Benign metastasizing leiomyoma: a cytogenetically balanced but clonal disease

Benign metastasizing leiomyoma (BML) is a rare condition, characterized by the occurrence of multiple smooth-muscle nodules, most often located in the lung after previous hysterectomy because of histologically benign appearing leiomyoma. Although the condition resembles a metastatic process, case studies provided evidence that it may be the result of an intravenous leiomyomatosis or an independent and multifocal smooth-muscle proliferation. Comparative genomic hybridization and X-chromosome inactivation analysis were used in a case of BML to determine whether pulmonary and uterine tumors are related one to another. A balanced karyotype, previously reported in leiomyomas and an identical X-chromosome inactivation pattern found in all tumorlets, is most consistent with a monoclonal origin of both uterine and pulmonary tumors and the interpretation that pulmonary lesions are metastatic.     

Gastric neuroendocrine neoplasms: tumour clonality and malignancy-associated large X-chromosomal deletions.

Type I gastric carcinoid tumours associated with corporal (body of stomach) atrophic gastritis (CAG) are benign tumours developing as the final step of a hyperplastic precursor sequence. The neoplastic nature of these tumours has been assumed but never proved. Type III gastric carcinoid tumours and neuroendocrine carcinomas are malignant neoplasms without known precursor lesions. To assess the neoplastic nature of type I carcinoids, the clonal status of 35 tumours from 23 female patients was investigated using the human androgen receptor (HUMARA) gene test, which is based on the pattern of X-chromosome inactivation. For comparison, the same test was also performed on four type III carcinoids and two neuroendocrine carcinomas. DNA extracted from paraffin sections was digested with Hha I restriction enzyme and then amplified by polymerase chain reaction (PCR) using established HUMARA primers. The PCR products were analysed in an automated DNA sequencer. In a complementary analysis of the same tumours, loss of heterozygosity (LOH) on the X chromosome was studied using three polymorphic markers (DXS989, DXS1003, DXS1192) in a PCR-microsatellite-based technique. After exclusion of non-informative cases, 14 of 16 type I carcinoids were found to be monoclonal on the basis of the pattern of X-chromosome inactivation. Monoclonality was also documented in one of three type III carcinoids and in the single neuroendocrine carcinoma, on the basis of LOH at the HUMARA locus, which per se can be regarded as evidence for clonality. Extensive LOH of the X chromosome involving at least two markers, was found in all metastasizing tumours (two type III carcinoids and two neuroendocrine carcinomas), but in none of the 27 benign carcinoids of types I and III. These results indicate that most type I carcinoids are true monoclonal neoplasms and that malignant evolution in gastric neuroendocrine tumours is associated with extensive allelic deletion of one X chromosome.     

Intravenous leiomyomatosis: molecular and cytogenetic analysis of a case.

Apart from its hormone responsiveness, little about the pathobiology of intravenous leiomyomatosis (IVL), a rare smooth muscle proliferation, is known. We investigated the cytogenetics and molecular biology of IVL in a 40-year-old female who presented with an abrupt onset of dyspnea. In addition to the intracaval tumor mass composed of histologically benign smooth muscle, four distinct retroperitoneal "fibroids" were cytogenetically investigated. An identical abnormal karyotype, 45,XX,der(14)t(12; 14)(q15;q24),-22, was observed in all five specimens. Fluorescence in situ hybridization revealed three copies of HMGIC (alias HMGA2), two on the normal chromosomes 12 at 12q15, as well as another on the der(14) in the breakpoint region, suggesting that the 12q breakpoint occurred 5' (centromeric) to HMGIC (HMGA2), as has been frequently observed in uterine leiomyoma. Such similarity in chromosomal rearrangements suggests that there may be a pathogenetic relationship between IVL and uterine leiomyomata with t(12;14). Skewed X inactivation was observed in each tumor sample, but not in the myometrium. In each tumor, the lower molecular weight allele of HUMARA was nonrandomly inactivated. This pattern of X inactivation is most consistent with origin from a single transformation event, and in this regard, IVL more closely resembles disseminated peritoneal leiomyomatosis than typical uterine leiomyomata.     

