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Results for “clonal analysis”

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PubMed2000

Clonal analysis of micronodules in virus C-induced liver cirrhosis using laser capture microdissection (LCM) and HUMARA assay.

Most hepatocellular carcinomas (HCC) arise from malignant transformation of regenerative cirrhotic nodules. Because HCC has a very poor prognosis, detection of these premalignant lesions may improve the management of patients with cirrhosis. In this regard, clonal analysis of liver micronodules should be of particular interest in order to differentiate polyclonal regenerative micronodules from monoclonal neoplastic potentially malignant micronodules. To address this issue, 112 micronodules from 15 cases of explanted liver cirrhosis were carefully microdissected from paraffin-embedded tissue using a laser capture microscopy system. Clonal analysis was performed by analyzing X-chromosome inactivation, as indicated by the methylation status of the human androgen receptor gene (HUMARA). For each microdissected micronodule, a large set of pathological features was evaluated and correlated with their clonal status. Clonal analysis showed that 57 micronodules (51%) were monoclonal and 55 (49%) were polyclonal. Prevalence of monoclonal nodules ranged from 25% to 71% according to cases. In all cases, mono- and polyclonal nodules were randomly distributed in the cirrhotic liver. Although the clonal status was not significantly affected by the presence or absence of macronodules in the adjacent liver, size of monoclonal micronodules was significantly larger than size of polyclonal micronodules (mean size of the monoclonal nodules: 3 + 0.1 mm vs mean size of the polyclonal nodules: 2.5 +/- 0.1 mm, p = 0.007). Among the elementary pathological features evaluated, only the presence of iron overload was correlated with a monoclonal status (p = 0.04). In conclusion, clonal analysis of liver cirrhosis shows that 51% of micronodules are monoclonal lesions, supporting the notion that liver cirrhosis is a multineoplastic lesion. Because monoclonality is a marker of neoplasia, cirrhosis with accumulation of monoclonal nodules may be carefully followed, and monoclonal nodules should be screened for additional markers to assess their biological behavior.

Adult↗

PubMed1983

Clonal analysis of corn plant development. I. The development of the tassel and the ear shoot.

The development of the tassel and the ear shoot has been investigated in corn (Zea mays L.). X irradiation of dry kernels and seedlings heterozygous for anthocyanin markers or for factors altering tassel and ear morphology results in the formation of clones (sectors) from cells of the apical meristem. Most tassels develop from 4 +/- 1 cells of the mature embryo. The expression of ramosa-1, tunicate, tassel seed-6, and vestigial is cell autonomous in the tassel. These genes act late in development and modify the developmental fate or decision of an individual clone and not of the whole group of cells producing a tassel. The ear shoot develops from lineages of one to three cells derived each from the L-I (outmost cell layer) and L-II (second cell layer) of the apical meristem. Typically the clones start in the ear shoot (in the husks and possibly in the cob), extend upward in an internode, continue along the margin of the leaf sheath or leaf blade at the node above, and terminate in this or the next higher leaf. The separation of lineages for ear shoot and internode occurs in the period around 13 days after sowing. The analysis of clonal boundaries shows that a small number of embryonic cells become isolated in their developmental capacity. This commitment process appears to be analogous to the process of compartmentation in the imaginal disks of fruit flies. The extent of proliferation of individual cells within a group of highly flexible and any particular clone does not generate a specific part of a tassel or an ear shoot. There must be cellular communication between various clones so that the overall size and morphology of an organ remain normal and more or less fixed. Thus the process of development in plants is also highly regulative in nature and shares many features in common with development in fruit flies.

Anthocyanins↗

PubMed2004

Clonality analysis of alveolar B lymphocytes contributes to the diagnostic strategy in clinical suspicion of pulmonary lymphoma.

