Correlation of HER2 Protein Level With mRNA Level Quantified by RNAscope in Breast Cancer.

Trastuzumab deruxtecan (T-DXd) has been approved by the US Food and Drug Administration (FDA) to treat patients with metastatic HER2-positive and HER2-low breast cancer, and clinical trials are examining its efficacy against early-stage breast cancer. Current HER2 immunohistochemical (IHC) assays are suboptimal in evaluating HER2-low breast cancers and identifying which patients would benefit from T-DXd. HER2 expression in 526 breast cancer tissue microarray (TMA) cores was measured using the FDA-approved PATHWAY and HercepTest IHC assays, and the corresponding RNA levels were evaluated by RNAscope. HER2 protein levels by regression analysis using a quantitative immunofluorescence score against cell line arrays with known HER2 protein levels determined by mass spectrometry were available in 48 of the cores. RNAscope was also performed in 32 metastatic biopsies from 23 patients who were subsequently treated with T-DXd, and the results were correlated with response rate. HER2 RNA levels by RNAscope strongly correlated with HER2 protein levels (P < .0001) and with HER2 IHC H-scores from the PATHWAY and HercepTest assays (P < .0001). However, neither protein levels nor RNA levels significantly differed between cases scored 0, ultralow, and 1+ by PATHWAY and HercepTest. The RNA levels were significantly higher (P = .030) in responders (6.4 ± 8.2 dots/cell, n = 12) than those in nonresponders (2.6 ± 2.2, n = 20) to T-DXd. RNAscope is a simple assay that can be objectively quantified and is a promising alternative to current IHC assays in evaluating HER2 expression in breast cancers, especially HER2-low cases, and may identify patients who would benefit from T-DXd.

Galectin-3 and PTEN expression in pancreatic ductal adenocarcinoma, pancreatic neuroendocrine neoplasms and gastrointestinal tumors on fine-needle aspiration cytology.

OBJECTIVE: Galectin-3 has been implicated in the carcinogenesis of pancreatic ductal adenocarcinoma (PDAC). Its applicability in pancreatic fine-needle aspiration (FNA) in separating malignant from benign lesions has never been addressed. In addition, a correlation between Galectin-3 and tumor suppressor phosphatase and tensin homolog (PTEN) and their potential diagnostic value has never been tested. STUDY DESIGN: This study analyzed Galectin-3 immunohistochemical expression in FNA cell blocks of PDAC, pancreatic neuroendocrine neoplasms (PNEN), gastrointestinal stromal tumors (GIST) and non-tumor pancreatic tissue. In parallel, Galectin-3 and PTEN levels were evaluated in a tumor tissue microarray (TMA). RESULTS: Forty-four of 46 PDAC FNA and 32 of 33 PDAC TMA demonstrated tumor-specific Galectin-3 positivity. In contrast, Galectin-3 was not detected in PNEN and GIST. Total loss of PTEN was displayed by 26 of 33 PDAC, while non-neoplastic tissues all retained PTEN expression. CONCLUSION: Galectin-3 could be a valuable marker to help diagnose PDAC and rule out PNEN and GIST. In addition, PTEN positivity strongly argues against a diagnosis of PDAC. These data also advocate their potential diagnostic roles in the work up of challenging cytologic cases requiring ancillary test confirmation.

Automated and manual human papilloma virus in situ hybridization and p16 immunohistochemistry: comparison in metastatic oropharyngeal carcinoma.

