Is monitoring of FOXP3 Treg cells in renal transplants during acute cellular rejection episodes useful?

BACKGROUND: The FOXP3 (forkhead Box p3) transcription factor is a marker for T regulatory cells (Treg). During cellular immune responses, Treg are expected to increase in number to ultimately control and limit this response. In renal transplants massive infiltration by T cells is often seen during rejection crises. This prompted us to examine changes in the numbers of FOXP3 positive T cells accompanying acute cellular rejection events. METHODS: A total of 32 transplant biopsies from 23 patients were studied retrospectively, these 16 protocol biopsies and 16 biopsies taken during rejection episodes included 9 serial pairs (protocol-rejection). To quantify FOXP3 positive T cells, frozen sections were double immunostained with anti-CD3 and anti-FOXP3 antibodies. Areas revealing T cell infiltrates were measured morphometrically and the number of FOXP3 positive cells per 1,000 µm2 of CD3 positive cells was taken as an FOXP3 index. RESULTS: This index was 0.46 (median, range 0.00-1.00) in the 16 protocol biopsies and 0.48 (median, range 0.16-2.31) in rejection episode biopsies. The highest values were seen during rejection crises, exceeding 1.00 in 6/16 biopsies, whereas no protocol biopsies had values greater than 1.00 (0/16) (difference significant p<0.02). In serial biopsies no consistent behavior was observed; the FOXP3 index remained unchanged, fell slightly or rose to a maximum of 13 fold. Expression levels of FOXP3 could vary within weeks. No correlations were found between donor type, initial therapy, therapy at biopsy, serum creatinine at the time of biopsy, at 3 months or 1 year later, and any of the morphometric parameters (CD3 and FOXP3) studied. CONCLUSIONS: During rejection of renal allografts the fraction of FOXP3+ Treg cells within the infiltrating T-cell population can increase transiently. This phenomenon was not consistently seen in acute cellular rejection and the information does not appear to be of value for individual patient management in such cases.

Effect of antiretroviral therapy on apoptosis markers and morphology in peripheral lymph nodes of HIV-infected individuals.

BACKGROUND: CD4+ T cell depletion and destruction and the involution of the lymphoid tissue are hallmarks of HIV infection. Although the underlying mechanisms are still unclear, apoptosis appears to play a central role. The objective of this study was to investigate the effect of antiretroviral therapy on the lymph node tissue, particularly with respect to morphology and apoptosis. PATIENTS AND METHODS: Between 1997 and 1999, two inguinal lymph nodes were excised from 31 previously untreated individuals who were in an early stage of HIV infection, the first one prior to treatment and the second after 16 to 20 months of treatment. Paraffin sections were investigated for lymph node architecture, distribution of cellular and viral markers, apoptosis, and expression of apoptotic key molecules which indirectly reflect apoptotic processes. RESULTS: After 16-20 months of antiretroviral therapy, a significant decrease in highly activated HIV-driven immune response was observed in the lymph node tissue as a marked reduction in follicular hyperplasia, a normalization of the follicular dendritic cell network, a significant increase in the number of CD4+ T cells, and a significant decrease in the number of CD8+ T cells. The expression of several proapoptotic (Fas, TRAIL, and active caspase 3) and antiapoptotic (Bcl-2 and IL-7Ralpha) molecules that were reconstituted in the tissues during therapy resembled their expression in lymph nodes of HIV-negative individuals. Limitations of the study are (a) the lack of untreated patients in the late stages, (b) for ethical reasons, the lack of a control group with untreated patients, and (c) for methodological reasons, the restriction of sequential measurements of apotpotic markers to one-third of the patients. CONCLUSION: Antiretroviral therapy initiated in the early stages in HIV infection may halt the irreversible destruction of the lymph node tissue and may partially normalize apoptotic processes.

Vitamin D receptor expression in human muscle tissue decreases with age.

