Cellular Imaging System Research Articles

Health evaluation and dangerous reptile detection using a novel framework powered by the YOLO algorithm to design high‐content cellular imaging systems

AbstractThis novel approach in animal biology could revolutionize identifying endangered species, addressing the issue of misclassifying potentially harmful animals based solely on visual characteristics. Particularly impactful for farmers in agricultural fields, it aims to reduce the heightened risk of venomous animal attacks, ultimately improving safety. Due to a lack of accessible education, illiterate farmers are more susceptible to adopting superstitious beliefs, which tragically leads to fatal snakebites even when medical treatment is readily available. Furthermore, environmental factors can unexpectedly hold typically non‐threatening animals responsible for a large number of human deaths each year. However, the complexity of human recognition of these hazards has prompted the development of a novel design approach aimed at simplifying the process. Integration of the ResNet learning algorithm in conjunction with You Only Look Once (YOLOv5) within the framework is recommended to facilitate real‐time processing and improve accuracy. This combined approach not only speeds up animal recognition but also takes advantage of ResNet's deep learning capabilities. The first phase entails deploying YOLOv5 to detect the presence of snakes in the proposed study, achieving a remarkable 87% precision in snake detection thanks to the synergistic fusion of ResNet and YOLOv5.

Modification of Breast Cancer Milieu with Chemotherapy plus Dendritic Cell Vaccine: An Approach to Select Best Therapeutic Strategies.

The addition of dendritic cell vaccines (DCV) to NAC could induce immune responses in those patients with residual disease (RD) by transforming the tumor microenvironment. Core diagnostic biopsies and surgical specimens from 80 patients (38 in the vaccinated group plus NAC (VG) and 42 in the control group (CG, treated only with NAC) were selected. We quantify TILs (CD8, CD4 and CD45RO) using immunohistochemistry and the automated cellular imaging system (ACIS III) in paired samples. A CD8 rise in TNBC samples was observed after NAC plus DCV, changing from 4.48% in the biopsy to 6.70% in the surgical specimen, not reaching statistically significant differences (p = 0.11). This enrichment was seen in up to 67% of TNBC patients in the experimental arm as compared with the CG (20%). An association between CD8 TILs before NAC (4% cut-off point) and pathological complete response in the VG was found in the univariate and multivariate analysis (OR = 1.41, IC95% 1.05-1.90; p = 0.02, and OR = 2.0, IC95% 1.05-3.9; p = 0.03, respectively). Our findings suggest that patients with TNBC could benefit from the stimulation of the antitumor immune system by using DCV together with NAC.

Integrated Analytical System for Clinical Single-Cell Analysis.

High‐dimensional analyses of cancers can potentially be used to better define cancer subtypes, analyze the complex tumor microenvironment, and perform cancer cell pathway analyses for drug trials. Unfortunately, integrated systems that allow such analyses in serial fine needle aspirates within a day or at point‐of‐care currently do not exist. To achieve this, an integrated immunofluorescence single‐cell analyzer (i2SCAN) for deep profiling of directly harvested cells is developed. By combining a novel cellular imaging system, highly cyclable bioorthogonal FAST antibody panels, and integrated computational analysis, it is shown that same‐day analysis is possible in thousands of harvested cells. It is demonstrated that the i2SCAN approach allows comprehensive analysis of breast cancer samples obtained by fine needle aspiration or core tissues. The method is a rapid, robust, and low‐cost solution to high‐dimensional analysis of scant clinical specimens.

3D Microscopy Keeps Getting Faster, Smarter, Leaner

3D Microscopy Keeps Getting Faster, Smarter, Leaner

A novel biocompatible zwitterionic polyurethane with AIE effect for cell imaging in living cells.

In this work, a novel and stable zwitterionic polymer (TPE-CB PUs) was prepared to realize a cellular imaging system. TPE was conjugated into the backbone of zwitterionic polyurethane, which could be well dispersed in aqueous solution and emitted strong blue fluorescence because the TPE segment was aggregated in the core of TPE-CB PUs micelles. More importantly, the TPE-CB PUs micelles showed significant stability in a large pH window and with different storage times. In addition, the micelles exhibited low cytotoxicity in HeLa cells and mainly distributed in the cytoplasm after being incubated with cells. The outstanding properties of TPE-CB PUs combining the merits of AIE and a zwitterionic segment highlight its potential for use as a cell imaging material with remarkable capability.

