Molecular analysis of clonality of sporadic angiomyolipoma

Angiomyolipoma, which consists of three intimately intermixed components, smooth muscle, blood vessels, and adipose tissue, is variably considered a hamartoma, a choristoma or a true neoplasm. This study has investigated the clonality of sporadic angiomyolipomas in seven women, each with a single lesion, by determining the pattern of X-chromosome inactivation. Polymerase chain reaction (PCR) amplification of the highly polymorphic human androgen receptor gene (HUMARA) was performed on the DNA extracted from the paraffin-embedded lesional tissue microdissected to sample the admixed smooth muscle and blood vessel component (SMC/BV) and the adipose tissue component. All seven patients were heterozygous for HUMARA polymorphism upon amplification of undigested DNA from non-lesional tissue and were therefore informative for further analysis. In all patients, lesional DNA, representative of the components, was predigested with HpaII restriction enzyme for amplification of the methylated allele. In six patients, the lesions were clonal, while in one, polyclonal. The polyclonal lesion was small and had less than 20 per cent SMC/BV component. Microdissected SMC/BV component was clonal in 6/7 lesions; the scanty SMC/BV in the remaining lesion did not yield amplifiable DNA. Microdissected adipose tissue was polyclonal in all seven lesions. Angiomyolipomas are three clonal lesions due to a clonal smooth muscle cell and blood vessel component, while the polyclonal adipose tissue is probably metaplastic or reactive. Copyright © 1999 John Wiley & Sons, Ltd.     

Genotypic and clinicopathological characterization of Kaposi's sarcoma‐associated herpesvirus infection in Japan

Kaposi's sarcoma‐associated herpesvirus (KSHV) is related causally to Kaposi's sarcoma, primary effusion lymphoma, and a subset of cases of multicentric Castleman's disease. As the numbers of acquired immunodeficiency syndrome (AIDS) patients have increased, KSHV‐associated diseases have also increased in Japan. Sporadic cases of classic Kaposi's sarcoma have also been reported in Japan. In the present study, the clinicopathological characteristics of 75 samples, comprising 68 cases of Kaposi's sarcoma, 5 cases of primary effusion lymphoma, and 5 cases of multicentric Castleman's disease were investigated. All of these cases were positive for KSHV by immunohistochemistry or PCR analysis. All fifty‐two of the AIDS‐associated Kaposi's sarcoma cases were males, whereas 7 of the 13 non‐AIDS‐associated Kaposi's sarcoma cases were females. The mean age of patients with AIDS‐associated Kaposi's sarcoma or primary effusion lymphoma was 46 years, whereas the mean age of patients with non‐AIDS‐associated Kaposi's sarcoma or primary effusion lymphoma was 71.8 and 97.5, respectively. KSHV genotypes were determined based on the sequence of variable region 1 in the K1 gene. Genotypes A and C of KSHV were detected in both AIDS‐ and non‐AIDS‐associated Kaposi's sarcoma. Genotype A was detected more frequently in AIDS‐associated cases than non‐AIDS‐associated cases, suggesting that genotype C is broadly distributed in Japan, and genotype A spreads among AIDS patients. Genotype D was detected only in non‐AIDS‐associated Kaposi's sarcoma. These data confirmed the difference between AIDS‐ and non‐AIDS‐associated KSHV diseases with regard to age of onset, gender, and genotypes in Japan. J. Med. Virol. 82:400–406, 2010. © 2010 Wiley‐Liss, Inc.     

Disseminated peritoneal leiomyomatosis. Clonality analysis by X chromosome inactivation and cytogenetics of a clinically benign smooth muscle proliferation.

