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J Sklar

Publications and source records attributed to J Sklar.

At least 55 records · Page 3Linked to original sources
PubMed1993

Clonal analysis by study of X chromosome inactivation in formalin-fixed paraffin-embedded tissue.

Analysis of clonality by X chromosome inactivation has proven to be a powerful strategy in the study of neoplastic and preneoplastic disorders (P. J. Fialkow, Biochim. Biophys. Acta, 458: 283-321, 1976; B. Vogelstein et al., Cancer Res., 47: 4806-4813, 1987). Recently, the gene for the androgen receptor has been shown to be a highly polymorphic locus in which methylation of DNA correlates with inactivation of one or the other X homologue (R. C. Allen et al., Am. J. Hum. Genet., 51: 1229-1239, 1992). Unlike other loci used for analysis of X inactivation, the methylation sites within the androgen receptor gene lie close to the region of DNA containing the polymorphism. Consequently, it should be possible to use methylation-sensitive restriction enzymes and polymerase chain reaction to study differential methylation among alleles of this gene in formalin-fixed and paraffin-embedded archival tissue specimens. To investigate this question, we performed clonal analysis on a variety of randomly selected, formalin-fixed, paraffin-embedded tumor tissues from 15 women. Thirteen of the women were found to be heterozygous for polymorphisms at the androgen receptor locus. Among these women, 11 tumors were clearly clonal in assays of methylation of the androgen receptor gene. Interpretation of results for the remaining two cases was complicated because of a skewed pattern of X chromosome inactivation found in normal control tissues. We conclude that analysis of methylation in the androgen receptor gene should allow study of clonality in most formalin-fixed, paraffin-embedded tissue specimens from women, including small preneoplastic lesions or rare conditions for which sufficient material is not available for analysis by other techniques.

Base Sequence↗
PubMed1993

An epidemiologic study of non-occupational lifting as a risk factor for herniated lumbar intervertebral disc. The Northeast Collaborative Group on Low Back Pain.

An epidemiologic case-control study of herniated lumbar intervertebral disc was conducted in Springfield, Massachusetts, New Brunswick, New Jersey, and New York, New York, to evaluate the role of several possible risk factors in the etiology of this disorder. Patients with signs and symptoms of herniated lumbar disc (N = 287) were matched to control subjects without back pain by age, sex, source of care, and geographic area. Of the total case-subject group, 177 were confirmed by surgery, computed tomographic scan, myelogram, or magnetic resonance imaging. This article focuses on non-occupational lifting, an activity not previously reported on. Frequent lifting of objects or children weighing 25 or more pounds with knees straight and back bent was associated with increased risk of herniated lumbar disc. This association was particularly strong among confirmed case subjects (relative risk = 3.95). Positive associations among confirmed case subjects were also seen for frequent lifting with arms extended (relative risk = 1.87) and twisting while lifting (relative risk = 1.90). No associations were found for frequent stretching or carrying. If confirmed in other investigations, these data suggest that instruction in lifting techniques should be extended into the home.

Adult↗
PubMed1993

Establishment of cell lines from B-cell precursor acute lymphoblastic leukemia.

In an attempt to establish permanent cell lines from children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), 123 clinical samples from 117 patients were cultured in vitro. Using a method which was successful for the growth of ALL with T-cell phenotype, 3% (2/74) of BCP-ALL samples from patients at diagnosis and 31% (9/29) of BCP-ALL samples from patients at relapse were established as cell lines. However, in most cultures, leukemic cells survived for only a few weeks and the majority of viable cells present after 28 days of culture were esterase-positive mononuclear cells. Based on the hypothesis that mononuclear cells inhibited leukemic cell growth, we evaluated the effect of a monocyte toxin, L-leucine methyl ester (Leu-OMe), on the growth of four frozen BCP-ALL samples. Thawed leukemic cells treated with Leu-OMe, but not untreated control cells, proliferated in three samples and one new cell line was established. Subsequently, when Leu-OMe was added to fresh leukemia cells in culture, leukemic cell lines were grown from 29% (4/14) of samples at diagnosis and 66% (4/6) of relapse samples. Overall, 20 BCP-ALL cell lines were established, all were Epstein-Barr virus (EBV)-negative, and authenticity of each cell line was verified by a direct comparison of the immunophenotype, karyotype, and genotype with the patient's tumor cells. This improved method of cell culture permits a higher success rate of cell line establishment from patients with BCP-ALL thereby aiding in analysis of B-lymphocyte transformation and neoplasia.