UNLABELLED: Intracellular 1,25-dihydroxyvitamin D receptor (VDR) is expressed in human skeletal muscle tissue. However, it is unknown whether VDR expression in vivo is related to age or vitamin D status, or whether VDR expression differs between skeletal muscle groups. INTRODUCTION: We investigated these factors and their relation to 1,25-dihydroxyvitamin D receptor (VDR) expression in freshly removed human muscle tissue. MATERIALS AND METHODS: We investigated biopsy specimens of the gluteus medius taken at surgery from 20 female patients undergoing total hip arthroplasty (mean age, 71.6 +/- 14.5; 72% > 65 years) and biopsy specimens of the transversospinalis muscle taken at surgery from 12 female patients with spinal operations (mean age, 55.2 +/- 19.6; 28% > 65 years). The specimens were obtained by immunohistological staining of the VDR using a monoclonal rat antibody to the VDR (Clone no. 9A7). Quantitative VDR expression (number of VDR positive nuclei) was assessed by counting 500 nuclei per specimen and person. Serum concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were assessed at day of admission to surgery. RESULTS: All muscle biopsy specimens stained positive for VDR. In the univariate analyses, increased age was associated with decreased VDR expression (r = 0.5: p = 0.004), whereas there were no significant correlations between VDR expression and 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D levels. VDR expression did not differ between patients with hip and spinal surgery. In the multivariate analysis, older age was a significant predictor of decreased VDR expression after controlling biopsy location (gluteus medius or the transversospinalis muscle), and 25-hydroxyvitamin D levels (linear regression analysis: beta-estimate = -2.56; p = 0.047). CONCLUSIONS: Intranuclear immunostaining of the VDR was present in muscle biopsy specimens of all orthopedic patients. Older age was significantly associated with decreased VDR expression, independent of biopsy location and serum 25-hydroxyvitamin D levels.

Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four-color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates.

Current polymerase chain reaction (PCR) methods for the molecular diagnosis of B- and T-cell lymphomas by determination of clonality of immunoglobulin heavy chain (IgH) and T-cell receptor-gamma rearrangements and by detection of the chromosomal translocations t(14;18) and t(11;14), require several laborious and costly PCR assays for each of these diagnostic tests. We have developed a multiplex PCR assay for the simultaneous determination of B- and T-cell clonality and the detection of the chromosomal translocations t(14;18) and t(11;14) in a single reaction, using four-color fluorescence and automated high-resolution fragment analysis. The 26 primers combined in the multiplex PCR correspond to the sequences of >90% of the 69 variables and 6 join IgH genes and 100% of the T-cell receptor-gamma variables and join genes that could participate in the respective rearrangements. In addition, they detect the major and the minor breakpoint regions of the t(14;18) and the major breakpoint region of the t(11;14), and amplify the beta-globin gene as an internal control. The specificity of the multiplex PCR was confirmed by analysis of 39 T-cell lymphomas and 58 B-cell lymphomas, including 11 mantle cell lymphomas bearing the t(11;14) and 25 follicular lymphomas bearing the t(14;18), with known rearrangements and/or translocations. Fifteen samples of reactive lymphadenitis remained negative.

Alterations on the 5' noncoding region of the BCL-6 gene are not correlated with BCL-6 protein expression in T cell non-Hodgkin lymphomas.

The BCL-6 proto-oncogene is expressed in germinal center B lymphocytes, in their neoplastic counterparts, and in a subpopulation of germinal center and perifollicular T lymphocytes. Rearrangements and/or mutations of the 5' noncoding region of the bcl-6 gene have been demonstrated in a large majority of diffuse large B cell lymphomas. Some, but not all, of these genetic alterations lead to dysregulation of the protein. Recently, anaplastic large cell lymphomas with T and null cell phenotypes, as well as T lymphoblastic lymphomas, have also been reported to exhibit immunoreactivity to the anti-BCL-6 antibody. We collected 33 T cell non-Hodgkin lymphomas (T-NHLs) and analyzed their expression of the BCL-6 protein by immunohistochemistry and investigated the organization of the bcl-6 gene by Southern blot and single strand conformation polymorphism (SSCP). The expression of BCL-6 was demonstrated in 37.5% of lymphoblastic (LBL), 40% of anaplastic large cell (ALCL), and 33% of peripheral T cell lymphomas (PTCL). BCL-6-positive malignant cells exhibited the CD4+ or CD4+/CD8+ phenotype. The bcl-6 gene was in a germline configuration in all T-NHLs examined, and a mutation at the first exon-intron boundary region structure of the wild-type bcl-6 gene was detected in 3 of 12 PTCL. One case of PTCL with mutations of the 5' noncoding region expressed BCL-6. In conclusion, expression of the BCL-6 protein is demonstrable independently of bcl-6 alterations in T-NHLs. This further suggests that molecular mechanisms other than rearrangements and/or mutations of the 5' noncoding region of the bcl-6 gene can result in expression of the protein. Whether these lymphomas arose from T cells expressing BCL-6 or expressed BCL-6 as part of the malignant transformation process needs to be determined. Finally, structural alterations of bcl-6 are rare in T-NHLs, but mutations do occur in the 5' noncoding region. We suggest that expression of BCL-6 in T cells may facilitate lymphomagenesis by repressing critical cytokines and cell cycle regulators.

