Allred Scoring System Research Articles

ANP32A modulates cell growth by regulating p38 and Akt activity in colorectal cancer

Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) possesses multiple biochemical activities, has been found to be decreased or absent in malignant tumors. However, new findings have shown that it is expressed in greater amounts in advanced cancers than in early-stage tumors. The role and clinical significance of ANP32A in colorectal cancer (CRC) is still unknown. In the present study, the expression of ANP32A was assessed in 68CRC patients by IHC, and then the correlation of its expression with clinicopathological factors was investigated using the Allred, Klein and immune response scoring system analysis. Western blot and real-time PCR analyses were used to assess ANP32A expression and the activity of Akt and p38 in cancer and normal tissues. These data indicated a significant association between ANP32A expression and the activity of Akt and p38, besides the tumor differentiation status in CRC patients. IHC and western blotting data revealed that ANP32A was overexpressed in CRC patients, and ANP32A levels were higher in poorly differentiated tumors. Protein and mRNA analysis revealed that with a high expression of ANP32A, the activation of Akt was enhanced, while the p-38 phosphorylation level was decreased in CRC tissues. MTT assay and functional studies revealed that knockdown of ANP32A inhibited cell growth and induced p38 phosphorylation and Akt dephosphorylation. The present study indicated that ANP32A promoted CRC proliferation by inhibition of p38 and activation of Akt signaling pathways and suggested that ANP32A may play a potential role in CRC diagnosis and therapy.

Effect of Hydrochloric Acid Decalcification on Expression Pattern of Prognostic Markers in Invasive Breast Carcinomas.

In the United States, it is estimated that 100,000 people are living with metastatic breast cancer (BC) with bone representing the most common site of involvement. However, patients with isolated bone metastasis at presentation may have a longer survival. Therapeutic options for BC bone metastases often include systemic anticancer therapy (endocrine, chemotherapy, monoclonal antibodies, and/or other targeted therapies), which is largely dependent on the immunohistochemical (IHC) repertoire of the cancer for the prognostic markers [estrogen (ER) and progesterone receptors (PR), Ki-67, p53, and Her-2/neu] at its osseous metastatic site. Traditionally, specimens obtained from the bone metastasis require decalcification, which may affect the immunoreactivity of these prognostic markers. To the best of our knowledge, limited studies describe the effect of decalcification on immunoexpression of the above-mentioned markers. A detailed illustration of the effect of decalcification on BC specimens in a real-time manner is lacking in the literature. Herein, we sought to determine the impact of decalcification on the IHC expression pattern of the above listed markers on BC tissue following decalcification. After Institutional Review Board approval, sections from the residual tumor specimens were collected prospectively from 15 BC excision specimens and 1 curetting from a BC bone metastasis. The sections (3 to 6 sections/case) for decalcification were collected following routine submission for pathologic evaluation. The sections were subjected to hydrochloric acid (HCl)-based Decal Stat decalcifying solution for 2, 12, 18, and 24 hours in each case. IHC studies for ER, PR, Ki-67, p53, and Her-2/neu were performed on 1 representative section of the regularly processed tumor block and 1 decalcified tumor block from each time point. Scoring of ER and PR were performed according to the Allred scoring system. Scoring of Her-2/neu was performed according to CAP/ASCO guidelines. The tumors comprised 11 grade 3 invasive ductal carcinomas, 2 grade 2 invasive ductal carcinomas, 2 grade 3 invasive lobular carcinoma, and 1 metastatic BC to bone. Nine cases showed Allred score 8 for ER, 1 case showed Allred score 4, 1 case showed Allred score 2, and the remaining 5 were ER negative. For PR, 1 showed Allred score 8, 2 Allred score 7, 4 Allred score 6, 1 each Allred score 5 and score 2 with the remainder negative for PR. Ki-67 ranged from 5% to 95%. Five cases showed p53 overexpression ranging from 35% to 95%. Five cases each showed 3+, 6 cases showed 2+ Her-2/neu, 3 cases showed 1+ Her-2/neu, and the remaining 2 were negative. All specimens demonstrated decline in ER, PR, Ki-67, and p53 immunoreactivity after 2 hours of decalcification, with additional decline up to 24 hours. The most significant declines in immunoreactivity occurred with Ki-67 and p53. Most of the Her-2/neu cases with an equivocal score declined to zero after 24 hours of decalcification. However, 3 out of 11 cases showing Her-2/neu overexpression remained at the baseline scoring even after extended (24 h) decalcification. Our results demonstrated that the decalcification process affects the immunoreactivity of the prognostic BC markers. There is progressive loss of reactivity at 2 hours and beyond for markers with lower degrees of expression. In addition, heterogeneity in marker distribution progressed from diffuse to more focal beyond 1 hour.

