[Multicenter retrospective study of 38 cases with fumarate hydratase deficiency uterine leiomyoma].

Objective: To investigate the clinicopathological features of fumarate hydratase (FH) deficiency uterine leiomyoma. Methods: The data of 38 patients with FH deficiency uterine leiomyoma were screened and analyzed. The expressions of FH, S-(2-succino)-cysteine (2SC), desmin, p16, p53, CD10 and cell proliferation associated nuclear antigen (Ki-67) proteins were detected by immunohistochemistry, and their clinicopathological features were analyzed retrospectively. Results: (1) Clinical features: the median age of the patients was (42.5±7.4) years old. Twenty-one cases (55%) of them were myomas found in physical examination, and the median maximum diameter of the tumor was 6.0 cm (range: 5.0-7.5 cm); myomectomy was performed in 23 cases (61%), total hysterectomy with or without bilateral appendages in 15 cases (39%); laparoscopic surgery in 27 cases (71%), open surgery in 11 cases (29%); none of the patients had renal cell carcinoma. (2) Histological features: atypical nuclear cells were distributed locally or diffusely, eosinophilic nucleoli and intranuclear inclusion bodies could be seen, glass like globules could be seen in the cytoplasm, nuclear division was 0-4/10 high power field (HPF), and antler like blood vessels and pulmonary edema-like changes could be seen in the stroma. Among 38 patients with FH deficiency uterine leiomyoma, FH was negative in 37 cases (97%), and positive in 1 case (3%); 2SC, desmin, p16, p53, CD10 and Ki-67 showed focal positive expression in 38 cases (100%), including 35 cases (92%) with Ki-67 index<10% and 3 cases (8%) with Ki-67 index ≥10%. (3) Follow-up: 4 cases (11%) recurred, and there was no death. There were significant differences in age, family history, distribution of atypical nuclei and mitosis number between recurrent group and non-recurrent group (all P<0.05). Conclusions: FH deficiency uterine leiomyoma is a rare tumor, which needs pathological examination,immunohistochemical examination and clinical history. Patients younger than 43 years old, with family history, histologically atypical diffuse nuclear distribution and mitotic number ≥3/10 HPF should be alert to the risk of recurrence.

Tryptophan 2,3-Dioxygenase-2 in Uterine Leiomyoma: Dysregulation by MED12 Mutation Status.

Uterine leiomyomas (fibroids) are common benign tumors in women. The tryptophan metabolism through the kynurenine pathway plays important roles in tumorigenesis in general. Leiomyomas expressing mutated mediator complex subunit 12 (mut-MED12) were reported to contain significantly decreased tryptophan levels; the underlying mechanism and the role of the tryptophan metabolism-kynurenine pathway in leiomyoma tumorigenesis, however, remain unknown. We here assessed the expression and regulation of the key enzymes that metabolize tryptophan. Among these, the tissue mRNA levels of tryptophan 2,3-dioxygenase (TDO2), the rate limiting enzyme of tryptophan metabolism through the kynurenine pathway, was 36-fold higher in mut-MED12 compared to adjacent myometrium (P < 0.0001), and 14-fold higher compared to wild type (wt)-MED12 leiomyoma (P < 0.05). The mRNA levels of other tryptophan metabolizing enzymes, IDO1 and IDO2, were low and not significantly different, suggesting that TDO2 is the key enzyme responsible for reduced tryptophan levels in mut-MED12 leiomyoma. R5020 and medroxyprogesterone acetate (MPA), two progesterone agonists, regulated TDO2 gene expression in primary myometrial and leiomyoma cells expressing wt-MED12; however, this effect was absent or blunted in leiomyoma cells expressing G44D mut-MED12. These data suggest that MED12 mutation may alter progesterone-mediated TDO2 expression in leiomyoma, leading to lower levels of tryptophan in mut-MED12 leiomyoma. This highlights that fibroids can vary widely in their response to progesterone as a result of mutation status and provides some insight for understanding the effect of tryptophan-kynurenine pathway on leiomyoma tumorigenesis and identifying targeted interventions for fibroids based on their distinct molecular signatures.

Histone deacetylase inhibition by suberoylanilide hydroxamic acid: a therapeutic approach to treat human uterine leiomyoma.

