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Differentiation and clonality of lesional lymphocytes in small plaque parapsoriasis.

BACKGROUND: Small plaque parapsoriasis is an idiopathic chronic dermatosis characterized by patches on the trunk and extremities that are often smaller than 5 cm in diameter and that sometimes have a digitate contour. These latter cases are often referred to as digitate dermatosis. Histopathologic examination reveals a mild superficial perivascular lymphocytic infiltrate associated with mild spongiosis and parakeratosis. To characterize this disease more completely, we analyzed the differentiation and clonality of lesional lymphocytes using immunohistologic and molecular biologic methods. OBSERVATIONS: We studied five cases using a frozen-section immunoperoxidase technique. In each case, there was a predominantly CD4+ T-cell infiltrate admixed with CD8+ T cells, Langerhans cells/indeterminate cells, and macrophages. In three cases, the clonality of lesional T cells was studied by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified T-cell receptor-gamma gene rearrangements. Two cases showed a dominant clonal pattern, while one case exhibited a polyclonal pattern. Clinical follow-up disclosed persistent disease in one of the two clonal cases, while lesions in the other clonal case and the polyclonal case gradually resolved. CONCLUSIONS: Our findings indicate that small plaque parapsoriasis is a clinically indolent, histopathologically nonspecific, predominantly CD4+ T-cell-mediated disease that, at least in some cases, contains a dominant T-cell clone. These features put small plaque parapsoriasis into a category with certain other members of the parapsoriasis group, namely, pityriasis lichenoides and lymphomatoid papulosis, which have been shown to be clonal T-cell disorders despite their clinically benign course. It remains to be determined if the dominant T-cell clones identified in some cases of small plaque parapsoriasis can ever be the direct precursors of overt cutaneous T-cell lymphomas.

Adult↗

Clonal analysis of predominantly intraductal carcinoma and precancerous lesions of the breast by means of polymerase chain reaction.

Clonality of predominantly intraductal carcinoma (PIC) and precancerous lesions of the breast was analyzed by a method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. The application of polymerase chain reaction to this method enabled clonal analysis of small lesions. In order to eliminate the contamination by normal stromal cells, intraductal components were microdissected from the frozen sections of PIC under a dissection microscope. Clonal analysis of the intraductal components from seven PICs revealed that all were monoclonal in origin. In three PICs with intraductal spreading of carcinoma cells over nearly a whole breast gland, the intraductal components were collected from eight widely separated sites in each case. Clonal analysis of these samples showed that every sample was monoclonal and the same allele of the phosphoglycerokinase gene was consistently inactivated in each case. These results suggest that PIC arises as a single monoclonal carcinoma and spreads through the ducts over the gland rather than having multicentric origins. Clonality of precancerous lesions such as atypical ductal hyperplasia and intraductal papilloma arising in the terminal ducts was also studied. Intraductal components were microdissected from the paraffin sections of these lesions and subjected to clonal analysis. Both atypical ductal hyperplasia and intraductal papilloma were found to be monoclonal in origin, suggesting that certain genetic changes had already occurred in the precancerous lesions. A further study is needed to elucidate these genetic changes, which would greatly help our understanding of the mechanism of carcinogenesis.

Base Sequence↗

Differentiation of primary and secondary breast cancer with clonal analysis.

BACKGROUND: It is often difficult to draw a firm conclusion as to whether the second breast cancer is primary or secondary (metastasis from the initial breast cancer) in a patient with metachronous bilateral breast cancer. In this study we have applied clonal analysis of breast cancer to distinguish whether the second breast cancer is primary or secondary. METHODS: A 54-year-old woman underwent modified radical mastectomy of the right breast as a result of breast cancer. Five years later she had tumors in the right chest wall and left breast. Fine-needle aspiration cytologic examination revealed that both tumors were adenocarcinoma. To elucidate the origin of these tumors, clonal analysis was done on DNA samples prepared from cryostat sections of the initial right breast cancer and from fine-needle aspirates of the tumors in the right chest wall and left breast. The method for clonal analysis was based on restriction fragment length polymorphism of X chromosome-linked phosphoglycerokinase gene and on differential methylation of the gene caused by random inactivation of one of two X chromosomes. RESULTS: Clonal analysis revealed that clonal origin of the right breast cancer was different from that of the left breast cancer and identical to that of the chest wall tumor. Therefore it was concluded that the left breast cancer was primary and the chest wall tumor was concluded that the left breast cancer was primary and the chest wall tumor was a recurrence of the initial breast cancer. CONCLUSIONS: Clonal analysis appears to be a useful method in discriminating a primary from a secondary cancer.

