A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found thatHpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is l10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days, about the time of X-chromosome inactivation of the inner cell mass.An assay based on the use of HpaII prior to the polymerase chain reaction (PCR) (20,22) allowed us to study DNA methylation in individual mouse embryos at the time of X-chromosome inactivation. We probed methylation in the CpG-rich island at the 5' end of the X-linked gene for phosphoglycerate kinase (Pgk-J), at a HpaII site (H-7) showing female-specific methylation in adult somatic tissues (Fig. 1). We found that site H-7 is not significantly methylated in male embryos but that in female embryos DNA methylation begins near 5.5 days and then rises, in both the embryo proper and the extraembryonic tissues, reaching nearly the adult level by 6.5 days.X-chromosome inactivation occurs in the inner cell mass of female mouse embryos between 5.5 and 6.5 days, at about the time of implantation (3, 10), a stage when the embryo consists of fewer than a thousand cells and the sex is not easily distinguishable. For this reason, only one prior study has been done correlating methylation with X-chromosome inactivation in the early embryo. Lock et al. (9) pooled many nonsexed embryos and found that methylation at sites in the first intron of the Hprt gene takes place between 9.5 and 13.5 days, well after the time of X-chromosome inactivation and the methylation we observe in the Pgk-1 gene.The promoter and first exon of Pgk-J are part of a CpG island that has been well characterized in both mice and humans (Fig. 1) (15) (Fig. 2). To assay for methylation, we made use of the fact that PCR amplification occurs only if the DNA between the two primer sites is intact. We chose PCR primers that flank H-7 and used a quantitative PCR procedure (20) to assay for HpaII-resistant (methylated) molecules. We had previously found, using this assay, that while male-embryo DNA was not amplified by PCR after HpaII digestion, female-embryo DNA was amplified, giving a signal 50% that of undigested DNA. Our interpretation was that site H-7 is methylated only on the inactive X chromosome (22). Figure 3 shows results of the HpaII-PCR assay on DNA from ectoplacental cone, extraembryonic ectoderm, and embryonic ectoderm dissected from two 6.5-day embryos, one male and one female. HpaII-resistant genomic molecules were present (giving band G in lanes marked +) in all three tissues of the female but not the male embryo. The larger fragments seen in the lanes without HpaII treatment came from M13 DNA, which was added as a carrier and also as a control for completeness of HpaII digestion. Band I in Fig. 3 is the PCR product of an internal standard, amplified by the same primers used for genomic DNA. The internal standard was a DNA fragment identical to genomic DNA except for a deletion internal to the primer binding sit...
Postoperative LEL incidence increased over time. The results of the present study showed a significant correlation with removal of circumflex iliac lymph nodes and cellulitis with the incidence of LEL. Multicenter or prospective studies are required to clarify treatment efficacies.
The results of this study suggest that prognosis for patients with AGCT depends on the MI and LVSI. During the follow-up period of patients, they need to be examined for distant metastasis including liver.
Diffuse large B-cell lymphomas (DLBCL) are a biologically and clinically heterogeneous entity. Although some DLBCL represent transformation of follicular lymphomas (FL), the proportion that is of follicular center cell (FCC) origin remains uncertain. Immunophenotypic and genotypic markers used to suggest a FCC origin for a lymphoma (bcl-6 and CD10 expression, lack of CD138 expression, bcl-2 rearrangements [R]) or to subdivide DLBCL (bcl-2 expresssion, bcl-6 R) were therefore investigated in 22 FL and 44 DLBCL using paraffin section immunostains and Southern blot/polymerase chain reaction analysis. All FL tested were bcl-6؉ (19) and CD138؊ (22) with 16/19 also bcl-2 and CD10؉ (classic phenotype), one bcl2؉, CD10؊ (grade III) and two bcl2؊, CD10؉ (grade II or III). Bcl-2R was identified in 4/5 FL-GrI, 3/6 FL-GrII, and 1/3 FL-GrIII. Bcl-6R was found in 0/5, 2/4, and 0/3 FL, respectively. All but 3/41 DLBCL were bcl-6؉ with 17/37 also bcl-2؉ and CD10؉. Three of these cases were also CD138؉. Twelve bcl-6؉ cases were bcl-2؉, CD10؊, six bcl-2؊, CD10؉, and two bcl-2؊, CD10؊. The three bcl-6؊ cases were bcl-2؉, CD138؊ and two were CD10؉. Bcl-2R was identified in 5/27 DLBCL with 4/5 bcl-2؉, 3/4 tested CD10؉ and 4/4 bcl-6؉. Bcl-6R was identified in 7/26 including three with a classic FL phenotype. The vast majority of DLBCL in this study have an immunophenotype that supports a FCC origin. Although the proportion of DLBCL that co-expressed bcl-6, CD10 and bcl-2 was lower than for the FL, absence of bcl-2 or CD10 may be associated with higher grade FL. It is also possible that bcl-6 expression is not completely specific for a FCC origin. Only a minority of cases suggested postfollicular differentiation. Only a minority of DLBCL show bcl-2R, suggesting that many have a different molecular pathogenesis than most low-grade FL. Bcl-6R did not exclude a FCC origin. KEY WORDS: BCL-2; BCL-6; CD10; CD138; diffuse large B-cell lymphoma; follicular lymphoma; germinal center Mod Pathol 2000;13(11):1219 -1231Diffuse large B-cell lymphomas (DLBCL), as defined in the REAL and upcoming WHO lymphoma classifications, represent a heterogeneous entity that includes not only transformed (noncleaved or centroblastic) follicular center cell (FCC) lymphomas but also all other lymphomas composed of large and transformed B cells (1-3). The proposed WHO classification recognizes three relatively uncommon subtypes of DLBCL [mediastinal (thymic), intravascular, and primary effusion lymphoma] but the immunoblastic lymphomas, transformed/"high grade" marginal zone lymphomas of mucosaassociated lymphoid tissue (MALT) and presumably nodal types and all other types of transformed B-cell lymphomas are not distinguished from one another. The DLBCL were grouped together for two main reasons. First, it was difficult to reproduce some of the previously described morphologic distinctions (such as those between transformed FCC lymphomas and immunoblastic lymphomas). Second, it was unclear how, for example, large noncleaved (LNC) FCC/centroblastic lymphomas should ...
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