OBJECTIVES: We compared the efficacy of automated and manual human papilloma virus (HPV) in situ hybridization (ISH) and p16 immunohistochemical staining (IHC) in fine needle aspiration (FNA) of metastatic oropharyngeal carcinoma. STUDY DESIGN: A total of 41 FNA cell blocks (CB) were evaluated. HPV ISH was interpreted as positive if a minimum of one tumor cell showed punctate dot-like nuclear positivity. p16 was interpreted as positive if ≥70% of tumor cells showed brown nuclear and cytoplasmic staining. RESULTS: Thirty of 41 CB (73%) were positive by automated HPV ISH, 25 of 41 CB (60%) with manual HPV ISH. Eighteen of 41 CB (43%) were positive for p16 IHC. Twelve of 41 CB (29%) with automated HPV ISH and 2 of 41 CB (4%) with the manual method were positive at 10× magnification. Three of 41 CB (7%) with automated HPV ISH and 14 of 41 CB (34%) with the manual method were positive at 20× magnification. Fifteen of 41 CB (36%) with automated HPV ISH and 9 of 41 CB (21%) with the manual method were positive at 40-60× magnification. CONCLUSION: Automated HPV ISH plays a more significant role in determining the HPV status in CB. However, the failure to use high magnification in the evaluation can give false-negative results.

Phosphohistone H3 and Ki-67 labeling indices in cytologic specimens from well-differentiated neuroendocrine tumors of the gastrointestinal tract and pancreas: a comparative analysis using automated image cytometry.

BACKGROUND: Ki-67 proliferation index was recently incorporated in the grading of neuroendocrine neoplasms (NENs) of the gastrointestinal tract (GIT) and pancreas. These are now divided into well-differentiated neuroendocrine tumors (WDNETs, grades 1 and 2) and poorly differentiated neuroendocrine carcinomas (grade 3). While Ki-67 is an established proliferation marker in NENs, phosphohistone H3 (PHH3), a newer marker of mitotic activity, is not. METHODS: We determined Ki-67 and PHH3 indices on cytologic samples from WDNETs of the GIT and pancreas using an automated cellular imaging system (ACIS®). RESULTS: There was a strong correlation between Ki-67 and PHH3 indices generated by ACIS on cytologic samples. However, in some cases the two stains caused conflicting grades within the same tumor. CONCLUSION: Both antibodies stain cells in different phases of the cell cycle which may cause discordant grades, thus affecting patient management and prognostication. Ki-67 staining is stronger than PHH3, making 'hot spots' easier to identify on ACIS. Ki-67 is more ideal than PHH3 for staining NENs, especially in tumors with borderline grades. Because PHH3 generates lower mitotic indices it should not be used as a proliferation marker in NENs until its expression has been further characterized.

Inference of the Basal epithelial phenotype in breast carcinoma from differential marker expression, using tissue microarrays in triple negative breast cancer and women younger than 35.

Basal-cell phenotype breast carcinoma has been associated with high-grade and metaplastic morphology, expression of basal-type cytokeratins, uniform negativity for ER and HER2, and decreased overall survival. Breast cancers occurring in young women are usually T2 disease at presentation, high-grade and of poor prognosis. We compared two groups of breast cancers, (a) ER-, PR-, HER2- (triple negative) [TNBrCa] and (b) non-triple negative breast cancers (non-TNBrCa) occurring in women under 35, using tissue microarray technology to characterize expression of the basal/myoepithelial cytokeratins (CK5/6, CK7, and CK14), luminal cytokeratins (CK8, CK18, and CK19), EGFR, p-cadherin, c-kit, p63, and p53. We also sought to identify characteristic histomorphologic features indicative of basal-like phenotype. The triple negative group showed preferential staining versus the age <35 group for CK5/6 (22% versus 4% p = 0.05), CK14 (44% versus 15%, p = 0.013), EGFR (83% versus 24%, p < 0.0001) and c-kit (19% versus 0% p = 0.026). Conversely, non-TNBrCa in women younger than 35 demonstrated increased expression of the luminal CK8 (92% versus 60%) compared with the triple negative patients (p = 0.006). The TNBrCa have characteristic histologic features including higher tumor grade, pushing tumor border, geographic necrosis, syncytial growth pattern, brisk mitotic activity, lack of/minimal in situ component, medullary-like and metaplastic differentiation. Invasive carcinomas in women younger than 35 usually have an associated in situ component, prominent nucleoli, central acellular fibrotic zone, and infiltrative tumor border. Triple negativity for ER/PR/HER2 coupled with EGFR, c-kit, and basal/myoepithelial cytokeratins (CK5/6, CK14) expression, and distinctive histomorphologic features predict morphology consistent with basal-cell phenotype.

