Regulatory T cell frequencies are increased in preterm infants with clinical early-onset sepsis.

The predisposition of preterm neonates to invasive infection is, as yet, incompletely understood. Regulatory T cells (Tregs ) are potential candidates for the ontogenetic control of immune activation and tissue damage in preterm infants. It was the aim of our study to characterize lymphocyte subsets and in particular CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) Tregs in peripheral blood of well-phenotyped preterm infants (n = 117; 23 + 0 - 36 + 6 weeks of gestational age) in the first 3 days of life in comparison to term infants and adults. We demonstrated a negative correlation of Treg frequencies and gestational age. Tregs were increased in blood samples of preterm infants compared to term infants and adults. Notably, we found an increased Treg frequency in preterm infants with clinical early-onset sepsis while cause of preterm delivery, e.g. chorioamnionitis, did not affect Treg frequencies. Our data suggest that Tregs apparently play an important role in maintaining maternal-fetal tolerance, which turns into an increased sepsis risk after preterm delivery. Functional analyses are needed in order to elucidate whether Tregs have potential as future target for diagnostics and therapeutics.

[Study of HRas1 minisatellite frequencies in children with thyroid papillary cancer]. / Izuchenie chastot minisatellita HRas1 sredi detei s papilliarnym rakom shchitovidnoi zhelezy.

The "rare" alleles of HRas1 gene minisatellite are well-known factor of predisposition to many kinds of cancer. We have studied HRas1 minisatellite frequencies among patients with papillary thyroid cancer which is related to consequences of Chernobyl accident. The HRas1 minisatellite was analysed in 32 patients who suffered from papillary carcinoma and underwent operation in 1996-2001 and in 75 Belorussian residents. Of 64 HRas1 alleles revealed in patients 14 were defined as "rare" (21.9%); in the control group we have detected 17 "rare" alleles (11.3%) of the examined 150 alleles. The higher frequency of "rare" HRas1 minisatellite alleles in patient group was statistically significant (p < 0.01). We can suppose that the "rare" alleles of HRas1 minisatellite are associated with increased risk of papillary thyroid cancer formation in children and adolescents after Chernobyl accident.

Diagnosis of metabolic acid-base disturbances in critically ill patients.

We compare two commonly used diagnostic approaches, one relying on plasma bicarbonate concentration and "anion gap," the other on "base excess," with a third method based on physicochemical principles, for their value in detecting complex metabolic acid-base disturbances. We analyzed arterial blood samples from 152 patients and nine normal subjects for pH, PCO(2), and concentrations of plasma electrolytes and proteins. Ninety-six percent of the patients had serum albumin concentration < or = 3 SD below the mean of the control subjects. In about one-sixth of the patients, base excess and plasma bicarbonate were normal. In a great majority of these apparently normal samples, the third method detected simultaneous presence of acidifying and alkalinizing disturbances, many of them grave. The almost ubiquitous hypoalbuminemia confounded the interpretation of acid-base data when the customary approaches were applied. Base excess missed serious acid-base abnormalities in about one-sixth of the patients; this method fails when the plasma concentrations of the nonbicarbonate buffers (mainly albumin) are abnormal. Anion gap detected a hidden "gap acidosis" in only 31% of those samples with normal plasma bicarbonate in which such acidosis was diagnosed by the third method; when adjusted for hypoalbuminemia, it reliably detected the hidden abnormal anions. The proposed third method identifies and quantifies individual components of complex acid-base abnormalities and provides insights in their pathogenesis.

[ret/PtC1 and ret/PTC3r1 rearrangement in thyroid cancer cells, arising in residents of Belorus in the period after the accident at the Chernobyl nuclear power plant]. / Perestroiki ret/PTC1 i ret/PTC3r1 v kletkakh raka shchitovidnoi zhelezy, voznikshie u zhitelei Belorussii v period posle avarii na ChAES.