Clonal analysis of focal nodular hyperplasia of the liver.

Recent evidence suggests that focal nodular hyperplasia of the liver (FNH) may represent a hyperplastic response to a vascular malformation, but the precise etiology remains unclear. We performed a clonal analysis of ten FNHs from nine patients by patterns of X chromosome inactivation. DNA isolated from paraffin-embedded specimens was subjected to polymerase chain reaction amplification for a highly polymorphic region of the human androgen receptor gene (HUMARA). Predigestion of tumor DNA with the methylation-sensitive, restriction enzyme HpaII allowed for selective amplification of the methylated (inactivated) allele. Of the nine patients analyzed, seven were heterozygous for the HUMARA polymorphism and informative for analysis. One informative patient had two lesions, for a total of eight FNHS. Amplification of lesional DNA after HpaII digestion demonstrated clonality in six of the eight informative cases. Paired tissue samples from different lesional areas were available in four of the six FNHs with evidence of clonality. In three of the four cases, DNA extracted from the two tissue samples showed both evidence of clonality and an identical pattern of X chromosome inactivation. In the remaining case, one sample showed evidence of clonality whereas the other was nonclonal. Three hepatic adenomas from two informative patients were also analyzed for comparative purposes, all of which showed evidence of clonality after HpaII digestion. The current study illustrates that most cases of FNH show a uniform pattern of X chromosome inactivation consistent with clonality.     

A polymerase chain reaction assay for non-random X chromosome inactivation identifies monoclonal endometrial cancers and precancers.

We hypothesize that endometrial carcinoma and their precursors share a monoclonal growth pattern and tested this thesis with archival paraffin-embedded tissues using a polymerase chain reaction-based assay for non-random X chromosome inactivation. Of the 10 well-differentiated endometrial adenocarcinoma cases with heterozygous markers (HUMARA, X-linked androgen receptor gene), 9 had skewed X inactivation consistent with a monoclonal process, and one contained a structurally altered HUMARA gene. X inactivation skewing similar to that of the tumor was seen in matched control polyclonal tissues of 4 (of 9) cases, caused by the small number of endometrial stem cells at the time of embryonic X inactivation. When the polymerase chain reaction assay was applied to four potential endometrial precancers (atypical endometrial hyperplasia) and matched control tissues, two were inconclusive, and two were found to be monoclonal. We conclude that 1) it is essential to include polyclonal control tissues in X inactivation analyses to determine whether skewing is a specific indicator of monoclonality; and 2) endometrial adenocarcinomas and some putative precancers, atypical endometrial hyperplasia, are monoclonal.     

Disseminated peritoneal leiomyomatosis: a case report

Disseminated peritoneal leiomyomatosis (DPL) is an exceedingly rare condition that simulates metastatic tumour and mostly occurs in women of reproductive age group subjected to an altered hormonal milieu, usually pregnancy. Here we report a case of a pregnant 28 year female with DPL, spontaneously regressing after pregnancy.     

Skewed X inactivation in manifesting carriers of Duchenne muscular dystrophy

We studied X inactivation patterns in manifesting carriers of familial and sporadic Duchenne muscular dystrophy (DMD) or unaffected carriers of DMD by analysis of the methylation of Hpall sites in the first exon of the human androgen-receptor gene (HUMARA) from peripheral blood samples. Three of the four manifesting carriers, four of the five asymptomatic carriers, and 31 of the 32 female controls were heterozygous for the CAG repeat of HUMARA. All manifesting carriers showed skewed X inactivation, while all unaffected carriers showed almost symmetrical inactivation. One family studied over three generations is noteworthy because it includes two mother/daughter pairs, one an affected pair with skewed X inactivation, and the other a phenotypically normal carrier pair with random X inactivation. On the other hand, the extent of X inactivation for each X chromosome in 31 female controls was widely distributed. These data suggest that in carriers of DMD, both affected and unaffected, it is valuable to analyze the pattern of skewed X inactivation because it provides important prognostic information. Carriers of DMD with skewed X inactivation might show slowly progressive myopathy with advancing age.     