Collaboration to partnerships.

Partnerships are at the center of the Hospital of the University of Pennsylvania Nursing Excellence Professional Practice (HUP-NEPP) model. Through the use of collaboration, skilled communication, and respectful workplace, partnerships can be formed, leading ultimately to world-class patient care. At HUP, interdisciplinary partnerships are evidenced by the clinical nurses through shared governance. This article describes the components necessary to form successful partnerships.

Automated RNA In Situ Hybridization for 18 High Risk Human Papilloma Viruses in Squamous Cell Carcinoma of the Head and Neck: Comparison With p16 Immunohistochemistry.

Detection of human papilloma virus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is important, as HPV-associated HNSCCs respond better to therapy. The RNAscope HPV-test is a novel RNA in situ hybridization (ISH) technique which strongly stains transcripts of E6 and E7 mRNA in formalin-fixed, paraffin-embedded tissue, with the potential to replace the indirect immunohistochemical (IHC) marker for p16 protein. A direct clinical comparison between p16 IHC and an automated RNA ISH using 18 probes has not been established. Samples from 27 formalin-fixed, paraffin-embedded HNSCC cases from the Emory University Hospital archives were stained using 18 individual RNA ISH probes for high-risk HPV (RNAscope 2.5 LS Probe ) on a Leica autostainer (Buffalo Grove, IL) and were compared with p16 IHC. Two pathologists reviewed and reached a consensus on all interpretations. The RNAscope technique was positive in 89% (24/27) and the p16 IHC was positive in 78% (21/27). The RNAscope was negative in 11.1% of samples (3/27) and the p16 IHC-negative in 22.2% (6/27). The RNA ISH detected 100% of the p16-positive IHC-stained slides and had a concordance of 88.9% (24/27). This easy to interpret automated staining method for 18 high-risk HPV genotypes is a feasible replacement for the indirect p16 IHC method.

RNA In Situ Hybridization for Epstein-Barr Virus and Cytomegalovirus: Comparison With In Situ Hybridization and Immunohistochemistry.

The RNAscope utilizes in situ hybridization (RISH) technology to detect single RNA molecules in a variety of tissue samples, including formalin fixed paraffin embedded (FFPE) tissues. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are found in association with neoplastic tissues and inflammatory lesions, and immunohistochemistry (IHC) or other techniques (ISH) are utilized to identify them. We compared the RNAscope RISH to ISH and IHC in the detection of EBV and CMV respectively to determine RNAscope utility in a clinical setting. Thirty-one FFPE tissues were stained by RISH to detect EBV and 24 samples of tissue for CMV. The RISH used the RNAscope (Leica BioSystems, Buffalo Grove, IL), the Bond III autostainer (Leica), and probes V-EBV and V-CMV (Advanced Cell Diagnostics, Newark, CA) as well as negative (DapB) and positive probe (PPIB) for RNA. Results were compared with those by ISH (Leica, EBV RNA probe), and IHC (CMV Dako, 1/160), respectively. RISH and ISH were concordant in 100% of cases positive for EBV by ISH (19/19). Of the cases negative for EBV by ISH, RISH showed positivity in an additional 25% of the samples (3/12). Overall concordance was 90.3% (28/31). RISH and IHC were concordant in 100% of cases positive for CMV by IHC (8/8). Of the cases negative for CMV by IHC, RISH detected positivity in an additional 50% of the samples (8/16). Overall concordance was 66.7% (16/24). RISH demonstrates increased sensitivity in the clinical setting, especially for CMV, detecting positive cells not stained by EBV ISH and CMV IHC.

