Objective
This study aimed to determine whether 1p deletion defines a subset of cellular leiomyomata (CL), which is a hypercellular variant of uterine leiomyomata that may have delayed malignant potential, and to correlate this genetic change with clinical and pathologic characteristics including those present in uterine sarcomas.
Study Design
Available CL cases at the Mayo Clinic (n = 101) and variant cases reported in another article (n = 16) were identified. Each case with sufficient tissue that met histologic criteria for CL when reviewed by a single pathologist underwent interphase fluorescence in situ hybridization to determine the presence of 1p deletion. Clinical characteristics of women with confirmed CL were compared on the basis of 1p deletion status using univariate analysis.
Results
Of the Mayo Clinic cohort of histologically confirmed CL, 23% had deletion of 1p. Women with this subset of CL, when compared to those without 1p deletion, were more likely to be postmenopausal ( P = .049) and their uteri tended to be heavier ( P = .039) with a larger dominant leiomyoma ( P = .030). The pathologic features associated with 1p deletion were high cellularity ( P = .036) and hyaline necrosis ( P = .047), which remained significant after inclusion of the CL cases from a previously published series.
Conclusion
Deletion of 1p occurs in approximately one-quarter of CL cases. This genetic alteration is potentially associated with clinicopathologic features that are present in uterine sarcomas, which suggests a distinct clinical entity that may have malignant potential. Our findings are particularly pertinent considering the increased preference for uterine-sparing options in leiomyoma treatment, suggesting assessment of 1p deletion status in CL may influence clinical surveillance decisions.
Uterine leiomyomata (UL), commonly referred to as fibroid tumors, represent a major concern in women’s health because of their high prevalence, morbidity, and impact on the healthcare system. UL arise from the smooth muscle layer of the uterus known as the myometrium in up to 77% of women based on a systematic review of hysterectomy specimens. These benign tumors result in significant morbidity in many of the approximately 25% of reproductive-age women in whom they are detected clinically with symptoms that include abdominal pain, heavy and prolonged menstrual bleeding, urinary incontinence, constipation, and fertility impairment. In the United States, such frequency and high morbidity result in UL accounting for one-third of hysterectomies and ever increasing expenditures, up to an estimated $5.9-34.4 billion annually.
UL are typically comprised of whorled bundles of smooth muscle cells that form well-circumscribed nodules with abundant pink cytoplasm, uniform spindle-shaped nuclei, and scant and normal mitoses. In contrast, cellular leiomyomata (CL) represent a variant that is significantly more cellular and occurs in <5% of cases.
Although historically CL has been approached clinically as being within the benign spectrum of typical UL, developing evidence suggests that CL may have long-term malignant potential. A recent case-control analysis that defined CL solely by pathologic review versus typical UL demonstrated that CL cases have a distinct clinical phenotype with a subset of characteristics that overlap those in leiomyosarcoma. Three of 18 women with CL or atypical leiomyomata (AL) variants were also shown to have disease-related deaths (ie, deaths directly connected to the variant tumors) that occurred >6 years after diagnosis in contrast to the benign outcome of typical UL or the poorer prognosis of leiomyosarcoma. In addition, chromosome and loss-of-heterozygosity analyses identified a limited number of CL with chromosome 1p deletion, and the expression profile of one such tumor segregated with leiomyosarcoma rather than the normal uterine tissue (myometrium) or typical UL in a hierarchic cluster analysis.
The current study demonstrates that 1p deletion is a common genetic abnormality in CL and suggests a correlation between 1p deletion status with certain pathologic characteristics and the clinical phenotype of the women in whom these variant tumors arise.
Materials and Methods
Study design and participants
Approval for this study was obtained from the Mayo Clinic Institutional Review Board. Formalin-fixed paraffin-embedded tissues of pathologist-designated CL were acquired from Mayo Clinic surgical specimens between January 1989 and December 2008; this represents the same cohort as our case-control analysis of women with a CL (n = 94, because 5 blocks were not available) and CL cases that were identified subsequent to that study (n = 7). A single CL tumor was analyzed from each patient. In addition, formalin-fixed paraffin-embedded tissues from the leiomyoma variant cases that were studied by Giuntoli et al were included (n = 16, because 2 blocks were not available). A fresh hematoxylin and eosin–stained section from each case was reviewed by a single pathologist who used the accepted criteria by Bell et al, which includes the assessment of cytologic atypia (degree: mild, moderate, or severe; and extent: focal or diffuse), mitotic index (mitotic figures per 10 high-power fields) and the presence or absence and quality of necrosis (coagulative, degenerative/hyaline or hydropic). Additional histologic characteristics were recorded such as epithelioid features, myxoid change, and cellularity (mild, moderate, high). CL was diagnosed when the specimen was highly cellular but lacked significant cytologic atypia or coagulative type necrosis. The diagnosis of CL was accepted in the presence of elevated mitotic index (up to 10 mitotic figures per 10 high-power fields) if cytologic atypia or coagulative necrosis was not identified. A mitotic index of 4 mitotic figures per 10 high-power fields in combination with moderate or severe atypia or any mitotic activity in the presence of coagulative necrosis was not classified as CL (ie, diagnosed as leiomyosarcoma). The hematoxylin and eosin–stained slides were also used to delineate the region of interest for subsequent determination of 1p deletion status by interphase fluorescence in situ hybridization (FISH).