The diagnostic procedure of chronic pulmonary opacities may envisage the search for non-Hodgkin lymphoma (NHL). Previous retrospective studies have shown that clonality analysis of bronchoalveolar B lymphocytes could reflect the clonality of pulmonary lymphocytes. Our objective was to define the diagnostic usefulness of bronchoalveolar lavage (BAL) B-lymphocyte clonality analysis in the setting of a clinical suspicion of both primary and secondary pulmonary lymphoma. A prospective BAL fluid B-cell clonality analysis was performed by polymerase chain reaction (PCR) in 106 consecutive patients presenting with a clinical suspicion of pulmonary NHL. Diagnosis was pulmonary B-cell lymphoma for 22 patients (13 primary and 9 secondary). When compared, pulmonary biopsy and BAL fluid have clonal identity. The detection of a strong B-cell clonal population in BAL fluid was associated with the diagnosis of pulmonary NHL (P <.0001), with a 97% specificity and a 95% negative predictive value. Thus, the absence of a dominant B-cell clone detection in BAL fluid could help to dismiss invasive investigations of pulmonary lesions. The detection of a dominant B-cell clone would lead to the performance of a pulmonary biopsy to get histologic diagnosis in primary pulmonary lymphoma and, by contrast, would avoid the need for biopsy in the setting of a secondary pulmonary lymphoma.

B-Lymphocytes↗

PubMed1988

Comparison of clonal analysis and DNA restriction analysis for typing of Neisseria meningitidis.

We have formerly described a clonal analysis of 423 serogroup A isolates of Neisseria meningitidis which distinguished 21 clones and 34 electrophoretic types. The clones were found to be uniform within distinct epidemiological situations and distinct between different sets of epidemics. Strains representative of the 34 electrophoretic types have now been analyzed by restriction endonuclease digestion of their genomic DNA's. The results correlated partially with the clonal assignments; the discrepancies were epidemiologically more consistent with the interpretation of the clonal analysis than with that of the restriction endonuclease analysis.

Bacterial Typing Techniques↗

PubMed1994

Clonal analysis of human gynecologic cancers by means of the polymerase chain reaction.

Clonality of human gynecologic cancers was analyzed in small DNA samples prepared from cryostat sections, by means of the polymerase chain reaction (PCR). The method used for clonal analysis was based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase (PGK) gene and on the differential methylation of the PGK gene due to random inactivation of 1 of 2 X-chromosomes by methylation in females. Among 52 gynecologic cancers tested, 25 were found to be heterozygous for the BstXI polymorphism of the PGK gene. All the 25 gynecologic cancers (4 cervix, 11 endometrium, 7 ovary and 3 fallopian tube) analyzed by the PCR-based method were monoclonal in origin while adjacent normal tissues were polyclonal. When DNA samples were prepared from widely separated sites of tumors and/or metastatic lesions, every sample was found to be monoclonal, and the same allele of the PGK gene was inactivated in each case. These results demonstrate that clonal analysis by PCR offers a good method for studying clonality in small DNA samples prepared from cryostat sections of tumors. This method could be applied to distinguish between benign and malignant gynecologic lesions.

Adult↗

PubMed1996

Clonal analysis of precancerous lesion of hepatocellular carcinoma.

BACKGROUND & AIMS: It remains to be established whether precancerous lesion (dysplastic nodule) of hepatocellular carcinoma (HCC) is a neoplastic or hyperplastic lesion. Clonal analysis of this lesion was conducted to elucidate this important issue on histogenesis. METHODS: The method for clonal analysis was based on restriction fragment length polymorphism of the X chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. Clonal somatic mutations were also analyzed by DNA fingerprinting with two multilocus probes (33.6 and 33.15), and loss of heterozygosity was studied using five single-locus probes (MS1[1p33-p35], MS31[7p22-pter], MS43a[12q24.3-qter], MS8[5q35-qter], and g3[7q36-qter]). RESULTS: Clonal analysis by the phosphoglycerokinase gene-based method showed that all of the five dysplastic nodules and seven HCCs were monoclonal in origin. DNA fingerprinting showed clonal somatic mutations in six of 10 dysplastic nodules and in six of nine HCCs. Loss of heterozygosity was found in one dysplastic nodule (7p22-pter) and two HCCs (1p33-p35 and 12q24.3-qter). CONCLUSIONS: These results confirm monoclonality of HCC and show that hepatic precancerous lesion (dysplastic nodule) is not a hyperplastic but a neoplastic lesion, consisting of monoclonal cells with genetic alterations.