Utility of Tru-Cut Biopsy of Breast Lesions - An Experience in a Regional Cancer Center of a Developing Country.

Tru-cut Needle Biopsy (TCB) is an integral part of triple assessment of breast cancer, which includes clinical assessment, mammography and TCB or Core Needle Biopsy (CNB). The technique is reliable, simple, and reproducible, and inexpensive, which can be adapted even for low-income group of patients and in developing countries. This study was done to establish the efficacy of TCB of palpable breast lesions in a developing country where mammography is not possible in all cases. A retrospective analysis of 892 TCBs was done in AH Regional Cancer Centre, Cuttack, Odisha, India where TCBs were performed in patients presenting to outpatient department with palpable breast lesions. The H&E stained sections were interpreted by pathologists of the same centre. Diagnosis was classified into different categories. Immunohistochemistry (IHC) for Estrogen Receptor (ER), Progesterone Receptor (PR) and Her-2/neu was done and interpreted by Allred scoring system. A total 892 TCBs were analysed with 23 repeat TCBs. There were 13 (1.4%) male patients. A total of 747 cases (83.6%) were diagnosed as malignant, including 735 carcinomas, nine malignant phyllodes tumour, two angiosarcoma and one case of Non-Hodgkin' Lymphoma (NHL). It was possible to diagnose special histological types such as lobular carcinoma, metaplastic carcinoma and mucinous carcinoma on TCB. A total of 21 cases were diagnosed as carcinoma on repeat biopsy. Eight of the 735 TCBs diagnosed as carcinoma were bilateral breast cancers, hence actual number of carcinoma cases were 727. IHC was done successfully on the paraffin blocks in 260 cases. In this series out of 727 patients of carcinomas 30% were in young, i.e., below 40 years of age, including four cases of carcinoma below 20 years. There were no false positive case in this study giving a specificity of 100% and sensitivity was 97%. TCBs are well tolerated by patients, can be done in OPDs and reduce cost. It is possible to give histological diagnosis of carcinoma, lymphoma, phyllodes tumour and sarcomas on TCBs. The paraffin blocks of TCBs can be used for IHC study which helps the oncologists for preoperative adjuvant therapy.

Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) association with lymph node metastasis predicts poor survival in oral squamous cell carcinoma patients.

Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) is a multifunctional protein aberrantly expressed in various types of cancers. However, its expression pattern and clinical significance in oral squamous cell carcinoma (OSCC) remains unclear. In this study, we immunohistochemically investigated the expression pattern of ANP32A in 259 OSCC patients and the results were correlated with clinicopathological factors using Allred, Klein and Immunoreactive scoring (IRS) system. Our data indicated that high expression of ANP32A was significantly associated with N stage and tumor differentiation status in OSCC patients. High ANP32A expression with N2/N3 stage had an increased mortality risk than low ANP32A expressing OSCC patients with N0/N1 stage. Functional studies revealed that knockdown of ANP32A significantly decreased the migration and invasion ability thereby concomitantly increasing E-cadherin and decreasing Slug, Claudin-1 and Vimentin expression in vitro. These results suggest that ANP32A is commonly increased in oral squamous cell carcinoma and ANP32A protein could act as a potential biomarker for prognosis assessment of oral cancer patients with lymph node metastasis.

Hormone receptor (ER, PR, HER2/neu) status and proliferation index marker (Ki-67) in breast cancers: Their onco-pathological correlation, shortcomings and future trends

Estrogen receptor (ER) and progesterone receptor (PR) are hormone receptors found on breast cells that pick up hormone signals resulting in cell growth. Similarly, positive human epidermal growth factor receptor-2 (HER2/neu) status of the breast carcinoma means that HER2/neu gene is making too many HER2/neu proteins, which acts as receptors on the cell surface and helps the cells to grow and divide. Hormone receptor studies such as ER, PR, and HER2/neu are routinely done in breast carcinoma. It not only helps in the prognosis of the tumor but also helps in deciding its treatment. The goal of doing this receptor status is to provide right treatment to the right patient. This hormone receptor status is graded using Allred scoring and grading system. Depending on the hormone status, breast carcinomas can be divided into a number of different categories ranging from triple positive through triple negative. Another marker of proliferation (Ki-67) is also being used which is a proliferation index marker. This scoring system has its own limitation and shortcomings, which depends on a lot of pre- and post-analytical factors. Certain new techniques such as genomic assays, PAM50, and HALO screening test are being used nowadays for breast cancer detection.

