Molecular evidence that the stromal and epithelial cells in pleomorphic adenomas of salivary gland arise from the same origin: clonal analysis using human androgen receptor gene (HUMARA) assay

Salivary gland pleomorphic adenomas are characterized by a biphasic growth of "epithelial" and "stromal" regions. The "epithelial" region is a compactly organized mixture of both luminal and nonluminal cells, whereas the stromal region is composed predominantly of the nonluminal cells. Using the polymerase chain reaction (PCR)-based HUMARA assay on DNA from formalin-fixed, paraffin-embedded tissues from pleomorphic adnomas of female patients, we intend to clarify the clonal relation between the luminal and nonluminal cells and the clonal nature of the morphologically diverse nonluminal cells in this tumor. HUMARA, the human androgen receptor gene, is located on the X chromosome and contains a segment of polymorphic CAG tandem repeats in exon 1. Several methylation-sensitive HhaI restriction sites are located 5' to these CAG repeats. It is an ideal tool to study clonality of female tissues by examining the methylation pattern. Of the 13 cases analyzed, 3 were homozygous at the HUMARA locus and therefore noninformative. The remaining 10 cases were informative. All 10 cases showed a monoclonal pattern in the stromal area, indicating that the morphologically diverse nonluminal cells are monoclonal. Eight of the 10 cases showed monoclonality in the "epithelial" areas, suggesting a common clonality between luminal and nonluminal cells. Of the remaining 2 samples, 1 was polyclonal for the "epithelial" region, and the other was not amplifiable. Our data provide the first molecular evidence that the luminal and nonluminal cells in pleomorphic adenomas arise from the same clone in most cases, and the morphologically diverse nonluminal cells are monoclonal.     

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.     

Warthin's tumor with malignant lymphoma. DNA analysis of paraffin-embedded tissue

The authors report a case of malignant lymphoma, small lymphocytic type, involving the lymphoid stroma of a Warthin's tumor of the parotid gland. This was confirmed by the presence of a monoclonal immunoglobulin heavy chain gene rearrangement, demonstrated by Southern blot hybridization of DNA extracted from paraffin-embedded tissue. Similar techniques showed only germline immunoglobulin gene bands in two control cases of Warthin's tumor.     

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.     

卵巢癌抑癌基因P53的初步研究

本研究采用聚合酶链反应一单链构象多态性分析(PCR-SSCP)新技术首先对16例卵巢癌及5例胎盘组织抑癌基因P53的第5、6外显于(Exon 5、6)PCR扩增产物进行SSCP分析，对其中3／16例异常表达产特DNA测序验证表明：1例核苷酸插入，2例核苷酸突变，5例胎盘组织对照检测无异常发现。进而同法补充追加了7例卵巢良性肿瘤，及5例正常卵巢组织P53的检测，无异常产物表达。从而推论抑癌基因P53在卵巢癌的启动．形成与进展有一定的生物效应。     

Molecular assessment of allelic loss in Warthin tumors.

Warthin's tumors are benign lesions of the head and neck that have a characteristic morphologic appearance. The etiology of Warthin's tumors is controversial and whether they are true neoplasms or developmental malformations continues to be debated. In this study, we examined 12 Warthin tumors with a molecular and immunohistochemical approach. Immunostains for p53 and p16ink were performed. The epithelial and lymphoid components of each lesion were microdissected and PCR was performed for 13 microsatellite markers at or near common tumor suppressor genes. The results were analyzed semiquantitatively using capillary electrophoresis. Frequency of allelic loss was calculated. The epithelial component of all tumors was negative for p53 and p16ink. By molecular genotyping there was only one case that had one locus with allelic imbalance, while the remainder had no evidence of clonal allelic loss. The immunohistochemical and molecular results in this study lend support to the hypothesis that Warthin tumors are non-neoplastic, as there was no evidence of aberrant staining for tumor suppressor gene protein products and no evidence of consistent clonal allelic losses.     

False aneuploidy in benign tumors with a high lymphocyte content: a study of Warthin's tumor and benign thymoma.