After the accident at the Chernobyl nuclear power plant, a considerable increase in the incidence of thyroid cancer among children in Belarus was observed. In the present study, the frequency of the c-ret protooncogene rearrangements in samples of thyroid carcinomas resected and diagnosed in 1998 from individuals in Belarus was investigated. The ret/PTC1 oncogene was detected in 19% of the samples, and the ret/PTC3r1 oncogene, in 14%. The number of ret/PTC1 rearrangements observed in tumor cells from the patients whose age at the time of the accident was from 1 to 10 years, was greater compared to those whose age at the time was from 10 to 20 years, irrespective of the year of surgery (1996 or 1998). The majority of the patients with ret/PTC3r1 rearrangements lived in Gomel oblast, which was contaminated by the Chernobyl meltdown.

Molecular alterations involving p53 codons 167 and 183 in papillary thyroid carcinomas from chernobyl-contaminated regions of belarus.

After the Chernobyl accident in 1986, there was a significant increase in the incidence of papillary thyroid carcinoma in fallout-exposed children from Belarus. We studied the p53 gene from 24 papillary thyroid carcinoma cases presenting in 1996. All subjects lived in contaminated regions of Belarus at the time of the accident and were under age 20 when exposed to fallout. Exons 5 through 9 of p53 were amplified from genomic tumor DNA using the polymerase chain reaction (PCR). The PCR products were analyzed by direct DNA sequencing using an automated sequencer. Five cases each exhibited two molecular alterations within exon 5. Alterations were confirmed by sequencing in both directions. One alteration, involving codon 167 (CAG-->CAT) in all five cases, resulted in the substitution of HIS for GLN. The second alteration, involving codon 183 (TCA-->TGA) in all five cases, resulted in a premature termination codon. Leukocyte DNA from each of the positive cases was analyzed and found to contain only wild-type p53 sequence. These results suggest that mutations involving codons 167 and 183 in the p53 locus are important in the pathogenesis of a subset (21%) of radiation-induced papillary thyroid carcinomas from Belarus.

[CD44 gene expression in cancerous thyroid cells]. / Ekspressiia gena CD44 v rakovykh kletkakh shchitovidnoi zhelezy.

The peculiarities of alternative CD44 mRNA splicing in thyroid cancer tissue of children from radiocontaminated areas was investigated. CD44 gene expression in thyroid cancer tissues of children exposed to radiation resembled that in spontaneously emerged cancers. It was concluded that CD44 gene expression is not the primary target of radioactive irradiation. Probably, the CD44 mRNA splicing deregulation is the consequence of cancer.

Low prevalence of the ret/PTC3r1 rearrangement in a series of papillary thyroid carcinomas presenting in Belarus ten years post-Chernobyl.

After the Chernobyl accident in 1986, there was a significant increase in the incidence of papillary thyroid cancer in fallout-exposed children from Belarus. Radiation-induced rearrangements of chromosome 10 involving the c-ret proto-oncogene have been implicated in the pathogenesis of these cancers. The ret/PTC3r1 rearrangement was the most prevalent molecular lesion identified in post-Chernobyl papillary thyroid cancers arising in 1991 and 1992. We identified the ret/PTC1 rearrangement in 29% of 31 papillary thyroid cancers presenting in Belarus in 1996. In the present report, we examined 14 cases from this series (plus 1 additional case) and found a ret/PTC3r1 rearrangement in only 1 (7%). The prevalence of ret/PTC3r1 in this series is significantly lower than previously reported (p = 0.0006, Fisher exact test). This result suggests a switch in the ratio of ret/PTC3 to ret/PTC1 rearrangements in late (1996) versus early (1991-1992) post-Chernobyl papillary thyroid cancers.

Anion gap and hypoalbuminemia.