OBJECTIVE: To evaluate the effect of inhibition of histone deacetylases (HDACs) by suberoylanilide hydroxamic acid (SAHA) treatment of human uterine leiomyoma primary (HULP) cells in vitro on cell proliferation, cell cycle, extracellular matrix (ECM) formation, and transforming growth factor ß3 (TGF-ß3) signaling. DESIGN: Prospective study comparing uterine leiomyoma (UL) vs. adjacent myometrium (MM) tissue and cells with or without SAHA treatment. SETTING: Hospital and university laboratories. PATIENT(S): Women with UL without any hormone treatment. INTERVENTION(S): Myomectomy or hysterectomy surgery in women for leiomyoma disease. MAIN OUTCOME MEASURE(S): HDAC activity was assessed by enzyme-linked immunosorbent assay, and gene expression was assessed by quantitative real-time polymerase chain reaction. Effects of SAHA on HULP cells were analyzed by CellTiter (Promega, Madison, Wisconsin), Western blot, and quantitative real-time polymerase chain reaction. RESULT(S): The expression of HDAC genes (HDAC1, fold change [FC] = 1.65; HDAC3, FC = 2.08; HDAC6, FC = 2.42) and activity (0.56 vs. 0.10 optical density [OD]/h/mg) was significantly increased in UL vs. MM tissue. SAHA decreased HDAC activity in HULP cells but not in MM cells. Cell viability significantly decreased in HULP cells (81.68% at 5 µM SAHA, 73.46% at 10 µM SAHA), but not in MM cells. Proliferating cell nuclear antigen expression was significantly inhibited in SAHA-treated HULP cells (5 µM SAHA, FC = 0.556; 10 µM SAHA, FC = 0.622). Cell cycle markers, including C-MYC (5 µM SAHA, FC = 0.828) and CCND1 (5 µM SAHA, FC = 0.583; 10 µM SAHA, FC = 0.482), were significantly down-regulated after SAHA treatment. SAHA significantly inhibited ECM protein expression, including FIBRONECTIN (5 µM SAHA, FC = 0.815; 10 µM SAHA, FC = 0.673) and COLLAGEN I (5 µM SAHA, FC = 0.599; 10 µM SAHA, FC = 0.635), in HULP cells. TGFß3 and MMP9 gene expression was also significantly down-regulated by 10 µM SAHA (TGFß3, FC = 0.596; MMP9, FC = 0.677). CONCLUSION(S): SAHA treatment inhibits cell proliferation, cell cycle, ECM formation, and TGF-ß3 signaling in HULP cells, suggesting that histone deacetylation may be useful for treatment of UL.

Tryptophan catabolism is dysregulated in leiomyomas.

OBJECTIVE: To determine the expression and functional roles of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) in leiomyoma. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): Blockade of IDO1 and TDO2. MAIN OUTCOME MEASURE(S): Expression of IDO1 and TDO2 in leiomyoma and the effects of their inhibitors on the extracellular matrix. RESULT(S): Leiomyoma expressed significantly higher levels of IDO1 and TDO2 messenger ribonucleic acid (mRNA; 60.3%, 35/58 pairs and 98.3%, 57/58 pairs, respectively) and protein (54%, 27/50 pairs and 92%, 46/50 pairs, respectively) as well as the enzyme activity marker kynurenine (78.3%, 36/46 pairs for IDO1/TDO2) compared with levels in matched myometrium. The expression of TDO2 but not IDO1 mRNA was significantly higher in fibroids from African American compared with that in Caucasian and Hispanic patients. The TDO2 but not the IDO1 protein and mRNA levels were more abundant in fibroids bearing the MED12 mutation compared with results in wild-type leiomyomas. Treatment of leiomyoma smooth muscle cell and myometrial smooth muscle cell spheroids with the TDO2 inhibitor 680C91 but not the IDO1 inhibitor epacadostat significantly repressed cell proliferation and the expression of collagen type I (COL1A1) and type III (COL3A1) in a dose-dependent manner; these effects were more pronounced in leiomyoma smooth muscle cells compared with myometrial smooth muscle cell spheroids. CONCLUSION(S): These results underscore the physiological significance of the tryptophan degradation pathway in the pathogenesis of leiomyomas and the potential utility of anti-TDO2 drugs for treatment of leiomyomas.

