Frozen Section Immunohistochemistry Research Articles

Lymphatic and Blood Vessels in Normal Rhesus Monkey Organs by Frozen Section Immunohistochemistry: A Conclusive Report

Lymphatic and Blood Vessels in Normal Rhesus Monkey Organs by Frozen Section Immunohistochemistry: A Conclusive Report

A New Intraepithelial \u03b3\u03b4 T-Lymphocyte Marker for Celiac Disease Classification in Formalin-Fixed Paraffin-Embedded (FFPE) Duodenal Biopsies

BackgroundThe histopathologic diagnosis of celiac disease (CD) may be challenging when the duodenal biopsies mucosal injury is limited. Intraepithelial T-lymphocytes (IELs) can be useful to characterize the degree of mucosal inflammation. A small fraction of IELs expresses the γδ T-cell receptor (named γδ-IELs), whose density, determined by flow cytometry or frozen section immunohistochemistry (IHC), is a specific marker for CD.AimTo establish a new IHC assay for γδ-IELs applicable to formalin-fixed paraffin-embedded (FFPE) duodenal biopsies.MethodsWe analyzed γδ-IELs using IHC in 138 duodenal biopsies using a standard IHC staining protocol with a new monoclonal antibody H-41. IELs were quantitated with digital image analysis.ResultsCompared to those in non-celiac controls (n = 51), γδ-IEL density was significantly increased in newly diagnosed celiac disease patients (n = 22, p < 0.0001). In ROC-curve analysis, the cutoff of 6.5 γδ-IELs/100 enterocytes distinguished optimally active CD patients from non-celiac controls (sensitivity 96%, specificity 95%). γδ-IEL density in CD patients on a gluten-free diet (n = 53) were also higher than in controls (p < 0.0001), but lower than those in newly diagnosed CD (p < 0.0001). The diagnostic value of γδ-IELs outperformed that of CD3 + IELs in both patient groups. γδ-IELs were better than CD3 + IELs distinguishing between celiac disease and conditions histologically mimicking celiac disease (n = 12).ConclusionsIntraepithelial γδ T-lymphocytes can be stained and quantitated reliably in FFPE duodenal biopsies. The results showed excellent specificity and sensitivity for celiac disease. The new IHC method of detection of γδ-IELs is a promising addition to the routine histopathologic assessment methodology of celiac disease.

SOX10 commonly stains scar in Mohs sections

Sox10 immunostaining is used for the diagnosis and margin evaluation of melanocytic lesions. Sox10 was initially thought not to stain fibrohistiocytic processes. Consequently, it was believed to reliably distinguish desmoplastic melanoma from scar. However, recent data from formalin sections suggest Sox10 is less specific than previously thought. In this report, we demonstrate that Sox10-stained Mohs sections commonly show strong, fractional staining of scar. When using Sox10 with frozen section immunohistochemistry, Mohs practitioners should recognize the potential of this marker to stain scar to avoid overdiagnosis of desmoplastic melanoma.

Reduced response of human meniscal cells to Osteogenic Protein 1 during osteoarthritis and pro-inflammatory stimulation

Many cell types lose responsiveness to anabolic factors during inflammation and disease. Osteogenic Protein 1 (OP1/BMP7) was evaluated for the ability to enhance extracellular matrix synthesis in healthy and OA meniscus cells. Mechanisms of cell response to OP1 were explored. Meniscus and cartilage tissues from healthy tissue donors and osteoarthritis (OA) patients undergoing total knee arthroplasties were acquired. Primary cell cultures were stimulated with OP1 and/or inflammatory factors (IL1α, IL1β, or fibronectin fragments (FnF)) and cellular responses were analyzed by RT-qPCR and immunoblots. Frozen section immunohistochemistry (IHC) was conducted to assess OP1 and receptor proteins in normal and OA meniscus. OP1 treatment of normal meniscus cells resulted in significant, dose-dependent increases in ACAN (aggrecan) and COL2A1, and decreased MMP13 gene transcription, while only ACAN was upregulated (P<0.01) at the highest dose of OP1 in OA meniscus cells. OP1 induced significantly more ACAN gene transcription in normal meniscus than normal articular cartilage (P=0.05), and no differences between normal and OA cartilage were detected. Receptor expression and kinetics of canonical signaling activation were similar between normal and OA specimens. Normal meniscus cells treated with inflammatory factors were refractory to OP1 stimulation. Smad1 phosphorylation at an inhibitory site was induced (P=0.01 for both normal and OA meniscus) by inflammatory cytokine treatment. The meniscus demonstrates resistance to OP1 stimulation in OA and in the presence of inflammatory mediators. MAPK-mediated Smad1 linker phosphorylation is a possible mediator of the loss of anabolic extracellular matrix production in the inflammatory cytokine affected meniscus.