OBJECTIVES: To show how hypoalbuminemia lowers the anion gap, which can mask a significant gap acidosis; and to derive a correction factor for it. DESIGN: Observational study. SETTING: Intensive care unit in a university-affiliated hospital. SUBJECTS: Nine normal subjects and 152 critically ill patients (265 measurements). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Arterial blood samples analyzed for pH, PCO2, and concentrations of plasma electrolytes and proteins. Marked hypoalbuminemia was common among the critically ill patients: 49% of them had serum albumin concentration of <20 g/L. Each g/L decrease in serum albumin caused the observed anion gap to underestimate the total concentration of gap anions by 0.25 mEq/L (r2 = .94). CONCLUSIONS: The observed anion gap can be adjusted for the effect of abnormal serum albumin concentrations as follows: adjusted anion gap = observed anion gap + 0.25 x ([normal albumin] [observed albumin]), where albumin concentrations are in g/L; if given in g/dL, the factor is 2.5. This adjustment returns the anion gap to the familiar scale of values that apply when albumin concentration is normal.

The ret/PTC1 rearrangement is a common feature of Chernobyl-associated papillary thyroid carcinomas from Belarus.

An increase in the incidence of papillary thyroid cancer has been documented in individuals exposed to Chernobyl fallout in 1986. Experiments using cultured human cells have suggested that radiation can induce the ret/PTC1 rearrangement involving the ret proto-oncogene. To test the hypothesis that the ret/PTC1 rearrangement is involved in the pathogenesis of Chernobyl-associated papillary thyroid carcinomas, we studied a panel of 31 cases from Belarus. All individuals lived in fallout-contaminated oblasts (regions) of Belarus at the time of the accident: Gomel (n = 13), Brest (n = 12), Minsk (n = 4), and Grodno (n = 2). All were under age 20 at the time of the accident; 20 were born between 1982 and 1986. Individual thyroid radiation doses were estimated at 1.1 to 110 rem. Patients underwent surgery in Minsk in 1996. Fifteen patients had locally advanced disease (stage T4). The majority had regional lymph node involvement (stage N1, n = 27). There were no distant metastases. Surgical specimens were frozen at -80 degrees C, RNA was extracted and cDNA prepared. The polymerase chain reaction (PCR) was performed with specific primers for ret/PTC1, and c-ret and GAPDH as controls. Controls were positive in all 31 cases. Nine cases yielded a positive PCR product for the ret/PTC1 rearrangement (29%). Thus, the ret/PTC1 rearrangement is a feature of some Chernobyl-associated papillary thyroid cancers, and is one possible mechanism involved in the pathogenesis of these cancers.

MDM2 and p53 protein expression in the histologic subtypes of endometrial carcinoma.

The frequency of p53 protein overexpression differs among the histologic subtypes of endometrial carcinoma, from 21 to 48% in endometrioid carcinoma but from 80 to 86% in papillary serous carcinoma. Although p53 gene mutation can closely correlate with p53 protein overexpression in papillary serous carcinomas, high expression of p53 protein may also occur without detectable gene mutation in endometrioid carcinomas. Because MDM2 protein can bind mutant and wild-type p53 protein, we examined MDM2 and p53 protein expression in 27 endometrioid carcinomas and compared their expression patterns with those of 25 uterine papillary serous carcinomas. We detected p53 protein in 14 (52%) of the 27 endometrioid carcinomas but in 21 (84%) of the 25 papillary serous tumors (P = 0.02). MDM2 expression was more commonly detected in the endometrioid carcinomas. Nineteen (70%) of the 27 cases showed expression compared with 9 (36%) of the 25 papillary serous carcinomas (P = 0.03). p53 Expression correlated closely with MDM2 expression in endometrioid carcinomas but not in the papillary serous carcinomas. In endometrioid carcinomas, MDM2 was detected in 13 (93%) of 14 p53-positive carcinomas but in only 6 (46%) of 13 p53-negative tumors (P = 0.01). In papillary serous carcinomas, MDM2 was detected in 9 (43%) of 21 p53-positive carcinomas but in none of the 4 p53-negative tumors (P value not significant). These findings suggest that although high rates of p53 protein overexpression are most frequently associated with p53 gene mutation in uterine papillary serous carcinoma, p53 protein overexpression in endometrioid carcinoma is frequently associated with MDM2 overexpression. The selective correlation of MDM2 expression with p53 expression in endometrioid carcinomas but not in papillary serous carcinomas suggests an active role for MDM2 in binding and inactivating p53 in endometrioid carcinomas, leading to its overaccumulation and potentially impeding repair to damaged DNA. Additional study as to the cause of increased MDM2 expression in endometrial carcinoma, e.g., gene amplification, enhanced translation, or rearrangement, is indicated.