Mechanical stiffness augments ligand-dependent progesterone receptor B activation via MEK 1/2 and Rho/ROCK-dependent signaling pathways in uterine fibroid cells.

OBJECTIVE: To test whether mechanical substrate stiffness would influence progesterone receptor B (PRB) signaling in fibroid cells. Uterine fibroids feature an excessive extracellular matrix, increased stiffness, and altered mechanical signaling. Fibroid growth is stimulated by progestins and opposed by anti-progestins, but a functional interaction between progesterone action and mechanical signaling has not been evaluated. DESIGN: Laboratory studies. SETTING: Translational science laboratory. PATIENT(S)/ANIMAL(S): Human fibroid cell lines and patient-matched fibroid and myometrial cell lines. INTERVENTION(S): Progesterone receptor B-dependent reporter assays and messenger RNA quantitation in cells cultured on stiff polystyrene plates (3GPa) or soft silicone plates (930KPa). Pharmacologic inhibitors of extracellular signal-related protein kinase (ERK) kinase 1/2 (MEK 1/2; PD98059), p38 mitogen-activated protein kinase (SB202190), receptor tyrosine kinases (RTKs; nintedanib), RhoA (A13), and Rho-associated coiled-coil kinase (ROCK; Y27632). MAIN OUTCOME MEASURE(S): Progesterone-responsive reporter activation. RESULT(S): Fibroid cells exhibited higher PRB-dependent reporter activity with progesterone (P4) in cells cultured on stiff vs. soft plates. Mechanically induced PRB activation with P4 was decreased 62% by PD98059, 78% by nintedanib, 38% by A13, and 50% by Y27632. Overexpression of the Rho-guanine nucleotide exchange factor (Rho-GEF), AKAP13, significantly increased PRB-dependent reporter activity. Collagen 1 messenger RNA levels were higher in fibroid cells grown on stiff vs. soft plates with P4. CONCLUSION(S): Cells cultured on mechanically stiff substrates had enhanced PRB activation via a mechanism that required MEK 1/2 and AKAP13/RhoA/ROCK signaling pathways. These studies provide a framework to explore the mechanisms by which mechanical stiffness affects progesterone receptor activation.

Q192R variant in paraoxonase 1 gene confers susceptibility to leiomyoma.

OBJECTIVE: Paraoxonase 1 (PON1) plays a defensive role against oxidative stress by destroying oxidized lipids. Q192R single nucleotide polymorphism of PON1 gene alters the enzyme's activity. Several investigations reported a link between Q192R and an increased risk of developing tumors including uterine leiomyomas. We assessed the antioxidant effects of Q192R on myoma which fluctuate in frequency between populations. STUDY DESIGN: The cohort consisted of 68 unrelated uterine leiomyoma patients and 93 healthy controls that were randomly selected from women with no ultrasonographic evidence of myoma. MATERIALS AND METHODS: Genotyping was performed using tetra-primer amplification refractory mutation system-polymerase chain reaction. Chi-square test was selected to evaluate differences between the groups. RESULTS: To analyze the correlation between PON1 Q192R and leiomyoma risk, the AA genotype was given as a reference genotype then the two other genotypes were compared with the reference. A significantly (P < 0.05) increased risk of myoma was observed with both Q192R homozygote GG and heterozygote AG genotypes. The odds ratio (OR) of AG genotype was calculated 1.8 (confidence interval [CI]: 0.94-3.62). A higher OR was seen with GG genotype (OR: 2.8; 95% CI: 0.98-8.18). CONCLUSION: Oxidative stress has been suspected of having a link with tumor development, and the role of endogenous-free radical scavenger is taken into consideration. Increased protein oxidative stress status and reduced antioxidant capacity have been observed in leiomyomas patients. Our study indicates that the low-antioxidant PON1 R192 allele correlates to leiomyoma development.

Activation of protein kinase B by WNT4 as a regulator of uterine leiomyoma stem cell function.