Prognostic value of tumor-infiltrating lymphocytes (TILs) and expression of PD-L1 in cholangiocarcinoma.

294 Background: Cholangiocarcinoma is a malignancy arising from the epithelial cells of the biliary tract with poor prognosis. Tumor-infiltrating lymphocytes (TILs) have a prognostic impact in various solid tumors. We aimed to investigate TIL expression and programmed cell death ligand PD-L1 and their clinical relevance in cholangiocarcinoma. Methods: Formalin-fixed paraffin-embedded tumor samples from thirty seven patients with resected and histologically verified cholangiocarcinoma between 1990 and 2011 were identified and immunohistochemically (IHC) stained with anti CD8, anti CD45RO and the anti-PDL1 mouseIgG1 (clone 5H1; Thompson) antibodies on a Leica automated IHC platform. The stains were semiquantitatively analyzed using the AllRed score system (range 1 to 8). Cases with IHC score > 3 were considered positive. The association between PDL1, CD45R0, overall survival (OS) and progressive free survival (PFS) was investigated using Kaplan-Meier survival and COX proportional hazard regression analyses. Results: The median age of patients was 64 (41-85) with 51% male. 25%, 59%, 10% and 5% were stage I, II, III, and IV respectively. CD8 was positive in 10/37 (27%) with median IHC of 5, CD45R0 (specimen with lymph node like structure) was positive in 16/37 (43%) with median IHC score of 4, PD-L1 was positive in 35/37 (94 %) with median IHC score of 8. Pts with tumors exhibiting lymph node (LN) like structures (CD45RO+) has better median OS (63 months vs 18 months, P = 0.0065) and median PFS (29 months vs 14.6 months, P = 0.05) than patients lacking LN like structures (CD45RO-). 5 yrs. OS in CD45RO+ was 64% vs 20% in CD45RO- (P 0.006) whereas 5 yrs. PFS in CD45RO+ was 41% vs. 8% in in CD45RO- pts (P value 0.05). Correlating PDL1 with OS and PFS was not done due to high expression of PD-L1 in most of the samples (94%). Conclusions: Presence of lymph node like structures (CD45RO+) was associated with significant better outcome reflecting the immune-mediated killing of tumor cells. The high expression of PD-L1 on cholangiocarcinoma cells represents a potential therapeutic target which may improve the prognosis for this dismal cancer. Future trials involving PD-L1 inhibitors in cholangiocarcinoma are warranted.

Assessment of the impact of the concentration of formalin on receptor studies in surgical specimens of breast carcinoma: a pilot study

Introduction: Assessment of oestrogen, progesterone and HER2 receptor status are routinely performed on specimens fixed in 10% formalin. Objective: To assess the impact of the concentration of formalin on the oestrogen, progesterone & HER2 receptor status in surgical specimens excised for carcinoma of breast. Method: A descriptive, cross sectional study was done on excision biopsy and mastectomy specimens of breast cancer over a period of 18 months. A 1x1cm piece of tumour tissue was fixed in 50% diluted formalin and the remainder in 10% formalin. Following routine tissue processing, staining for ER, PR and HER2 receptor status was done. Allred scoring system was used to interpret ER and PR receptor status and CAP (College of American Pathologists) guidelines were used to interpret HER2 receptor status. Results: In view of the small sample size (n=20), binomial sign test was selected to assess the differences between the two groups (10% formalin vs 50% diluted formalin). There were statistically significant differences in the results with a marked downgrading of the total score of ER and PR and HER 2 receptor studies with 50% diluted formalin. Conclusion: It is vital for the specimen to be adequately fixed in 10% buffered formalin before commencing on receptor studies. DOI: http://dx.doi.org/10.4038/jdp.v9i1.7651 Journal of Diagnostic Pathology 2014; 9(1): 24-32

Expression of BMP6 is Associated with its Methylation Status in Colorectal Cancer Tissue but Lacks Prognostic Significance

