanes A and B). Both of these markers are
on the long arm of chromosome 10. Loss of heterozygosity for D10S1435,
a marker near the 10p telomere, was present in only one of two tumor
samples (lane A). These results are consistent with two tumor subclones
in this case.
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As a comparison to leiomyosarcomas, we tested 13 benign leiomyomas for LOH for markers on both 10p and 10q. These benign tumors included eight usual type leiomyomas, two cellular leiomyomas, one leiomyoma with epithelioid differentiation, one lipoleiomyoma, and one symplastic leiomyoma with significant nuclear atypia. No LOH was detected. By Fischer's Exact test, the difference in frequency of LOH between leiomyomas and leiomyosarcomas was significant at a value of P < 0.001.
Analysis of Microsatellite Instability
MI was found only in three cases, and each occurred in different leiomyosarcomas and at different loci on different chromosomes (data not shown). No MI was detected in leiomyomas.
Correlation of Loss of Heterozygosity with Clinicopathological Features
Pathological and clinical parameters for the leiomyosarcomas are
shown in Table 1
. Some of the cases (cases 8–16) have been analyzed
previously with respect to flow cytometric parameters.32,33
Consequently, the duration of clinical follow-up of these cases has
increased. Correlation between LOH and these clinicopathological
features was not apparent. In particular, LOH did not appear to be a
strong prognostic factor. Slightly more than one-half (3 of 5) of
leiomyosarcomas with long term follow-up and LOH for any marker on
chromosome 10 had unfavorable clinical outcomes. A similar fraction (4
of 6) of leiomyosarcomas with long term follow-up but without LOH for
any marker on chromosome 10 also had unfavorable clinical outcomes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies using a different approach (comparative genomic hybridization) to identify changes in genomic DNA copy number have also suggested that leiomyosarcomas are characterized by frequent losses and gains in chromosomal copy number.43,44 In a study of uterine leiomyosarcomas, the most commonly observed genetic aberrations were gains on either arm of chromosome 1 (5 of 8 tumors).43 Chromosomal gains (amplifications) and the corresponding allelic imbalance(s) potentially might be interpreted as LOH when analyzed by non-quantitative microsatellite PCR analysis. In contrast to our results, consistent losses were not detected by comparative genomic hybridization in that tumor collection and loss of material from chromosome 10 was found only in 1 of 8 leiomyosarcomas in that study. The chromosomal loss in this particular tumor did, however, involve the entire chromosome 10. In a study of extrauterine leiomyosarcomas, the most frequent loss was detected in 10q (20 of 29 tumors), with a minimal common overlapping region corresponding to 10q11-q24.44 Frequent loss of 10q in leiomyosarcomas from a number of anatomical sites including the uterus suggests a common pathogenetic mechanism.
Significantly, LOH for markers on chromosome 10 was found in none of the benign leiomyomas in our study. Rearrangements of chromosome 10 band q22 has been described as a distinct subgroup of those non-random rearrangements found in benign leiomyomas.24,25,45 Interestingly, rearrangements of 10q22 by translocation to 17p13 as the sole cytogenetic abnormality in one leiomyosarcoma and by translocation to 11p15 among other complex aberrations in another leiomyosarcoma have also been reported.46,47 These translocations in leiomyomas and leiomyosarcomas are ostensibly balanced and would not be detectable by LOH analysis except for possibly at or near translocation breakpoints. Such an alternative mechanism potentially might account for tumors lacking LOH for chromosome 10 loci. The fact that LOH for this or any region on chromosome 10 is not frequent in leiomyomas, however, suggests that leiomyomas and leiomyosarcomas are the products of two different mechanisms.
It is possible that chromosome 10 deletion(s) occur later in a hypothetical progression from normal myometrium to leiomyoma, and ultimately in a small number of cases, to leiomyosarcomas. The heterogeneous loss of chromosome 10 observed in one of our malignant tumors (case 3) raises the possibility that deletion of chromosome 10 material is acquired after malignant transformation. LOH involving much or all of chromosome 10 has also been observed in glioblastoma and a gene, PTEN, on chromosome 10 in band q23 has been implicated.48,49 PTEN has also been implicated in another tumor of the female genital tract, endometrial adenocarcinoma.50 Frequent LOH on chromosome 10 in leiomyosarcomas may likewise point to this or another tumor suppressor gene and this observation merits further study.
Progressive acquisition of chromosomal aberrations potentially might explain the variable phenotypes found in leiomyosarcomas. For example, tumors with losses on chromosome 10 might have had greater histological pleomorphism, more frequent necrosis, or more aggressive clinical behavior. When we reviewed these features, however, we were unable to discern any correlation between these morphological or prognostic parameters and LOH involving chromosome 10. It therefore seems unlikely that any potential leiomyosarcoma-associated gene on chromosome 10 is sufficient to determine histopathological or clinical phenotypes. Nevertheless, detection of LOH for loci on chromosome 10 may complement histological criteria for distinguishing between biologically benign and malignant uterine smooth muscle neoplasms. Such a diagnostic adjunct could be helpful both as a marker influencing clinical management and classifying smooth muscle tumors of uncertain malignant potential for further study of these neoplasms.
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Footnotes |
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Supported in part by National Cancer Institute Grant CA72594–02 (to B.J.Q.). A.P.P. is a recipient of a Ph.D. fellowship from Conselho National de Desenvolvimento em Pesquisa, Brazil.
Accepted for publication December 8, 1998.
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This article has been cited by other articles:
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  S. D. P. Moore, S. R. Herrick, T. A. Ince, M. S. Kleinman, P. D. Cin, C. C. Morton, and B. J. Quade Uterine Leiomyomata with t(10;17) Disrupt the Histone Acetyltransferase MORF Cancer Res., August 15, 2004; 64(16): 5570 - 5577. [Abstract] [Full Text] [PDF]
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,
loss of heterozygosity; 
, retention of heterozygosity; —,
uninformative
(homozygosity); *,
microsatellite instability; blank, not determined. Numbers above the
550 band resolution idiogram of chromosome 10 label cytogenetic bands
and bars below show the approximate cytogenetic localization of
microsatellite markers. The diagonal hash marks on the ideogram
correspond to the centromere between the short
(p) and long
(q) chromosomal arms. The
double lines beneath the idiogram mark the cytogenetic localization of
the PTEN gene in band q23. Rows 3a and
3b correspond to different portions of the same tumor and
analysis of selected microsatellites is illustrated in Figure 2