OBJECTIVE: To investigate the functional interaction between the Wnt/ß-catenin and protein kinase B (Akt) pathways in leiomyoma stem cells (LSC). DESIGN: Laboratory study. SETTING: Research laboratory. PATIENT(S): Premenopausal women (n = 36; age range: 28 to 49 years) undergoing hysterectomy or myomectomy for leiomyoma. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Gene expression, protein phosphorylation, and cell proliferation. RESULT(S): Cells from human leiomyoma tissues were sorted by fluorescence-activated cell sorting (FACS) into three populations: LSC, intermediate cells (LIC), and differentiated cells (LDC) with the function of the Wnt/ß-catenin and Akt signaling pathways in leiomyoma cells evaluated using real-time quantitative polymerase chain reaction and immunoblot analyses. The Wnt/ß-catenin signaling pathway components were differentially expressed in each leiomyoma cell population. WNT4 was distinctly overexpressed in LIC, and its receptor FZD6 was primarily expressed in LSC. WNT4 stimulated Akt phosphorylation, activated ß-catenin, and increased primary leiomyoma cell proliferation. These stimulatory effects were abolished by cotreatment with the Akt inhibitor, MK-2206. WNT4 up-regulated the expression of pro-proliferative genes, c-Myc and cyclin D1, specifically in LSC; this was also abrogated by Akt inhibition. CONCLUSION(S): Our data suggest that WNT4 regulates LSC proliferation via Akt-dependent ß-catenin activation, representing a key step toward a better understanding of LSC regulation and potentially novel therapeutic targets.

Prevalence of somatic and germline mutations of Fumarate hydratase in uterine leiomyomas from young patients.

AIMS: Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is caused by germline mutations in the Fumarate hydratase (FH) gene. In young women, the syndrome often presents with symptomatic uterine leiomyomas, leading to myomectomy or hysterectomy. In this study, we aimed to investigate the incidence and mutational profiles of FH-negative leiomyomas from young patients, thus allowing for early identification and triage of syndromic patients for surveillance. METHODS AND RESULTS: We evaluated 153 cases of uterine leiomyomas from women aged up to 30 years for loss of FH expression by tissue microarray (TMA)-based immunohistochemical staining. Mutational analysis of tumours with loss of FH was carried out by polymerase chain reaction (PCR) amplification of 10 exons within the FH gene and subsequent Sanger sequencing. The status of promoter methylation was assessed by bisulphite sequencing. Loss of FH protein expression was detected in seven (4.6%) of 153 tested uterine leiomyomas from young patients. All FH-negative leiomyomas displayed staghorn vasculature and fibrillary/neurophil-like cytoplasm. We found that six (86%) of seven FH-negative tumours detected by immunohistochemistry harboured FH mutations, 50% of which contained germline mutations. In particular, the germline mutational rate in FH gene was 2.0% (three of 153 cases). Bisulphite sequencing analysis failed to detect promoter methylation in any of the seven tumours. CONCLUSION: Our study showed a relatively high rate of FH germline mutation in FH-negative uterine leiomyomas from patients aged up to 30 years. While genetic mutations confer protein expression loss, epigenetic regulation of the FH gene appears to be unrelated to this phenotype.

Cross-talk between miR-29c and transforming growth factor-ß3 is mediated by an epigenetic mechanism in leiomyoma.

OBJECTIVE: To determine the expression of miR-29c and its target gene transforming growth factor-ß3 (TGF-ß3) in leiomyoma and the mechanisms of their reciprocal regulation. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): Overexpression and underexpression of miR-29c; blockade of DNA methyltransferase 1 (DNMT1). MAIN OUTCOME MEASURE(S): The miR-29c and its target gene TGF-ß3 in leiomyoma and the effects of TGF-ß3 and blockade of DNMT1 on miR-29c expression. RESULT(S): Leiomyoma expressed significantly lower levels of miR-29c, but higher expression of TGF-ß3 compared with matched myometrium. The expression of TGF-ß3 and miR-29c were independent of race/ethnicity. Using 3' untranslated region luciferase reporter assay we confirmed that TGF-ß3 is a direct target of miR-29c in leiomyoma smooth muscle cells (LSMCs). Gain-of-function of miR-29c in LSMCs inhibited the expression of TGF-ß3 at protein and messenger RNA levels, whereas loss-of-function of miR-29c had the opposite effect. Treatment of LSMCs with TGF-ß3 inhibited the expression of miR-29c, whereas it stimulated DNMT1 expression. Knockdown of DNMT1 through transfection with small interfering RNA significantly decreased the expression of TGF-ß3, and induced miR-29c expression. Knockdown of DNMT1 also attenuated the inhibitory effect of TGF-ß3 on miR-29c expression. Furthermore, we demonstrated that TGF-ß3 increased the methylation level of miR-29c promoter in LSMCs. CONCLUSION(S): There is an inverse relationship in the expression of TGF-ß3 and miR-29c in leiomyoma. The TGF-ß3 is a direct target of miR-29c and inhibits the expression of miR-29c through an epigenetic mechanism. The cross-talk between miR-29c and TGF-ß3 provides a feed forward mechanism of fibrosis in leiomyoma.