Multiple angiomyolipomata of the liver: a case report.

Angiomyolipoma (AML) is a rare benign tumor that occurs most commonly in the kidney. Occasionally it may be found in the liver. Lesions in the liver are usually solitary. Multiple AMLs are extremely rare and are typically seen in patients with tuberous sclerosis. We now report an unusual case of a 46-year-old woman with multiple hepatic AMLs. There were more than 15 lesions distributed predominantly in the right hepatic lobe. The tumors ranged from 0.2 to 6 cm in size and consisted of a variable admixture of proliferating blood vessels, adipose tissue, and smooth muscle. There was no clinical evidence of tuberous sclerosis in this patient. Polymerase chain reaction amplification of the highly polymorphic human androgen receptor gene (HUMARA) was performed and the pattern of X chromosome inactivation was analyzed. Three of the five representative AML nodules showed a preferential loss of one of the two HUMARA alleles indicating a clonal proliferation with involvement of different alleles. Histologic examination of the corresponding lesions showed clonal lesions to be predominantly composed of epithelioid myoid cells while the polyclonal lesions were predominantly composed of adipose tissue. While the histologic diagnosis of AML in a surgical resection specimen is often straightforward, the radiographic, cytologic and intra-operative interpretation of a case with multiple lesions presents a considerable challenge.     

Disseminated peritoneal leiomyomatosis with malignant change,in a male

Summary Disseminated peritoneal leiomyomatosis (DPL) is a rare disorder, characterized by the occurrence of multiple leiomyomas scattered throughout the peritoneal cavity. Until this report DPL had been observed only in women and there is only one previous case with malignant change. A case of DPL in a male, complicated by sarcomatous transformation is reported. DPL has a definite malignant potential; patients with the disease should be followed closely for signs of progression.     

Pathogenetic mechanism for a female patient with hemophilia A北大核心CSCD

Objective: To explore the pathogenetic mechanism for a female patient affected with hemophilia A (hemophilia A, HA). Methods: Potential genetic defect was detected with inverse shifting-polymerase chain reaction (IS-PCR). The pattern of X chromosome inactivation was determined with a human androgen receptor assay (HUMARA assay). G-banded karyotyping was carried out to exclude potential chromosome aberrations. Results: IS-PCR showed that the defect of FVIII gene was the distal type of intron 22 inversion. The HUMARA assay showed that the X chromosome inactivation was non-random, and that the mother's X chromosome activity was lower than that of the father's X chromosome which has carried the inverted FVIII gene. No abnormalities were found with G-banded chromosomes. Conclusion: The prevalence of female HA patient may be caused by non-random inactivation of X chromosomes. © 2016, West China University of Medical Sciences. All rights reserved.     

Assessing the inactivation pattern in chromosome X among symptomatic carriers and women with haemophilia

X chromosome inactivation is a stochastic event that occurs early in female embryo development to achieve dosage compensation with males. Certain genetic mechanisms affect the normal process causing a skewed X inactivation pattern which has clinical relevance in female carriers of X-linked recessive disorders, like haemophilia. The most commonly used assay to evaluate the X inactivation pattern is the PCR amplification of the human androgen receptor gene (HUMARA). The use of this technique in bleeding carriers and women with haemophilia allows identifying if their hemorrhagic symptoms are due to an unfavourable lyonization. Furthermore, these studies are important for understanding the X chromosome inactivation process in humans.     

Disseminated peritoneal leiomyomatosis postmorcellated resection of uterine leiomyomatous tissue

In a retrospective review, we identified six cases of disseminated peritoneal leiomyomatosis (DPL) that occurred after resection for uterine leiomyoma(ta) using a morcellation procedure between 2010 and 2016. DPL occurred in less than 1% of all patients who underwent a prior hysterectomy with morcellation, and DPL never occurred without having underwent such a resection. The median age of women at the time of their original resection of uterine tissue was 38.6 years; the median time interval until resection of DPL after the primary morcellation procedure was 73 months and the median age was 48 years. At the time of DPL resection, a median of 6.5 individual lesions was present per patient, with each lesion having a median size of 1.2 cm in the greatest dimension. The most common peritoneal sites of involvement included the sigmoid colon serosa, right pelvis/pelvic side wall, and anterior abdominal parietal peritoneum. The same parameters are described for previously reported cases of DPL in the literature developing after a morcellated resection of uterine leiomyoma(ta). The use of morcellating hysterectomy specimens with leiomyomata may lead to the development of DPL by seeding, may involve numerous peritoneal sites, and often presents 2 years after the original resection.     