The study aimed to evaluate the incidence of CpG island promoter methylation of BMP6, a member of the transforming growth factor beta family, in tissue samples from colorectal cancers (CRC) and look for its association with BMP6 expression and clinicopathological correlation. Methylation specific PCR for the BMP6 promoter region was performed with 85 frozen tissue samples of CRC and 45 of normal colon. Methylation status of MLH1 was also determined by the same method. Expression of BMP6 was evaluated by immunohistochemistry (IHC), using Allred's scoring system. The methylation status was analyzed against clinical and pathological parameters in CRC. The study revealed BMP6 hypermethylation in 34 of 85 tumor specimens (40%), and 15 out of 45 normal tissue samples from CRC (33%). The incidence of hypermethylation was inversely correlated with IHC score. Allred's scores of 7 or more were correlated with lower frequency of BMP6 hypermethylation (29% compared to 50% in the remaining, p-value 0.049). However, there was no association between hypermethylation status and any clinicopathological parameters. The methylation status of BMP6 was not correlated with that of MLH1, a key methylation determinant in CRC. On survival analysis, there was no significant difference in progress-free survival (PFS) between the cases with and without hypermethylation (2-year PFS 74% and 76%, respectively). CpG island methylation of BMP6 is found in high frequency in CRC and this epigenetic event is associated with suppressed protein expression in the tumor tissue. However, the marker is not associated with tumor progression of the disease.

Expression of Acetylated and Dimethylated Histone H3 in Colorectal Cancer

Aim: The expression of acetylated and dimethylated histone H3 in colorectal cancer was examined by immunohistochemistry and chromatin immunoprecipitation (ChIP)/Western blot (WB) assay. The correlation between the expression of histone H3 and clinicopathological findings was analyzed. Methods: Formalin-fixed and paraffin-embedded sections obtained from 80 operated cases of colorectal cancer were immunostained with anti-acetylated histone H3 (H3Ac) antibody and anti-dimethylated histone H3 lysine 4 (H3K4) antibody. Positive immunoreactivity was evaluated using the Allred scoring system. Furthermore, the expression was confirmed by ChIP/WB assay using formalin-fixed and paraffin-embedded sections. Results: There was good correlation between immunostaining and expression on ChIP/WB assay (p = 0.0005). There was a significant difference between the Allred score of H3K4 and the depth of tumor invasion (p = 0.0003) and the pathological stages (p = 0.0065). In overall survival classified by Allred scores of H3Ac (p = 0.0072) and H3K4 (p = 0.0187), the highest scores represented significantly worse prognoses than the other scores. Specifically, in stages II and III, the highest scores represented significantly worse prognoses than the other scores (p < 0.0001 and p = 0.0173, respectively). Conclusion: The expression of H3Ac and H3K4 may estimate patient prognosis.

Overexpression of epidermal growth factor receptor as a prognostic factor in colorectal cancer on the basis of the Allred scoring system

BackgroundOverexpression of epidermal growth factor receptor (EGFR) is found in many types of neoplasms. The aim of the study was to evaluate EGFR expression in colorectal cancer (CRC) specimens and to determine whether EGFR expression correlates with clinicopathological data and overall survival.Patients and methodsTissue specimens from 181 consecutive CRC patients treated at the Military Institute of Medicine in 2006–2010 were collected and examined for EGFR expression, by immunohistochemistry staining. The staining intensity and percentage of cells with membranous EGFR expression were scored and then grouped according to the parameters of the Allred Scoring system. Cutoff values were subjected to further statistical analysis. Univariate tests and a multivariate Cox proportional hazards model were used in data analysis.ResultsEGFR was overexpressed in 96 of 181 CRC specimens (53%). EGFR expression was not correlated with other clinicopathological variables. On univariate analysis, overexpression of EGFR, determined by PS (percentage score) (>3) and total score (sum of PS and intensity score) (>4), was associated with poor overall survival. On multivariate analysis, EGFR overexpression (PS > 3) was an independent adverse prognostic factor (hazard ratio [HR] 1.62; 95% confidence interval [CI]: 1.03–2.53). Elevated carcinoembryonic antigen (CEA) serum concentration before treatment, performance status (Word Health Organization [WHO]-2), and tumor localized in colon and liver metastases were also independent unfavorable prognostic factors.ConclusionEGFR overexpression (PS > 3) in a CRC patient population was an independent adverse prognostic factor. Implementation of the Allred Scoring system criteria into clinical practice might facilitate treatment decisions in CRC patients.

PTU-143 Digital Image Analysis Enhances Quantitative Immunohistochemistry in the Squamous-Metaplasia-Dysplasia-Carcinoma Sequence

IntroductionWe have previously shown how the Allred scoring system may be used to semi-quantify expression of nuclear biomarkers in the Barrett’s (BE) to oesophageal adenocarcinoma (OA) sequence. Recently, a number...