Base Sequence↗

In situ hybridization analysis of lymphoproliferative disorders. Assessment of clonality by immunoglobulin light-chain messenger RNA expression.

Determination of clonality in B-cell lymphomas is a useful diagnostic adjunct. In situ hybridization (ISH) for the detection of kappa and lambda mRNAs has the potential to overcome some common specimen-related limitations in clonal assessment. Tritium-labeled antisense cRNA probes directed at conserved segments of the constant regions of the kappa and lambda mRNAs were used in an autoradiographic method to detect B-cell clonality. Using these probes, we analyzed 103 formalin-fixed, paraffin-embedded biopsy samples, and the results were subsequently compared to available immunophenotypic (all cases) and genotypic (50 cases) data. Of 103 samples, 82 (80%) had adequate RNA preservation as determined by actin RNA signals, and 73 (89%) of the 82 cases demonstrated concordant clonality assignment by both ISH and immunophenotyping. The remaining nine cases showed a specific form of discordance in that each exhibited no protein (Ig) expression but had evidence of mRNA immunoglobulin light-chain expression. Forty-five (90%) of 50 cases evaluated for immunoglobulin and T-cell receptor beta-gene rearrangements demonstrated concordant results with respect to clonality assignment by ISH. Thus, ISH demonstrates adequate sensitivity with respect to traditional methods of clonality assessment. However, its practical utility awaits the development of nonradioactive detection methods with adequate sensitivity to improve turnaround time.

Gene Rearrangement↗

Neoplastic progression of rat tracheal epithelial cells is associated with a reduction in the number of growth factors required for clonal proliferation in culture.

Factors regulating the proliferation of normal, preneoplastic, and neoplastic rat tracheal epithelial (RTE) cells were investigated to identify changes taking place during the progression of RTE cells to neoplasia. Normal RTE cells exhibit clonal proliferation in a serum-free medium containing pituitary extract, serum albumin, cholera toxin, epidermal growth factor, hydrocortisone, and insulin. All combinations of these six factors were examined for their abilities to support clonal proliferation of normal, preneoplastic, and neoplastic RTE cells. In general, preneoplastic RTE cells required fewer factors for proliferation than normal RTE cells, and neoplastic cells required fewer factors than preneoplastic cells. A common pattern of reductions has been identified in the growth factors required for the clonal proliferation of preneoplastic vs. normal RTE cells and for neoplastic vs. preneoplastic and normal RTE cells. Normal RTE cells exhibit clonal proliferation in a serum-free medium supplemented with a minimum of six factors: bovine serum albumin, bovine pituitary extract, cholera toxin, epidermal growth factor, hydrocortisone, and insulin. Preneoplastic RTE cells exhibit clonal proliferation in a serum-free medium supplemented with four factors: bovine serum albumin, bovine pituitary extract, hydrocortisone, and insulin. Finally, neoplastic RTE cells exhibit clonal proliferation in a serum-free medium supplemented with two factors: bovine serum albumin and bovine pituitary extract. These results suggest that the progression of RTE cells to neoplasia is associated with a series of changes in regulatory pathways that control cell proliferation.

Animals↗

A large proportion of patients with a diagnosis of essential thrombocythemia do not have a clonal disorder and may be at lower risk of thrombotic complications.