In situ detection of 1,25-dihydroxyvitamin D3 receptor in human skeletal muscle tissue.

Growing evidence suggests that intracellular vitamin D receptors are present in skeletal muscle tissue mediating vitamin D hormone response. The aim of the work reported here was to investigate the in situ expression of 1,25-dihydroxyvitamin D3 receptor in human skeletal muscle tissue. Intraoperative periarticular muscle biopsies were taken from 20 female orthopaedic patients (17 middle-aged and elderly patients receiving total hip arthroplasty due to osteoarthritis of the hip or an osteoporotic hip fracture and 3 young patients who received back surgery). The immunohistological distribution of the vitamin D3 receptor was investigated using a monoclonal rat antibody to the receptor (Clone Nr. 9A7). The receptor-positive nuclei were quantified by counting 500 nuclei per biopsy. Strong intranuclear immunostaining of the vitamin D receptor was detected in human muscle cells. Biopsies of hip patients had significantly fewer receptor-positive nuclei compared to those of back surgery patients (Mann-Whitney U-test: p = 0.0025). VDR expression (number of antigen-positive nuclei) was significantly correlated with age (coefficient of correlation = 0.46; p = 0.005), but not with 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D levels. The data clearly demonstrate presence of nuclear 1,25-dihydroxyvitamin D3 receptor in human skeletal muscle. To our knowledge this is the first in situ detection of the receptor in human skeletal muscle. The difference in the expression of the receptor between hip and spinal muscle biopsies might be explained by age or location. Further research is needed in order to evaluate whether vitamin D3 receptor expression in human skeletal muscle is age-dependent and varies between different muscles.

No complement receptor 1 stumps on podocytes in human glomerulopathies.

BACKGROUND: Type one complement receptor (CR1) is the only physiological inhibitor of complement on podocytes. CR1 is lost in different glomerulopathies, in particular in lupus nephritis, in which it has been suggested that CR1 is removed by proteolysis from the cell membrane. METHODS: To define whether proteolytic cleavage of CR1 on podocytes is a general phenomenon, we analyzed the expression of CR1 in different glomerulopathies using a monoclonal antibody against epitopes present on the extracellular portion of the molecule and a polyclonal antibody directed at the intracellular tail of CR1. The two antibodies were applied on sequential serial histologic sections of renal biopsy. RESULTS: In normal glomeruli, the two antibodies provided similar results, that is, strong staining of podocytes, and both were shown to recognize specifically CR1. Decreased expression of the extracellular portion of CR1 was observed in lupus nephritis (8/8), focal and segmental glomerulosclerosis (FSGS; 7/7), IgA nephritis (6/6), membranous glomerulonephritis (3/3), and minimal change disease (3/3). In each case, the decreased expression was accompanied by a simultaneous decrease of the expression of the intracellular tail of CR1 (Spearman's correlation coefficient rs = 0.951, P < 0.001). This observation was confirmed by analyzing focal glomerular lesions on sequential serial sections. CONCLUSION: These data indicate that there are no CR1 stumps on podocytes, even in lupus nephritis, and suggest that the CR1 loss on podocytes is not due to consumption but to decreased synthesis. A loss of CR1 synthesis might render podocytes highly sensitive to complement attack.

Severe bronchiolitis in acute Mycoplasma pneumoniae infection.

We report on a 17-year-old patient with severe bronchiolitis due to Mycoplasma pneumoniae infection. Despite an early 10-day course of clarithromycin, she developed progressive dyspnea, cough, fever, and severe obstructive ventilatory impairment. Sixteen days after onset of the disease a severe hemolytic anemia developed with only cold agglutinins positive at serologic screening. Thoracoscopic lung biopsy revealed diffuse bronchiolitis with suppurative intrabronchiolar inflammation, lymphohistiocytic "cuffing" of the bronchioli, and foam cell aggregates within neighboring alveoli. The infiltrate consisted mainly of CD3+, CD8+ lymphocytes and CD68+ macrophages. The diagnosis of Mycoplasma pneumoniae bronchiolitis was based on repeated complement fixation tests, which turned strongly positive only at day 74 after onset of the disease. Pulmonary function improved slowly under long-term prednisone treatment.

