Aziza Nassar

Immune-induced epithelial to mesenchymal transition in vivo generates breast cancer stem cells

The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell-dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24(-/lo)CD44(+) phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse.

Intratumoral heterogeneity of immunohistochemical marker expression in breast carcinoma: a tissue microarray-based study.

Core needle biopsies of breast carcinomas provide diagnostic, prognostic, and predictive information before neoadjuvant therapy. Possible intratumoral heterogeneity of biomarker expression questions the validity of core needle biopsy interpretation in small biopsy specimens. Using tissue microarray (TMA) technology, we studied intratumoral heterogeneity of 7 immunomarkers. Five TMAs were constructed from 44 breast carcinomas and 5 normal breast tissues, each represented by 1-mm cores in triplicate from each of 3 foci. TMAs were immunostained for monoclonal estrogen receptor (ER), monoclonal progesterone receptor (PR), polyclonal human epidermal growth factor receptor 2 (HER2), monoclonal E-cadherin (E-cad), monoclonal epidermal growth factor receptor (EGFR), monoclonal p53, and monoclonal MIB-1. Expression was quantified visually by light microscopy and by image cytometry as intensity, percentage of cells positive, and score. Using intraclass correlation coefficient, heterogeneity in the expression of the immunomarkers within subjects was compared with the overall variance. Intratumoral heterogeneity was seen with 5 immunomarkers: ER, PR, HER2, p53, and MIB-1. E-cad and EGFR failed to show intratumoral heterogeneity. Intratumoral heterogeneity in ER, PR, HER2, p53, and MIB-1 indicates their problematic interpretation in small biopsy specimens as indicative of the status of the entire tumor. A negative result does not exclude the expression of these markers in the remainder of the tumor. E-cad (positive in ductal carcinomas) and EGFR lacked heterogeneity.

Utility of glypican-3 and survivin in differentiating hepatocellular carcinoma from benign and preneoplastic hepatic lesions and metastatic carcinomas in liver fine-needle aspiration biopsies.

Glypican‐3 (GPC‐3), a membrane‐anchored heparin sulfate proteoglycan, has been shown to be expressed in ∼80% of hepatocellular carcinoma (HCC) but not in benign hepatic lesions. Survivin, a novel inhibitor of apoptosis, and a prognostic marker, has also been expressed in HCC. We evaluated these two immunomarkers (GPC‐3 and survivin) in differentiating HCC from benign and preneoplastic hepatic lesions and metastatic carcinomas, comparing them to HepPar‐1 (hepatocyte paraffin‐1) in liver fine‐needle aspiration biopsies (FNAB).

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

BackgroundSurvivin 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. DesignA 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. ResultsEighty-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). ConclusionsSurvivin and caspase-3 expression correlate with poor prognostic parameters (higher histologic grade and high proliferation), but not with outcome, in breast carcinoma patients.

Utility of alpha-methylacyl coenzyme A racemase (p504s antibody) as a diagnostic immunohistochemical marker for cancer.

α-Methylacyl-coenzyme A racemase (AMACR; P504S) is a mitochondrial and peroxisomal enzyme involved in the metabolism of branched-chain fatty acid and bile acid intermediates. Recently, AMACR has been demonstrated to be overexpressed in localized and metastatic prostate cancer and in high-grade prostatic intraepithelial neoplasia but not in normal prostatic glands, suggesting that it may be an important tumor marker. This study examines AMACR expression in a variety of human cancers to assess its viability as a tumor marker in the clinical setting. Two hundred sixty-three cancers from different sites were examined in three multitumor tissue micro arrays, which included two or three tissue cores (1.0 mm in diameter) from each neoplastic and normal tissue specimen. Cancers studied included breast (94 cases), prostate (38), lung (28), endometrium (27), colon (29), ovary (26), and melanoma (21). Normal tissues in the microarray were prostate (15), lung (6), endometrium (5), colon (4), ovary (2), and skin (3). Sections were immunostained, after prior pressure cooker antigen retrieval, using rabbit monoclonal AMACR antibody (1:40) (Zeta Corp, Sierra Madre, CA) and horseradish peroxidase-labeled polymer conjugated secondary antibody (Envision, Dako, Carpinteria, CA). A section of prostate cancer and prostatic intraepithelial neoplasia was used as positive control. Protein expression was scored as negative, weak (faint cytoplasmic or granular apical staining), moderate (diffuse granular cytoplasmic stain), and strong (diffuse intense cytoplasmic stain). Only moderate and strong staining was considered as positive staining, based on prior work. AMACR protein overexpression was found in several cancers, including prostate (34/38 [89.5%]), colon (13/29 [44.8%]), lung (4/28 [14.3%]), melanoma (2/21 [9.5%]), endometrium (2/27 [7.4%]), and breast (3/94 [3.2%]). None of the ovarian cancers (26 cases) demonstrated AMACR overexpression. AMACR expression was not present in any of the normal tissues nor in benign prostatic tissue associated with prostate carcinomas. This study suggests that AMACR is potentially an important tumor marker, particularly for prostate and colon cancer. It may be a useful adjunct to an immunohistochemical panel employed in the differential diagnosis of colon versus ovarian and breast carcinoma; the latter two infrequently express AMACR.