Embryonic poly(A)-binding protein stimulates translation in germ cells.

The function of poly(A)-binding protein 1 (PABP1) in poly(A)-mediated translation has been extensively characterized. Recently, Xenopus laevis oocytes and early embryos were shown to contain a novel poly(A)-binding protein, ePABP, which has not been described in other organisms. ePABP was identified as a protein that binds AU-rich sequences and prevents shortening of poly(A) tails. Here, we show that ePABP is also expressed in X. laevis testis, suggesting a more general role for ePABP in gametogenesis. We find that ePABP is conserved throughout vertebrates and that mouse and X. laevis cells have similar tissue-specific ePABP expression patterns. Furthermore, we directly assess the role of ePABP in translation. We show that ePABP is associated with polysomes and can activate the translation of reporter mRNAs in vivo. Despite its relative divergence from PABP1, we find that ePABP has similar functional domains and can bind to several PABP1 partners, suggesting that they may use similar mechanisms to activate translation. In addition, we find that PABP1 and ePABP can interact, suggesting that these proteins may be bound simultaneously to the same mRNA. Finally, we show that the activity of both PABP1 and ePABP increases during oocyte maturation, when many mRNAs undergo polyadenylation.

Maspin and Mutant p53 expression in malignant melanoma and carcinoma: use of tissue microarray.

Maspin (mammary serine protease inhibitor), a member of the serpin family, has been shown to inhibit angiogenesis, tumor invasion, and metastasis. Previous studies suggest a p53-dependent regulatory pathway of maspin protein expression. Its loss correlates with progression of disease in both breast and prostate cancer. We studied the in vivo correlation of maspin expression with p53 mutation in malignant melanoma (MM) with and without use of tissue microarray (TMA). Seventy-seven MMs were immunostained on individual slides for maspin and p53 expression. Results were validated in 1 slide for each marker on a TMA system (TARP-2) with 498 tissue cores (0.6-mm diameter) from MM, other tumors, and normal tissue. The relationship between maspin and p53 in MM and carcinomas of other sites (breast, ovary, colon, lung, and prostate) was delineated using Pearson chi analysis. The inverse relationship between maspin and p53 expression predicted by hypothesized p53 regulation of maspin transcription, or any other correlation between these 2 markers, is not demonstrated in MM cases, using either classic individual slide (P=0.20) or TMA (P=0.85) methods when cutoffs for both markers are set at 10% or greater of cells staining. Even when cutoffs are altered with respect to either intensity or percentage of cells staining, no relationship is demonstrated between these markers, with either TMA or the conventional slide method. TMA immunostaining also showed no such relationship in carcinomas of the various other sites sampled-including breast and prostate, where previous studies have suggested a linkage. Despite published experimental evidence linking these 2 markers, this study failed to demonstrate correlation between maspin loss and p53 expression in MM using both individual slides and TMA, or in TMA of other carcinomas. Use of TMA is a quick, easy, and inexpensive method of immunohistochemical analysis of large numbers of cases, both to validate results obtained from individual slides and to assess specificity in a variety of neoplasms. However, heterogeneity and minimal tumor may lead to variable results.

Survivin and caspase-3 expression in breast cancer: correlation with prognostic parameters, proliferation, angiogenesis, and outcome.