Disseminated peritoneal leiomyomatosis (DPL, leiomyomatosis peritonealis disseminata) is a rare condition in which multiple histologically benign smooth muscle tumorlets diffusely stud peritoneal and omental surfaces in females, predominantly of reproductive age. Although the distribution of these lesions suggests a metastatic process, DPL generally has a benign clinical course and has been regarded as a metaplastic process. We assessed clonality of 42 tumorlets and 15 normal tissues from four females with DPL by analyzing X chromosome inactivation as indicated by the methylation status of the androgen receptor gene (HUMARA). In each of the four patients, the same parental X chromosome was nonrandomly inactivated in all tumorlets, consistent with a metastatic unicentric neoplasm, or alternatively, selection for an X-linked allele in clonal multicentric lesions. Anomalous demethylation of the marker for X inactivation (HUMARA) was associated with loss of heterozygosity for markers spanning the X chromosome, or monosomy X, in part of one leiomyomatous lesion. Biallelic demethylation of the HUMARA microsatellite polymorphism was also found in one intramural leiomyoma. Two of six DPL lesions karyotyped had cytogenetic abnormalities involving chromosomes 7, 12, and 18, suggesting a pathogenesis in common with uterine leiomyomas.     

Vitiligo Associated with Kaposi's Sarcoma

A patient who had Kaposi''s sarcoma and an associated vitiligo is reported. This is the first known case of Kaposi''s sarcoma associated with vitiligo.     

Consistent polymerse chain reaction–single-strand conformation polymorphism pattern of human herpesvirus-8 in the course of classical Kaposi's sarcoma assumes its clonal origin

There is emerging evidence that Kaposi's sarcoma–associated herpesvirus (KSHV or HHV-8) has a central role in the pathogenesis of Kaposi's sarcoma (KS). The occurrence of HHV-8 in classical KS biopsies is reported irrespective of its clinical stage (patch, plaque, nodular). HHV-8 was detected in 25 of 28 formalin-fixed paraffin-embedded classical KS samples by nested polymerase chain reaction. In addition, in six patients multiple tumors were available (n = 21). Single-strand conformation polymorphism (SSCP) analysis of the amplicons showed uniform SSCP pattern of samples belonging to the same patient regardless of whether the KS was multiplex or developed again years after the first excision. Most of the SSCP patterns were confirmed by further sequence analysis. The presence of the same sequence variant of HHV-8 in various samples of the same patient supports the clonal origin of classical Kaposi's sarcoma. J. Med. Virol. 54:300–304, 1998. © 1998 Wiley-Liss, Inc.     

Clonality as a criterion in the differential diagnosis of chronic neutrophilic leukaemia

Chronic neutrophilic leukaemia (CNL) is a rare BCR/ABL negative myeloproliferative disorder of elderly patients, showing sustained neutrophilia and splenomegaly. Differentiation between CNL and leukaemoid reactions (LR) is problematic since both conditions share similar morphological features but is essential because CNL patients generally have a poor prognosis. We studied blood samples from 10 female patients with CNL or LR using the HUMARA assay to determine clonality patterns in neutrophils. T-lymphocytes of the patients were investigated as an internal control cell population. In all five CNL patients the neutrophils, and in four of them also T-lymphocytes were monoclonal, indicating that the latter may also originate from the neoplastic clone. In LR patients the neutrophils and T-lymphocytes were generally polyclonal except in one patient showing monoclonal neutrophils suggesting that this patient might be in the process of developing a myeloproliferative disorder. In females clonality studies of blood neutrophils using HUMARA aid in distinguishing patients with monoclonal CNL from polyclonal LR.     

Clonality Analysis of Benign Parathyroid Lesions by Human Androgen Receptor (HUMARA) Gene Assay

Benign conditions of the parathyroid gland have been classified as adenomas and hyperplasias. These entities however are difficult to distinguish when only a single gland is enlarged. Adenomas are defined as neoplastic clonal growths whereas hyperplasias are considered to be reactive processes of polyclonal origin. In order to analyze the clonal pattern of these lesions, we have studied hyperplasias and adenomas of parathyroid glands from women by the human androgen receptor (HUMARA) assay, a recently reliable and highly-lnformative technique based on the X-chromosome inactivation pattern in females. Samples consisted of formalin-fixed as well as frozen tissues. Informativeness with HUMARA marker was 87% (13/15 cases). All hyperplasias (5/5) and 6/8 adenomas yielded polyclonal results, since two alleles of similar intensity appeared when the lesion was HpaIl-digested. Two parathyroid adenomas had a loss of one X-alIeIe for the HUMARA gene and they were interpreted as monoclonal. These results show that parathyroid hyperplasias and adenomas, considered as multigland or monogland involvement diseases respectively, may be both polyclonal in origin, and that only a small subset of adenomas is found to be clonal. Consequently, clonality analysis cannot allow a clear distinction between these two entities as classically diagnosed. A different approach should be considering hyperplasia or adenoma when a polyclonal or monoclonal result has been obtained by clonality analysis.     