Adolescent↗
PubMed1993

The impact of new DNA diagnostic technology on the management of cancer patients. Survey of diagnostic techniques.

Revolutionary advances in technology have enhanced our understanding of the genetic changes that occur in cancer cells. This article summarizes some of the basic features of these techniques and describes their application to the identification of specific types of genetic alterations in cells. The emphasis is on their use in obtaining information that is of diagnostic and prognostic importance. The techniques fall into two broad categories; the first is the direct analysis of the chromosome pattern in metaphase cells or the indirect assessment of chromosome abnormalities in interphase nuclei. The second general category involves the isolation of DNA, RNA, or protein from the tumor cells and the analysis of these components for abnormalities related to the presence, absence, or amplification of a specific gene or its products or other alterations, eg, those due to chromosome translocations. The techniques described in this article have broad applicability to medicine in general and some familiarity with these techniques is critical for the practice of modern medicine.

Blotting, Southern↗
PubMed1993

Molecular genetic analysis of post-transplant lymphoproliferative disorders.

Lymphoproliferative disorders developing in the post-transplant setting are a devastating set of diseases with major implications for the patients who develop them. On the other hand, they have provided us with an opportunity to better understand the basic biology of these disorders and malignant lymphomas in general. Molecular gene rearrangement studies and the Epstein-Barr virus, which is found in the genome of most of the abnormal lymphocytes in these disorders, have been investigated to further our understanding of the development and progression of these diseases.

Gene Rearrangement↗
PubMed1993

Extrarenal Wilms' tumors. A study of their relationship with classical renal Wilms' tumor using expression of WT1 as a molecular marker.

BACKGROUND: Wilms' tumors (WT) are renal malignancies typically of childhood with classic histopathologic features. A candidate WT gene (WT1) has been described, expression of which is largely restricted to the developing genitourinary system. WT1 is highly expressed in most sporadic childhood renal WT, and inactivating mutations have been described, consistent with its role as a tumor suppressor gene. WT1, therefore, may be used as a molecular marker for renal WT in most cases. Rarely, do tumors, that histologically resemble WT, develop in adult kidneys or exceptionally at nonrenal sites. This study addresses the question, are extrarenal tumors that have the morphology of renal WT really a type of WT? We investigated this question by using WT1 expression as a molecular marker for WT. EXPERIMENTAL DESIGN: We studied WT1 expression in eight well-documented cases of extra-renal WT by RNA-RNA in situ hybridization using an 35S-labeled probe derived from a cloned WT1 gene sequence. RESULTS: WT1 mRNA expression was detected in 5 of 5 childhood cases of sporadic childhood renal WT and 2 of 8 extrarenal WT. Both WT1-positive extrarenal WT were endometrial primaries. The WT1-negative extrarenal WT included retroperitoneal (3), pararenal or paravesical (2), and paraspinal (1) primaries. The seven non-WT uterine malignancies (carcinomas and sarcomas) studied were WT1-negative. CONCLUSIONS: These findings suggest that some cases of extrarenal WT are related to classical renal WT by more than mere morphologic resemblance, as indicated by the detection of WT1 mRNA expression in at least a subset of these rare tumors. Furthermore, the results suggest different pathogeneses of subsets of extrarenal WT.

Adult↗
PubMed1992

Interallelic V(D)J trans-rearrangement within the beta T cell receptor gene is infrequent and occurs preferentially during attempted D beta to J beta joining.

Previous work has demonstrated that intergenic V(D)J rearrangement, a process referred to as trans-rearrangement, occurs at an unexpectedly high frequency. These rearrangements generate novel V(D)J combinations which could conceivably have some role in the normal immune system, and since they probably arise through chromosomal rearrangements akin to those associated with lymphoid neoplasia, they may also serve as a model for investigating recombinational events which underlie oncogenesis. In view of the existence of a mechanism that permits relatively frequent intergenic trans-rearrangements, it seems reasonable that interallelic trans-rearrangements involving segments belonging to each of the two alleles of a single antigen receptor gene might also occur. To determine the frequency of such rearrangements, we examined thymocytes of F1 progeny of a cross between SWR mice, which have a deletion spanning 10 of the known V beta segments, and NZW mice, which have a deletion involving all J beta 2 segments. Rearranged TCR-beta genes containing V beta segments from the NZW chromosome and J beta segments from the SWR chromosome were amplified from the DNA of F1 thymocytes with the polymerase chain reaction. Using this approach, we found that such rearrangements are relatively uncommon, being present in about 1 in 10(5) thymocytes, a frequency lower than that of V gamma/J beta intergenic trans-rearrangements. The ratio of conventional cis-rearrangement to interallelic trans-rearrangement for any particular V beta segment appears to be about 10(4):1. The structure of the junctions in all trans-rearrangements analyzed closely resembles conventional cis-rearrangements, indicating involvement of V(D)J recombinase in the ultimate joining event. However, in contrast to cis-rearrangements, a strong bias for inclusion of D beta 1 segments over D beta 2 segments was noted, suggesting that interallelic trans-rearrangement may occur preferentially during attempted D-J joining. J beta 2 segment usage in trans-rearrangements also appeared to differ from that expected from previously studied cis-rearrangements. The results have implications with respect to the events and timing of conventional cis-rearrangement during thymocyte differentiation, and the prevalence of various types of trans-rearrangements.