Crosstalk-eliminated quantitative determination of aflatoxin B1-induced hepatocellular cancer stem cells based on concurrent monitoring of CD133, CD44, and aldehyde dehydrogenase1.

Cancer stem cells (CSCs), known as tumor initiating cells, have become a critically important issue for cancer therapy. Although much research has demonstrated the induction of hepato cellular carcinoma by aflatoxin B1, the formation of hepatocellular CSCs and their quantitative determination is hardly reported. In this work, it was found that hepatocellular CSCs were produced from HepG2 cells by aflatoxin B1-induced mutation, and their amount was quantitatively determined using crosstalk-eliminated multicolor cellular imaging based on quantum dot (Qdot) nanoprobes and an acousto-optical tunable filter (AOTF). Hepatocellular CSCs were acquired via magnetic bead-based sorting and observed using concurrent detection of three different markers: CD133, CD44, and aldehyde dehydrogenase1 (ALDH1). The DNA mutation of HepG2 cells caused by aflatoxin B1 was quantitatively observed via absorbance spectra of aflatoxin B1-8, 9-epoxide-DNA adducts. The percentages of hepatocellular CSCs formed in the entire HepG2 cells were determined to be 9.77±0.65%, 10.9±1.39%, 11.4±1.32%, and 12.8±0.7%, respectively, at 0 μM, 5 μM, 10 μM, and 20 μM of aflatoxin B1. The results matched well with those obtained utilizing flow cytometry. This study demonstrates that aflatoxin mediated mutation induced the conversion of hepatic cancer cell to hepatic CSCs by using a Qdot based constructed multicolor cellular imaging system.

N-Cadherin Immunoexpression in Patients with Acute Myeloid Leukemia

Introduction:N-cadherin (NCAD) is a member of the cadherin family which is involved in calcium ion dependent adhesion between cells by interaction with catenin on neighboring cells. In our previous work, we have shown differential patterns of N-cadherin expression in acute myeloid leukemia (AML) cell lines when cultured in traditional 2-dimensional (2-D) culture conditions (over monolayer of stromal cells) compared to 3-D culture conditions. In addition, we observed that AML cell lines which were more resistant to chemotherapy in vitro had higher expression of N-cadherin. In order to further examine the role of NCAD in AML, we studied patterns of NCAD immuno-expression in bone marrow samples taken from AML patients prior to induction therapy and compared them to control bone marrow samples. We then compared patterns of NCAD immuno-expression among different AML cytogenetic risk groups as well as by their response to treatment.Methods:Bone marrow aspirate/biopsy sections of leukemia patients and controls comprising of lymphoma patients were stained for NCAD by immunohistochemistry. The percentage of NCAD positive cells and the intensity of nuclear and cytoplasmic NCAD staining were determined using Automated Cellular Imaging system. A four-tier grading system from grade 0 (negative) to grade 3(highest) was used. Control bone marrow biopsies were chosen from lymphoma patients with negative staging bone marrow.Results:A total of 33 AML bone marrow samples were examined and compared with 16 controls. The percentage of nuclear NCAD expression was significantly higher in AML compared to controls (58.36% vs 39.75%, p=0.000636). Though grade 1 and grade 2 NCAD expression was statistically significantly higher in AML samples compared to controls (grade 1: 38.10% vs 27.13%, p=0.000044; grade 2: 17.40% vs 10.38%, p=0.034), there was no statistically significant difference in grade 3 expression between AML cases and controls (2.91% vs 2.25%, p=0.607). Similarly, cytoplasmic NCAD staining was quantified. The percentage of cytoplasmic NCAD expression was significantly higher in AML samples compared to controls (84.30% vs 65%, p=<0.00001). In the case of cytoplasmic NCAD expression, grade 2 and grade 3 immuno-expression was significantly higher in AML cases compared to controls (grade 2: 24.70% vs 7.38%, p=0.000142; grade 3: 2.79% vs 0.81%, p=0.023), while grade 1 NCAD cytoplasmic immuno-expression was not significantly different between AML cases and controls (56.67% vs 56.81%, p=0.9692). NCAD expression between different cytogenetic risk categories (favorable vs intermediate, intermediate vs poor, favorable vs poor) was compared; no significant difference in NCAD expression between different cytogenetic risk groups was detected. Similarly when we compared NCAD expression between AML cases and the response to chemotherapy, there was no significant difference in NCAD expression between AML cases that achieved complete remission with chemotherapy and AML cases that did not achieve complete remission.Conclusion.Based on these results, we conclude that nuclear and cytoplasmic N-cadherin expression is significantly higher in AML patients than control bone marrow samples. We did not see a difference in level of NCAD expression among different AML cytogenetic risk groups or by response to chemotherapy. These results should be further evaluated in a larger cohort to determine the significance of N-cadherin expression in AML. DisclosuresNo relevant conflicts of interest to declare.