Decreased levels of CD44 protein and mRNA in prostate carcinoma. Correlation with tumor grade and ploidy.

BACKGROUND: CD44, a transmembrane protein, is associated with cell-cell and cell-matrix interaction and with tumor growth and metastasis. Expression of both standard form and variant isoforms of CD44 protein has been associated with aggressive behavior and metastasis in various tumors, but has not been characterized in prostate adenocarcinoma (PAC). METHODS: The expression of CD44 standard (CD44s) and splice variant v3, v4/5, v6, v7/8, and v10 proteins were studied in 109 PACs and correlated with tumor grade, DNA ploidy, and mRNA levels. Monoclonal antibodies against the various CD44 proteins were applied to microwave irradiated, formalin fixed, paraffin embedded sections. The DNA content of the tumors was evaluated by the Feulgen method with the CAS200 Image Analyzer. Total RNA exhibiting 18s and 28s bands was derived from two benign prostatic tissues and 5 PACs exhibiting decreased levels of CD44 protein by immunohistochemistry. The RNA was analyzed with reverse transcriptase polymerase chain reaction using CD44 specific primers. RESULTS: The basal cells of the benign prostatic acini revealed uniform membranous staining for CD44s, v3, and v6 in 95-97% of cases. Similar staining was observed for v4/5, v7/8, and v10 in 40%, 30%, and 2% of cases, respectively. Secretory epithelial cells of the benign prostatic acini showed predominant expression of CD44s (97% of cases). Staining for CD44 variant proteins (v3, v4/5, v6, v7/8, and v10) in this location ranged from 9-22% of cases. Approximately 70% of the PACs showed significant loss of CD44s expression, which correlated with high tumor grade (Gleason > or = 7) (P = 0.01) and aneuploid status (P = 0.002). In 93-98% of the PACS, there was a complete lack of membranous expression for all CD44 variant isoforms. The metastatic PACS did not show preferential expression of either the standard form or any variant isoform. The cDNA from the normal prostates yielded a prominent CD44 standard form polymerase chain reaction product at 482 base pair (bp) and variant isoforms at approximately 650 and 850 bp. No CD44 products could be amplified from the subset of five PAC cDNAs, even when present at four-fold excess. CONCLUSIONS: PACS exhibit down-regulation of CD44 protein expression, which correlates with high tumor grade and aneuploidy. v6 and v3 isoforms were preferentially expressed in the basal cells of benign prostatic acini. Based on a subset of cases, loss of CD44 protein expression is associated with decreased abundance of CD44 mRNA.

Predicted and trifluoroethanol-induced alpha-helicity of polypeptides.

The alpha-helix stabilizing solvent 2,2,2-trifluoroethanol (TFE) is frequently used as a medium for determining the average alpha-helicity of polypeptides by CD spectroscopy. CD spectra measured in solutions containing 10, 15, 20, 50, and 90% (vol/vol) TFE are presented for 5 peptides that were selected to demonstrate possible variations in the effect of TFE concentration on the secondary structure. The analysis is extended to 6 further peptides whose CD spectra as measured in TFE are documented in the literature. The observed alpha-helicity at a high TFE concentration is compared with the alpha-helicity determined by a structure prediction method that combines conformational filtering [S. Vajda, (1993) Journal of Molecular Biology, Vol. 229, pp. 125-145], and a Monte Carlo simulation [J. Figge et al. (1993) Protein Science, Vol. 2, pp. 155-164]. For the set of 11 peptides we find a correlation of 0.84 between the predicted [theta]222 values and the corresponding values observed by CD spectroscopy in a high concentration of TFE (p < 0.01). Although we generally find a good correlation at high TFE concentration between observed and predicted alpha-helicity, there are several peptides that do not follow the predicted behavior. An analysis of the TFE titration curves in one such case revealed that TFE can induce a sharp transition from a partial beta-sheet conformation to an alpha-helical conformation as the TFE concentration is increased above a critical value.