Leiomyomatous neoplasms of the lung: a clinical, histologic, and immunohistochemical study

A series of 12 pulmonary lesions initially diagnosed as leiomyomatous in origin were reviewed. The original slides were analyzed for a series of histological features, the patients' charts were reviewed for clinical presentation and follow-up, and the usefulness of immunohistochemistry was assessed. Three groups were noted after analysis. Group 1, or benign lesions (three cases), all occurred in premenopausal female patients who remain alive and well. Group 2, or leiomyosarcomas (five cases), mostly occurred in older females, were thought to originate in the uterus, and resulted in death in four patients. In one of three cases of metastatic leiomyosarcoma, estrogen receptor was identified. Group 3 (4 cases) consisted of other diagnoses, including leiomyomatosis, fibrous histiocytoma, metastatic malignant melanoma, and metastatic synovial sarcoma. Immunohistochemistry was useful in separating nonsmooth muscle tumors and demonstrated muscle marker expression in all benign and most malignant smooth muscle lesions. Criteria are proposed for the diagnosis of pulmonary leiomyosarcoma, whether primary or secondary. In addition, if strict criteria are applied, the term "benign metastasizing leiomyoma" should be abandoned.     

掌纤维瘤病的克隆性分析

目的通过人雄激素受体（HUMARA）基因位点克隆性分析技术确定掌纤维瘤病是否为肿瘤性增生。方法收集12例女性掌纤维瘤病患者的组织蜡块,连续切片、HE染色后,采用激光显微切割技术获取梭形细胞丰富的区域,1例女性直肠腺癌组织蜡块作为阳性对照。酚-氯仿抽提DNA后,经甲基化敏感的HpaⅡ限制性内切酶消化,聚合酶链反应（PCR）扩增HUMARA基因,PCR产物经8％非变性聚丙烯酰胺凝胶电泳分离,凝胶成像系统分析。结果作为阳性对照的直肠腺癌被验证是单克隆起源的,说明了本实验的克隆性分析方法的有效性。12例掌纤维瘤病石蜡组织标本中,3例标本未扩增成功;1例标本的HUMARA基因为纯合子,不适于克隆性分析;其余8例标本在HpaⅡ酶切前后均显示2条条带,经凝胶成像系统软件分析2条条带的亮度相近,说明掌纤维瘤病是多克隆性增生。结论掌纤维瘤病是多克隆性增生,属于非肿瘤性增生。     

X chromosome inactivation and X-linked mental retardation

The expression of X-linked genes in females heterozygous for X-linked defects can be modulated by epigenetic control mechanisms that constitute the X chromosome inactivation pathway. At least four different effects have been found to influence, in females, the phenotypic expression of genes responsible for X-linked mental retardation (XLMR). First, non-random X inactivation, due either to stochastic or genetic factors, can result in tissues in which one cell type (for example, that in which the X chromosome carrying a mutant XLMR gene is active) dominates, instead of the normal mosaic cell population expected as a result of random X inactivation. Second, skewed inactivation of the normal X in individuals carrying a deletion of part of the X chromosome has been documented in a number of mentally retarded females. Third, functional disomy of X-linked genes that are expressed inappropriately due to the absence of X inactivation has been found in mentally retarded females with structurally abnormal X chromosomes that do not contain the X inactivation center. And fourth, dose-dependent overexpression of X-linked genes that normally “escape” X inactivation may account for the mental and developmental delay associated with increasing numbers of otherwise inactive X chromosomes in individuals with X chromosome aneuploidy. © 1996 Wiley-Liss, Inc.     