The roles of differential branches of ER stress signaling in determining cell fate of autophagic hepatoma cells (737.2)

Autophagy is a conserved lysosomal degradative pathway in eukaryotic cells and its dual effects on cell fate are controversial. The present study was designed to clarify the switching effect of IRE1 activity on ER stress‐induced autophagic cell fate and monitor the dynamic changes of autophagic processing. Firstly, by Micro Cellular Imaging and Analysis System, the fluorescent signals were dynamically recorded in HepG2 cells which were pretransfected with mRFP‐EGFP‐LC3 plasmid. With this system, cytosolic yellow puncta indicated the formation of autophagosome, while the red fluorescence puncta indicated the turnover of autolysosome in these cells. In the presence of tunicamycin (Tm) (5µg/mL) for 24h, it was found that the number of yellow puncta was markedly elevated in HepG2 cells. Interestingly, not only the intracellular yellow puncta but also red puncta were significantly increased in these cells exposured to thapsigargin (Tg) (500nM). Secondly, by western blotting, both Tm and Tg evidently induced an increment of IRE1 protein, an early attenuated branch of ER stress. However, another critical ER stress signaling molecule PERK was found to persistently increase in these cells in response to Tg, but not Tm. Thirdly, inhibiting the IRE1 activity by siRNA substantially decreased the LC3II conversion, and dramatically promoted the Tg‐induced apoptosis of HepG2 cells with about 40%. Consistently, Tg‐induced red puncta signal was apparently enhanced with IRE1 siRNA treatment. Taken together, these results suggested that IRE1 as well as PERK, the hallmarks of ER stress signaling, importantly govern the autophagic cellular outcome, which may serve as a novel potential therapeutic target for HCC.Grant Funding Source: Supported by NSFC Grants 881172260, 30630145, 81360077 &amp; 2010GXNSFC013020 to G.Z.

N-Cadherin Immunoexpression In Patients With Acute Myeloid Leukemia

IntroductionN-cadherin is a member of the cadherin family which is involved in calcium ion dependent adhesion between cells by interaction with catenin on other cells. In our previous work, we have shown differential patterns of N-cadherin expression in acute myeloid leukemia (AML) cell lines when cultured in traditional 2-dimensional (2-D) culture conditions (over monolayer of stromal cells) compared to 3-D culture conditions. In addition, we have observed that AML cell lines which are more resistant to chemotherapy in vitro had higher expression of N-cadherin, suggesting potential translational applications to patients with AML. In order to further examine the role of N-cadherin in AML, we studied patterns of N-cadherin immunoexpression in bone marrow samples taken from patients with untreated AML and compared them to control bone marrow samples. MethodsIn this retrospective study, we evaluated bone marrow biopsy specimens of 10 AML patients for N-cadherin expression by immunohistochemistry. Bone marrow biopsy specimens from 10 negative staging lymphoma patients were used as control. Automated Cellular Imaging system (ACIS) was used to quantify and grade N-cadherin immunoexpression in the nucleus as well as in the cytoplasm of evaluated cells. ACIS uses advanced color detection software in order to evaluate N-cadherin positive cells and measure the intensity of staining. Grades of N-cadherin positivity were subclassified as grade 0 (no staining) 1+, 2+, 3+. 10 different areas of each sample were evaluated in order to estimate the median intensity of staining per slide. Student's t-test was used to compare the generated medians and a P-value of <0.05 was considered statistically significant. ResultsNuclear N-cadherin staining was graded and compared between bone marrow biopsy specimens of patients with AML and control bone marrow biopsies from patients with negative staging bone marrow examinations for lymphoma. Interestingly, the percentage of N-cadherin grade 0 (negative) cells was higher in controls than AML samples (54% vs. 37%, p=0. 01). Also, the percentage of grade 1 and grade 2 expressing cells was significantly higher in AML cases compared to controls (grade 1: 39% vs. 29%, p=0.005; grade 2: 21% vs. 13%, p=0.029). However, there was no statistical difference seen in between AML cases and controls in terms of levels of grade 3 expression. Similarly, cytoplasmic N-cadherin staining was quantified. Grade 0 expressing cells were significantly higher in control samples compared to AML samples (34% vs. 16%, p=0.01). There was no statistical difference seen in terms of levels of grade 1 expression. However, grade 2 and 3 expression levels quantifying the higher levels of N-cadherin expression were significantly higher in bone marrow biopsies from patients with AML versus those from negative staging bone marrows (grade 2: 26% vs. 9%, P=0.01; grade 3: 4% vs. 1%, p=0.04). ConclusionBased on these results, we conclude that cytoplasmic N-cadherin expression is significantly increased in AML bone-marrow samples compared to control samples. These results should be further evaluated in the future to determine the prognostic significance of N-cadherin expression in AML. Disclosures:No relevant conflicts of interest to declare.