Expression of the CD44 cell adhesion molecule in urinary bladder transitional cell carcinoma.

CD44 cell adhesion molecule (PgP-1, ECM III, Hermes antigen) is a polymorphic integral membrane glycoprotein associated with cell matrix adhesion, lymphocyte activation, recirculation, and homing. CD44 expression has been reported in in malignant lymphoma and in a variety of epithelial human cancers but has not been studied as a prognostic marker in urinary bladder transitional cell carcinoma. CD44s (standard 85- to 95-kDa macromolecule) expression was measured by qualitative and image analysis-quantitated immunohistochemical techniques using the A3D8 monoclonal antibody. CD44v6 (a splice variant exon of CD44s) expression was measured by qualitative immunohistochemical techniques using the 2F10 monoclonal antibody. The results of CD44s and CD44v6 expression were compared with tumor grade, pathologic stage, and DNA content analysis on Feulgen-stained tissue sections in 44 cases of urinary bladder transitional cell carcinoma. The mean percentage-positive area of staining intensity for CD44s expression in Grade 1 tumors was 61%, compared with 30% for the Grade 3 tumors (P < 0.001). Non-invasive tumors featured a 59%-positive area of staining intensity, compared with the 30% staining percentage for the deeply invasive tumors (P < 0.001). There was significant correlation of aneuploid DNA content with loss of CD44s staining (P < 0.05). The staining results for CD44v6 paralleled those for the CD44s, with a significant loss of staining in high-grade and high-stage aggressive tumors in comparison with the low-grade nonaggressive tumors (P < 0.01). In urinary bladder transitional cell carcinoma, CD44s and CD44v6 expression parallels that for other cell adhesion molecules, such as E-cadherin, that feature a significant progressive loss of immunoreactivity in association with tumor dedifferentiation, advancing pathologic stage, and abnormal DNA content.

Novel CD44 messenger RNA isoforms in human thyroid and breast tissues feature unusual sequence rearrangements.

CD44 is a family of cell surface proteins implicated in adhesion interactions and tumor metastasis. Multiple CD44 mRNA isoforms arise from alternative splicing of variant exons (termed v1-v10). We recently discovered a novel CD44 mRNA isoform in human papillary thyroid cancers featuring a junction between subsegments of exons 4 and 13 (v8). The sequence ACAG was repeated at both the donor and acceptor sites in the genomic DNA (G. Ermak et al., Cancer Res., 56: 1037-1042, 1996). We used reverse transcription-PCR to characterize expression of this isoform in a panel of thyroid lesions. In addition, we assayed three cryopreserved human breast cancers and two samples of normal breast tissue (from female subjects who had undergone cosmetic mammoplasty) to determine whether a similar isoform is present in breast carcinomas. Levels of the novel isoform were up-regulated in 88% of the goiters, adenomas, and papillary cancers, but were undetectable in cases of thyroiditis and absent or low-level in four samples of normal thyroid tissue. The three breast cancers each yielded a 546-bp PCR product that was not detected in normal breast tissue. The PCR product from one of the breast cancers was cloned, and sequence analysis revealed a novel mRNA isoform featuring a junction between exon 3 and an internal site within exon 13 (v8). The sequence GCTTCAG was repeated at both the donor and acceptor sites in the genomic DNA. These results show that human thyroid and breast tissues contain novel CD44 mRNA isoforms featuring unusual rearrangements at repeated sequences. Further studies are warranted to determine whether the expression of this class of isoforms correlates with growth status.

Restricted patterns of CD44 variant exon expression in human papillary thyroid carcinoma.