COX-2 expression in invasive breast cancer: correlation with prognostic parameters and outcome.

Lipoxygenases (LOX) and cyclooxygenases (COX) are key mediators of arachidonic acid metabolism. Recently, studies have reported that human breast carcinomas aberrantly express LOX and cyclooxygenase-2 (COX-2), and that decreased levels of 15-lipoxygenase (15-LOX) and raised levels of COX-2 and 12-LOX have prognostic value in patients with breast cancer. 15-LOX was significantly reduced with increasing stage, and in patients who developed metastatic disease, local recurrence, and/or died. With high COX-2, patients developed local recurrence, died from breast cancer and had reduced disease-free and disease-related overall survival in estrogen receptor (ER)-negative but not ER-positive disease. COX-2 expression is also associated with increased angiogenesis, lymph node metastasis, and Her2-neu overexpression. The purpose of this study is to evaluate COX-2 expression in breast cancer and to determine its correlation with prognostic parameters and outcome. Five tissue microarrays were constructed from 43 breast carcinomas and 5 normal breast tissues, represented by 1 mm cores in triplicate from each of 3 foci. Tissue microarray cores were immunostained with monoclonal COX-2. Expression was assessed as intensity and scored as percentage of cells positive. Prognostic parameters and follow-up information were obtained from the hospital records of Mexican Oncology Hospital, Mexico, where the carcinomas were diagnosed. Ninety-five percent (41/43) of the breast carcinomas showed cytoplasmic COX-2 expression. COX-2 intensity and percentage of cells positive correlated significantly with size of carcinoma (P=0.0271; P=0.0539, respectively). COX-2 intensity correlated significantly with histologic grade (P=0.0182). COX-2 did not correlate with outcome (disease-free and overall survival). There was no significant correlation between COX-2 and ER. In conclusion, COX-2 correlates with poor prognostic markers in breast cancer (large tumor size and high tumor grade), but not with outcome. The therapeutic value of COX-2 inhibitors in COX-2 positive breast cancer patients requires further investigation.

The state of cell block variation and satisfaction in the era of molecular diagnostics and personalized medicine.

Background: In the recent past, algorithms and recommendations to standardize the morphological, immunohistochemical and molecular classification of lung cancers on cytology specimens have been proposed, and several organizations have recommended cell blocks (CBs) as the preferred modality for molecular testing. Based on the literature, there are several different techniques available for CB preparation-suggesting that there is no standard. The aim of this study was to conduct a survey of CB preparation techniques utilized in various practice settings and analyze current issues, if any. Materials and Methods: A single E-mail with a link to an electronic survey was distributed to members of the American Society of Cytopathology and other pathologists. Questions pertaining to the participants’ practice setting and CBs-volume, method, quality and satisfaction-were included. Results: Of 95 respondents, 90/95 (94%) completed the survey and comprise the study group. Most participants practice in a community hospital/private practice (44%) or academic center (41%). On average, 14 CBs (range 0-50; median 10) are prepared by a laboratory daily. Over 10 methods are utilized: Plasma thrombin (33%), HistoGel (27%), Cellient automated cell block system (8%) and others (31%) respectively. Forty of 90 (44%) respondents are either unsatisfied or sometimes satisfied with their CB quality, with low-cellular yield being the leading cause of dissatisfaction. There was no statistical significance between the three most common CB preparation methods and satisfaction with quality. Discussion: Many are dissatisfied with their current method of CB preparation, and there is no consistent method to prepare CBs. In todays era of personalized medicine with an increasing array of molecular tests being applied to cytological specimens, there is a need for a standardized protocol for CB optimization to enhance cellularity.