Retrospective review of Mayo Clinic patient medical records resulted in ascertainment of clinical, intraoperative, and pathologic information. Premenopausal status was defined as the occurrence of at least one menstrual period in the 12 months before surgery. Leiomyoma recurrence is based on either radiographic or pathologic evidence of new tumors after the initial surgery. The mean clinical follow-up time in the entire 67 cases of the Giuntoli et al series was 3.7 years/44.4 months and the median follow-up time for uterine disease–specific survivors was 15.6 years/187.2 months; the mean of the Mayo cohort was 4.85 years/58.2 months.
Probe design
The enumeration probe set included a 1p21.1 target region probe, labeled in Spectrum Orange-dUTP (Abbott Molecular, Abbott Park, IL) which gives a red-appearing signal, that consisted of RP11-729K11, RP11-701D1, CTD-2306N18, RP11-10C14, and CTD-2310O4 (572 kb total size). The control probe at 1q25.2, which was labeled in Spectrum Green-dUTP (Abbott Molecular), contained RP5-990P15, RP5-1098D14, RP11-18E13, and RP4-595C2 (470 kb total size with a 75 kb gap). The second control probe at 1q21.3, which was labeled in Spectrum Aqua-dUTP (Dyomics, Jena, Germany), contained RP11-48P22, CTD-2147N16, RP11-204K12, CTD-3011N3, RP11-936M7, and RP11-68O11 (572 kb total size; Appendix , Supplemental Materials and Methods ).
FISH
All samples, including both those that met the criteria for CL and those that were given an alternate designation by a single pathologist, were sorted randomly and analyzed in a blinded manner (without knowledge of the pathologic diagnosis). Each of 2 technologists independently scanned the entire tissue section to assess tumor heterogeneity and then scored 50 representative nuclei (100 nuclei total). Cases were considered to have “1p deletion” that had loss of the 1p signal in >15% of nuclei while cases were called “relative 1p deletion” that had fewer 1p signals compared to additional copies of the 1q control probes in >15% of nuclei. The data were decoded subsequently and compared to the pathologic and clinical findings. The conservative false-positive cutoff of 15% for 1p deletion was established by approximately doubling the positive rate found in typical UL ( Supplemental Materials and Methods ).
Statistical analyses
Data were coded and entered into an Excel database (Microsoft Corporation, Redmond, WA). Statistical analyses were performed with JMP for Windows (version 7.0.1; SAS Institute, Cary, NC). For non-normally distributed continuous variables, means and standard deviations are reported while differences between groups of women were assessed with Wilcoxon rank-sum tests. For nominal or categoric variables, frequency counts and percentages are reported while differences between groups of women were assessed using Pearson’s χ 2 or Fisher exact test, as appropriate. A probability value of < .05 was considered statistically significant in all analyses.
Results
Frequency of 1p deletions in Mayo Clinic cohort of CL
UL arise from the uterine myometrium and usually are defined on hematoxylin and eosin stain by fascicles of smooth muscle cells with abundant pink cytoplasm, uniform spindle-shaped nuclei, and a low mitotic index ( Figure 1 , A). In contrast, the pathologic variant CL is similar but significantly more cellular ( Figure 1 , B). Of cases originally diagnosed as CL that had available tissue from the Mayo Clinic cohort (n = 101), the majority were confirmed by a single pathologist as CL (n = 85 that included 17 cases with hyaline necrosis) while a subset of cases were classified with other designations (n = 16); specifically, typical UL (n = 4 for those with hyaline necrosis and n = 7 for those without hyaline necrosis), AL (n = 1), AL with low recurrence risk (n = 1), smooth muscle tumor of unknown malignant potential (n = 2), and mixed endometrium stromal and smooth muscle neoplasm (n = 1).
To identify 1p deletions, an enumeration FISH probe set was developed that targeted 1p21.1 with control probes at 1q21.3 and 1q25.2 ( Figure 2 , A). Interphase nuclei from tumors with the normal complement of 2 copies of chromosome 1 exhibit a 2R2G2A signal pattern ( Figure 2 , B). A typical deletion of the 1p target region results in a 1R2G2A pattern ( Figure 2 , C) while the presence of additional copies of the control probes with relative loss of the 1p target probe appears with multiple patterns such as 2R4G4A ( Figure 2 , D). The target region on 1p was selected as 1p21.1 based on the smallest region of overlapping loss-of-heterozygosity in 10 cases of CL as defined by the polymorphic microsatellite markers that were used in a recent study.