Aged↗

PubMed1994

Discrimination between multicentric and multifocal carcinomas of the breast through clonal analysis.

BACKGROUND: An unanswered, important question concerning multiple breast carcinomas is whether they arise independently in the breast (multicentric) or are metastatic deposits from a single, primary carcinoma (multifocal). This issue was studied by clonal analysis of each focus of multiple breast carcinomas. METHODS: First, the clonality of 30 breast carcinomas was analyzed by the method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase (PGK) gene and on random inactivation of the gene. Second, the clonality of each focus of three multiple breast carcinomas was analyzed by the same method. RESULTS: Clonal analysis of the 30 breast carcinomas revealed that every carcinoma was monoclonal in origin, and one of two alleles of the PGK gene was inactivated at random in these carcinomas. Three patients with multiple breast carcinomas had three, three, and four histologically separate foci of carcinoma in the breast, respectively. Clonal analysis showed that each focus was monoclonal in origin and, in addition, the same allele of the PGK gene was inactivated consistently at each focus in every patient. CONCLUSIONS: The thesis that multiple carcinoma foci arise independently is unlikely to be true, because the probability that every independent focus happens to inactivate the same allele of PGK gene in every patient is very low. It seems more likely that a single primary carcinoma spreads throughout the breast to culminate in multiple secondary carcinoma foci. Therefore, it is concluded that multiple breast carcinomas are multifocal and not multicentric in origin.

Breast Neoplasms↗

PubMed1994

Clonal analysis of regenerative nodules in hepatitis C virus-induced liver cirrhosis.

BACKGROUND/AIMS: Based on histological criteria, regenerative nodules in cirrhotic liver have been generally considered to result from hyperplastic proliferation of hepatocytes. Whether these nodules are hyperplastic or neoplastic has not been determined definitively. This study examined the issue by clonal analysis of each nodule. METHODS: The method for clonal analysis was based on restriction fragment length polymorphism of the X chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. RESULTS: Clonality of hepatocellular carcinoma (n = 7) and regenerative nodules (n = 76) induced by hepatitis C virus infection was analyzed. All carcinomas were monoclonal. Clonal analysis of regenerative nodules showed that 43% (33 of 76) were monoclonal in origin. Adjacent monoclonal nodules showed inactivation of the same allele of phosphoglycerokinase gene. Because the gene allele is inactivated at random, it is unlikely that each nodule happens to inactivate the same allele; it is more likely that monoclonal cell expansion is initiated before the nodule is established by septum formation. CONCLUSIONS: Monoclonal cell expansion is seen in a considerable number of regenerative nodules in cirrhotic liver. The results indicate that certain genetic changes, which are required for hepatocarcinogenesis, have already occurred in these nodules.

Base Sequence↗

PubMed1995

Progression of fibroadenoma to phyllodes tumor demonstrated by clonal analysis.