ProEx C Immunocytochemistry and High-Risk Human Papillomavirus DNA Testing in Papanicolaou Tests With Atypical Squamous Cell (ASC-US) Cytology: Correlation Study With Histologic Biopsy

CONTEXT Papanicolaou tests with atypical squamous cells of undetermined significance (ASC-US) cytology and adjunct testing for high-risk human papillomaviruses (hr-HPV) are helpful in detecting high-grade disease. Detection of disease may be further improved with molecular markers known to be overexpressed in cervical carcinoma. ProEx C detects 2 such molecular markers, minichromosome maintenance protein 2 and topoisomerase II, which are associated with abnormal cell cycle regulation. OBJECTIVE To determine the utility of ProEx C as a marker for high-grade cervical intraepithelial neoplasia 2+ disease when compared with hr-HPV status in Papanicolaou tests with ASC-US cytology. DESIGN A SurePath slide was prepared on all ASC-US cases from the residual SurePath vial pellet and stained using the ProEx C reagent prediluted with water-bath antigen retrieval, using a Dako autostainer. Nuclear staining of cytologically atypical squamous cells was considered a positive result. Adjunct testing for hr-HPV used Digene Hybrid Capture 2. Follow-up biopsy results were available for review following the Papanicolaou test. RESULTS Two hundred patients with ASC-US diagnoses were part of this study. The sensitivities of ProEx C and hr-HPV testing in detecting high-grade cervical intraepithelial neoplasia 2+ disease were 98.04% and 82.35%, respectively, whereas the specificity for detecting high-grade disease was 74.50% and 73.15%, respectively. CONCLUSIONS ProEx C staining is a more sensitive and specific biomarker for detecting cervical disease than adjunct testing for hr-HPV status in Papanicolaou tests with ASC-US.

Novel Breast Tissue Feature Strongly Associated With Risk of Breast Cancer

PURPOSE Accurate, individualized risk prediction for breast cancer is lacking. Tissue-based features may help to stratify women into different risk levels. Breast lobules are the anatomic sites of origin of breast cancer. As women age, these lobular structures should regress, which results in reduced breast cancer risk. However, this does not occur in all women. METHODS We have quantified the extent of lobule regression on a benign breast biopsy in 85 patients who developed breast cancer and 142 age-matched controls from the Mayo Benign Breast Disease Cohort, by determining number of acini per lobule and lobular area. We also calculated Gail model 5-year predicted risks for these women. RESULTS There is a step-wise increase in breast cancer risk with increasing numbers of acini per lobule (P = .0004). Adjusting for Gail model score, parity, histology, and family history did not attenuate this association. Lobular area was similarly associated with risk. The Gail model estimates were associated with risk of breast cancer (P = .03). We examined the individual accuracy of these measures using the concordance (c) statistic. The Gail model c statistic was 0.60 (95% CI, 0.50 to 0.70); the acinar count c statistic was 0.65 (95% CI, 0.54 to 0.75). Combining acinar count and lobular area, the c statistic was 0.68 (95% CI, 0.58 to 0.78). Adding the Gail model to these measures did not improve the c statistic. CONCLUSION Novel, tissue-based features that reflect the status of a womans normal breast lobules are associated with breast cancer risk. These features may offer a novel strategy for risk prediction.

Arginase-1: A novel immunohistochemical marker of hepatocellular differentiation in fine needle aspiration cytology †‡§

Arginase‐I is a key urea cycle metalloenzyme that has been used as an immunohistochemistry (IHC) marker for hepatocellular carcinoma (HCC). Previous studies have demonstrated the efficacy of HepPar‐1 and glypican‐3 (GPC‐3) IHC in liver fine needle aspiration (FNA) cytology.