BACKGROUND: Survivin is an inhibitor of apoptosis protein that is overexpressed in most human cancers, including breast, but is not expressed in normal tissue. Survivin is associated with more aggressive behavior and decreased survival in a variety of tumor types. It regulates the G2/M phase of the cell cycle by associating with mitotic spindle microtubules, and it directly inhibits caspase-3 and caspase-7 activity. We used a breast cancer tissue microarray to assess survivin and caspase-3 expression in breast cancer and to correlate both markers with proliferation (MIB-1), angiogenesis (CD31), and prognosis. DESIGN: A breast cancer tissue microarray with a total of 190 1-mm tissue samples (2 from each specimen) were immunostained for survivin, caspase-3, MIB-1, and CD31. The microarray contains 91 cases of breast carcinoma diagnosed at Emory University Hospital between 1992 and 2000, and 4 normal breast tissue controls. Follow-up information was obtained from hospital records and the Winship Cancer Center database. RESULTS: Eighty-four percent of breast carcinoma showed nuclear survivin expression. Normal breast tissue was immunonegative. Fifty-seven percent and 43% of breast cancer showed reduced and absent caspase-3 expression, respectively. Survivin (nuclear) and caspase (nuclear/cytoplasmic) expression showed significant correlation with histologic grade (P=0.008 and 0.041) and MIB-1 expression (P=0.033 and 0.012). Survivin nuclear expression also correlated significantly with tumor stage (P=0.012) and tended to correlate with estrogen receptor (P=0.050). There was no significant correlation between survivin and caspase expression. Furthermore, there was no correlation of both markers with other clinicopathologic parameters (age, tumor size, histologic type, progesterone receptor, Her-2 neu status, lymph node status), angiogenesis (CD31), or outcome (overall and disease-free survival). CONCLUSIONS: Survivin and caspase-3 expression correlate with poor prognostic parameters (higher histologic grade and high proliferation), but not with outcome, in breast carcinoma patients.

GATA-4 and GATA-6 expression in human ovarian surface epithelial carcinoma.

GATA-4 and GATA-6 are zinc finger transcription factors named for their recognition motif and involved in ovarian development and function. GATA factors are strongly expressed and primarily localized within the nuclei of ovarian surface epithelial cells. GATA factors have been previously shown to be expressed in sex-cord stromal ovarian tumors and may contribute to the tumor phenotype. Differential expression of GATA-4 within serous and mucinous ovarian carcinomas has been reported. Using immunohistochemistry, we studied GATA-4 and GATA-6 expression in 50 ovarian surface epithelial carcinomas and examined the relationship to clinicopathologic parameters and outcome. We found that the majority of the carcinomas retained GATA-4 expression, whereas approximately two-thirds of the carcinomas had mislocalization or loss of GATA-6 expression. No statistically significant correlations were found between histologic type, histologic grade, or patient outcome and GATA-4. Cytoplasmic GATA-6 expression tended to correlate with overall survival (P=0.0756). These findings suggest that although GATA factors play a role in ovarian surface epithelial carcinoma oncogenesis, they do not seem to affect clinicopathologic parameters.

The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6+/- mouse model of epithelial wound-healing delay.

BACKGROUND: Congenital aniridia caused by heterozygousity at the PAX6 locus is associated with ocular surface disease including keratopathy. It is not clear whether the keratopathy is a direct result of reduced PAX6 gene dosage in the cornea itself, or due to recurrent corneal trauma secondary to defects such as dry eye caused by loss of PAX6 in other tissues. We investigated the hypothesis that reducing Pax6 gene dosage leads to corneal wound-healing defects. and assayed the immediate molecular responses to wounding in wild-type and mutant corneal epithelial cells. RESULTS: Pax6+/- mouse corneal epithelia exhibited a 2-hour delay in their response to wounding, but subsequently the cells migrated normally to repair the wound. Both Pax6+/+ and Pax6+/- epithelia activated immediate wound-induced waves of intracellular calcium signaling. However, the intensity and speed of propagation of the calcium wave, mediated by release from intracellular stores, was reduced in Pax6+/- cells. Initiation and propagation of the calcium wave could be largely decoupled, and both phases of the calcium wave responses were required for wound healing. Wounded cells phosphorylated the extracellular signal-related kinases 1/2 (phospho-ERK1/2). ERK1/2 activation was shown to be required for rapid initiation of wound healing, but had only a minor effect on the rate of cell migration in a healing epithelial sheet. Addition of exogenous epidermal growth factor (EGF) to wounded Pax6+/- cells restored the calcium wave, increased ERK1/2 activation and restored the immediate healing response to wild-type levels. CONCLUSION: The study links Pax6 deficiency to a previously overlooked wound-healing delay. It demonstrates that defective calcium signaling in Pax6+/- cells underlies this delay, and shows that it can be pharmacologically corrected. ERK1/2 phosphorylation is required for the rapid initiation of wound healing. A model is presented whereby minor abrasions, which are quickly healed in normal corneas, transiently persist in aniridic patients, compromising the corneal stroma.