Alleles↗
PubMed1992

Changing antigen receptor gene rearrangements in a case of early pre-B cell leukemia: evidence for a tumor progenitor cell with stem cell features and implications for monitoring residual disease.

A case of acute lymphoblastic leukemia (ALL) was encountered in which the two clonal gamma T-cell receptor gene (TCR gamma) rearrangements found in bone marrow (BM) samples at relapse both differed from the single clonal TCR gamma rearrangement present in BM obtained at diagnosis 5 years previously. In contrast, two clonal Ig heavy chain gene (IgH) rearrangements present at relapse were identical to those present at diagnosis. Comparison of the DNA sequences of the relapse TCR gamma rearrangements with that of the diagnostic TCR gamma rearrangement indicated that they must have been generated de novo from TCR gamma loci in germline configuration. By polymerase chain reaction using clonotypic N-region oligonucleotide primers (N-PCR), cells bearing the diagnosis or relapse TCR gamma rearrangements were undetectable in the sample from the opposite time point. Two BM samples obtained at different times in clinical remission were both devoid of detectable residual tumor when analyzed by N-PCR, indicating a depth of remission of less than 1 tumor cell per 4 x 10(5) BM mononuclear cells. The tumor cells showed a primitive phenotype: T-cell antigen-negative, CALLA/CD10-negative, CD20-negative, CD19-positive, and positive for the myeloid marker My9. This case, which appears to represent a tumor arising from a progenitor cell with both early B-lineage and certain stem cell features, has implications for monitoring residual ALL and possibly also for treatment of the disease.

Adolescent↗
PubMed1992

Acute lymphoblastic leukemia occurring as a second malignant neoplasm in childhood: report of three cases and review of the literature.

PURPOSE: The long-term effects of childhood cancer and its therapy are a problem of increasing concern. One of the most important of these late effects is the development of second malignant neoplasms (SMNs), which occur in approximately 8% of children within 20 years of diagnosis of a malignancy. These secondary cancers may result (individually or in combination) from increased genetic susceptibility, the mutagenic effects of chemotherapy and/or radiation therapy, or chance. Whereas the development of acute nonlymphocytic leukemia (ANLL) as an SMN is a well-recognized phenomenon, acute lymphoblastic leukemia (ALL) has been infrequently described as an SMN in either adults or children. PATIENTS AND METHODS: We report three patients treated at our institution in whom ALL developed as an SMN after treatment for neuroblastoma, Wilms' tumor, and Hodgkin's disease. These cases prompted us to review the published literature for cases of secondary ALL in childhood. Patients whose initial malignancy was diagnosed at age less than 16 years were classified as pediatric patients. SMNs were defined as cancers of clearly distinct histologic type occurring 6 or more months after diagnosis of the first malignant neoplasm. RESULTS: Including the three index cases, a total of 18 children with secondary ALL are reviewed, and the clinical features are discussed and compared with those of secondary ANLL. CONCLUSIONS: This review summarizes the published case histories of secondary ALL. The data suggest that ALL represents approximately 5% to 10% of the cases of acute leukemia that arise as SMNs in both adults and children.

Adolescent↗
PubMed1992

Detection of the t(14;18) at similar frequencies in hyperplastic lymphoid tissues from American and Japanese patients.