Intralipid-based short-term total parenteral nutrition does not impair small intestinal mucosa-related cellular immune reactivity in the healthy rat.

BACKGROUND: The lipid component of total parenteral nutrition (TPN) has reportedly been associated with trophic effects on the intestinal mucosa and suppressive effects on the immune system. METHODS: We have challenged these hypotheses using a 7-day TPN rodent model comparing the effects of isocaloric, isonitrogenous lipid-based (TPN-lipid, 50% of calories as long-chain triacylglycerol) and carbohydrate-based TPN (TPN-CH, 100% of calories as carbohydrates) on mucosal morphology and immune function. Enterally fed animals were included to establish a baseline for immunologic read-outs. The study was performed in healthy, metabolically stable animals to avoid interference by septic or trauma-related stress factors. RESULTS: Both TPN regimens resulted in a significantly smaller weight gain (TPN-lipid, 29.8 +/- 4.0 g; TPN-CH, 30.3 +/- 4.4 g) compared with enterally fed reference animals (49.2 +/- 3.2 g; p = .007), with no difference in nitrogen balance between the TPN groups. Mucosal sucrase activity was significantly lower in both TPN groups (TPN-lipid, 8.8 +/- 1.0 x 10(-7) katal per gram (kat/g) of protein; CH: 11.9 +/- 1.6 x 10(-7) kat/g of protein) compared with enteral feeding (17.4 +/- 0.9 x 10(-7) kat/g of protein; ANOVA: p = .0007). Morphometric analysis of the small intestine revealed no differences between the two TPN groups although a significantly depressed villus height in the TPN-lipid group could be observed in comparison to enterally fed reference rats (TPN-lipid, 0.47 +/- 0.02; TPN-CH, 0.50 +/- 0.01; enteral, 0.56 +/- 0.02 mm; ANOVA: p = .0298). Light and electron microscopy revealed a normal surface architecture in all three groups of rats. Cellular immune reactivity was evaluated using a novel specific immunization protocol: animals were immunized against OVA 4 weeks before TPN. OVA-induced lymphoproliferative responses and phenotypic data from draining popliteal and mesenteric lymph nodes were evaluated after the different regimens. Results did not differ among the three groups. CONCLUSIONS: In healthy rodents, short-term lipid-based and carbohydrate-based TPN regimens lead to limited mucosal atrophy with preserved surface architecture compared with enteral feeding. However, peripheral and mesenteric cellular immune responsiveness after both TPN regimens remained comparable to enterally fed reference animals. Therefore, mesenteric and systemic cellular immune reactivity does not appear to be impaired by lipid-based or carbohydrate-based TPN.

NY-ESO-1 tumour associated antigen is a cytoplasmic protein detectable by specific monoclonal antibodies in cell lines and clinical specimens.

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients.

BK-virus nephropathy in renal transplants-tubular necrosis, MHC-class II expression and rejection in a puzzling game.

We review BK-virus nephropathy (BKN) as a new complication that increasingly affects renal allografts and causes dysfunction. Since starting in 1996, we have seen 11 cases. Currently, the prevalence of BKN is 3% in our graft biopsies. The diagnosis can only be made histologically. The virus affects tubular epithelial cells that show characteristic intranuclear inclusion bodies. The major reason for impaired graft function and a possible way for viral particles to gain access to the blood via peritubular capillaries is necrosis of infected epithelial cells. BK-virus DNA in the plasma, which can be detected by PCR, is closely associated with nephropathy. BK-virus does not stimulate tubular MHC-class II expression as judged by immunofluorescence double labelling. The inflammatory response is inconsistent and the frequency of rejection episodes is not increased during disease. Clinical manifestation of viral nephropathy evolves in several stages. (i) Initial, asymptomatic and reversible activation of the virus, judged by the presence of inclusion bearing cells in the urine. (ii) High dose immunosuppressive drug regimens, often including tacrolimus. (iii) Tubular injury and viraemia as additional promoting conditions. BKN nephropathy was associated with graft loss in 45% of our patients. The remaining patients with persistent viral nephropathy showed renal dysfunction (serum creatinine levels on average 150% above baseline readings). Currently, no established antiviral therapy is available. We discuss attempts to lower immunosuppression as a means to control viral replication. We propose a diagnostic algorithm for screening and monitoring the disease.