Cell surface glycoconjugate abnormalities and corneal epithelial wound healing in the pax6+/- mouse model of aniridia-related keratopathy.

PURPOSE: Congenital aniridia due to heterozygosity for Pax6 is associated with ocular surface disease, including keratopathy. This study investigated how defects in glycoconjugate component of the cell surface of Pax6+/- could cause the abnormal cellular migration phenotypes associated with the disease. METHODS: Immunohistochemistry, lectin-based histochemistry, conventional staining techniques, and proteomic assays were performed on eyes and cultured corneal epithelial cells from wild-type and Pax6+/- littermates. Wild-type cells were manipulated in culture to replicate the glycoconjugate abnormalities found in Pax6 heterozygotes and determine the consequences for wound healing. RESULTS: Multiple glycoconjugate defects were found in Pax6-mutant cells. Lectin cytochemistry of corneal epithelial cells suggested a partial failure of glycoprotein trafficking. Blocking cell surface carbohydrate moieties in wild-type corneal cells caused wound-healing delays similar to those seen in untreated Pax6+/- cells. CONCLUSIONS: Alterations to the cell surface glycoconjugate signature of Pax6+/- corneal epithelia restrict the ability of cells to initiate migration in response to wounding. This underlies the observed wound-healing delay in cultured Pax6+/- epithelia.

Rapid intraoperative immunohistochemical evaluation of sentinel lymph nodes for metastatic breast carcinoma.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is an integral part of the surgical management of patients with breast cancer. Rapid immunohistochemistry (RIHC) has the potential to increase detection of metastatic carcinoma at the time of frozen section consultation. The authors assessed the accuracy and turnaround time of a newly developed RIHC method for pancytokeratin (RIHC-CK). METHODS: Sixty-six SLNs from 32 patients with breast carcinoma were examined for metastasis using the Zymed Sentinel Lymph Node Rapid IHC Kit. Intraoperative frozen sections (6 mum) of the SLNs were incubated with Zymed anti-pan-cytokeratin/HRP conjugate, diaminobenzidine (DAB), and stained with hematoxylin. Slides were ready within 8 minutes and were interpreted as positive or negative for metastatic carcinoma. Results were compared with previous intraoperative touch preparations, frozen sections, hematoxylin and eosin (Perm H&E), and AEl/3-immunostained permanent sections (Perm CK). RESULTS: Fourteen lymph nodes (19%) in 13 patients tested positive for metastatic carcinoma in Perm H&E, the gold standard. RIHC-CK had the highest sensitivity (92%) of the intraoperative tests, compared with touch preparations (64%) and frozen sections (80%). RIHC-CK showed 94% accuracy, compared with 96% (frozen section) and 93% (touch preparation). The RIHC technique took 8 minutes and was easy to perform and interpret. CONCLUSIONS: Zymed RIHC is a sensitive method for detecting breast cancer metastases in SLNs. The speed, accuracy, and ease of interpretation of the test allow for recognition of micrometastases (<2 mm) that might otherwise be undetectable by current methods of intraoperative evaluation. The prognostic significance and effect on surgical management of micrometastases in SLNs have yet to be determined.

Proliferation (MIB-1 expression) in oligodendrogliomas: assessment of quantitative methods and prognostic significance.