X inactivation as a mechanism of selection against lethal alleles: further investigation of incontinentia pigmenti and X linked lymphoproliferative disease.

Thirty-one females with incontinentia pigmenti (IP), 42 controls, and 11 females from four families segregating for X linked lymphoproliferative disease (XLP) were studied for evidence of skewed X inactivation by analysis of methylation at sites in the HPRT, PGK, and M27 beta (DXS255) regions of the X chromosome. Extensive skewing of X inactivation was present in blood from 4/42 (9.5%) control females and 11/31 (35%) of those with IP. This frequency of skewed inactivation was seen in both familial and sporadic cases of IP. Analysis of inactivation in mother/daughter pairs, both affected and control subjects, showed no familial consistency of pattern, arguing against specific mutations being associated with particular patterns of inactivation. In the only informative family where both mother and daughter were affected by IP and showed skewed inactivation, the IP mutation was on the active X chromosome. This argues against cell selection during early embryogenesis being the explanation for the skewed inactivation observed. These data confirm that skewed inactivation of one X is observed in lymphocytes from a significant minority of normal females, and is seen with raised frequency in IP heterozygotes. It is not, however, a universally observed phenomenon, and the relationship of X inactivity to the IP mutation appears to be complex. In the case of XLP, though skewed X inactivation patterns are seen in most disease carriers, the frequency with which this phenomenon occurs in normal females renders it an unreliable diagnostic marker for XLP carriers.     

X chromosome inactivation patterns in haematopoietic cells of female carriers of X-linked severe combined immunodeficiency determined by methylation analysis at the hypervariable DXS255 locus

The patterns of X chromosome inactivation were determined in 14 females from three unrelated X-linked severe combined immunodeficiency (XSCID) pedigrees. All the females were found to be heterozygous for the hypervariable DXS255 locus, enabling analysis of differential methylation of this locus in peripheral blood haematopoietic cells. All six obligate carriers manifested a unilateral X chromosome inactivation in the T lymphocyte population. Differential methylation analysis of T lymphocytes was subsequently applied to establish the carrier status of females at risk in the XSCID pedigrees. In the B lymphocyte population of four XSCID carriers a unilateral X chromosome inactivation was observed. Four other carriers had minor fractions and one carrier had a substantial fraction of B lymphocytes with the XSCID gene defect on the active X chromosome. Within single XSCID pedigrees the carriers manifested different patterns. In two pedigrees the granulocyte populations of all carriers showed a random distribution of X chromosome inactivation. In the third pedigree the granulocytes of the three carriers analyzed manifested complete inactivation of the X chromosome that carried the XSCID mutation, exposing a selective disadvantage of granulocytes that express the XSCID defect. The pedigree-dependent differences in the involvement of the granulocyte population suggest the existence of two distinct XSCID defects.     

Molecular analysis of clonality in Kaposi's sarcoma.

BACKGROUND: Kaposi''s sarcoma is considered to be an angioproliferative disease associated with a novel herpesvirus (KSHV/HHV8), but the precise pathophysiology of the lesion remains unclear. The study of clonality in Kaposi''s sarcoma using X linked DNA polymorphism has been difficult so far, because of a very strong prevalence of the disease in males. AIMS: To study the clonality of Kaposi''s sarcoma lesions. METHODS: An assay based on a methyl sensitive restriction digest followed by polymerase chain reaction (PCR) amplification of the highly polymorphic human androgen receptor (HUMARA) gene was used. Tissues from Kaposi''s sarcoma lesions and control tissues from the same patients were obtained from seven females, four with classic Kaposi''s sarcoma and three with AIDS associated Kaposi''s sarcoma. A cutaneous angiosarcoma was also analysed, for comparative purposes, and showed evidence of clonality after HpaII digestion. RESULTS: All patients were heterozygous for the HUMARA polymorphism and informative for analysis. In all patients, including four with a nodular form of Kaposi''s sarcoma and more than 70% spindle cells in the lesion, a polyclonal pattern of inactivation could be demonstrated. CONCLUSIONS: The Kaposi''s sarcoma lesion is first of all a polyclonal cell proliferation.