Evidence of clonality in chronic neutrophilic leukaemia

BACKGROUND: Chronic neutrophilic leukaemia (CNL) is a rare myeloproliferative disorder of elderly patients characterised by sustained neutrophilia and splenomegaly. The diagnosis of CNL requires the exclusion of BCR/ABL positive chronic myelogenous leukaemia (CML) and of leukaemoid reactions (LRs). The differentiation between CNL and LR is problematic because both conditions share similar morphological features; it is also important because patients with CNL generally have a poor prognosis. AIMS: To determine whether CNL and LR could be distinguished on the basis of different clonality patterns. METHODS: Blood samples from 52 women were studied using the human androgen receptor gene assay (HUMARA). RESULTS: Monoclonality was found in the neutrophils in all 17 patients with different myeloproliferative syndromes (MPSs), including those with CNL. In four of the patients with CNL, autologous T cells were also monoclonal, suggesting that they belonged to the neoplastic clone. This finding was in contrast to other MPSs in which T cells were almost always polyclonal. Of nine patients with clinically suspected LR, the neutrophils of five were polyclonal, whereas three patients had monoclonal neutrophils, suggesting that they might be in the process of developing an MPS. Among 26 healthy blood donors, 20 had polyclonal neutrophils and five showed skewed clonality patterns. One case of LR and one normal blood donor were scored "not informative" at the HUMARA locus. CONCLUSIONS: Clonality studies of blood neutrophils using HUMARA aid in distinguishing female patients with monoclonal CNL from those with LR. For the diagnosis of CNL, monoclonality of the neutrophils should be demonstrated whenever possible.     

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.     

Clonality analysis of benign parathyroid lesions by human androgen receptor (HUMARA) gene assay

Benign conditions of the parathyroid gland have been classified as adenomas and hyperplasias. These entities however are difficult to distinguish when only a single gland is enlarged. Adenomas are defined as neoplastic clonal growths whereas hyperplasias are considered to be reactive processes of polyclonal origin. In order to analyze the clonal pattern of these lesions, we have studied hyperplasias and adenomas of parathyroid glands from women by the human androgen receptor (HUMARA) assay, a recently reliable and highly-informative technique based on the X-chromosome inactivation pattern in females. Samples consisted of formalin-fixed as well as frozen tissues. Informativeness with HUMARA marker was 87% (13/15 cases). All hyperplasias (5/5) and 6/8 adenomas yielded polyclonal results, since two alleles of similar intensity appeared when the lesion wasHpaII-digested. Two parathyroid adenomas had a loss of one X-allele for the HUMARA gene and they were interpreted as monoclonal. These results show that parathyroid hyperplasias and adenomas, considered as multigland or monogland involvement diseases respectively, may be both polyclonal in origin, and that only a small subset of adenomas is found to be clonal. Consequently, clonality analysis cannot allow a clear distinction between these two entities as classically diagnosed. A different approach should be considering hyperplasia or adenoma when a polyclonal or monoclonal result has been obtained by clonality analysis. Presented in part at the 1998 Annual Endocrine Society Companion Meeting of the United States and Canadian Academy of Pathology. Boston. MA Feb. 28, 1998.     

Monoclonality of Atypical Adenomatous Hyperplasia of the Lung

Atypical adenomatous hyperplasia (AAH) of the lung has been postulated as a possible precursor lesion of bronchioloalveolar carcinoma (BAC). The clonality of AAHs from seven female patients was analyzed to determine whether AAH is a monoclonal expansion. All AAHs were identified in lungs surgically resected for BAC. The clonality of the BAC and bronchiolar metaplasia in each case was also analyzed. Approximately 500 cells in each lesion were precisely microdissected from methanol-fixed sections. Adjacent normal lung tissue was collected as a normal control. DNA was extracted for clonal analysis based on an X-chromosome-linked polymorphic marker, the human androgen receptor gene (HUMARA). HUMARA was found to be amplified with or without previous digestion by the methylation-sensitive restriction endonuclease HpaII. Five cases were informative. All 10 AAHs and 7 BACs obtained from the informative cases showed monoclonality, whereas the control cells showed polyclonality. Three different AAH lesions in a single case showed both possible patterns of monoclonality. BAC and contiguous AAH showed identical monoclonality in two cases. Two lesions of bronchiolar metaplasia, which was considered reactive, were polyclonal. Our results demonstrated the monoclonal nature of AAH, and this finding suggests that AAH is a precursor of BAC or a preneoplastic condition.     