Immunohistochemical Detection of Hairy Cell Leukemia in Paraffin Sections Using a Highly Effective CD103 Rabbit Monoclonal Antibody

Detection of the integrin subunit CD103 is a useful diagnostic tool in the diagnosis of hairy cell leukemia (HCL). Currently, flow cytometric analysis (FC) and frozen section immunohistochemistry (IHC) represent the only available methods of detection. This study is the first to describe the successful use of a CD103 antibody to identify HCL and HCL-variant in paraffin sections of formalin- or Bouin solution- fixed specimens (n = 68) using an immunoperoxidase technique. In other B-cell lymphoproliferative disorders that morphologically may resemble HCL, including chronic lymphocytic leukemia/small lymphocytic lymphoma (n = 32), mantle cell lymphoma (n = 23), lymphoplasmacytic lymphoma (n = 27), follicular lymphoma (n = 7), and marginal zone lymphoma (n = 13), lymphoid cells are nonreactive for CD103. In HCL, the CD103 staining pattern is predominantly membranous with delineation of delicate cytoplasmic projections. This CD103 antibody is an extremely valuable addition to the IHC panel for the diagnosis of HCL, especially in cases lacking FC analysis.

Development of an ELISA to detect the secreted prostate cancer biomarker AGR2 in voided urine

Comparative transcriptomics between sorted cells identified AGR2 as one of the highest up-regulated genes in cancer. Overexpression in primary tumors was verified by tissue microarray analysis. AGR2 encodes a 19-kDa secreted protein that might be found in urine. Monoclonal antibodies were generated against AGR2. One antibody pair, P1G4 (IgG1) to capture and P3A5 (IgG2a) to detect, showed good performance characteristics in a sandwich ELISA. This assay could detect AGR2 at sub ng/ml quantities. AGR2 was detected in tissue digestion media of tumor specimens and culture media of AGR2-secreting prostate cancer cell lines. Additional testings involved frozen section immunohistochemistry, immunoprecipitation, and Western blot analysis. Voided urine samples were collected from pre-operative cancer patients, and urinary protein was desalted and concentrated by filtration. The amount of AGR2 detected was scored as pg/100 µg total protein, and then converted to pg/ml urine. The developed ELISA detected AGR2 protein, ranging from 3.6 to 181 pg/ml, in an initial cohort of samples. AGR2 was not detected in the urine of non-cancer and a bladder cancer patient. For prostate cancer, an AGR2 urine test could be used for diagnosis. The data, although derived from a small number of samples assayed, showed that developing such a test for clinical application is viable because AGR2 is specific to cancer cells, and apparently secreted into urine.

Gene Expression Profiling, Frozen and Paraffin Section Immunohistochemistry, and In Situ Hybridization for Determination of Monoclonality in Diffuse Large B-Cell Lymphoma.