The quality of results of flow cytometric DNA content analysis of formalin-fixed, paraffin-embedded tissue may be affected by a number of preanalytical variables. We performed flow cytometric DNA content analysis on two types of benign tumors to investigate the effect of a prominent lymphocytic component: Warthin's tumor (N = 20) and benign thymoma (N = 8). Malignant tumors (N = 23) were included as DNA aneuploid controls. All tissues studied were archival material processed using Hedley's technique either without prolonged rehydration in water (day 0 samples) or with 24- or 48-hour rehydration (day 1 and day 2 samples, respectively). Image cytometric DNA ploidy analysis was also performed on most cases. Eight cases (40%) of Warthin's tumor and five cases (63%) of benign thymoma showed either hyperdiploid peaks or marked asymmetry on the day 0 DNA histograms; nine of the malignant tumors were aneuploid. The DNA histogram abnormalities of the benign tumors could be gated out by excluding the lymphocyte nuclei. None of the DNA indices of the benign tumors corresponded with expected deviations based on published chromosomal studies. All of the DNA histogram abnormalities of the benign tumors disappeared and/or fused with the main peaks on the day 1 or day 2 samples, except for one case of benign thymoma. All the DNA aneuploid peaks on the malignant tumors persisted with prolonged rehydration. Image cytometric DNA analysis showed a diploid pattern in all benign tumors. We conclude that a high lymphocyte content may be a cause of false aneuploidy in these benign tumors. Furthermore, the degree of rehydration appears to be an important factor in achieving optimum fluorochrome staining of DNA.     

Mycobacterium tuberculosis infection within Warthin's tumor: report of two cases

We report two patients with Warthin's tumor who were also infected with Mycobacterium tuberculosis. Case 1 was a 75-year-old woman with Warthin's tumor and multiple small epithelioid granulomas with caseous necrosis involving the submandibular gland. This patient died of tuberculous meningitis 4 months after biopsy. Case 2 was a 78-year-old man with a 10-year history of a parotid mass which had enlarged rapidly over 2 months. Surgical excision revealed Warthin's tumor and epithelioid granulomas involving the left parotid gland. DNA extracted from paraffin sections was amplified by nested polymerase chain reaction (PCR) with primer sets for the mycobacterial 65-KDa antigen gene. Restriction enzyme digestion of the PCR products could differentiate Mycobacterium tuberculosis from other mycobacteria in both cases. Although the histogenesis of lymphoid components of Warthin's tumor is controversial, the frequent prevalence of inflammation or necrosis and our present findings suggest these components have a similar behavior to regional lymph nodes.     

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。     

Mucoepidermoid carcinoma arising in Warthin's tumor: a case report

Malignant transformation of the epithelial component of Warthin's tumor is extremely rare. We describe our experience of mucoepidermoid carcinoma arising in Warthin's tumor of the parotid gland in a 35 year old female. The tumor removed from the parotid region was well encapsulated and histologically comprised ofmucoepidermoid carcinoma along with areas of Warthin's tumor. The pathogenesis and differential diagnosis of this rare occurrence have been discussed.     

Monoclonality of normal human colonic crypts

The monoclonality of human colonic crypts was demonstrated by human androgen receptor (HUMARA) gene assay following application of the crypt isolation method. DNA was extracted from an isolated single crypt, Hpa II digestion was performed before polymerase chain reaction (PCR) by primers spanning the HUMARA exon 1 region. The PCR product of a single crypt clearly showed allelic exclusion based on methylation status, while PCR product from a mixture of 40 crypts or colonic mucosa as a whole that included epitheliums and interstitial connective tissue had two bands. This method will facilitate the non-isotopic analysis not only of tumor clonality, but also of the nonnal structures derived from a single progenitor cell.     

Monoclonality of both pale cells and cuboidal cells of sclerosing hemangioma of the lung.

Sclerosing hemangioma of the lung remains poorly understood, and it is still unclear whether this lesion is neoplastic or not. It consists of two major cell types, pale cells and cuboidal cells. We analyzed the clonality of each cell types from six female cases of surgically resected sclerosing hemangioma. The pale cells and cuboidal cells were separated by microdissection from methanol-fixed sections, and DNA was extracted for clonal analysis based on an X-chromosome-linked polymorphic marker, the human androgen receptor (HUMARA) gene or the phosphoglycerate kinase (PGK) gene. The HUMARA and PGK genes were found to be amplified with or without digestion by the methylation-sensitive restrictive endonuclease HpaII. Five of six cases were informative. Pale cells and cuboidal cells showed the same monoclonality in all of the informative cases, whereas the control cells showed a polyclonal pattern. Our results demonstrated that sclerosing hemangioma is caused by monoclonal expansion of cells, confirming that it is a neoplasia. Moreover, the present data indicate that both pale cells and cuboidal cells are derived from the same cell.     

Adenomyoepithelioma of the breast with exaggerated proliferation of epithelial cells: Report of a case

A case of adenomyoepithelioma of the breast in a 58-year-old woman Is described. The tumor was a solitary, solid mass measuring 35X30 mm. Hlstologically, the tumor was composed of a proliferation of both epithelial and myoeptthelial cell components. The epithelial cell component showed extensive columnar cell change with clear cytoplasm and focal exaggerated proliferation with cytologic atypia and increased mitotic figures. DNA ploldy analysis revealed aneuploidy. DNA ploldy analysis might be used to predict the biological behavior of adenomyoepithelioma.     

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.     