BACKGROUND: The histogeneses of fibroadenoma and phyllodes tumor of the breast appear to be closely related, but it is still unclear whether fibroadenoma can progress directly to phyllodes tumor. METHODS: This issue was studied by conducting clonal analysis of fibroadenoma and phyllodes tumors that were obtained sequentially from the same patient. One patient developed local recurrence of phyllodes tumor twice, and the other two patients each developed a phyllodes tumor after excision of a primary fibroadenoma. The method for clonal analysis was based on trinucleotide repeat polymorphism of the X chromosome-linked androgen receptor (AR) gene and on random inactivation of the gene by methylation. RESULTS: Clonal analysis revealed that all the three primary fibroadenomas were monoclonal and all four recurrent phyllodes tumors were also monoclonal in origin. In addition, the same allele of the AR gene was inactivated in fibroadenoma and phyllodes tumor(s) in each patient. The probability that phyllodes tumors of different origin happen to inactivate the same allele of the AR gene as fibroadenomas in every case is quite low. Rather, it is more reasonable to assume that the phyllodes tumor has the same origin as fibroadenoma. CONCLUSIONS: These results identified monoclonal fibroadenomas that can progress to phyllodes tumors.

Adult↗

PubMed2001

Hepatocellular carcinoma associated with focal nodular hyperplasia. Report of a case with clonal analysis.

We describe a hepatocellular carcinoma partially surrounded by focal nodular hyperplasia in a 65-year-old female patient. In order to clarify the relationship of the hepatocellular carcinoma and the adjacent focal nodular hyperplasia, clonal analysis was conducted. The clonal analysis was based on the methylation pattern of the polymorphic X-chromosome-linked androgen receptor gene (HUMARA). The allelic bands from the amplification of the focal nodular hyperplasia and of the hepatocellular carcinoma showed a significant reduction in the intensity of one of the two alleles as compared with two alleles of equal intensity in the buff coat after HhaI digestion, which indicated that these two parts were monoclonal. However, the inactivated allele in the focal nodular hyperplasia and that in the hepatocellular carcinoma were not identical. Therefore, the focal nodular hyperplasia and hepatocellular carcinoma probably derived from the clonal expansion of two different clones.

Aged↗

PubMed1999

Clonality analysis of granulocytes and T lymphocytes in healthy females by the PCR-based HUMARA method.

Clonality analysis utilizing X-chromosome inactivation has been used in the study of various diseases, including hematological malignancies. The human androgen receptor gene (HUMARA) assay is the newest of such methods, and the majority of the female population can be assessed by this relatively simple procedure. One problem in using these clonality analysis methods, however, is that there may be significant variation in Lyonization in blood cells in normal individuals. To determine the diversity in X-chromosome methylation patterns, which reflect Lyonization, assessed by the HUMARA assay in the supposedly normal population, we analyzed granulocytes and T cells from 97 relatively young (18- to 35-year-old) healthy female volunteers. We found that the methylation patterns in the two HUMARA alleles were distributed even more widely, both in granulocytes and in T cells, than previously reported with other methods. We also found that the deviation of methylation in granulocytes and T cells was well correlated. Thus, we conclude that appropriate controls from the same individuals, such as T cells in the case of stem cell disorders, should always be employed to conclusively determine whether certain cells of hematopoietic origin are clonal.

Adolescent↗

PubMed1997

[Clonal analysis of blood cells using X-linked HUMARA gene polymorphism].

OBJECTIVE: To explore the method for clonal analysis of blood cells. METHODS: Thirty female AML patients and 20 normal female subjects were analysed by the analysis of X-linked HUMARA gene polymorphism and methylation pattern. RESULTS: The heterozygote rate of HUMARA gene in Chinese women was 88% and the gene had a stable methylation pattern. Out of 20 control females, 17 were heterozygotes and among them 15 were polyclonal, 2 (11.8%) showed skewed X-inactivation. All heterozygote AML were clonal hematopoiesis. CONCLUSION: HUMARA gene polymorphism is a good marker for clonality analysis of blood cells.

Adult↗

PubMed1999

Consecutively Occurring Multiple Fibroadenomas of the Breast Distinguished from Phyllodes Tumors by Clonality Analysis of Stromal Tissue.