Glomerulonephritis in a patient with complement factor I deficiency.

Complement factor I deficiency is known to be associated with recurrent pyogenic infections. The patient described here had recurrent attacks of otitis, sinusitis, and bronchopneumonia since childhood. At the age of 24 years, he had an acute episode of systemic vasculitis with purpura, but no nephritis. A factor I deficiency was diagnosed when he was 36 years old. Because of the uncontrolled activation of the alternative pathway of complement, several other components were depleted, in particular C3, which explained the predisposition for pyogenic infections. A progressive loss of renal function accompanied by proteinuria and hematuria started after the age of 40 years. Renal biopsy showed a focal segmental glomerulonephritis (GN) with glomerular deposits of immunoglobulins and complement C3 and C4 fragments. The glomerular podocytes showed an almost complete loss of complement receptor 1 (CR1; CD35). The expression of CR1 was very low on erythrocytes, as well. Thus, CR1, the most efficient cell-bound cofactor for the inactivation of C4b/C3b by factor I, appears to be consumed when factor I is missing. Although this is the first report of factor I deficiency associated with GN, it is unlikely that the development of the nephritis was fortuitous because GN has been found in many other diseases characterized by uncontrolled activation of the alternative pathway.

Polyomavirus infection of renal allograft recipients: from latent infection to manifest disease.

Polyomavirus (PV) exceptionally causes a morphologically manifest renal allograft infection. Five such cases were encountered in this study, and were followed between 40 and 330 d during persistent PV renal allograft infection. Transplant (Tx) control groups without PV graft infection were analyzed for comparison. Tissue and urine samples were evaluated by light microscopy, immunohistochemistry, electron microscopy, and PCR. The initial diagnosis of PV infection with the BK strain was made in biopsies 9+/-2 mo (mean +/- SD) post-Tx after prior rejection episodes and rescue therapy with tacrolimus. All subsequent biopsies showed persistent PV infection. Intranuclear viral inclusion bodies in epithelial cells along the entire nephron and the transitional cell layer were histologic hallmarks of infection. Affected tubular cells were enlarged and often necrotic. In two patients, small glomerular crescents were found. In 54% of biopsies, infection was associated with pronounced inflammation, which had features of cellular rejection. All patients were excreting PV-infected cells in the urine. PV infection was associated with 40% graft loss (2 of 5) and a serum creatinine of 484+/-326 micromol/L (mean +/- SD; 11 mo post-Tx). Tx control groups showed PV-infected cells in the urine in 5%. Control subjects had fewer rejection episodes (P<0.05) and stable graft function (P = 0.01). It is concluded that a manifest renal allograft infection with PV (BK strain) can persist in heavily immunosuppressed patients with recurrent rejection episodes. PV mainly affects tubular cells and causes necrosis, a major reason for functional deterioration. A biopsy is required for diagnosis. Urine cytology can serve as an adjunct diagnostic tool.

Polyomavirus disease under new immunosuppressive drugs: a cause of renal graft dysfunction and graft loss.

BACKGROUND: Manifest polyomavirus (PV) renal graft infection is a rare complication. We diagnosed 5 cases among 70 kidney recipients undergoing transplants since December 1995; however, there were no cases at our institution before December 1995. METHOD: To identify risk factors promoting manifest PV graft infection, we compared those 5 patients with kidney recipients who had signs of PV replication but no manifest graft infection (n=23, control group). PV replication was judged by the presence of intranuclear inclusion cells in the urine. RESULTS: Before the infection, five of five patients had recurrent rejection episodes. All were switched from cyclosporine A to high dose tacrolimus as rescue therapy. Infection was diagnosed histologically 9+/-2 months posttransplantation; it persisted and led to graft loss in four of five patients. In control patients, graft function was stable, 1 of 23 patients were switched to tacrolimus as rescue therapy, and graft loss occurred in 4 of 23 patients. CONCLUSION: Recurrent rejection episodes and high dose immunosuppressive therapy, including tacrolimus, are risk factors for manifest PV kidney graft infection, which has an ominous prognosis.

Molecular diagnosis of Ewing tumors: improved detection of EWS-FLI-1 and EWS-ERG chimeric transcripts and rapid determination of exon combinations.