Follicular lymphoma shows a wide geographic variation in incidence, occurring more frequently in the U.S. than in Japan. A translocation involving the bcl-2 gene on chromosome 18 and the immunoglobulin heavy chain gene on chromosome 14 is frequently found in follicular lymphomas and is believed to play a critical role in the pathogenesis of these tumors. Recently, bcl-2/IgH rearrangements have been detected in reactive lymphoid tissue obtained from European patients, indicating that such rearrangements occur at some low but measurable background rate. In non-malignant tissues, the polymerase chain reaction was used to study the frequency of bcl-2/IgH rearrangements in reactive lymphoid tissue obtained from American and Japanese patients to find out whether geographic variation in the incidence of follicular lymphoma was caused by differences in sporadic occurrence of the t(14;18). We found such rearrangements in 5 of 15 American hyperplastic tonsils and lymph nodes and 5 of 10 Japanese tonsils, an incidence close to that previously seen in European patients. These data suggest that the background incidence of such rearrangements is similar in all populations, regardless of the incidence of follicular lymphoma.

Adult↗
PubMed1991

Transrearrangements between antigen receptor genes in normal human lymphoid tissues and in ataxia telangiectasia.

The polymerase chain reaction (PCR) was used on DNA obtained from various normal lymphoid tissues to amplify chimeric TCR gene rearrangements involving J segments of the beta gene and V segments of the gamma or delta genes. As found previously for the transrearrangements between the gamma and delta genes, transrearrangements involving the beta gene were more abundant in DNA of the thymus than in DNA of the spleen, lymph node, bone marrow, or PBL. In addition, transrearrangements between Ig H chain V region segment and J segment of TCR delta chain were also found in DNA of normal thymus. Sequence analysis of the trans-rearrangement PCR products revealed structures closely resembling normal intragenic rearrangements, with N insertions and often D segments at the junctions between segments. The sequences analyzed suggest that transrearrangements arise through the action of normal lymphocyte recombinase, involve trans recognition of heptamer/nonamer recombination signals, and follow the 12 + 23 spacer rule. To test whether transrearrangements result from chromosomal rearrangements with breakpoints at the sites of Ag receptor genes, PCR was performed on the DNA of PBL from patients with ataxia telangiectasia, a disorder in which circulating lymphocytes often have numerous karyotypic abnormalities with breakpoints at the cytogenetic positions of these genes. Comparison of the results of PCR on this DNA and that of normal tissues demonstrated a substantially increased frequency for most types of transrearrangements investigated. These results support the interpretation that transrearrangement among TCR genes may occur by chromosomal rearrangement.

Ataxia Telangiectasia↗
PubMed1991

Chromosomal translocations joining LCK and TCRB loci in human T cell leukemia.

A case of T lymphoblastic leukemia (T-ALL) showing t(1;7)(p34;q34) as the sole karyotypic abnormality was investigated at the molecular level. Screening of a phage library of tumor DNA with a probe for the beta T cell receptor gene (TCRB), which maps to chromosomal band 7q34, resulted in the isolation of a clone containing DNA spanning the translocation breakpoint of the der(1) chromosome. This clone contained chromosome 1 DNA juxtaposed upstream of a D beta-J beta joint. Cloning of the corresponding germline region of chromosome 1 resulted in the isolation of a phage containing the breakpoint from the reciprocal, der(7), product, which showed chromosome 1 DNA joined downstream to a V beta segment. Comparison of germline and translocation clones demonstrated that breakage of chromosome 1 had occurred at the border of a tandem repeat of Alu sequences. To search for transcripts from DNA near the breakpoint, a chromosomal walk was initiated along chromosome 1. A probe consisting of chromosome 1 DNA from 24-30 kb upstream of the breakpoint hybridized to a transcript derived from the gene encoding the lymphocyte-specific tyrosine kinase p56lck, previously mapped to chromosomal band 1p34. The nonrandom nature of the breakpoints in this case was confirmed by the analysis of a second independent case of T-ALL containing a t(1;7) translocation, which was also found to show breakage within the LCK locus. The chromosomal breakpoint in the first case was localized 2 kb upstream of the lck upstream promoter and first nontranslated exon, while the breakpoint of the second case lay between the two alternative lck promoters, upstream of the second exon. Relative to normal thymus and activated T cells, levels of lck mRNA were greatly elevated in the first case and moderately elevated in the second. The existence of these translocations raises the possibility that alterations in the promoter region of the LCK locus may play a role in human cancer.

Base Sequence↗
PubMed1991

Sensitive detection of clonal antigen receptor gene rearrangements for the diagnosis and monitoring of lymphoid neoplasms by a polymerase chain reaction-mediated ribonuclease protection assay.