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.

Prospective Analysis of Ki-67 as an Independent Predictor of Oncologic Outcomes in Patients with High Grade Upper Tract Urothelial Carcinoma

We determined the association of the proliferation marker Ki-67 with pathological parameters and oncologic outcomes in patients with high grade upper tract urothelial carcinoma. Immunohistochemical staining for Ki-67 was done prospectively in 101 consecutive patients undergoing radical nephroureterectomy/ureterectomy for high grade upper tract urothelial carcinoma. Data were compared based on Ki-67 status (normal vs over expressed). Survival was assessed by the Kaplan-Meier method. Cox regression analysis was done to identify independent predictors of time dependent outcomes. Median patient age was 70.0 years and median followup was 22.0 months (range 1 to 77). Overall, 30.2% of the population experienced recurrence and 24.8% died of upper tract urothelial carcinoma. Organ confined disease (T2 or less and lymph node negative), lymphovascular invasion and sessile architecture were present in 56.3%, 33.3% and 20.8% of patients, respectively. Ki-67 was over expressed in 73.3% of patients and associated with adverse pathological features. Patients with over expressed Ki-67 had significantly worse recurrence-free survival (43.2 vs 69.0 months, p = 0.006) and cancer specific survival (48.9 vs 68.9 months, p = 0.031) than patients with normal Ki-67. Patients with nonmetastatic disease similarly had worse recurrence-free survival (40.7 vs 71.8 months, p = 0.003) and cancer specific survival (41 months vs not attained, p = 0.008) for over expressed vs normal Ki-67. After adjusting for the effects of organ vs nonorgan confined disease Ki-67 over expression was an independent predictor of recurrence-free survival in the total cohort (HR 4.3, p = 0.05) and in patients with nonmetastatic disease (HR 8.5, p = 0.038). Ki-67 over expression was associated with adverse pathological features in cases of upper tract urothelial carcinoma. It was also an independent predictor of recurrence-free survival in patients with high grade upper tract urothelial carcinoma.

Markers suggest reduced malignant potential of subsquamous intestinal metaplasia compared with Barrett's esophagus

Esophageal subsquamous intestinal metaplasia (SSIM) is frequently observed in patients with Barrett's esophagus (BE) and can also be found in patients after endoscopic ablative treatments for dysplastic BE. While these 'buried glands' appear identical to BE glands, features of SSIM and its malignant potency have yet to be fully elucidated. To determine differences in malignant potential between nondysplastic BE and SSIM, the Automated Cellular Imaging System was used to assess and compare changes in DNA content between SSIM and BE. Samples were further immunostained for Ki67 and Lgr5 to gauge general proliferative and possible stem cell features, respectively, in SSIM cells compared with BE glands. No significant differences were found between SSIM and BE with regards to DNA ploidy aberrance. However, significant differences were noted between SSIM and BE upon immunohistochemical analysis. SSIM was found to be negative for both Ki67 and Lgr5 while BE was positive for both markers. SSIM cells appear to be relatively quiescent and behave differently from BE, suggesting a reduced proclivity toward cancer progression.

Enzyme-catalyzed oxidation facilitates the return of fluorescence for single-walled carbon nanotubes.