Most Ewing tumors (ET), including Ewing sarcomas, peripheral primitive neuroectodermal tumors (PNET), and Askin's tumors, can be defined according to the specific chromosomal translocations t(11;22)(q24;q12) (EWS-FLI-1) or t(21;22)(q21;q12) (EWS-ERG). Detection of the chimeric RNA transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) has greatly facilitated the diagnosis of ET. Because of variable chromosomal breakpoint locations, however, the EWS gene fusions with FLI-1 and ERG genes are highly heterogenous, resulting in different sizes of the amplification products. To improve the diagnostic usefulness of the RT-PCR assay, we have developed an assay to detect chimeric mRNA transcripts by nested RT-PCR, followed by digestion of the PCR fragments with three different restriction endonucleases. This allows confirmation of the specificity of the PCR product and provides a rapid method to determine the combination of exons present in a transcript. In the 12 Ewing tumors tested, five different exon combinations were detected. In nine repeat biopsies of four patients, the case-specific translocation remained unchanged. One additional central PNET had no ET-specific translocation. In conclusion, the suggested combination of RT-PCR and restriction analysis of the PCR products allows a rapid and specific determination of ET-specific translocations.

[Non-Hodgkin lymphoma of the conjunctiva (MALT lymphoma)--clinical and pathological markers]. / Das Non-Hodgkin-Lymphom der Conjunctiva (MALT-Lymphom)--Klinische und pathologische Merkmale.

BACKGROUND: Lymphoid tumors of the conjunctiva are extremely rare. Primarily such tumors can appear as isolated neoplasm or secondarily within a systemic disease. PATIENTS: The difficulties relating to dignity of the clinical diagnosis and the histological examination of the biopsies are presented. Both patients received orbital radiation therapy. RESULTS: The therapeutically success and clinical course are reported.

Angiogenesis in cervical neoplasia: microvessel quantitation in precancerous lesions and invasive carcinomas with clinicopathological correlations.

Recently, angiogenic properties have been shown in preinvasive cervical lesions. Our goal was to determine the angiogenesis in cervical intraepithelial neoplasia (CIN) and the relationship between microvessel counts, histopathological parameters, and clinical outcome in invasive cervical carcinoma. One hundred thirty-eight cervical specimens were evaluated; among these 20 were designated normal epithelium, 20 low-grade CIN, 40 high-grade CIN, and 58 invasive carcinoma. Histological sections immunostained for CD31 were quantitatively evaluated for microvessel density. The tumor proliferation rate was determined by the Ki-67 Labeling Index. Comparison of microvessel counts from normal epithelium with those from CIN and invasive carcinoma showed significant increases in precancerous lesions and invasive cancer (P < 0.0001). Microvessel density was found to be associated with the overall survival in women with invasive carcinoma (P < 0.01). There was a significant correlation of microvessel density (P < 0.05) with relapse-free survival in patients with regional lymph node metastasis. A Cox stepwise regression analysis revealed microvessel density, together with depth of invasion, regional lymph node status, and vascular invasion, to be a strong independent prognostic indicator for overall survival in patients with clinical stage IB cervical carcinoma.

Altered expression of mdm-2 and its association with p53 protein status, tumor-cell-proliferation rate and prognosis in cervical neoplasia.

Recent results suggest that p53 inactivation is required for cervical-carcinoma development. The mdm-2 oncogene, which forms an auto-regulatory feedback loop with the normal p53 protein, has been found amplified in human carcinomas, thus abolishing the anti-proliferative function of p53. To investigate whether the mdm-2/p53 interaction plays a role in cervical neoplasms, we performed an immunohistochemical study in archival fixed, embedded specimens that included 178 pre-cancerous lesions (CIN) and invasive squamous-cell carcinomas of clinical stage IB. In addition to p53, we assessed the p53-associated protein, mdm-2, and the Ki-67 labelling index (LI). The presence of HPV was assessed by in situ DNA hybridization. Tumor expression of all nuclear proteins was scored as fraction of positive CIN or cancer nuclei. The analysis demonstrated a significant association of the Ki-67 LI with grade of atypia in cervical neoplasms. p53 accumulation and mdm-2 expression are higher in invasive carcinomas than in pre-cancerous lesions. No correlation was observed with HPV status. An inverse correlation was found between increased tumor-cell proliferation and mdm-2 expression in invasive carcinomas (p < 0.0001). mdm-2 expression was significantly associated with p53 accumulation (p < 0.02). However, the investigated nuclear proteins were not associated with overall survival in patients with invasive carcinomas. Cox stepwise-regression analysis revealed regional lymph node status and depth of invasion to be independent parameters.