Abstract 4760: Evaluation of the progesterone receptor status using three different antibodies: A comparison by Allred score system.

Abstract Abstract: Breast cancer (BC) hormonal receptors (progesterone, PgR and estrogen) status is assessed by immunohistochemistry (IHC), a specific, sensitive, and accessible method that guide BC treatment. We evaluated PgR expression in 53 BC cases using 3 anti-PgR antibodies (AB): monoclonal (SP42 and RP636) and polyclonal ab62621. Methods: Primary BC cases (with signed informed consent) were used to generate tissue microarray platforms, where PgR expression was accessed by IHC and evaluated by the Allred score. Categorical and quantitative data are shown in percentage and mean, respectively. Concordance (CON) and correlation among ABs were analyzed by Kappa factor (K), Spearman's correlation coefficient (ρ) or intraclass correlation coefficient. Staining patterns of each AB were compared by paired T-Test. Findings: We noted poor CON and K between ab62621 vs SP42 (CON=64.1%; K=0.247), and ab62621 vs RP636 (CON=62.3%; K=0.204), but higher CON between SP42 vs RP636 (CON 90.6%; K =0.738). Data were corroborated by Mc Nemar statistical test (p=0.019, p=0.014 and p&amp;gt;0.05, respectively). Regarding staining intensity among PgR+ samples, we found higher proportion of weak staining and lower IS for ab62621 (48.3%; mean IS=1.6), when compared to SP42 (20.0%, mean IS=2.1, T-test p&amp;lt;0.01) and RP636 (2.3, 21.9%, T-test p&amp;lt;0.01). Within the entire sample, similar results were observed following ρ: SP42 vs RP636 (ρ=0.8103); ab62621 vs SP42 (ρ=0.3524); ab62621 vs RP636 (ρ=0.4075). As for proportion of stained cells and PS, among PgR+ samples, the mean values for ab62621 (75.4%; 4.8) were significantly higher than those of SP42 (56.3%, 4.3; T-test p&amp;lt;0.01) and RP636 (60.1%; 4.2; T-test p&amp;lt;0.01). Similar data were found after analyzing PS for the entire sample: SP42 vs RP636 (ρ=0.8588); SP42 vs ab62621(ρ=0.4832); RP636 vs ab62621(ρ=0.4050). Interpretation: Our data indicate that anti-PgR monoclonal ABs, RP636 and SP42, are, unlike ab62621, equally suitable to test BC PgR status by IHC due to their higher accuracy. Therefore, we suggest their clinical use during BC diagnosis; thus, enabling more precise therapeutic decisions to treat BC. Citation Format: Klesia P. Madeira, Renata Daltoe, Alex Carvalho, Lucas Rezende, Ian Silva, Leticia Rangel. Evaluation of the progesterone receptor status using three different antibodies: A comparison by Allred score system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4760. doi:10.1158/1538-7445.AM2013-4760 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The role of insulin-like growth factor 1 in the development of benign and malignant thyroid nodules.

This study aims to investigate the role of IGF-1 in the development of nodular thyroid disease. A total number of 100 consecutive patients operated for nodular thyroid disease in our institution were included in this prospective study. In addition to classical pathological examinations, nodules and extranodular healthy tissues were sampled and immunochemically stained for IGF-1. The materials were independently evaluated using an Allred Scoring System ranging from 0 to 8. If the score was ≥1, the tissue was accepted as IGF-1 positive. IGF-1 positivity was observed in 88% and 58% of the samples obtained from nodules and extranodular healthy tissues, respectively. Allred 8-unit scores were higher in benign nodules (n=89; 4.1±2.3) and papillary carcinomas (n=7; 6.7±1.3), than in extranodular healthy tissues in the same patients (2.3±2.3 and 3.3±1.9, respectively); and higher in papillary carcinomas than in benign nodules, when the scores were compared to each other (p<0.01 for all comparisons). Allred 8-unit scores for IGF-1 increase in the presence of benign thyroid nodules, papillary cancer. The results of our study support the findings of previous studies demonstrating the role of IGF-1 in the development of thyroidal nodules.

PWE-014 Polo-like kinase 1 expression predicts aneuploidy in the Barrett's metaplasia-dysplasia-carcinoma sequence

IntroductionFinding an accurate and convenient biomarker for cancer progression in Barrett's (BE) is of high clinical importance. DNA ploidy abnormalities (DNA-PA) are a reliable predictor of cancer risk in BE,...