Background: Determining monotypia of surface immunoglobulin (SIg) is often an important step in the diagnosis of B-cell lymphomas. Many competing methods have been developed and are commonly used in clinical practice. The role of gene expression profiling (GEP) in determining light chain restriction is yet to be clarified. The goal of this study was to compare 4 methods by which light chain restriction can be determined: frozen section immunohistochemistry (FS-IHC), paraffin section IHC (PS-IHC), in-situ hybridization (ISH), and GEP.Design: 40 cases of DLBCL, part of a previous GEP study of DLBCL (Rosenwald et al, NEJM 2002), were made into a tissue microarray (TMA). FS-IHC slides, previously stained using by-hand streptavidin and diaminobenzidine, were re-examined for κ:λ restriction. PS-IHC was done on the TMA using the Benchmark system according to manufacturer's protocols for κ/λ staining (Ventana Medical Systems VMSI, Tucson, AZ). ISH was performed on the TMA with a newly developed sensitive ISH procedure from VMSI which uses a purified streptavidin reagent to reduce background and increase sensitivity. The FS-IHC, PS-IHC, and ISH arrays were reviewed and scored as κ/λ monotypic, SIg-negative or indeterminate by 3 pathologists. The averaged gene expression ratios for microarray elements probing for κ and λ on each case were plotted on a log2 scale. Monoclonality was determined using 2 different GEP criteria: (1) κ-monoclonal if κ:λ expression was >2log2 or λ-monoclonal if expression was λ>κ or (2) κ and λ light chain relative expression on either side of the median. These data from all 4 techniques were used to determine a consensus clonality in which the majority of the results agreed. The results for each technique were then compared to the consensus for that case.Results: 7 Cases had a 4/4 consensus, 9 cases a 3/4, 11 cases a 3/3, 7 cases a 2/3, and 1 case a 2/2. 5 cases were excluded from the study because there was no majority consensus. 19 cases (47.5%) were κ monoclonal, 10 (25%) λ monoclonal, 6 (15%) SIg-negative, and 5 cases (12.5%) indeterminate. Compared to the consensus clonality FS-IHC was accurate in 21/26 (81%), PS-IHC in 28/32 (87%), sensitive ISH in 29/29 (100%) cases. GEP was accurate in 31/35 (89%) cases using either criteria (1) or (2); in 27/35 (77%) using criterion (1) alone, in 28/35 (80%) using criterion (2) alone, and 24/35 (69%) of cases when both criteria (1) and (2) were met. 6 SIg-negative cases failed all GEP criterion, but were considered correctly classified as neitherκ nor λ monoclonal.Discussion: The technique with the highest accuracy compared to consensus was the sensitive ISH assay, in part because samples were eliminated if the mRNA control (polyT probe) staining was suboptimal. GEP was also accurate using either of our criteria. The SIg-negative cases were not classified as either κ or λ monoclonal using any of our criteria. Given the increasing role GEP is likely to play in hematopathology, the application of GEP to B cell clonality may have diganostic utility.

Mohs Micrographic Surgery of Primary Cutaneous Mucinous Carcinoma Using Immunohistochemistry for Margin Control

BACKGROUND Primary cutaneous mucinous carcinoma is a rare adnexal malignancy with a high recurrence rate following conventional excision and the potential for aggressive local invasion. OBJECTIVE To enhance the microscopic detection of mucinous carcinoma in Mohs micrographic surgical sections by incorporating rapid immunohistochemical staining. METHODS Standard Mohs micrographic surgical technique was used in conjunction with frozen section immunohistochemistry using an antibody to low-molecular-weight cytokeratin. RESULTS Rapid immunoperoxidase staining using low-molecular-weight cytokeratin detected residual foci of mucinous carcinoma that were difficult to identify on routine frozen sections. Immunostaining was strongly positive in areas with clear evidence of tumor by routine histology, as well as in adjacent areas on a subsequent stage where frozen sections were equivocal or negative. Immunostaining was distinctly negative at the final surgical margin, which was shown by en face permanent sections to be free of tumor. The patient has been free of recurrence for 3 years. CONCLUSION Immunoperoxidase-guided Mohs micrographic surgery using low-molecular-weight cytokeratin enhances the sensitivity for detection of mucinous carcinoma, and may help contribute to complete tumor removal.