This report describes a novel assay involving the polymerase chain reaction (PCR) and RNase protection for the rapid and sensitive detection of malignant lymphoid cells by nucleotide sequences within their individual rearranged gamma T-cell receptor (TCRG) genes. In this assay, clonal rearrangements are amplified from the DNA of diagnostic tumor specimens using a consensus V segment primer and a consensus J segment primer to which the promoter for T7 RNA polymerase has been appended. The PCR product from this amplification is transcribed into a radiolabeled RNA probe. Test RNA transcribed from the opposite DNA strand is synthesized by similar methods from TCRG genes of a subsequent biopsy specimen. The test RNA is hybridized with the probe, and mismatched nucleotide sequences in the RNA hybrids are digested by RNase A. Detection of fully protected probe by means of polyacrylamide gel electrophoresis and autoradiography indicates the presence of malignant cells in the test specimen. Dilution experiments with DNA of cell lines from acute lymphoblastic leukemias (ALLs) show that detection of one tumor cell among 10(5) normal bone marrow cells is usually possible. Residual disease was also successfully detected in several cases of ALL during clinical remission, including detection in one case at the 10(-5) level. The procedure described here may provide a simplified and rapid method for the sensitive diagnosis and monitoring of lymphoid malignancies. This procedure should be applicable to most antigen receptor genes, and unlike most comparable methods, requires neither analysis of nucleotide sequence nor synthesis of tumor-specific oligonucleotide probes or primers.

Autoradiography↗
PubMed1991

TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms.

Previously we described joining of DNA in the beta T cell receptor gene to DNA of an uncharacterized locus in a t(7;9)(q34;q34.3) chromosomal translocation from a case of human T lymphoblastic leukemia (T-ALL). We now show that the locus on chromosome 9 contains a gene highly homologous to the Drosophila gene Notch. Transcripts of the human gene, for which we propose the name TAN-1, and its murine counterpart are present in many normal human fetal and adult mouse tissues, but are most abundant in lymphoid tissues. In t(7;9)(q34;q34.3) translocations from three cases of T-ALL, the breakpoints occur within 100 bp of an intron in TAN-1, resulting in truncation of TAN-1 transcripts. These observations suggest that TAN-1 may be important for normal lymphocyte function and that alteration of TAN-1 may play a role in the pathogenesis of some T cell neoplasms.

Amino Acid Sequence↗
PubMed1991

Chimeric gamma-delta signal joints. Implications for the mechanism and regulation of T cell receptor gene rearrangement.

Rearrangement of Ag receptor genes requires recognition by the lymphocyte recombinase of heptamer-nonamer signal sequences followed by two endonucleolytic cleavages and two DNA ligations to form the coding and signal joints. The phenomenon of trans-rearrangement, in which Ag receptor gene segments located on different chromosomes recombine to yield chimeric products, provides an in vivo system in which to investigate the ability of the recombinase to carry out each of these functions in trans. Trans-rearrangements between TCRG and TCRD loci, similar in structure and frequency to those observed previously in human lymphoid tissues, were demonstrated in normal mouse thymus by PCR with crossed V gamma/J delta and V delta/J gamma primer pairs. A simple mechanistic model for trans-rearrangement was then tested. This model posits an ability of the recombinase to catalyze the formation of both coding and signal joints in trans and therefore predicts that trans-rearrangements will generate chimeric signal joints. In adult thymus, chimeric D delta 2-J gamma 1 and D delta 2-J gamma 2 signal joints, containing fused heptamer-nonamer sequences, could be detected by PCR and were each present at frequencies sufficient to account for a large proportion of the corresponding TCRG/TCRD trans-rearrangements. In agreement with the predictions of the model, chimeric signal joints were found as both linear chromosomal and circular episomal DNA. The results provide a framework for understanding the formation of chromosomal translocations in normal and neoplastic lymphoid cells and support the possibility of a looping mechanism for standard gene rearrangement. To test the form of regulation of TCRG rearrangement, the frequencies of specific signal joints from standard and trans-rearrangements were compared. Although J gamma 1 and J gamma 2 segments participated with equal frequency in trans-rearrangement with D delta 2, only the J gamma 1 segment participated in standard rearrangement with V gamma 5. The results suggest that V-J recombination in the TCRG locus is regulated directly at the DNA level by cis-acting constraints which do not affect the accessibility of individual TCRG gene segments to recombination in trans.

Animals↗