We present a woman with consecutively occurring multiple fibroadenomas of thebreast distinguished from phyllodes tumor by clonality analysis of stromal tissue. Fifty-three masses developed in her right breast in an 11.5 year period since first onset. A clonality analysis, performed by polymerase chain reaction (PCR), focused on the Hpa II site within exon 1 of the androgen receptor (AR) gene. Estrogen receptor (ER) alpha immunohistochemistry was performed with a specific polyclonal antibody against the hormone binding domain of the protein. Since all of the nodules were polyclonal in stromal tissues, they were hyperplastic rather than true neoplasms such as phyllodes tumors. The co-existence of abundant adenosisfoci and numerous sub-clinical small nodules in the adjacent breast tissue alsosuggested proliferative stimuli throughout the breast. ER alpha protein was expressed only in the nuclei of epithelial cells. The possible influence of ER positive epithelial cells on stromal cell growth is discussed.

Journal Article↗

PubMed2003

Molecular immunoglobulin/T- cell receptor clonality analysis in cutaneous lymphoproliferations. Experience with the BIOMED-2 standardized polymerase chain reaction protocol.

BACKGROUND AND OBJECTIVES: Molecular clonality analysis of immunoglobulin (Ig) and T-cell receptor (TCR) genes is a widely used diagnostic tool for discrimination between polyclonal, oligoclonal, and monoclonal lymphoproliferative skin lesions. We studied Ig/TCR clonality in a series of 60 patients with an initial suspicion of (primary) cutaneous B- or T-cell lymphoma (CBCL/CTCL). Clonality of Ig/TCR gene rearrangements was assessed by Southern blot (SB) and polymerase chain reaction (PCR) analysis using standardized PCR primers and protocols of the BIOMED-2 Concerted Action BMH4-CT98-3936. The obtained PCR products were subjected to heteroduplex (HD) and GeneScan (GS) analysis. We compared the data of 154 samples with the histopathologic diagnosis, based on the EORTC classification of skin lymphomas. DESIGN AND METHODS: Molecular results were largely concordant with histopathology. In 12 CBCL patients PCR analysis of Ig gene rearrangements detected clonality in 83% of cases whereas SB did so in 92%. Clonal TCR gene rearrangements were detected by SB in 68% of CTCL patients, whereas TCRG and TCRB PCR analysis detected clonality in 76% and 66% of cases, respectively. PCR GS analysis of TCR rearrangements appeared to be slightly more informative than HD analysis. Clonality assessment was particularly informative for studying involvement of extracutaneous sites, such as regional lymph nodes, peripheral blood, and bone marrow. INTERPRETATION AND CONCLUSIONS: Our study shows that the BIOMED-2 multiplex PCR analysis strategy is a reliable and useful technique in the diagnostic process of patients with an initial suspicion of (primary) CBCL/CTCL and for assessment of extracutaneous dissemination, provided that the results are interpreted in the context of clinical, histologic and immunophenotypic data.

Clone Cells↗

PubMed2003

Clonal analysis of granulocyte-monocyte colony-forming unit cells with the human androgen receptor gene in chronic myeloid leukemia.

Coexistence of Philadelphia chromosome-negative (Ph-) progenitors with the Ph+ clone in the early chronic phase of chronic myeloid leukemia (CML) has been documented in previous reports. A different evaluation of methods is needed to justify the clonality of the residual Ph- progenitors. Therefore, the X chromosome inactivation patterns in individual granulocyte-monocyte colony-forming unit (CFU-GM) colonies were studied with the clonality assay for the human androgen receptor gene. A prerequisite for this evaluation was the validation of T-lymphocytes and buccal cells as control cells representing the constitutional lyonization. The percentages of polyclonal CFU-GM cells were determined in 9 Ph+ women with CML and in 5 healthy women. Results of the clonal analysis of CFU-GM colonies were compared with those from reverse transcriptase-polymerase chain reaction analysis of single colonies for BCR/ABL transcripts. Both methods of CFU-GM cell analysis were in agreement regarding the presence of variable proportions (0%-94%) of normal cells in CML. Our results suggest that (a) T-cells and buccal cells have potential for use as controls for the clonal analysis of CML cases and (b) this method can evaluate the frequency of polyclonal/clonal CFU-GM cells in CML cases and is applicable to the analysis of myeloid clonal disorders that lack specific molecular markers.