ALCAM/CD166 is up-regulated in low-grade prostate cancer and progressively lost in high-grade lesions.

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, including malignant melanoma, prostate cancer cell lines, and prostate cancer. We aimed to clarify the expression patterns of ALCAM in normal and malignant prostate tissue. RNA from 54 matched pairs of microdissected prostate tissue (tumor and normal) was hybridized to a custom built 4K GeneChip. Frozen section immunohistochemistry was used to confirm the ALCAM expression. Chip-based transcript analysis revealed an up-regulation of ALCAM in prostate cancer in 22% of cases. Immunohistochemistry showed a focally raised ALCAM expression in 81% and a decreased expression in 19% of cases. These expression patterns correlated significantly with Gleason tumor grade: ALCAM up-regulation was found in most low-grade tumors (Gleason grade 1-3), whereas down-regulation occurred preferentially in high-grade tumors (Gleason grade 4 and 5), although up-regulation of ALCAM expression was preserved in two Gleason grade 5 tumors. ALCAM expression is commonly disturbed in prostate cancer, which might indicate a role of ALCAM in the progression of this disease.

Is Fluorescence In Situ Hybridization Really Superior to HercepTest?

Is Fluorescence In Situ Hybridization Really Superior to HercepTest?

Cardiac allograft vascular (microvascular) rejection

Acute vascular (microvascular) rejection is a term that has been applied to the morphologic appearance of rejection responses involving the allograft vasculature. Recent studies have highlighted the diverse mechanisms that can be responsible for this morphologic appearance. Features of microvascular injury caused by reperfusion injury are related to complement mediated effects that are operative in both reperfusion as well as humorally mediated allograft rejection. Recent work has documented the importance of antibody and complement in mediating antigen-dependent microvascular damage and its relation to cardiac allograft vasculopathy. Strategies to monitor patients with acute vascular rejection include monitoring of serum antibody titers coupled with frozen section immunohistochemistry. Treatment strategies include plasmapheresis and immunosuppressive regimens containing cyclophosphamide or mycophenolate mofetil.

Overexpression of MDM2 oncoprotein correlates with possession of estrogen receptor alpha and lack of MDM2 mRNA splice variants in human breast cancer.

To evaluate the significance of murine double minute 2 (MDM2) oncoprotein in human breast cancer as a nuclear-cytoplasmic shuttling protein, an estrogen receptor (ER) alpha regulator, and a prognostic marker and to study how MDM2 is overexpressed, we investigated its status in tissue samples and examined the correlation between overexpression and MDM2 gene abnormalities, status, and clinicopathological parameters. We detected MDM2 oncoprotein in both nucleus and cytoplasm by frozen-section immunohistochemistry. There was a significant correlation between MDM2 overexpression and low-grade nuclear atypia, absence of lymph node involvement, and increased levels of ER alpha protein. Our molecular assays found no point mutations in Ser17, but there was a correlation between MDM2 overexpression and the lack of splice variant mRNAs. These results suggest that the distribution of MDM2 reflects its nuclear-cytoplasmic shuttling ability; that interaction between p53 and MDM2 for tumor progression is not enhanced by point mutations at codon 17; and that the expression of MDM2 splice variants is a reason for the lack of its overexpression. MDM2 overexpression correlates with favorable prognostic parameters. A decreased level of MDM2 will lead to a deviation from the ER alpha signaling pathway.

THE EFFECT OF ANTISENSE BCL-2 OLIGONUCLEOTIDES ON BCL-2 PROTEIN EXPRESSION AND APOPTOSIS IN HUMAN BLADDER TRANSITIONAL CELL CARCINOMA