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.     

Three distinct commonly deleted regions of chromosome arm 16q in human primary and metastatic prostate cancers

Human prostate cancers frequently show loss of heterozygosity (LOH) at loci on the long arm of chromosome 16 (16q). In this study, we analyzed prostate cancer specimens from 48 patients (Stage B, 20 cases; Stage C, 10 cases; cancer death, 18 cases) for allelic loss on 16q, using either restriction fragment length polymorphism (RFLP)- or polymerase chain reaction (PCR)-based methods. Allelic losses were observed in 20 (42%) of 48 cases, all of which were informative with at least one locus. Detailed deletion mapping identified three distinct commonly deleted regions on this chromosome arm: q22.1–q22.3, q23.2–q24.1, and q24.3-qter. On the basis of a published sex-averaged framework map, the estimated sizes of the commonly deleted regions were 4.7 (16q22.1–q22.3), 17.2 (16q23.2–q24.1) and 8.4 cM (16q24.3-qter). Allelic losses on 16q were observed more frequently in the cancer-death cases (11 of 18; 61%) than in early-stage tumor cases (9 of 30; 30%; P < 0.05). In 7 of 11 patients from whom DNA was available from metastatic cancers as well as from normal tissues and primary tumors, the primary cancer foci had no detectable abnormality of 16q, but the metastatic tumors showed LOH. These results suggest that inactivation of tumor suppressor genes on 16q plays an important role in the progression of prostate cancer. We also analyzed exons 5–8 of the E-cadherin gene, located at 16q22.1, in tumor DNA by means of PCR-single strand conformation polymorphism and direct sequencing, but we detected no somatic mutations in this candidate gene. Genes Chromosom Cancer 17:225–233 (1996). © 1996 Wiley-Liss, Inc.     

Hemizygous deletions of chromosome band 16q24 in Wilms tumor: detection by fluorescence in situ hybridization

Loss of heterozygosity (LOH) for markers on chromosome arm 16q in Wilms tumor has been linked to an increased risk of treatment failure. We therefore postulated that fluorescence in situ hybridization (FISH) with probes from this region might enhance current strategies for identifying high-risk patients at diagnosis. In a blinded comparative pilot study of 19 Wilms tumor samples from 18 patients with favorable histology, FISH and DNA polymorphism analysis yielded concordant results in 14 cases, either retention (n = 6) or loss (n = 8) of chromosome arm 16q markers. Discordant findings in 4 of the 5 remaining cases resulted from detection of LOH, but no loss by FISH. Two of these cases, directly comparable at marker D16S422, appeared to have tumor-specific uniparental disomy, in that 2 copies of D16S422 and the 16 centromere were evident, despite LOH. In 2 other cases, the discrepancies could be explained by LOH confined to loci distal to the D16S422 locus. In the fifth case, FISH detected 2 distinct populations of tumor cells, one characterized by normal diploidy and the other by monosomy 16, whereas DNA polymorphism analysis failed to indicate LOH altogether. Thus, FISH confirmed the presence of allelic loss (hence, the possible location of biologically important tumor suppressor genes) on the distal long arm of chromosome 16 in cases of favorable-histology Wilms tumor, with the advantages of technical simplicity, successful analysis of samples that were otherwise uninformative by analysis of DNA polymorphisms, and the addition of internal controls for chromosomal aneusomy. We suggest that combined analysis of the chromosome 16q region in Wilms tumor by FISH and DNA polymorphism analysis would improve evaluations to identify high-risk patients who might benefit from alternative therapy.     

Clonality studies in sacral chordoma

Chordomas are rare, slow-growing, primary malignant skeletal neoplasms. Chromosome analysis, telomere reduction and telomere activity, DNA microsatellite, and loss of heterozygosity studies have been performed on chordomas; however, the clonality status (monoclonal versus polyclonal proliferation) is unknown. The primary purpose of this study was to determine whether sacral chordoma is monoclonal or polyclonal in origin with the use of a polymorphic X-linked gene (AR; alias HUMARA) and X-chromosome inactivation studies. DNA was harvested from tumor and corresponding normal tissue from eight women (37-71 years) with chordoma. Clonality was determined using an X chromosome inactivation protocol and a polymorphic human androgen receptor gene (AR) located on the X chromosome. The procedure required a methylation-specific polymerase chain reaction (PCR) and determination of the ratio of active to inactive X chromosomes. Results were informative for seven of the eight women, with two separate X-linked alleles seen for the AR gene in the normal tissue. Expression of AR gene alleles from each of the two X chromosomes was present in the chordoma tumor, indicating a polyclonal proliferation in all seven women. Most solid tumors and skeletal neoplasms are polyclonal in nature. Our study indicates that chordoma is polyclonal in its pattern of proliferation.     

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.