CD44 is a polymorphic family of cell surface proteoglycans and glycoproteins implicated in cell-cell and cell-matrix adhesion interactions, cell migration, and tumor metastasis. CD44 exists as a standard form and as multiple isoforms arising from alternative splicing of variant exons (termed v1-v10) encoding parts of the extracellular domain. We demonstrated previously that papillary thyroid carcinomas exhibit aberrant patterns of alternative CD44 mRNA splicing (G. Ermak et al., Cancer Res., 55: 4594-4598, 1995). In the present report, we use reverse transcription-PCR using a new high-performance polymerase formulation (Ex Taq; TaKaRa Shuzo Co., Ltd., Otsu, Japan) , followed by Southern hybridization, and demonstrate that alternative exon usage in papillary thyroid carcinomas is restricted primarily to exons v6, v7, v8, v9, and v10, with weak expression of v3. Expression of v8 is tightly linked to v9 and closely related to v10 expression. Also, v6 and v7 expression are closely related. Papillary thyroid cancers exhibit a marked increase in specific mRNA species containing combinations of exons v6 to v10. Several isoforms found in papillary cancers are not detectable in histologically normal tissue derived from the corresponding contralateral thyroid lobes. Examples include a 750-bp v6- and v7-containing PCR product and a 650-bp v8- and v9- containing PCR product. Finally, a novel 530-bp PCR product was discovered and shown to contain a subsegment from exon 4 joined to a subsegment of exon 13 (v8), followed by the complete sequence of exons 14 (v9) and 15 (v10). This novel isoform was present in both the papillary cancers and contralateral tissues. In conclusion, papillary thyroid cancers exhibit specific patterns of aberrant alternative CD44 splicing, distinguishing them from histologically normal thyroid tissue.

Increased bcl-2 protein levels in prostatic adenocarcinomas are not associated with rearrangements in the 2.8 kb major breakpoint region or with p53 protein accumulation.

bcl-2, an inhibitor of programmed cell death (apoptosis), is present in high levels in a subset of prostatic adenocarcinomas. In this study, 42 prostatic adenocarcinomas were analyzed to determine whether increased bcl-2 levels are associated with rearrangements in the 2.8-kb major breakpoint region, an association known to occur in certain follicular lymphomas featuring a t(14:18) translocation. Immunostaining for bcl-2 and p53 proteins was performed on formalin-fixed, paraffin-embedded tumor specimens using murine anti-human bcl-2 and p53 monoclonal antibodies in all 42 cases. Genomic DNA from paired frozen samples of each tumor was subjected to digestion with HindIII and EcoRI and the products analyzed on a Southern blot with a 2.8-kb-digoxigenin-labeled major breakpoint region probe. Comparisons between groups were evaluated with the Fisher exact test. Diffuse, strong cytoplasmic immunoreactivity for bcl-2 was present in the epithelial cells of tumor glands in 16 of 42 cases (38%), including 8 of 19 low grade (Gleason score 6 and below) and 8 of 23 high grade (Gleason score 7 and above) prostatic adenocarcinoma. Southern blotting demonstrated a normal 2.8-kb germline DNA fragment in every case, with no evidence of rearrangement. Nuclear p53 staining was present in 10 of 24 high grade and 0 of 18 low grade tumors (P < 0.001). Only four cases exhibited positivity for both bcl-2 and p53, and there was no association of bcl-2 positivity with co-expression of the p53 protein (P = 0.58). We conclude that aberrations in the function of either bcl-2 or p53 could possibly modify the apoptotic pathway resulting in the extended survival of tumor cells. Also, increased bcl-2 levels in prostatic adenocarcinomas occur in the absence of detectable rearrangements in the major breakpoint region.

Deregulated alternative splicing of CD44 messenger RNA transcripts in neoplastic and nonneoplastic lesions of the human thyroid.