Bcl-2 is an important determinant of transitional cell carcinoma of the bladder recurrence and progression as well as a factor in patient response to chemotherapy or radiotherapy. We determined Bcl-2 down-regulation after antisense oligonucleotide therapy and synergism with mitomycin C in transitional cell carcinoma of the bladder. Bcl-2 protein was quantified using flow cytometry and immunohistochemistry in 4 bladder cancer cell lines, in bladder washings from 6 patients with carcinoma in situ and in 16 patient tumor samples. The synergistic effects of antisense oligonucleotides G3139 and 2009, and mitomycin C were investigated in 4 cell lines, while 2009 down-regulation was examined in 20 tumor explants in an ex vivo model. Bcl-2 protein expression was found in all 4 cell lines and in 5 of the 6 cell populations derived from patients with carcinoma in situ. Of the 16 tumors 7 were classified positive by frozen section immunohistochemistry and quantitative flow cytometry. G3139 and 2009 down-regulated Bcl-2 protein expression in all 4 cell lines and 2009 down-regulated Bcl-2 protein expression in half of the Bcl-2 positive tumor specimens. There was only evidence in 1 cell line, T24/83, that Bcl-2 protein expression down-regulation enhanced mitomycin C induced apoptotic cell death. Bcl-2 was expressed in a significant proportion of bladder tumors and in carcinoma in situ. Therefore, antisense oligonucleotides represent a viable strategy for Bcl-2 protein down-regulation. However, it may not always translate into an increased level of mitomycin C induced apoptosis in transitional cell carcinoma of the bladder.

Demonstration of Distinct Antigenic Profiles of Small B-Cell Lymphomas by Paraffin Section Immunohistochemistry

The immunoperoxidase technique was used with antibodies against B-cell-associated antigens, including CD20, CD79a, CD10, CD23, CD43, cyclin D1, bcl-2, and kappa and lambda immunoglobulin light chains on formalin-fixed and B5-fixed tissue sections of follicular, small lymphocytic, mantle cell, and marginal zone lymphomas. Results obtained with paraffin section immunohistochemistry for CD20, CD10, CD23, and kappa and lambda light chains were compared with results obtained with flow cytometry or frozen section immunohistochemistry. Cells in all of the lymphoma types were positive for CD20 and CD79a. The antigenic profiles of the B-cell lymphomas demonstrated in paraffin sections were lymphoma type distinctive. Intrafollicular lymphocytes in follicular lymphomas were positive for CD10 and bcl-2. Small lymphocytic lymphomas expressed CD43 and CD23 and were negative for CD10 and cyclin D1. Mantle cell lymphomas characteristically expressed CD43 and cyclin D1 and were negative for CD23 and CD10. Marginal zone lymphomas were negative for CD23, CD10, and cyclin D1. All of the antibodies performed better in B5-fixed tissues, but formalin-fixed tissue immunophenotypes were always similar to those obtained on the B5-fixed tissue. These results were possible using well-fixed tissue, various antigen retrieval strategies, paraffin section reactive primary antibodies, and sensitive detection systems. Paraffin section immunohistochemistry on sections of routinely fixed tissue can be used similarly to flow cytometry and frozen section immunohistochemistry when classifying the lymphomas of small B lymphocytes.

Author reply

We appreciate the attention of Dr. Nakamura et al. to our article1 and simply apologize for the fact that we unfortunately did not refer to their article,2 although it mainly dealt with gastric B-cell lymphoma, especially mucosa-associated lymphoid tissue (MALT) lymphoma. However, to our surprise, "the detailed clinicopathologic features of gastric T-cell lymphoma" that they mentioned were comprised only of histologic classification, MIB-1 index, and overall survival rate. We believe clinicopathologic features of a certain type of lymphoma should include patient age and gender, and a description of the macroscopic and histologic features of the gastric lesions with either the study of frozen section immunohistochemistry or DNA analysis. These features should have included the birthplace and viral status of the patients because as we pointed out in our article, the southwestern part of Japan is an endemic region for human T-lymphotropic virus type 1 (HTLV-1). The objective of our article was not simply to describe clinicopathologic features of gastric T-cell lymphoma as a whole, but to document two types of gastric T-cell lymphoma associated with or without HTLV-1. We found that HLTV-1 negative T-cell lymphoma mainly is comprised of a distinct type of lymphoma with a natural killer (NK) cell phenotype accompanied by the prominent infiltration of eosinophils. We believe that the future studies of gastric T-cell lymphoma should cite this information, referring HTLV-1 status and NK cell phonotype. Motoko Shimada-Hiratsuka M.D.*, Masashi Fukayama M.D.