Expression of to nuclear antigen Ki-67 (MIB-1) has been linked to proliferative activity and prognosis in a variety of tumors. The authors assessed three techniques for quantitating MIB-1 (expression in oligodendrogliomas, correlating results with mitotic activity and prognosis. Formalin-fixed, paraffin-embedded sections of 38 oligodendrogliomas were immunostained using monoclonal MIB-l. Proliferation index (PI) was quantitated by visual estimation, CAS-200, and AC1S image analysis. MIB-1 expression and mitotic count were correlated with overall survival and recurrence (disease-free survival), defined clinically and radiographically as new tumor growth. Mean follow-up was 54 months (range 1-276). Mean PI quantitated by the three methods was statistically similar (Visual 10.5%, CAS-200, 12.2%, CAIS 11.2%). PI results by all three techniques correlated significantly with each other; visual and CAS-200 PI correlated with mitotic index. Overall and disease-free survivals were similar for patients with PIs above and below the mean by both image cytometric assays; visually estimated PIs below the mean, versus above the mean, correlated with improved disease-free survival. The authors show a significant correlation between MIB-1 PI using the visual method and recurrence in patients with oligodendrogliomas. The objectivity and speed of the image analysis systems make them an attractive alternative to visual estimation, and larger series should be analyzed for prognostic value.

Renal Medullary and Cortical Correlates in Fibrosis, Epithelial Mass, Microvascularity, and Microanatomy Using Whole Slide Image Analysis Morphometry.

Renal tubulointerstitial injury often leads to interstitial fibrosis and tubular atrophy (IF/TA). IF/TA is typically assessed in the renal cortex and can be objectively quantitated with computerized image analysis (IA). However, the human medulla accounts for a substantial proportion of the nephron; therefore, medullary scarring will have important cortical consequences and may parallel overall chronic renal injury. Trichrome, periodic acid-Schiff (PAS), and collagen III immunohistochemistry (IHC) were visually examined and quantitated on scanned whole slide images (WSIs) (N = 67 cases). When tuned to measure fibrosis, IA of trichrome and Trichrome-PAS (T-P) WSIs correlated for all anatomic compartments (among cortex, medulla, and entire tissue, r = 0.84 to 0.89, P all <0.0001); and collagen III deposition correlated between compartments (r = 0.69 to 0.89, P <0.0001 to 0.0002); however, trichrome and T-P measures did not correlate with collagen deposition, suggesting heterogeneous contributions to extracellular matrix deposition. Epithelial cell mass (EPCM) correlated between cortex and medulla when measured with cytokeratin IHC and with the trichrome red portion (r = 0.85 and 0.66, respectively, all P < 0.0001). Visual assessment also correlated between compartments for fibrosis and EPCM. Correlations were found between increasing medullary inner stripe (IS) width and fibrosis in all of the tissue and the medulla by trichrome morphometry (r = 0.56, P < 0.0001, and r = 0.48, P = 0.00008, respectively). Weak correlations were found between increasing IS width and decreasing visual assessment of all tissue EPCM. Microvessel density (MVD) and microvessel area (MVA) measured using a MVD algorithm applied to CD34 IHC correlated significantly between all compartments (r = 0.76 to 0.87 for MVD and 0.71 to 0.87 for MVA, P all < 0.0001). Overall, these findings demonstrate the interrelatedness of the cortex and medulla and the importance of considering the renal parenchyma as a whole.

Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization.