Fumarate Hydratase Mutations and Alterations in Leiomyoma With Bizarre Nuclei.

Leiomyoma with bizarre nuclei (LM-BN), is a variant of uterine smooth muscle tumor with atypical histologic features. Although some LM-BN share several significant genetic alterations with leiomyosarcoma, including p16 and p53, the underlying tumorigenesis of LM-BN remains largely unknown. As we previously reported, LM-BN can be divided into 2 subtypes, type I and type II, based on different nuclear features. Type I LM-BN have similar histologic features as uterine smooth muscle tumors with fumarate hydratase (FH) alterations. In this study, we examined FH expression and FH mutations in 77 LM-BN (40 type I cases and 37 type II cases). FH expression was examined by immunohistochemistry using S-(2-succino)-cysteine antibodies (2SC, a protein modification associated with FH inactivation and subsequent fumarate accumulation) and FH antibodies (FH gene products). Seventy-two LM-BN tumors underwent Sanger sequencing to detect FH mutations. We found that 51% (39/77) of LM-BN showed FH alterations detected by immunohistochemistry with both 2SC and FH. Mutational analysis showed that 21% (15/72) of LM-BN harbored FH gene mutations. Further analysis revealed that 85% (34/40) of those with FH alterations were type I LM-BN while 19% (7/37) were type II LM-BN. Our findings suggest that over half of histologically diagnosed LM-BN may be related to FH alterations or FH mutations and the majority of these have the characteristic histologic features of type I LM-BN.

Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases.

The role of the extracellular matrix (ECM) in uterine fibroids (UF) has recently been appreciated. Overhydroxylation of lysine residues and the subsequent formation of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) cross-links underlie the ECM stiffness and profoundly affect tumor progression. The aim of the current study was to investigate the relationship between ECM of UF, collagen and collagen cross-linking enzymes [lysyl hydroxylases (LH) and lysyl oxidases (LOX)], and the development and progression of UF. Our results indicated that hydroxyl lysine (Hyl) and HP cross-links are significantly higher in UF compared to the normal myometrial tissues accompanied by increased expression of LH (LH2b) and LOX. Also, increased resistance to matrix metalloproteinases (MMP) proteolytic degradation activity was observed. Furthermore, the extent of collagen cross-links was positively correlated with the expression of myofibroblast marker (α-SMA), growth-promoting markers (PCNA; pERK1/2; FAKpY397; Ki-67; and Cyclin D1), and the size of UF. In conclusion, our study defines the role of overhydroxylation of collagen and collagen cross-linking enzymes in modulating UF cell proliferation, differentiation, and resistance to MMP. These effects can establish microenvironment conducive for UF progression and thus represent potential target treatment options of UF.

Ovarian steroid hormones modulate the expression of progesterone receptors and histone acetylation patterns in uterine leiomyoma cells.

Uterine leiomyomas are the most common benign smooth muscle cell tumors in women. Estrogen (E2), progesterone (P4) and environmental factors play important roles in the development of these tumors. New treatments, such as mifepristone, have been proposed. We evaluated the gene expression of total (PRT) and B (PRB) progesterone receptors, and the histone acetyltransferase (HAT) and deacetylase (HDAC) activity after treatment with E2, P4 and mifepristone (RU486) in primary cell cultures from uterine leiomyoma and normal myometrium. Compared to myometrium, uterine leiomyoma cells showed an increase in PRT mRNA expression when treated with E2, and increase in PRB mRNA expression when treated with E2 and P4. Treatment with mifepristone had no significant impact on mRNA expression in these cells. The HDAC activity was higher in uterine leiomyoma compared to myometrial cells after treatment with E2 and E2 + P4 + mifepristone. HAT activity was barely detectable. Our results suggest that ovarian steroid hormones modulate PR, and mifepristone was unable to decrease PRT and PRB mRNA. The higher activity of HDAC leiomyoma cells could be involved in transcriptional repression of genes implicated in normal myometrium cell function, contributing to the maintenance and growth of uterine leiomyoma.