A descriptive study of Kaposi's sarcoma in south Florida

A larger-than-expected incidence of Kaposi''s sarcoma in women is observed in south Florida. The possibility of a shift in cancer experience to groups previously considered at low risk (eg, women and blacks) or tumors (eg, Kaposi''s sarcoma) is suggested.     

Profound reduction of CD4+ lymphocytes without HIV infection: two cases from the horn of Africa

Idiopathic CD4+ lymphocytopenia is a disorder associated with low CD4+ T cell count and opportunistic infections resembling AIDS. Most cases are described in developed countries. We report two HIV-negative patients with idiopathic CD4+ lymphocytopenia and AIDS-defining events diagnosed in Djibouti. The first patient developed lesions of Kaposi''s sarcoma and the second one presented with pulmonary tuberculosis. Both patients died with severe immunodepression. In poor resource-areas where HIV testing may not be available it is important to bear in mind that severe immunodepression and a clinical presentation compatible with AIDS do not necessary carry the diagnosis of AIDS.     

Erdheim-Chester disease: case report, PCR-based analysis of clonality, and review of literature.

Erdheim-Chester disease (ECD) is a rare, distinct clinicopathologic entity with nearly pathognomonic radiographic features. The lesions consist of lipid-storing CD68 (+), CD1a (-) non-Langerhans' cell histiocytes, either localized to the bone or involving multiple organ systems in the body. Whether these histiocytic proliferations represent monoclonal neoplastic populations or are part of a polyclonal reactive process is unclear. We present a case report of ECD in a 35-year-old African-American woman with a progressive course over 6 years. We investigated the clonality of the histiocytes using the HUMARA assay on paraffin-embedded tissue sections but did not find any evidence that these cells represent a monoclonal population. In this report, the characteristics of ECD are reviewed, the genetic basis of the HUMARA assay is discussed, and our results in the context of other clonality investigations reported in the literature to date are summarized.     

Human herpesvirus 8 in Kaposi's sarcoma and Kaposi's sarcoma-mimicking vascular tumors.

Kaposi''s sarcoma (KS) had been a rare and unusual vascular tumor until a recent epidemic of a disseminated and fulminant form of KS in AIDS patients. Infectious agents have been suspected of causing KS, and recently partial genomic DNA sequences of human herpesvirus 8 (HHV8) have been identified in AIDS-associated KS lesions. Since then, genomic DNA sequences of HHV8 have been isolated in other forms of KS. Although the partial genomic DNA sequence of HHV8 was reported to be, if rare, identified in vascular tumors other than Kaposi''s sarcoma (KS), the presence of HHV8 in a very large fraction of KS indicates that detection of HHV8 by PCR is a useful auxiliary tool in differentiating KS from other KS-mimicking vascular tumors. We examined whether the 233-bp segment of the viral DNA was detected in Korean patients with KS and other KS-mimicking vascular tumors. HHV8 sequences were identified in all of nine classic type of KS but not in three epithelioid hemangioendotheliomas and seven angiosarcomas. Our results confirm the relatively restricted distribution of HHV8 and also argue against the likelihood of secondary colonization of KS cells by HHV8.     