Comparison of cell adhesion molecule expression between glioblastoma multiforme and autologous normal brain tissue

We investigated glioblastoma multiforme (GBM) for a pattern of consistent alterations in cell adhesion molecules (CAM) expression that might distinguish tumor from normal autologous brain tissue. We used frozen section immunohistochemistry with anti-CAM and computerized image analysis to quantify staining intensity which we expressed as relative intensity units (RIU). Our results showed that normal brain tissue generally did not express α 1 β 1, intercellular CAM-1 (ICAM-1), and sialylated Lewis x, slightly expressed α 2, α 4, α 5, α 6 β 1, α v β 3, lymphocyte function-associated antigen-3 (LFA-3), Lewis x, sialylated LewisLewis x, had a good expression of α 3 β 1 rmand CD44, and strongly expressed neural CAM (NCAM). GBM expressed α 1, α, 3 α 5, α 6 β 1, α v β 3, ICAM-1, LFA-3, CD44, Lewis x, sialylated Lewis x, and sialylated LewisLewis x significantly higher (2-11-fold RIU) than normal brain tissue. ICAM-1 and LFA-3 were the most distinctive markers of GBM. The small blood vessel endothelial cells of the normal brain and the GBM showed a few differences. The tumor endothelium expression of α 2 β 1, α 4 β 1, and LFA-3 RIU appeared twice higher than in normal endothelium and α 6 β 1 showed an average of 40% RIU decrease in comparison to normal. These results show that the expression of several CAM is consistently altered in GBM and its microvasculature when compared with autologous normal brain tissue.

UCL3D3 and UCL4D12 reactivity in small B-cell neoplasms with special emphasis on monocytoid B-cell lymphoma

Two recently described monoclonal antibodies, UCL3D3 and UCL4D12, have been reported to have some specificity for mantle zone B lymphocytes and marginal zone/follicular center B lymphocytes, respectively, in the spleen. Forty-nine B-cell neoplasms, including 20 cases of monocytoid B-cell lymphoma (MBCL), were studied by frozen section immunohistochemistry with these antibodies to evaluate their utility. Tonsil, lymph node, and reactive spleen also were studied with the antibodies. Although a wide overlap was observed among the different lymphomas, a majority of cases of MBCL and half of cases of hairy cell leukemia (HCL) reacted with both markers, suggesting both marginal/follicular and mantle cell antigen expression. None of four cases of mantle cell lymphoma reacted with the proposed mantle cell marker UCL3D3, whereas three of these cases immunoreacted with UCL4D12. This marker is known to react with a subpopulation of follicular center cells and possibly with marginal zone lymphocytes. A comparison of nodal and extranodal neoplasms failed to show a significant difference in the pattern of immunoreactivity with these antibodies. Tonsil and lymph node controls showed some mantle zone staining with both antibodies, and there was a slight overlap in mantle and marginal zone staining of the spleen controls. These findings suggest an immunologic similarity between some cases of HCL and MBCL. However, the findings also would suggest that these antibodies, particularly UCL4D12, have less specificity than has been previously assumed, and UCL4D12 may not have practical utility in the evaluation of low grade B-cell lymphomas.

Cathepsin D by western blotting and immunohistochemistry: failure to confirm correlations with prognosis in node-negative breast cancer.