Dysfunctional MnSOD leads to redox dysregulation and activation of prosurvival AKT signaling in uterine leiomyomas.

AKT signaling promotes cell growth and survival and is often dysregulated via multiple mechanisms in different types of cancer, including uterine leiomyomas (ULMs). ULMs are highly prevalent fibrotic tumors that arise from the smooth muscular layer of the uterus, the myometrium (MM). ULMs pose a major public health issue because they can cause severe morbidity and poor pregnancy outcomes. ‬We investigate the mechanisms driving ULM growth and survival via aberrant activation of AKT. We demonstrate that an acetylation-mediated impairment of the manganese superoxide dismutase (MnSOD) activity is prevalent in ULM cells compared to the normal-matched MM from the same patients. This impairment increases the levels of superoxide and oxidative stress, which activate AKT via oxidative inactivation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Redox activation of AKT promotes ULM cell survival under conditions of moderate but persistent oxidative stress that are compatible with ULM's prooxidative microenvironment. Moreover, because of impaired MnSOD activity, ULM cells are sensitive to high levels of reactive oxygen species (ROS) and superoxide-generating compounds, resulting in decreased ULM cell viability. On the contrary, MM cells with functional MnSOD are more resistant to high levels of oxidants. This study demonstrates a causative role of acetylation-mediated MnSOD dysfunction in activating prosurvival AKT signaling in ULMs. The specific AKT and redox states of ULM cells provide a potential novel therapeutic rationale to selectively target ULM cells because of their defective ROS-scavenging system.‬‬‬‬‬‬‬‬.

[Atypical leiomyoma in a patient with cutaneous leiomyomatosis and mutation of the enzyme fumarate hydratase]. / Leiomioma cutáneo atípico en un paciente con leiomiomatosis cutánea y mutación de la enzima fumarato hidratasa.

We report the case of a 56 year-old male with an atypical leiomyoma in the context of a cutaneous leiomyomatosis and a family history of uterine leiomyomatosis. The genetic study revealed a mutation in the gene for the enzyme fumarate hydratase, but he has not had any renal malignancy so far. Atypical leiomyoma is a rare tumor that usually presents as a single lesion and is exceptional in patients with cutaneous leiomyomatosis. The relation between fumarate hydratase enzyme mutations with multiple leiomyomas, uterine leiomyomatosis and an increased risk of developing kidney cancer is widely known. However, the role of these mutations in the development of atypical leiomyomas is still impossible to clarify given the few cases reported in the literature.

Fumarase-deficient Uterine Leiomyomas: An Immunohistochemical, Molecular Genetic, and Clinicopathologic Study of 86 Cases.