Clonal analysis of the epithelial component of Warthin's tumor

The proliferation of the epithelial component of Warthin's tumor is generally considered to represent a neoplastic condition. There has been much controversy about the histogenesis of this tumor, and the clonality of the epithelial component has not been clarified. We examined the clonal status of epithelial cells of Warthin's tumor by using a polymerase chain reaction (PCR) method based on trinucleotide repeat polymorphism of the X chromosome-linked human androgen receptor gene (HUMARA) and on random inactivation of the gene by methylation. Total DNA was isolated from formalin-fixed, paraffin-embedded tissue from 16 women with Warthin's tumor. Of the 16 cases analyzed, 7 were heterozygous for the HUMARA polymorphism and informative. The epithelial components of the tumors from the 7 cases were microdissected under the light microscope, and were subjected to extraction of DNA and HUMARA analysis. Using a permanent aqueous mounting medium during microdissection, we succeeded in reducing the rate of contamination by lymphocytes in the samples to less than 10%. All 7 cases showed patterns of polyclonal proliferation in the HUMARA analysis. Our results showed the nonclonal nature of Warthin's tumor, suggesting that Warthin's tumor is a non-neoplastic tumor-like condition. HUM PATHOL 31:1377-1380.     

Treatment of classical Kaposi's sarcoma with intralesional injections of cidofovir: Report of a case

The effect of intralesional injections of cidofovir, a nucleotide analog with potent in vitro activity against human herpesvirus 8 (HHV-8), was studied in vivo in an HIV-negative patient with classical Kaposi's sarcoma (KS). After five weekly injections of the drug, no clinical, histological, immunohistological, or virological changes could be detected in comparison with saline-injected lesions. These findings suggest that, once the KS tumor has developed, active viral replication is no longer involved in the pathogenesis of the disease. Alternative hypotheses are that HHV-8 replication in blood-borne cells may foster growth of spindle cells in the skin, or that blocking HHV-8 may not affect existing lesions but may prevent new lesions from developing. J. Med. Virol. 55:215–218, 1998. © 1998 Wiley-Liss, Inc.     

Clonal analysis of a solitary follicular nodule of the thyroid with the polymerase chain reaction method.

Solitary follicular nodules of the thyroid occasionally create a diagnostic problem, especially in the differential diagnosis between adenoma and nodular hyperplasia To obtain confident histologic parameters of clonal lesions, we analyzed DNA samples prepared from paraffin-embedded archival tissue from 20 solitary follicular nodules of the thyroid for clonality with the polymerase chain reaction (PCR) method. On the base of X chromosome inactivation mosaicism, we tested restriction fragment-length polymorphism of the phosphoglycerate kinase (PGK) gene and a highly polymorphic short tandem repeat of the human androgen receptor (HUMARA) gene. Of 18 informative cases, 10 were monoclonal, 7 were polyclonal, and 1 showed microsatellite instability. All of the five completely encapsulated nodules were monoclonal. Four of the five unencapsulated nodules showed polyclonality. Of the seven partially encapsulated nodules, four were monoclonal, and the others were polyclonal. The former showed 50% or more of encapsulation degree, whereas the latter showed less than 50%. The capsule tended to be thicker in monoclonal nodules (mean, 0.33 mm) than in polyclonal nodules (mean, 0.13 mm). Other histologic features of the nodules and surrounding parenchymal changes had no significance with respect to predicting clonality. This study suggests that the degree of encapsulation and capsular thickness are morphologically important for predicting the clonality of the thyroid nodule.     

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。     

Chester-Erdheim disease: a neoplastic disorder.

Chester-Erdheim disease is a rare non-langerhans cell histiocytosis characterized by a xanthomatous infiltration of foamy macrophages. The cause and pathogenesis remain unclear. The aim of the present study was to determine whether Chester-Erdheim disease is a polyclonal reactive disease or a clonal neoplastic disorder. The clonal status of samples obtained from five patients with Chester-Erdheim disease was studied. DNA was extracted from fixed and paraffin-embedded sections after microdissection and clonal status was studied using the Xchromosome inactivation pattern of the human androgen receptor gene (HUMARA assay). One patient was homozygous for the HUMARA gene and noninformative. Three other cases were monoclonal. One was polyclonal, and this case showed a dense reactive infiltrate in association with spumous macrophages. This study suggests strongly that Chester-Erdheim disease is a monoclonal lesion consistent with neoplastic disorder. Thus, Chester-Erdheim disease may be considered as the "macrophage" counterpart of Langerhan's cell histiocytosis in the histiocytosis spectrum. Further studies are needed to establish the origin of this clonal proliferation.