CD44 is a polymorphic family of immunologically related cell surface proteoglycans and glycoproteins implicated in cell-cell and cell-matrix adhesion interactions, lymphocyte activation and homing, cell migration, and tumor metastasis. CD44 exists as a standard form and as multiple isoforms, each generated by alternative splicing of up to 10 variant exons (termed v1-v10) encoding parts of the extracellular domain. Using semiquantitative reverse transcriptase-PCR and Southern hybridization, alternative CD44 mRNA splicing was examined in 10 papillary thyroid carcinomas, 8 nodular goiters, 9 adenomas, 2 cases of thyroiditis, and 3 histologically normal thyroid controls. The amount of input cDNA for the CD44 PCRs was standardized against an internal control gene (glyceraldehyde phosphate dehydrogenase). Four papillary carcinomas showed significant overexpression of CD44 transcripts migrating between 750 and 1000 bp. These cases demonstrated reduced levels of the 482-bp standard isoform transcript. In six papillary cancers, we found a prominent v6-containing isoform at 750 bp that was present in only trace amounts in normal thyroid tissue. It is of interest that similar findings were seen in the majority of the goiters and adenomas but not in the cases of thyroiditis. These results show that deregulation of alternative CD44 splicing is a common feature of disordered thyroid follicular cell growth, both in neoplastic and nonneoplastic lesions. The data imply an important role for CD44, including CD44v6, in the pathogenesis of various thyroid lesions.

Nuclear accumulation of MDM2 protein in well-differentiated papillary thyroid carcinomas.

Papillary thyroid cancer is the most common endocrine malignancy. Of all solid cancers presenting in adults, papillary thyroid cancer generally carries the best long-term prognosis. However, very little is understood about the molecular pathogenesis of this neoplasm. We recently hypothesized that increased nuclear levels of MDM2 protein might occur in well-differentiated papillary thyroid carcinomas (Gerasimov et al., Exp. Mol. Pathol. 62, 52-62, 1995). MDM2 is known to complex with and inactive the p53 tumor suppressor protein. Since p53 inactivation by gene mutation has an established role in the pathogenesis of undifferentiated (anaplastic) thyroid carcinoma, we reasoned that abrogation of p53 function by nuclear MDM2 protein accumulation might participate in the pathogenesis of certain well-differentiated thyroid cancers such as papillary cancer. In the present report we present the first direct evidence of MDM2 protein accumulation in the nuclei of papillary thyroid carcinoma cells in a subset of tumors. Using the IF-2 monoclonal antibody, which reacts specifically with human MDM2 protein, we studied 24 well-differentiated papillary thyroid carcinomas and 26 benign lesions (nodular goiters, adenomas, thyroiditis). Nuclear staining was quantitated using the CAS computerized image analysis system. We found positive nuclear MDM2 immunoreactivity in 8 (33%) of the carcinomas. In contrast, MDM2 staining was negative in all benign lesions (P = 0.001, two-tailed Fisher exact test). Normal thyroid tissue was also negative. These data suggest that nuclear accumulation of MDM2 protein might be implicated in the pathogenesis of a subset of papillary carcinomas. Further studies to investigate this possibility are warranted.

p53 gene status in endometrial carcinomas showing diffuse positivity for p53 protein by immunohistochemical analysis.

Although detection of p53 protein by immunohistochemical testing was originally thought to indicate p53 gene mutation, recent analyses of human malignancies have shown that high expression of p53 protein may occur without detectable gene mutation. Several explanations have been proposed for this phenomenon, including mutation out of "hot spot" regions, overexpression of wild-type protein, sampling error in molecular analyses, and conformational changes of wild-type p53 protein. As discussed, it is unlikely that the first two possibilities contribute significantly to the occurrence of this phenomenon, and the current study examined the possibility that sampling error in molecular analyses might account for a lack of concordance between immunohistochemical and molecular analyses. Such a possibility exists because immunohistochemical studies frequently report high expression when staining is only focal or regional and molecular analyses are based on the polymerase chain reaction, which is highly exponential in nature and may not detect mutation if the target gene segment is not amplified early in the chain reaction. In the current report, p53 protein expression was examined by immunohistochemical testing in 45 cases of endometrioid carcinoma, and all cases showing diffuse positivity were then examined by polymerase chain reaction in combination with single-strand conformational analysis for exons 4 to 9 with the use of a microdissection technique to separate malignant from benign cells. Of the 45 cases, diffuse staining was found in four cases, and only two of the four were found to show evidence of gene mutation.(ABSTRACT TRUNCATED AT 250 WORDS)