Su1877 Polo-Like Kinase 1 Expression Predicts Aneuploidy in the Barrett's Metaplasia-Dysplasia-Carcinoma Sequence

Introduction Finding an accurate and convenient biomarker for cancer progression in Barrett9s (BE) is of high clinical importance. DNA ploidy abnormalities (DNA-PA) are a reliable predictor of cancer risk in BE, but measurement is expensive and scarcely available. We have shown polo-like kinase-1 (PLK1) may act as a surrogate marker of DNA-PA in oesophageal adenocarcinoma (OA) resection specimens. This study aimed to examine the potential of PLK1 in predicting DNA-PA in the metaplasia-dysplasia-OA sequence. Methods 36 paraffin embedded oesophageal tissue specimens were selected from patients with non-dysplastic BE (NDBE, n=5), low grade dysplasia (LGD, n=5), high grade dysplasia (HGD, n=12), intramucosal cancer (IMC, n=5) and invasive OA (IOA, n=9). Sections were mounted and immunostained with 2 PLK-1 antibodies PLK1-M and PLK1-L using an automated system (BOND-MAX, Leica) for consistency. Results were reported by two independent expert pathologists blinded to patient status with the Allred scoring system, a composite of the percentage and intensity of staining. Results Aneuploidy was present in LGD (20%), HGD (75%), IMC (20%) and IOA (56%) samples. Using linear regression analysis, Pearson coefficient was calculated for the correlation between DNA-PA and mean Allred expression scores for each PLK1 antibody. PLK1-M had a higher degree of correlation (r 2 =0.26, p=0.001) then PLK1-L (r 2 =0.22, p=0.004). Inter-observer analysis with linear κ scores confirmed good correlation of PLK1-M (κ=0.72, 95% CI 0.60 to 0.83) and PLK1-L (κ=0.53, 95% CI 0.38 to 0.68), but a Bland–Altman plot found pathologist 2 had a trend to score PLK-L more highly. However, intra-observer analysis confirmed both PLK1-L scores correlated with ploidy status (r 2 =0.14, p=0.023 and r 2 =0.27, p=0.0016). Pathologists took 90.3 s/slide (mean) to review and score with the Allred method. Using a mean Allred cut off score of 3.5, the sensitivity and specificity for the detection of aneuploidy were 81.3% and 75% for PLK1-M, 93.8% and 45% for PLK1-L. Conclusion This pilot study demonstrated a significant correlation between Allred reporting of PLK1 staining and DNA-PA. PLK1-L appears more sensitive and PLK1-M more specific. PLK1 immunostaining is relatively inexpensive, less work intensive and is more available than current methods to predict DNA-PA. Scoring staining with Allred is rapid and reproducible. In future, PLK1 staining may provide the basis of a more durable biomarker panel to predict DNA ploidy. Competing interests None declared.

Su1878 Risk Factors for the Development of Esophageal Precancerous Lesions: an Analysis of the Cases Registered for a Multicenter Prospective Cohort Study

Introduction: Finding an accurate and convenient biomarker for cancer progression in Barrett's oesophagus (BE) is of high clinical importance. DNA ploidy abnormalities (DNAPA) are a reliable predictor of cancer risk in BE, but measurement is expensive and not routinely available. We have previously shown in a sample of surgical oesophageal resection specimens that replication licensing factors (RLFs), particularly polo-like kinase-1 (PLK1) may act as surrogate markers of DNA-PA. This study aimed to examine the potential of PLK1 in predicting DNA-PA in the metaplasia-dysplasia-OA sequence. Method: 36 paraffin embedded oesophageal tissue specimens were selected from patients with non-dysplastic Barrett's (NDBE, n=5), low grade dysplasia (LGD, n=5), high grade dysplasia (HGD, n=12), intramucosal cancer (IMC, n=5) and invasive OA (IOA, n=9). Sections from these blocks were mounted and immunostained with two PLK-1 antibodies from Millipore (PLK1-M) and Leica (PLK1-L) with the automated BOND-MAX system (Leica Microsystems) for consistency. Staining was reported by 2 independent expert pathologists blinded to patient status with the Allred scoring system, a composite of the percentage (0-no cells, 1= 66%) and intensity of cells staining (0=negative, 1=weak, 2=moderate, 3=strong). Adjacent sections were analysed with image cytometry to identify DNA ploidy abnormalities. Results: Aneuploidy was present in LGD (20%), HGD (75%), IMC (20%) and IOA (56%) samples. Using linear regression analysis, Pearson coefficient was calculated for the correlation between DNA-PA and mean Allred expression scores for each PLK1 antibody. PLK1-M had a higher degree of correlation (r2=0.26, p=0.001) then PLK1-L (r2= 0.22, p=0.004). Inter-observer analysis with linear kappa scores confirmed good correlation of PLK1-M (κ=0.72, 95% CI:0.60-0.83) and PLK1-L (κ=0.53, 95% CI:0.38-0.68), but a Bland-Altman plot found pathologist 2 had a trend to score PLK-L more highly. However, intra-observer analysis confirmed both PLK1-L scores correlated with ploidy status (r2=0.14, p=0.023 and r2=0.27, p=0.0016). Pathologists took 90.3 seconds/slide (mean) to review and score with the Allred method. Using a mean Allred cut off score of 3.5, the sensitivity and specificity for the detection of aneuploidy were 81.3% and 75% for PLK1-M, 93.8% and 45% for PLK1-L. Conclusion: This pilot study demonstrated a statistically significant correlation between Allred reporting of PLK1 staining and DNA-PA. PLK1-L antibody appears more sensitive and PLK1-M more specific. PLK1 immunostaining is relatively inexpensive, less work intensive and more available than current methods to predict DNA-PA. Scoring staining with the Allred method is rapid and reproducible. In future, PLK1 staining may provide the basis of a more durable clinical biomarker panel to predict DNA ploidy.