Adult↗

PubMed1993

Use of X-linked clonal analysis in acute promyelocytic leukemia.

X-linked clonal analysis (XLCA) either using Glucose-6-phosphate dehydrogenase (G-6-P-D) polymorphisms or restriction fragment length polymorphisms (RFLP) and methylation analysis has provided considerable understanding of haematologic malignancy. Acute Promyelocytic Leukemia (APL) is characterized by a unique cytogenetic translocation t(15;17), frequent achievement of remission without a preceding phase of marrow hypocellularity after induction chemotherapy and a high rate of clinical response to all-trans retinoic acid (ATRA). In limited studies XLCA has provided insight into the pathogenesis and mechanism of drug action in this disease.

Humans↗

PubMed1997

Clonality analysis in tumours of women by PCR amplification of X-linked genes.

Modifications have been made to two polymerase chain reaction (PCR) methods for clonality analysis based on the inactivation patterns of two highly polymorphic X-linked genes encoding the androgen receptor (AR) and monoamine oxidase A (MAOA). These methods have been used to examine the clonal nature of frozen tissues from 42 tumours and 25 non-tumour controls from female subjects. Unbalanced inactivation patterns of the genes, which indicate monoclonality, were frequently observed in tumours of heterozygous (informative) cases (18/35 = 51.4 per cent for the AR gene, 9/30 = 30 per cent for the MAOA gene, and 21/38 = 55.2 per cent for both). Among 23 informative non-tumour controls, only one (4.3 per cent), a reactive lymph node, showed skewing in the AR gene. Successful detection of monoclonality was found to depend on the proportion of tumour cells in the tissues examined. None of the AR or MAOA informative cases containing less than 50 per cent of tumour cells showed imbalance in inactivation patterns. With more than 50 per cent of tumour cells in the samples, 66.6 per cent (18/27) of AR and 39.1 per cent (9/23) of MAOA informative cases showed allelic imbalance, with a combined frequency of 72.4 per cent (21/29) of both genes. Our results demonstrate that the methods described are useful for clonal analysis of tissue samples from female patients.

Breast Neoplasms↗

PubMedDate not supplied

Clonality analysis of archival cervical smears. Correlation of monoclonality with grade and clinical behavior of cervical intraepithelial neoplasia.

OBJECTIVE: To establish a polymerase chain reaction (PCR)-based clonality assay for archival cervical smears and examine its value in the detection of cervical intraepithelial neoplasia (CIN) and prediction of its clinical behavior. STUDY DESIGN: Dyskaryotic cells were microdissected from archival cervical smears of 33 cases and subjected to PCR-based clonality analysis of the androgen receptor gene. High-risk HPV subtypes were screened by PCR. RESULTS: Monoclonal patterns were found in 9/9 CIN 3 and 15/21 CIN 2, while polyclonal patterns were observed in the remaining 6 CIN 2 and 3/3 CIN 1. All patients with monoclonal CIN lesions, including 15 CIN 2, showed recurrence of the disease despite treatment. The original CIN 2 and recurrent CIN lesion in each of the 6 examined cases showed the same monoclonal pattern, suggesting a clonal link. In contrast, the patients with polyclonal CIN 1 or 2 became negative and remained disease free. High-risk HPV subtypes were found in all monoclonal CIN lesions, including 9 CIN 3 and 15 CIN 2, and in 4/6 polyclonal CIN 2 but not in CIN 1 lesions. CONCLUSION: Clonality analysis of cervical smears is potentially valuable in the identification of true neoplastic cells and prediction of clinical behavior of CIN 2 lesions.

Archives↗