Essential thrombocythemia (ET) is traditionally considered to be a clonal disorder. No specific karyotypic abnormalities have been described, but the demonstration of clonality using X-chromosome inactivation patterns (XCIPs) has been used to differentiate ET from a non-clonal reactive thrombocytosis. However, these assays may be difficult to interpret, and contradictory results have been reported. We have studied 46 females with a diagnosis of ET according to the Polycythemia Vera Study Group (PVSG) criteria. XCIP results in 23 patients (50%) were uninterpretable due to either constitutive or possible acquired age-related skewing. Monoclonal myelopoiesis could be definitively shown in only 10 patients. Thirteen patients had polyclonal myelopoiesis, and in 8, it was possible to exclude clonal restriction to the megakaryocytic lineage. Furthermore, there was no evidence of clonal progenitors in purified CD34(+)CD33(-) and CD34(+)CD33(+) subpopulations from bone marrow of 2 of these 13 patients. There was no difference between patients with monoclonal and polyclonal myelopoiesis with respect to age or platelet count at diagnosis, duration of follow-up, incidence of hepatosplenomegaly, or hemorrhagic complications. However, polyclonal patients were less likely to have experienced thrombotic events (P =.039). These results suggest that ET is a heterogeneous disorder, and the clinical significance of clonality status warrants investigation in a larger study.

Adolescent↗

Case Report: Immune-driven clonal selection underlying lineage switch from B-Precursor acute lymphoblastic leukemia to acute myeloid leukemia following inotuzumab ozogamicin.

Lineage switch (LS), defined as a change in leukemic lineage during the disease course, is a rare but clinically significant event in acute leukemia and is typically associated with poor prognosis. Although LS has been increasingly reported following targeted immunotherapies, the clonal mechanisms underlying this phenomenon remain incompletely understood, particularly in cases without KMT2A rearrangement. We report a case of LS from B-precursor acute lymphoblastic leukemia (BCP-ALL) to acute myeloid leukemia (AML) following treatment with the CD22-targeted antibody-drug conjugate inotuzumab ozogamicin. To elucidate the clonal architecture underlying LS, targeted next-generation sequencing was performed on bone marrow samples obtained at multiple time points throughout the disease course. Genomic analysis demonstrated that the lymphoid and myeloid disease phases shared ancestral genetic alterations but displayed distinct mutational profiles. At the time of LS, TP53 and SMC1A mutations newly emerged, whereas only a subset of mutations detected at ALL relapse was retained. These findings suggest that the AML phase most likely resulted from the selective expansion of a genetically distinct subclone derived from a common progenitor, rather than the direct transdifferentiation of the dominant ALL clone, consistent with immunotherapy-driven clonal selection. Longitudinal genomic profiling revealed stepwise clonal evolution during disease progression, supporting a model of immunotherapy-driven clonal selection leading to LS. This case provides molecular evidence suggesting that immune-targeted therapy can promote expansion of minor pre-existing subclones with alternative lineage potential within a common progenitor even in non-KMT2A-rearranged leukemia. Our findings highlight the importance of comprehensive genomic monitoring during immunotherapy to identify therapy-resistant subclones and better understand mechanisms of lineage plasticity in acute leukemia.

Humans↗

Maternal transfer of infectious mouse mammary tumor retroviruses does not depend on clonal deletion of superantigen-reactive V beta 14+ T cells.

Female C3H/HeJ mice maternally transmit through their milk an infectious mouse mammary tumor retrovirus (MMTV) which causes clonal deletion of T cell receptor (TcR)V beta 14+ T cells reactive to the retroviral superantigen (SAG). To test whether CD4+ or CD8+ T cells are crucial for intestinal infection and maternal transfer of exogenous retroviruses, newborn mice lacking CD4 or CD8 molecules after gene targetting were raised by surrogate C3H/HeJ mothers. In CD8-/- mice, clonal deletion of TcRV beta 14+ cells reactive to the SAG from this exogenous MMTV occurred with delayed kinetics. Deletion of TcRV beta 14+ cells was not observed in CD4-/- mice up to 12 months after exposure to the retrovirus. In both CD4-/- and CD8-/- mice TcRV beta 5+ and TcRV beta 11+ T cells were deleted in the presence of genomically integrated endogenous MMTV (Mtv), indicating that the lack of SAG-induced clonal deletion was not due to a general defect in these mutant mouse strains. Although TcRV beta 14+ T cells were not deleted in CD4-/- mice, female CD4-/- mice nursed on C3H/HeJ milk maternally transmitted the retrovirus to their offspring, albeit with delayed kinetics. These data demonstrate that CD4+ and CD8+ lymphocytes influence clonal deletion events and that the mechanisms responsible for clonal deletion of SAG-reactive TcRV beta 14+ T cells may be different from mechanisms which allow the mammary tumor virus to enter the mammary gland and complete its infectious cycle.