Combined evaluation of CK5/6, ER, p63, and MUC3 for distinguishing breast intraductal papilloma from ductal carcinoma in situ

The differentiation of intraductal papilloma (IDP) in the breast from ductal carcinoma in situ (DCIS) is sometimes difficult. Fifty papillary lesions (25 DCIS and 25 IDP) were immunohistochemically examined using a panel of antibodies, including CK5/6, ER, p63, Ki-67, chromogranin A, synaptophysin, neuron specific enolase, CD56, MUC1, MUC3, CD44, p21, p27, and p53. The immunohistochemical staining pattern of each antibody was evaluated using the Allred scoring system. Then, the area under curve (AUC) for each antibody was computed by receiver operating characteristic (ROC) analysis. DCIS typically showed high scores for ER and MUC3 reactivity compared with IDP, and the AUC for ER and MUC3 were 0.941 and 0.908, respectively. In contrast, IDP showed high scores for CK5/6 and p63 reactivity compared with DCIS, and the AUC for CK5/6 and p63 were 1.00 and 0.954, respectively. We devised a 'Differential Index' (DI) using the following formula: [S(ER) + S(MUC3)]/[S(CK5/6) + S(p63) + 1]. The distributions of the DI for IDP and DCIS did not overlap when the cutoff value was placed arbitrarily at DI = 1.0. From these results, it is concluded that a panel of four CK5/6, ER, p63, and MUC3 antibodies provide valuable information for differentiating IDP from DCIS.

Wnt pathway is affected by endocrine therapy in breast carcinomas

Background and aim: Identifying potential predictive biomarkers of response to endocrine therapy would benefit most estrogen receptor-positive (ER+) breast cancer patients. Our aim was to compare the expression of the wingless type (Wnt) pathway-related proteins (adenomatous polyposis coli [APC], E-cadherin, beta-catenin, cyclin D1, and c-myc) in postmenopausal women with ER+ invasive ductal carcinomas (IDC), prior to and after tamoxifen (n= 18) or anastrozole (n= 15) treatment, in a double-blind, placebo-controlled (n= 25), prospective study for 26 days prior to surgery. Methods: Tissue microarray blocks were constructed from pre- and post-treatment biopsy samples. Nuclear immunostaining of c-myc, APC, estrogen and progesterone receptor levels were assessed using the Allred scoring system, and cytoplasmic immunostaining of cyclin D1, beta-catenin and E-cadherin was assessed with the Hercep-Test system. An anova statistical analysis estimated general equations and analysis of variance with a significance level of 0.05. Results: Tamoxifen increased c-myc (P= 0.0061) and APC (P= 0.0452) expression. Anastrozole did not significantly affect expression of any Wnt-related proteins. Conclusions: In post-menopausal ER+ IDC, tamoxifen for 26 days prior to IDC surgery influenced statistically the expression of important cell cycle regulators as APC and c-myc, whereas anastrozole therapy did not interfere with this pathway during the same period. These Wnt related proteins may contribute to selective estrogen receptor modulator resistance.