We attempted to replicate and improve on our previous study (N Engl J Med 322:297-302, 1990) that showed that 34-kd cathepsin D levels as determined by Western blotting strongly correlated with disease-free survival (DFS) and overall survival (OS) of axillary node-negative (N-) breast cancer patients. We also examined the prognostic significance of cathepsin D measured by immunohistochemistry (IHC) in these patients. Western blotting was performed on cytosols from frozen tumor specimens of 927 N- breast cancer patients in the San Antonio Breast Tumor Bank. The monoclonal antibody M1G8 was used to detect cathepsin D (in previous study, a polyclonal antiserum had been used). The same monoclonal antibody was also used for frozen-section IHC staining of tumor specimens from 562 N- patients from the same tumor bank. Levels of cathepsin D expression were then correlated with DFS and OS. Although the levels of cathepsin D expression as measured by Western blotting and IHC correlated with each other and with levels of cathepsin D measured in previous work using Western blotting with the polyclonal antiserum, in this present study, using the monoclonal antibody M1G8, we were unable to demonstrate that cathepsin D expression (measured by either Western blotting or by IHC) correlates with DFS or OS. In this study, cathepsin D expression as determined by either Western blotting or IHC using the monoclonal antibody M1G8 failed to improve the prognostic evaluation of N- breast cancer patients. The role of cathepsin D expression as a prognostic factor is still not precisely defined, raising questions about its use in the routine evaluation of N- breast cancer patients.

Association of p53 Protein Expression With Tumor Cell Proliferation Rate and Clinical Outcome in Node-Negative Breast Cancer

The p53 (also known as TP53) tumor suppressor gene encodes for a nuclear phosphoprotein thought to regulate proliferation of normal cells. Most p53 mutations result in a nonfunctional protein that accumulates in tumor cell nuclei. These common mutations appear to be involved in the development and/or progression of several neoplastic diseases including human breast cancer. Our purpose was to investigate the relationships between levels of mutant p53 protein expression, tumor cell proliferation rate, and clinical outcome in patients with node-negative breast cancer. Expression of mutant p53 protein was evaluated by frozen-section immunohistochemistry (IHC) and light microscopy in 700 breast cancers from axillary lymph node-negative patients with long-term follow-up (median, 54 months). The immunostaining signal was expressed as the sum of scores representing the proportion and staining intensity of negative and positive tumor cell nuclei (ranges, 0 and 2-8, respectively). Statistical comparisons were made between levels of p53 protein expression and disease-free survival, overall survival, and tumor proliferation rate expressed as the percentage of cells in the S phase (%S phase) as determined by flow cytometry. Of the 700 tumors, 362 (52%) showed positive nuclear immunostaining (IHC score > 0). Proliferation rates were significantly higher (P = .0001) in positive tumors (median %S phase, 7.1%) than in negative tumors (4.1%). In a univariate cutpoint analysis, negative tumors (n = 388) versus low-positive tumors (IHC score = 2-6; n = 263) versus high-positive tumors (IHC score > 6; n = 99) showed progressively reduced disease-free survival (80% versus 72% versus 58% at 5 years, respectively; P < or = .05 for all pairwise comparisons). Analogous results for overall survival were 88% versus 84% versus 74%; only the result for negative versus high positive tumors was significant (P = .003). In a multivariate analysis, expression of p53 protein and high %S phase were independently associated with reduced disease-free survival (P = .008 and .01, respectively). Expression of mutant p53 protein was associated with high tumor proliferation rate, early disease recurrence, and early death in node-negative breast cancer. Despite the strong direct correlation between accumulation of p53 protein and tumor proliferation rate, both factors were independently associated with poor prognosis, suggesting that p53 may have other biological functions in addition to cell-cycle regulation. This test, when combined with other prognostic factors, may enhance our ability to identify node-negative breast cancer patients at high risk for early disease recurrence and/or death, for whom the use of adjuvant chemotherapy is unequivocally justified.

Aberrant expression of the tumour suppressor gene p53 is very frequent in Wilms' tumours.

A series of 34 Wilms' tumours have been analysed for abnormal expression of the tumour suppressor gene p53 using frozen section immunohistochemistry. All tumours showed immunoreactivity with at least one of the specific antibodies used (monoclonal antibody PAb240, polyclonal antibodies CM1 and JG8). Abnormalities of p53 expression are very frequent in this type of childhood tumour.