Animals↗

Retrotransposable element derepression distinguishes DNMT3A-mutant from TET2-mutant clonal haematopoiesis.

Clonal haematopoiesis (CH) is driven by somatic mutations in haematopoietic stem cells that generate clonal populations detectable in peripheral blood and is present in 10-20% of individuals over the age of 65. Mutations in DNMT3A and TET2 are the most common drivers and have been linked to inflammatory phenotypes and increased risk of haematologic and cardiovascular disease. However, the cell-intrinsic mechanisms connecting these mutations to inflammatory signalling remain incompletely understood. Because retrotransposable elements (RTEs) are epigenetically regulated and can activate innate immune pathways when derepressed, we hypothesised that RTE reactivation may represent a mutation-specific mechanism linking clonal haematopoiesis driver mutations to inflammatory pathways. We analysed RTE expression and clonal burden in peripheral blood mononuclear cell (PBMC) samples from 56 individuals with CH and 12 non-CH controls using integrated genomic and transcriptomic approaches, with complementary validation by TARGET-seq across haematopoietic lineages. High variant allele frequency (VAF; > 10%) DNMT3A-mutant clones exhibited widespread derepression of RTEs, particularly LINE and LTR families, whereas TET2-mutant clones showed a trend towards reduced RTE expression relative to controls. Transcriptomic analyses revealed that DNMT3A high-variant allele frequency clones with elevated RTE expression were enriched for inflammatory signalling pathways, including TNF-α/NF-κB signalling, interferon responses, and senescence-associated signatures. In contrast, TET2-mutant clones lacked these RTE-associated inflammatory signatures and instead showed enrichment of oxidative phosphorylation, reactive oxygen species signalling, and a mechanistic target of rapamycin complex 1 pathway. These findings were reproduced in an independent cohort. Collectively, our results highlight mutation-specific inflammatory mechanisms in clonal haematopoiesis and provide a foundation for future functional and preclinical studies to determine whether modulation of RTE activity can influence the inflammatory phenotype of DNMT3A-mutant CH and represent a potential therapeutic strategy.

DNMT3A↗

Clonal integration and expression of human T-cell lymphotropic virus type I in carriers detected by polymerase chain reaction and inverse PCR.

Adult T-cell leukemia (ATL) is a neoplasm of mature helper (CD4) T lymphocytes, and human T-cell lymphotropic virus type-I (HTLV-I) has been suggested to be the causative virus of ATL. HTLV-I integrates its proviruses into random sites in host chromosomal DNA. Clonal integration has been observed in patients with ATL, including smoldering, chronic, and acute states. However, random and/or polyclonal integration has only been reported in a few asymptomatic HTLV-I carriers. To clarify the clonality of HTLV-I-infected cells in carriers, we used an inverse polymerase chain reaction (IPCR), which is more sensitive than Southern blot analysis. We used the peripheral blood momonuclear cells (PBMC) from 16 asymptomatic carriers and the separated CD4-positive cells. No cases showed either a monoclonal or polyclonal integration of the HTLV-I provirus by Southern blot. But, using IPCR, 7 of 16 cases showed either mono- or oligoclonal integration. In addition, the populations of clonal provirus in the total PBMC were frequently different from those in the CD4-positive cells. Three cases showed expression of HTLV-I tax/rex mRNA in the total PBMC, but no such expression was found in CD4-positive cells. In this study, an unexpected frequency of clonal HTLV-I provirus DNA was observed in HTLV-I carriers. These findings indicate that the clonal but nonmalignant proliferation of HTLV-I-infected cells already occurs even in HTLV-I carriers, and therefore that some other step is necessary to induce malignant proliferation.

Adolescent↗

A polymorphism of the X-linked gene IDS increases the number of females informative for transcriptional clonality assays.