In this work, we studied enzyme-catalyzed oxidation of single-walled carbon nanotubes (SWCNTs) produced by the high-pressure carbon monoxide (HiPco) method. While oxidation via strong acids introduced defect sites on SWCNTs and suppressed their near-infrared (NIR) fluorescence, our results indicated that the fluorescence of SWCNTs was restored upon enzymatic oxidation, providing new evidence that the reaction catalyzed by horseradish peroxidase (HRP) in the presence of H2O2 is mainly a defect-consuming step. These results were further supported by both UV-vis-NIR and Raman spectroscopy. Therefore, when acid oxidation followed by HRP-catalyzed enzyme oxidation was employed, shortened (<300 nm in length) and NIR-fluorescent SWCNTs were produced. In contrast, upon treatment with myeloperoxidase, H2O2, and NaCl, the oxidized HiPco SWCNTs underwent complete oxidation (i.e., degradation). The shortened, NIR-fluorescent SWCNTs resulting from HRP-catalyzed oxidation of acid-cut HiPco SWCNTs may find applications in cellular NIR imaging and drug delivery systems.

Comparative analysis of bone marrow micrometastases with sentinel lymph node status in early-stage breast cancer.

570 Background: A definitive relationship between bone marrow (BM) micrometastases (M) and sentinel lymph node (SLN) status has not been established in Breast Cancer (BRCa). Hence, a retrospective study was done to examine the relationship between BMM and SLN status in BRCa patients (pts). Methods: T1/T2 BRCa pts underwent SLN biopsy and definitive surgery for primary tumors. At the operation, BM aspiration from bilateral post-superior iliac spines was performed. BM samples were examined using a Cytokeratin Detection Kit using CAM 5.2 monoclonal antibody and visualization achieved with a Ventana iView V-Red detection system. An Automated Cellular Imaging System was applied to identify metastatic BRCa cells. Pts with +ve BM underwent repeat BM analysis 6 months after completing initial treatments. Data was collected for T-stages, SLN, BM, ER/PR receptor and Her-2/neu statuses and analyzed using Chi-Square Analysis or Fischer’s Exact Test. Results: We analyzed 458 consecutive pts, of which SLN metastasesv(mets) were detected in 22.9% and BMM were detected in 8.1%. BMM were found 23% bilaterally and 77% unilaterally. BMM were detected in 11.4% of SLN +ve pts versus 7.1% of SLN -ve ptsv(p=0.15) and 6.8% of T1 pts versus 12.3% of T2 ptsvv(p=0.07). BMM were found in 8.7% of ER/PR +ve pts versus 4.5% of ER/PR –ve ptsvv(p=0.1) and 9.5% of HER2/neu +ve pts versus 7.7% of HER2/neu –ve ptsv (p=0.5) (Table). Repeat BM analysis detected BMM only in 6.6% pts. Conclusions: Bilateral BM aspirate resulted in a significant increase in detection of micrometastases. BM metastases may occur independently of lymphatic metastasis. Therefore, evaluation for the presence of BMM may help identify high risk pts with node negative disease in early stage BRCa. [Table: see text]

Correlation of bone marrow micrometastases with nodal status in gastrointestinal tumors.

e14518 Background: Presence of bone marrow (BM) micrometastases (M) is a prognostic factor for patients (pts) with solid tumors. Sentinel lymph node (SLN) mapping (M) has been found to upstage pts with gastrointestinal (GI) cancers. However, a direct correlation between the presence of BMM and nodal metastases (mets) is lacking. A retrospective study was done to determine the relationship between BMM and nodal status in GI cancers. Methods: A total of 385 consecutive pts with GI cancers were studied. Pts underwent bilateral posterior-superior-iliac-spine BM aspiration. A highly sensitive staining method was used to differentiate cytokeratin postitive cells of epithelial origin from normal hematopoetic cells. Cytospin slides were stained wtih monoclonal anti-CAM 5.2 antibody. Visualization was achieved with Ventana view-Red detection system. Slides were then counterstained with haematoxylin. Automated Cellular Imaging System was used to classify cells of interest by cellular color, size and shape. SLNM was done by peritumoral injection of 1% methylene blue in the subserosal layer. Results: A total of 385 pts were analyzed. Of these 11% were found to have BMM. The three highest sites of BMM were cholangiocarcinoma 33%, Stomach 22% and esophagus 13% (Table 1A). BMM was +ve bilaterally in 44% of pts, unilaterally in 56%. In pts who underwent SLNM, BMM were detected in 8.3% of SLN +ve vs. 12% for SLN -ve pts. For a subgroup of pts with colorectal cancer undergoing SLNM, BMM were detected in 9.6% of SLN +ve pts vs. 11% for SLN -ve pts. Conclusions: BMM did not correlate with nodal status for GI malignancies suggesting a possible different mechanism for metastases. Detection of BMM may have a significant clinical value in SLN negative pts who may benefit from adjuvant therapy. [Table: see text] [Table: see text]