PURPOSE: To determine the expression of angiogenic cytokines in macrophages and retinal pigment epithelium cells in choroidal neovascularization (CNV). METHODS: Ten surgically-excised subfoveal CNV specimens and ten eye bank eyes with subfoveal CNV were routinely processed, serially sectioned, and immunostained for factor VIII (F8), CD68 (KP1), cytokeratin 18 (CK18), vascular endothelial growth factor (VEGF), tissue factor (TF), and monocyte chemotactic protein (MCP). The CNV was classified as "inflammatory active" (more inflammation than fibrosis) or "inflammatory inactive" (morefibrosis than inflammation). The immunostaining was graded as none, mild (+), moderate (++), or heavy (+++). Five additional surgically-excised CNV specimens were dual labeled with CK18/MCP or CD68/TF and confocal scanning laser microscopy was performed. RESULTS: Vascular endothelium, macrophages, and RPE expressed F8, KP1, and CK18 respectively. Macrophages expressed + to ++ VEGF and ++ to +++ TF; RPE expressed ++ to +++ VEGF and ++ to +++ MCP. Staining for angiogenic cytokines was stronger in inflammatory active versus inflammatory inactive CNV. RPE dual labeled for CK18/MCP and macrophages dual labeled for CD68/TF. CONCLUSIONS: This study shows that RPE cells express MCP, a cytokine involved with macrophage recruitment, and that macrophages express TF in CNV. Macrophages and RPE express VEGF, thus perpetuating angiogenesis. TF is involved with fibrin formation and provides a scaffold effect for growth of the CNV complex. CNV likely represents a dynamic process with inflammatory active and inflammatory inactive (involutional) stages.

Rapid immunohistochemistry for cytokeratin in the intraoperative evaluation of sentinel lymph nodes for metastatic breast carcinoma.

The sensitivity and specificity of detecting metastatic breast carcinoma in sentinel lymph nodes using a rapid immunohistochemistry technique was determined and compared with methods currently used at the authors' institution. At the time of intraoperative consultation, after routine diagnostic touch preparations and frozen sections were prepared, 6-microm frozen sections of 72 sentinel lymph nodes from 32 patients with breast carcinoma were placed on plus slides, fixed in cold acetone for 2 or 3 minutes, and stored at -70 degrees C. These sections were immunostained with a prediluted broad-spectrum anticytokeratin monoclonal antibody coupled to an inert polymer with horseradish peroxidase (DAKO EPOS). Slides were ready for interpretation within 16 minutes and were scored as positive, negative, or equivocal for metastatic carcinoma. Results were compared with those of the intraoperative touch preparations and frozen sections and with paraffin-embedded, hematoxylin and eosin-stained, and AE1/AE3 immunostained permanent sections. Fourteen (19%) sentinel lymph nodes were positive for metastatic carcinoma in 13 patients. All methods tested were 100% specific. The rapid immunohistochemistry method was the least sensitive (57% sensitivity) of all methods used to detect metastasis. Routine diagnostic touch preparations, frozen sections, and permanent sections had sensitivities of 69%, 86%, and 100% respectively. In conclusion, this rapid immunohistochemistry method would not be helpful in intraoperative assessment of sentinel lymph nodes in breast cancer patients due to its low sensitivity.

Tumor stem cells (CD271, c-kit, SOX10) in Melanomas: prognostic and outcome implications.

Melanoma cells that express stem cell marker CD271 are shown to form tumors when transplanted into nude or immunodeficient mice. These tumors have a higher metastatic potential and worse prognosis than melanomas resulting from transplantation of CD271-negative cells. We studied stem cell markers (CD271, c-kit, SOX1O) in melanomas, correlating their presence with prognostic factors and outcome. A total of 82 melanomas in tissue microarrays were immunostained for CD271, c-kit, and SOX10. Results were correlated with clinicopathologic prognostic parameters (Breslow depth of invasion, Clark level, sentinel lymph node status, and pathologic stage) and outcome (recurrence, metastases, and death). Of the 82 melanomas, CD271 was expressed in 18 (21%), c-kit in 47 (57%), and SOX10 in all (100%). CD271 does show correlation with metastases (P=0.05). c-kit is associated with favorable prognostic parameters [Breslow depth (P<0.001) and pathologic stage (P=0.02)] and with improved outcome [recurrence (P=0.03) and metastases (P=0.004)]. Although SOX10 is a good diagnostic marker, it cannot be used for prognosis because it is expressed in all the melanomas studied. In conclusion, CD271 expression in melanomas is associated with increased frequency of metastases, and c-kit immunoreactivity is associated with favorable prognostic parameters and improved outcome.