Immunochemistry for oestrogen receptor, progesterone receptor and HER2 on cell blocks in primary breast carcinoma

Steroid receptors and human epidermal growth receptor 2 (HER2) have been used for predicting response to treatment in breast cancers. Fine needle aspiration cytology can provide highly cellular material and can be used for such analysis. The present study was undertaken to assess the reliability of oestrogen and progesterone receptor (ER, PR) status and HER2 as demonstrated by immunochemistry (IHC) on cell blocks from breast carcinoma cases, in comparison with histological sections. IHC for ER, PR and HER2 was performed on cell blocks and their corresponding tissue sections of 50 primary pre-chemotherapy breast carcinomas. Positivity for ER and PR was scored according to the Allred scoring system. Strong membranous positivity in more than 30% of tumour cells was considered positive for HER2. The tumours were classified as luminal A, luminal B, HER2-over-expressing and triple negative on the basis of ER, PR and HER2 status and results on cell blocks compared with histological sections. Correlation between immunostaining on cell blocks and the corresponding tumour tissues revealed a concordance rate for ER, PR and HER2 of 90% [Correlation coefficient (r) = 0.79], 94% (r = 0.86) and 90% (r = 0.76), respectively. Including five cases in which cell blocks were either ER or PR positive, 43/50 cases (86.0%) could be correctly classified on cell block immunostaining alone. The main reasons for seven discordant cases included technical errors (sampling error and staining error) and interpretational error in HER2 evaluation on cell blocks; the core biopsy was inadequate in one, and apparently false negative for HER2 in another. Cell blocks are useful in the assessment of hormone receptor status and HER2 by IHC, especially in cases of locally advanced breast cancer for planning neoadjuvant chemotherapy. It is highly recommended to have good quality cell blocks and quality control of their interpretation.

Abstract 4706: Gene and protein expression of TGF-beta signaling pathway molecules in women undergoing reduction mammoplasty

Abstract Gene and protein expression of TGF-beta signaling pathway molecules in women undergoing reduction mammoplasty. Introduction: The TGF- β signaling pathway is involved in growth, differentiation, apoptotic and senescence processes; it also participates in the malignant transformation events that lead to breast cancer. Hypothesis: TGF- β expression might contribute to the risk of breast cancer. Methods: As part of an ongoing epidemiology study conducted at the Lombardi Cancer Center in Georgetown University, we collected breast tissue samples from women undergoing reduction mammoplasty without history of cancer. We obtained histological slides from 92 women and performed immunohistochesmistry staining against TGF-β1, TGF-β2, TGF-β3, TGF-βR1 and TGF-βR2. The Allred scoring system was used to asses the intensity and distribution of the staining in each slide (in a scale of 0 to 6). Additionally, we extracted RNA from frozen tissue samples in RNA-later solution from 75 women and assessed the gene expression of the TGF-β signaling pathway by quantitative-PCR (relative quantification against endogenous RNase-p). Results: We found the staining highly specific and confined mainly to lobular structures; TGF-β2 (median A-score 6, SD 0.9009), TGF-βR1 (median A-score 6, SD 0.6603) and TGF-βR2 (median A-score 6, SD 0.7460) scores were higher than TGF-β1 (median A-score 0, SD 1.8485) and TGF-β3 (median A-score 5, SD 0.6450), and we noticed high levels of TGF-β1 in 9 women with proliferative lesions (atypical hyperplasia), when compared against women without lesions (Fisher's exact test P&amp;lt;0.0001). When we examined the localization, the staining was predominantly cytoplasmic among all the molecules studied, however, we identified also nuclear staining in TGF-βR1 and some TGF-βR2, moreover, we identified particularly intense TGF-βR2 staining in myoepithelial cells. We found a very uniform gene expression among the three ligands and two receptors of the TGF-β pathway: for TGF-β1 the median RQ was 1.321, and the SD 2.091; for TGF-β2 the median RQ was 1.361, and the SD 1.301; for TGF-β3 the median RQ was 1.291 and the SD 1.799; for TGF-βR1 the median RQ was 1.424, and the SD 2.266 and for TGF-βR2 the median RQ was 1.281 and the SD1.072. There were no significant differences in the expression of the TGF-β molecules (ANOVA P=0.3475), which in addition to the IHC analysis, indicates that the modulation on TGF-β signaling pathway occurs at the post-transcriptional level. Conclusion: There is an interindividual variation in the gene expression of TGF-β signaling pathway molecules in healthy women, and TGF-β1 protein expression is increased in women with proliferative lesions in their breast tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4706.