Versatile Fluorescent Conjugated Polyelectrolyte-Capped Mesoporous Silica Nanoparticles for Controlled Drug Delivery and Imaging.

Fluorescent conjugated polyelectrolytes (CPEs) with a large extinction coefficient, high quantum efficiency, good photostability, and efficient intramolecular/intermolecular exciton migration have recently received great research attention as a new generation of fluorescent probes. Herein, the unique properties of fluorescent conjugated polyelectrolyte and mesoporous silica nanoparticles (MSNs) are combined to prepare a pH-sensitive drug delivery and cellular imaging system using a simple and more general method. To the best of our knowledge, this is the first proof-of-concept investigation on the use of fluorescent conjugated polyelectrolyte as both the capping agent of MSNs and the cellular imaging probe in parallel. Moreover, the bifunctional nanomaterial exhibits low cytotoxicity and good biocompatibility demonstrated by a cytotoxicity assay against HepG2 cells and a hemolysis assay against human red-blood cells. It is a promising candidate for simultaneous diagnostics and therapeutics and can also provide a general strategy to develop novel multifunctional nanomedical systems.

Correlation of bone marrow micrometastases (BMM) with nodal status in gastrointestinal tumors (GI).

567 Background: Presence of BMM is an important prognostic factor for patients (pts) with solid tumors. Sentinel lymph node (SLN) mapping (M) has been found to upstage pts with GI tumors. However, a direct correlation between the presence of BMM and nodal metastases is lacking. Hence, a retrospective study was undertaken to determine the relationship between BMM and nodal status in GI tumors. Methods: A total of 385 consecutive pts with GI tumors were analyzed. All pts underwent bilateral posterior superior iliac spine BM aspiration. This protocol employs highly sensitive staining methods combined with automated microscopy to differentiate cytokeratin +ve cells of epithelial origin from normal hematopoetic cells in BM. The cytospin slides were stained with the monoclonal anti-CAM 5.2 antibody from Becton Dickinson at a 1:25 dilution. Visualization was achieved with Ventana view Red detection system. The slides were counterstained with Ventana haematoxylin. The Automated Cellular Imaging System is applied to detect, count, and classify cells of clinical interest based upon the recognition of cellular objects of particular color, size and shape. SLNM when applicable was done by peritumoral injection of 1% methylene blue. Results: Of these 385 pts, 11.2% were found to have BMM. When analyzed by sites, the 3 highest BMM were Cholangiocarcinoma 33%, Stomach 22% and esophagus 13%. The BMM was +ve bilaterally in 44%; unilaterally in 56%. In pts who underwent SLNM, BMM were found in 8.3% of SLN +ve vs. 12% for SLN -ve pts. For pts with colon cancer undergoing SLNM, BMM were found in 9.6% of SLN +ve pts vs. 11.1% for SLN -ve pts. Conclusions: BMM did not correlate with nodal status for GI malignancies suggesting a possible different mechanism for metastases. Detection of BMM may have a significant clinical value in SLN negative pts who may benefit from adjuvant therapy. [Table: see text]

High incidence of DNA ploidy abnormalities and increased Mcm2 expression may predict malignant change in oral proliferative verrucous leukoplakia

To assess the DNA content of cases of oral proliferative verrucous leukoplakia (PVL) and correlate the DNA ploidy findings with the expression of Mcm2, geminin, and Ki67, and with clinicopathological data. DNA quantification was performed by image cytometry using the ACIS III Automated Cellular Imaging System. Expression of Ki67, Mcm2 and geminin was determined by immunohistochemistry. There were 21 cases of PVL, the female/male ratio was 6:1, and the average age was 65.5years. Seventeen patients (81.0%) did not report tobacco and alcohol consumption. Nine patients (42.9%) developed verrucous or squamous cell carcinoma. Levels of Mcm2 expression showed a positive correlation with increasingly severe epithelial changes (P=0.03). Twenty patients had their DNA examined by ACIS III, and 19 (95%) showed aneuploidy. The frequency and severity of aneuploidy (P<0.0001), the mean values of the DNA heterogeneity index (P<0.0001) and the 5n-exceeding fractions (P= 0.0007) increased according to epithelial alterations. Abnormal DNA content was observed even in the more indolent lesions. Mcm2 expression and DNA ploidy analysis could be used to predict areas of malignant transformation. The clinicopathological findings associated with the immunohistochemical and DNA ploidy results support the distinct and aggressive profile of this entity.