Loss-of-function germline mutations in the fumarase (FH) gene of the Krebs cycle characterize hereditary leiomyomatosis and renal cell cancer syndrome. Fumarase (FH) deficiency can be diagnosed by the loss of immunohistochemical expression. In this study, we investigated the occurrence and clinicopathologic features of FH-deficient uterine smooth muscle tumors (SMTs). A total of 1583 uterine and 157 nonuterine SMTs were examined using a polyclonal FH antibody and automated immunohistochemistry, and 86 uterine leiomyomas with an FH loss were identified. The frequencies of FH deficiency for subcohorts of uterine SMTs were 1.6% for unselected nonatypical leiomyomas, 1.8% for cellular leiomyomas, 37.3% for atypical leiomyomas, and 0% for leiomyosarcomas. One extrauterine, retroperitoneal estrogen receptor-positive leiomyoma was also FH deficient. The patient age of FH-deficient uterine leiomyomas was 20 to 52 years (median, 38 y). Grossly, these tumors were often soft and amorphous resembling a fibrothecoma. Histologically, the FH-deficient nonatypical leiomyomas lacked cellular packeting and distinct collagenous zones and showed chain-like or palisading nuclear arrangements, prominent staghorn-shaped blood vessels, oval nuclei with no or at most mild atypia, small eosinophilic nucleoli, and a low mitotic rate (0 to 1/10 HPF). The FH-deficient atypical leiomyomas had nuclear atypia often manifesting as multinucleation, prominent eosinophilic nucleoli, and mitotic activity up to 7/10 HPF, with atypical mitoses seen in 32% of cases. However, similar histologic changes were seen in some non-FH-deficient atypical leiomyomas. Loss-of-function FH-gene mutations including 5 whole-gene deletions and 3 frameshift mutations were identified in 8 of 16 FH-deficient nonatypical leiomyomas using multiplex ligation-dependent probe amplification and Sanger sequencing, respectively. Follow-up data on patients with FH-deficient atypical uterine leiomyomas revealed 19 patients alive (median follow-up 27 y) and 5 patients dead. Deaths occurred 9 to 30 years after surgery at a median age of 72 years; causes of death could not be determined. These results indicate that FH-deficient uterine leiomyomas occur with a high frequency among atypical leiomyomas and infrequently in nonatypical leiomyomas and are often histologically distinctive. They seem to have a low biological potential and lack any significant association with leiomyosarcoma.

MED12 mutations and FH inactivation are mutually exclusive in uterine leiomyomas.

BACKGROUND: Uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer (HLRCC) patients are driven by fumarate hydratase (FH) inactivation or occasionally by mediator complex subunit 12 (MED12) mutations. The aim of this study was to analyse whether MED12 mutations and FH inactivation are mutually exclusive and to determine the contribution of MED12 mutations on HLRCC patients' myomagenesis. METHODS: MED12 exons 1 and 2 mutation screening and 2SC immunohistochemistry indicative for FH deficiency was performed on a comprehensive series of HLRCC patients' (122 specimens) and sporadic (66 specimens) tumours. Gene expression analysis was performed using Affymetrix GeneChip Human Exon Arrays (Affymetrix, Santa Clara, CA, USA). RESULTS: Nine tumours from HLRCC patients harboured a somatic MED12 mutation and were negative for 2SC immunohistochemistry. All remaining successfully analysed lesions (107/116) were deficient for FH. Of sporadic tumours, 35/64 were MED12 mutation positive and none displayed a FH defect. In global gene expression analysis FH-deficient tumours clustered together, whereas HLRCC patients' MED12 mutation-positive tumours clustered together with sporadic MED12 mutation-positive tumours. CONCLUSIONS: Somatic MED12 mutations and biallelic FH inactivation are mutually exclusive in both HLRCC syndrome-associated and sporadic uterine leiomyomas. The great majority of HLRCC patients' uterine leiomyomas are caused by FH inactivation, but incidental tumours driven by somatic MED12 mutations also occur. These MED12 mutation-positive tumours display similar expressional profiles with their sporadic counterparts and are clearly separate from FH-deficient tumours.

Human uterus myoma and gene expression profiling: A novel in vitro model for studying secretory leukocyte protease inhibitor-mediated tumor invasion.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that diminishes tissue destruction during inflammation. A recent report revealed high levels of SLPI expression in the oral carcinoma cell. In addition, overexpression of SLPI up-regulates metastasis in lung carcinoma cells. On the other hand, matrix metalloproteinases (MMPs) are proteinases that participate in extracellular matrix degradation. SLPI and MMPs are involved as accelerators of the tumor invasion process; however, their exact roles are not fully understood. Understanding the mechanism of tumor invasion requires models that take the effect of microenvironmental factors into account. In one such in vitro model, different carcinoma cells have been shown to invade myoma tissue in highly distinct patterns. We have used this myoma model, as it provides a more natural stroma-like environment, to investigate the role of SLPI in tumor invasion. Our results indicate that the model provides a relevant matrix for tumor invasion studies, and that SLPI is important for the invasion of oral carcinoma Ca9-22 cells in conjunction with MMPs. Furthermore, using bioinformatics analysis, we have identified candidates as key molecules involved in SLPI-mediated tumor invasion.