Studies of clonality have been essential for understanding the hierarchy of hematopoiesis and the biology of malignancies. Most clonality assays are based on the X chromosome inactivation phenomenon in females; these assays detect protein polymorphisms, differences in DNA methylation, or transcripts of the active X chromosome. Assays based on protein polymorphisms or DNA methylation have significant shortcomings. The major disadvantage of transcriptional assays is their limited applicability since only approximately half of females are informative for these studies. We have developed a new transcriptional assay based on an exonic polymorphism of the X-chromosome gene IDS. This gene is located in the same X-chromosome region (Xq28) as G6PD and p55, two genes with exonic polymorphisms for which we previously developed transcriptional assays. We developed non-radioactive PCR-based assays for rapid screening of genotype and determination of clonality. We also report reaction conditions for a quantitative ligase detection assay of IDS allelic transcripts. The frequency of the IDS polymorphism is 46% in Caucasian females and 39% in African-American females; in combination with G6PD and p55, 76% of Caucasian females and 62% of African-American females are informative for these assays. While this gene is highly polymorphic in Caucasian and African-American females, it is not informative in Oriental females. We established that the IDS gene is in linkage equilibrium with G6PD and p55. Unlike methylation-based assays, this assay is suitable for studying clonality in non-nucleated cells such as platelets and reticulocytes. With the discovery of exonic polymorphisms of other X-chromosome genes, all females should eventually be suitable for X-chromosome transcriptional clonality analysis.

Antigens, Bacterial↗

Clonality of B-cells in portal lymphoid infiltrates of HCV-infected livers.

Evidence has been accumulating in favour of a role for hepatitis C virus (HCV) in the pathogenesis of human lymphoproliferative disorders. HCV infection has been documented in the majority of patients with essential mixed cryoglobulinaemia type II (MC-II); in patients with HCV infection, B-cell clonal expansion have been detected in peripheral blood and bone marrow, and a high prevalence of B-cell non-Hodgkin's lymphomas has been documented. Liver biopsies in chronic hepatitis C frequently show portal lymphoid infiltrates with features of B follicles, whose clonality has not yet been investigated. This study has analysed the B-cell clonality of portal lymphoid infiltrates from 16 patients with chronic HCV hepatitis. Portal tracts showing obvious lymphoid infiltrates were microdissected from the paraffin-embedded liver tissue sections and the clonality of lymphoid B-cells was tested using a polymerase chain reaction (PCR) approach designed to identify immunoglobulin heavy chain gene (IgH) rearrangements. A successful IgH-PCR analysis was achieved in 35 lymphoid infiltrates from 11 patients (seven with the four without MC-II) and yielded a single band in 21 cases, two bands in ten cases, and three bands in four cases. Comparison of the IgH-PCR amplification bands obtained from the different lymphoid aggregates of the same biopsy revealed that they differed in size. This finding indicates that each aggregate derives from the proliferation of one or a few founder B-cells, which are not related to each other. The results obtained in patients with and without MC-II were similar, suggesting that the presence of B-cell clonal proliferations in liver biopsies is independent of the occurrence of B-cells producing monoclonal IgMk cryoglobulins.

B-Lymphocytes↗

Field cancerization, clonality, and epithelial stem cells: the spread of mutated clones in epithelial sheets.

There has been considerable debate about the origin of human tumours, whether they arise from a single cell and are clonal populations or whether there needs to be some sort of co-operativity between cells for the neoplastic process to begin. Current theories subscribe to the clonal view, where a series of mutations in one cell begins a process of selection and clonal evolution leading to the development of the malignant phenotype. This review approaches this problem by asking how mutated clones, once established, spread through tissues before becoming overtly invasive. While there is substantial evidence in favour of independent origins of each tumour from a unique mutated clone, there are instances where such clones expand and remain cohesive, often involving a large area of tissue. The main example is the movement of mutated clonal crypts through the colorectal epithelium, by the process of crypt fission. In passing, the clonal architecture of early, pre-invasive lesions is examined, often with some surprising results.

Animals↗

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.

Actins↗

Production and propagation of fully inbred clonal lines in the Nile tilapia (Oreochromis niloticus L.).

Fully inbred clonal lines of fish are likely to be of great value in research on immunology, sex determination, quantitative genetics, and toxicology. In this study on the Nile tilapia (Oreochromis niloticus), gynogenesis or androgenesis were used to produce a first generation of completely inbred fish, from which clonal lines were established using gynogenesis, androgenesis, hormonal sex reversal and intraline crosses. The clonal nature of these lines was verified by using multilocus DNA fingerprinting and the isozyme locus ADA*. Although these lines might be expected to be monosex in nature (all-female XX or all-male YY depending on the clone), one line did contain both sexes of fish. The presence of males in this gynogenetic clonal line and data from progeny testing of these males suggested that this line was homozygous for an allele or combination of alleles at an autosomal locus or loci which caused female to male sex reversal but with limited penetrance. Outbred clonal lines were also produced by crossing between different inbred clones. J. Exp. Zool. 284:675-685, 1999.