P53 protein profile by IHC may be helpful to define patient prognosis

We read with interest the article ‘‘Prognostic value of oncoprotein expressions in thyroid papillary carcinoma’’ by A. Z. Balta, A. I. Filiz, Y. Kurt, I. Sucullu, E. Yucel and M. L. Akin [1] because our most recent studies of p53 protein have also been showing that immunohistochemical (IHC) staining is augmented in patients with papillary thyroid carcinoma (PTC). However, we found some different associations that deserve further reflection, especially in what concerns clinical pathological aspects of PTC. We analyzed a large series of cases: 206 patients with differentiated thyroid cancer (DTC), including 164 PTC (106 classic form; 54 follicular variant; and 04 tall cell variant) and 42 follicular carcinomas, using monoclonal mouse anti-human p53 antibody (clone DO-7; monoclonal, DAKO, Carpenteria, CA, USA). In addition, we studied 105 benign thyroid tissues including: 50 nodular goiters, 55 follicular adenomas and 18 normal thyroid tissues. Our patients were all managed according to a standard protocol and followed up for 53.8 ± 41.0 months. Although our quantitative analysis using the automated cellular imaging system (ACIS-III) (Chroma Vision Medical Systems, Inc, DAKO) identified mean values of 5.11 % and medians of 1.71 % of p53 nuclei expression, we concur with Balta et al. interpretation and considered all the benign cases negative. Conversely, 28.67 % of our malignant tissues expressed p53 with a mean of 41.26 % and a median of 28.67 % stained nuclei. Balta et al. observed that p53 expression was significantly associated with the presence of lymph node metastasis and extrathyroidal invasion (p = 0.003 and p = 0.004, respectively). In contrast, we found higher expression of p53 in patients presenting better prognostic features, such as: females (p = 0.0367); smaller (\2 cm) and solitary tumors (p = 0.0105) that did not present extrathyroidal invasion (p = 0.0241). P53 was also more expressed in patients evolving free of disease than in patients with persistence or recurrence (p = 0.0310). A proportional hazard regression analysis (Cox’s multivariate proportional hazard model) identified age (p = 0.0070; HR = 1.0675; CI 95 %, 1.0180–1.1195) and the presence of metastasis at diagnosis (p = 0.0384; HR = 4.6889; CI 95 %, 1.0862–20.2417) as independent variables that influence relapse-free survival, excluding any role of p53. In addition, the Kaplan–Meier survival curve did not identify p53 as a factor affecting recurrence-free survival. The differences between our findings and those by Balta et al. [2] could be related to the patients investigated, since they have selected a group that does not correspond to the usual profile of DTC patients seen in clinical practice. In fact, they described a study population of 53 men and 34 women, whereas it is well known that 70–80 % of the DTC patients are females. Male patients are usually diagnosed in more advanced stages and may present more aggressive features and a worse outcome than females. In addition, Balta et al. classified 42 out of their 47 PTC patients as high-risk patients and only 5 as low-risk patients. Our study population (80.4 % women and 19.6 % men), with a large majority of low-risk patients (129 out of 206 DTC cases— 62.6 % were pTNM stages I and II), is more representative of the DTC usual patients. Although our results contrast with the ones described by Balta et al., they evidence the importance of clinical and M. A. Marcello (&) E. C. Morari L. S. Ward University of Campinas (FCM-Unicamp), Campinas, Sao Paulo, Brazil e-mail: marjoryam@gmail.com

Tu1497 Automated Cellular Imaging System for Aneuploidy Differentiates Chronic Pancreatitis From Pancreatic Cancer

Tu1497 Automated Cellular Imaging System for Aneuploidy Differentiates Chronic Pancreatitis From Pancreatic Cancer