Leiomioma cutáneo atípico en un paciente con leiomiomatosis cutánea y mutación de la enzima fumarato hidratasa / Atypical leiomyoma in a patient with cutaneous leiomyomatosis and mutation of the enzyme fumarate hydratase

Se presenta un varón de 56 años con un leiomioma atípico en el contexto de una leiomiomatosis cutánea, con antecedentes familiares de miomatosis uterina y con estudio genético que revela una mutación en el gen de la enzima fumarato hidratasa, sin que hasta el momento presente ningún tipo de neoplasia maligna renal. El leiomioma atípico es un tumor poco frecuente, que usualmente ocurre de forma aislada, siendo excepcional la presentación en pacientes con leiomiomatosis cutánea. Es ampliamente conocida la relación de la mutación de la enzima fumarato hidratasa con leiomiomas mútiples, miomas uterinos y el mayor riesgo de desarrollar cáncer renal; sin embargo, el papel de esta mutación en el desarrollo de leiomiomas atípicos es por hoy imposible de esclarecer debido a los escasos casos recogidos en la literatura.

We report the case of a 56 year-old male with an atypical leiomyoma in the context of a cutaneous leiomyomatosis and a family history of uterine leiomyomatosis. The genetic study revealed a mutation in the gene for the enzyme fumarate hydratase, but he has not had any renal malignancy so far. Atypical leiomyoma is a rare tumor that usually presents as a single lesion and is exceptional in patients with cutaneous leiomyomatosis. The relation between fumarate hydratase enzyme mutations with multiple leiomyomas, uterine leiomyomatosis and an increased risk of developing kidney cancer is widely known. However, the role of these mutations in the development of atypical leiomyomas is still impossible to clarify given the few cases reported in the literature.

Lichong decoction reduces Matrix Metalloproteinases-2 expression but increases Tissue Inhibitors of Matrix Metalloproteinases-2 expression in a rat model of uterine leiomyoma.

OBJECTIVE: To study the effect of Lichong decoction (LD) on expression of matrix metalloproteinase- 2 (MMP-2) and metalloproteinase-2 (TIMP-2) in a rat model of uterine leiomyoma (UL). METHODS: UL was induced in rats using exogenous estrogen and progesterone. LD was administered (p.o.) for 4 weeks, and mifepristone (RU-486) used as a control. To observe the effect of LD on the uterine coefficient and uterine transverse diameter, a radioimmunoassay method was used to detect serum levels of sex hormones. Light microscopic analyses of pathologic changes in the tissues of UL rats were evaluated. Expression of the proteins of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in uterine tissues was assessed by immunohistochemical staining and western blotting. RESULTS: A UL model in rats was established successfully. LD reduced uterine weight, uterine coefficient, and uterine transverse diameter compared with untreated controls. LD reduced levels of estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone in our UL models. LD improved the pathologic condition of uterine muscle. Expression of MMP-2 protein decreased to varying extents in LD-treated groups, but TIMP-2 levels were enhanced. LD appears to reduce MMP-2 expression and increase TIMP-2 expression in UL tissue. CONCLUSION: These data suggest that the mechanism of action of LD on ULs may involve reduction of MMP-2 expression and increase in TIMP-2 expression in rats.

Novel fumarate hydratase mutation in a family with atypical uterine leiomyomas and hereditary leiomyomatosis and renal cell cancer.

OBJECTIVE: To describe a novel mutation in the fumarate hydratase (FH) gene in a family with atypical uterine leiomyomas. DESIGN: Case report and review of the literature. SETTING: Academic community hospital. PATIENT(S): Three sisters who presented as nulligravidas aged 27-30 years with large atypical uterine leiomyomas. INTERVENTION(S): Abdominal myomectomy, robotic myomectomy, hysterectomy, gene sequencing. MAIN OUTCOME MEASURE(S): Identification of a family with hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome and a novel mutation in the FH gene. RESULT(S): Two of the three sisters tested positive for a novel FH mutation p.Leu99Glufsx6. The eldest sister was clinically diagnosed with HLRCC. The patients' father also carries the same mutation in the FH gene. The patients and their father are now undergoing yearly screening for renal cancer. CONCLUSION(S): Patients with HLRCC are at risk for developing renal cancer as well as losing their fertility via early hysterectomy. Physicians must be aware of this condition and refer at-risk individuals for genetic testing.