Animals↗

Molecular analysis reveals somatically mutated and unmutated clonal and oligoclonal B cells in T-cell-rich B-cell lymphoma.

This paper describes the immunohistology and molecular genetics of 18 cases of T-cell-rich B-cell lymphoma (TCRBL). In all cases, the large B cells stained strongly for CD20, with more variable expression of CD79a, and were negative for CD30 and CD15. The majority of T cells were predominantly positive for TIA-1 and negative for CD57; a large population of histiocytes was present in all cases. Epstein-Barr virus (EBV)-coded RNA (EBER) was found in B blasts from four cases and in one case was present among the background lymphoid cells. IgH PCR products were generated in 16/18 cases and revealed clonal, oligoclonal and polyclonal PCR products in 12, two and two cases, respectively. In addition, TCRG clonal gene rearrangements were identified in two cases. TCRB gene rearrangements were polyclonal. Sequence analysis of seven cases with clonal/oligoclonal IgH gene rearrangements revealed functional sequences with predominant V(H)3 gene usage associated with various D genes and J(H)4 or J(H)6 gene segments. Four cases displayed varying degrees of replacement and silent mutations (1.8-21%), with one case exhibiting intraclonal heterogeneity; the distribution of mutations was indicative of antigen selection in three cases. The remaining three cases, including two cases with functional oligoclonal IgH rearrangements, harboured unmutated V region genes. The EBV-positive cases were associated with clonal, oligoclonal and polyclonal PCR products and with mutated and germline clonal sequences. These data indicate that TCRBL may be a heterogeneous entity associated with clonal and oligoclonal B cells derived from both germinal centre and naïve B cells.

Adult↗

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↗

Genetic diversity among Mycoplasma species bovine group 7: clonal isolates from an outbreak of polyarthritis, mastitis, and abortion in dairy cattle.

A comprehensive genetic analysis of 60 Mycoplasma sp. bovine group 7 isolates from different geographic origins and epidemiological settings is presented. Twenty-four isolates were recovered from the joints of calves during sporadic episodes of polyarthritis in geographically distinct regions of Queensland and New South Wales, Australia, including two clones of the type strain PG5O. A further three Australian isolates were also recovered from the tympanic bulla, retropharyngeal lymph node and the lung and another three isolates had unconfirmed histories. Six isolates originated from Germany, Portugal, Nigeria, and France. Twenty-four epidemiologically related isolates of Mycoplasma sp. bovine group 7 were recovered from multiple tissue sites and body fluids of infected calves with polyarthritis, mastitic milk, and from the stomach contents, lung and liver from aborted foetuses in three large, centrally managed dairy herds in New South Wales, Australia. Restriction endonuclease analysis (REA) of genomic DNA differentiated 29 Cfol profiles among these 60 isolates and grouped all 24 epidemiologically related isolates in a defined pattern showing a clonal origin. Three isolates of this clonal cluster were recovered from mastitic milk and the synovial exudate of clinically-affected calves and appeared sporadically for periods up to 18 months after the initial outbreak of polyarthritis indicating a persistent, close association of the organism with cattle in these herds. The Cfol profile representative of the clonal cluster was distinguishable from profiles of isolates recovered from multiple, unrelated cases of polyarthritis in Queensland and New South Wales and from other countries. All 24 isolates from the clonal cluster possessed a plasmid (pBG7AU) with a molecular size of 1022 bp. DNA sequence analysis of pBG7AU identified two open reading frames sharing 81 and 99% DNA sequence similarity with hypothetical replication control proteins A and B respectively, previously described in plasmid pADB201 isolated from M. mycoides subspecies mycoides. Other isolates of bovine group 7, epidemiologically unrelated to the clonal cluster, including two clones of the type strain PG5O, possessed a similar-sized plasmid. These data confirm that Mycoplasma sp. bovine group 7 is capable of migrating to, and multiplying within, different tissue sites within a single animal and among different animals within a herd.

Abortion, Veterinary↗