With this FISH strategy, 23% (18 of 79) of the confirmed CL in the Mayo cohort had a 1p deletion, 6% (5 of 79) had relative loss of 1p, and 71% (56 of 79) were normal. FISH was not possible on a subset of confirmed CL cases because of failed hybridization (n = 6). All cases with pathologic designations other than CL (n = 16) were normal by FISH analysis.
Frequency of 1p deletions in the Giuntoli et al series of leiomyoma and uterine tumor variants
Sixteen of 18 leiomyoma variant cases presented in a series examining updated criteria for leiomyosarcoma diagnosis, which were originally reported as AL (n = 4) and CL (n = 12), were available for further study. Reexamination of these cases by a single pathologist in the current effort reclassified the AL tumors into CL (n = 3) and leiomyosarcoma with epithelioid features (n = 1) while the CL cases were described as CL (n = 7, which includes 1 with hyaline necrosis and 1 with epithelioid features), AL (n = 1), UL (n = 1), leiomyosarcoma (n = 1), mixed endometrial stromal/smooth muscle neoplasm (n = 1), and adenofibroma (n = 1).
FISH on the Giuntoli et al series demonstrated a typical deletion of 1p in 47% (7 of 15 cases), relative loss of 1p in 6% (1 of 15 cases), and a normal result in 47% (7 of 15 cases). FISH was not possible on a single CL case because of failed hybridization. The 7 cases with a typical deletion of 1p were classified as CL (n = 4, which included 1 with epithelioid features), leiomyosarcoma (n = 2, which included 1 with epithelioid features), and UL (n = 1). The single case with relative loss of 1p was a CL. The 7 cases with normal FISH results were categorized as CL (n = 4, including 1 with hyaline necrosis), AL (n = 1), mixed endometrial stromal/smooth muscle neoplasm (n = 1), and adenofibroma (n = 1).
Thus, in the combined Mayo cohort and Giuntoli et al series of tumors confirmed to be CL by a single pathologist that were evaluated successfully by FISH, 25% of cases (22 of the 88 total CL) had 1p deletion.
Univariate analysis
Univariate analysis was used to compare the clinical characteristics of women with CL based on 1p deletion status and confined to those with tumors confirmed by a pathologist using current criteria to be CL with successful FISH after removal of tumors with relative 1p deletion (n = 18 CL with 1p deletion and n = 56 CL without 1p deletion; Table 1 ). The number of women who were included in the analysis of each clinical characteristic was based on data availability, with a minimum of 16 women from the 1p deletion group and 52 women from the group without 1p deletion. In the Mayo cohort, women with a CL that had 1p deletion were more likely to be postmenopausal (27.8 vs 9.0%; P = .049) and had heavier uteri (1028.1 ± 1385.0 g vs 574.3 ± 613.0 g; P = .039) with a larger dominant leiomyoma (10.6% ± 4.9% vs 8.0% ± 4.6%; P = .030). The pathologic features associated with 1p deletion were high cellularity (38.9% vs 14.3%; P = .036) and hyaline necrosis (38.9% vs 17.9%; P = .047). It is of note that the single CL case with a 1p deletion that had a recurrence ( Table 1 ) has been classified clinically as a benign metastasizing leiomyoma (BML).
| Variable | Cellular leiomyomata | P value | |
|---|---|---|---|
| With typical 1p deletion (n = 18) | With no deletion (n = 56) | ||
| Clinical features | |||
| Age, y b | 50.72 ± 14.60 | 44.32 ± 10.08 | .114 |
| Uterine/compound c /tissue d weight, g b | 1028.06 ± 1384.96 | 573.95 ± 612.97 | .039 e |
| Leiomyomata, n b | 3.67 ± 3.40 | 4.32 ± 5.66 | .463 |
| CL, n (%) | 1.11 (0.47) | 1.36 (2.29) | .978 |
| Diameter of largest leiomyomata, cm b | 10.6 ± 4.86 | 8.02 ± 4.56 | .030 e |
| Postmenopausal, n (%) | 5 (27.80) | 5 (9.0) | .049 e |
| Postmenopausal bleeding, n (%) | 3 (16.67) | 2 (3.60) | .052 |
| CL recurrence, n (%) | 1 (5.56) | 4 (7.14) | .243 |
| Surgery for CL recurrence, n (%) | 1 (5.56) | 4 (7.14) | .243 |
| Largest leiomyomata is CL, n (%) | 18 (100) | 51 (91.10) | .416 |
| Surgical indication enlarging CL, n (%) | 5 (27.80) | 11 (19.64) | .803 |
| Increasing abdominal girth, n (%) | 1 (5.56) | 5 (9.0) | .650 |
| Pathologic features | |||
| High cellularity, n (%) | 7 (38.89) | 8 (14.29) | .036 e |
| Hyaline necrosis, n (%) | 7 (38.89) | 10 (17.86) | .047 e |
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