Juan P. Palazzo

MicroRNA Gene Expression Deregulation in Human Breast Cancer

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.

Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors

BackgroundAlthough numerous mouse models of breast carcinomas have been developed, we do not know the extent to which any faithfully represent clinically significant human phenotypes. To address this need, we characterized mammary tumor gene expression profiles from 13 different murine models using DNA microarrays and compared the resulting data to those from human breast tumors.ResultsUnsupervised hierarchical clustering analysis showed that six models (TgWAP-Myc, TgMMTV-Neu, TgMMTV-PyMT, TgWAP-Int3, TgWAP-Tag, and TgC3(1)-Tag) yielded tumors with distinctive and homogeneous expression patterns within each strain. However, in each of four other models (TgWAP-T121, TgMMTV-Wnt1, Brca1Co/Co;TgMMTV-Cre;p53+/- and DMBA-induced), tumors with a variety of histologies and expression profiles developed. In many models, similarities to human breast tumors were recognized, including proliferation and human breast tumor subtype signatures. Significantly, tumors of several models displayed characteristics of human basal-like breast tumors, including two models with induced Brca1 deficiencies. Tumors of other murine models shared features and trended towards significance of gene enrichment with human luminal tumors; however, these murine tumors lacked expression of estrogen receptor (ER) and ER-regulated genes. TgMMTV-Neu tumors did not have a significant gene overlap with the human HER2+/ER- subtype and were more similar to human luminal tumors.ConclusionMany of the defining characteristics of human subtypes were conserved among the mouse models. Although no single mouse model recapitulated all the expression features of a given human subtype, these shared expression features provide a common framework for an improved integration of murine mammary tumor models with human breast tumors.

MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets

Small non‐coding microRNAs (miRNAs) contribute to cancer development and progression, and are differentially expressed in normal tissues and cancers. However, the specific role of miRNAs in the metastatic process is still unknown. To seek a specific miRNA expression signature characterizing the metastatic phenotype of solid tumours, we performed a miRNA microarray analysis on 43 paired primary tumours (ten colon, ten bladder, 13 breast, and ten lung cancers) and one of their related metastatic lymph nodes. We identified a metastatic cancer miRNA signature comprising 15 overexpressed and 17 underexpressed miRNAs. Our results were confirmed by qRT‐PCR analysis. Among the miRNAs identified, some have a well‐characterized association with cancer progression, eg miR‐10b, miR‐21, miR‐30a, miR‐30e, miR‐125b, miR‐141, miR‐200b, miR‐200c, and miR‐205. To further support our data, we performed an immunohistochemical analysis for three well‐defined miRNA gene targets (PDCD4, DHFR, and HOXD10 genes) on a small series of paired colon, breast, and bladder cancers, and one of their metastatic lymph nodes. We found that the immunohistochemical expression of these targets significantly follows the corresponding miRNA deregulation. Our results suggest that specific miRNAs may be directly involved in cancer metastasis and that they may represent a novel diagnostic tool in the characterization of metastatic cancer gene targets. Copyright

Fragile genes as biomarkers: epigenetic control of WWOX and FHIT in lung, breast and bladder cancer.

This study aimed to (a) determine if DNA methylation is a mechanism of WWOX (WW domain containing oxidoreductase) and FHIT (fragile histidine triad) inactivation in lung, breast and bladder cancers; (b) examine distinct methylation patterns in neoplastic and adjacent tissues and (c) seek correlation of methylation patterns with disease status. Protein expression was detected by immunohistochemistry, and methylation status by methylation-specific PCR (MSP) and sequencing, in lung squamous cell carcinomas and adjacent tissues, invasive breast carcinomas, adjacent tissues and normal mammary tissues and bladder transitional cell carcinomas. Wwox and Fhit expression was reduced in cancers in association with hypermethylation. Differential patterns of WWOX and FHIT methylation were observed in neoplastic vs adjacent non-neoplastic tissues, suggesting that targeted MSP amplification could be useful in following treatment or prevention protocols. WWOX promoter MSP differentiates DNA of lung cancer from DNA of adjacent lung tissue. WWOX and FHIT promoter methylation is detected in tissue adjacent to breast cancer and WWOX exon 1 MSP distinguishes breast cancer DNA from DNA of adjacent and normal tissue. Differential methylation in cancerous vs adjacent tissues suggests that WWOX and FHIT hypermethylation analyses could enrich a panel of DNA methylation markers.

Reciprocity between membranous and nuclear expression of β-catenin in colorectal tumours

Abstract β-Catenin has a central role not only in linking the cadherin-mediated cell adhesion system but also in the intercellular signalling pathway. To investigate alterations of β-catenin in the development of colorectal carcinoma, the pattern of β-catenin expression was studied using immunohistochemistry in 74 sporadic colorectal adenomas, in histologically normal mucosa adjacent to 65 of these adenomas, and in 52 carcinomas arising in adenomas. All normal epithelia displayed cell boundary staining for β-catenin. Adenomas and carcinomas showed varying degrees of membranous staining. However, some tumours also showed nuclear staining of β-catenin protein. Decreased membranous and increased nuclear β-catenin staining were associated with increasing degrees of dysplasia in adenomas (P < 0.005, P < 0.05, respectively). Carcinomas manifested significantly reduced membranous, but enhanced nuclear β-catenin expression compared with their associated adenomas (P < 0.001, P < 0.005, respectively). An inverse correlation was found between decreased membranous and increased nuclear staining of β-catenin in both adenomas and carcinomas (P < 0.025, P < 0.05, respectively). The data confirm that reduced membranous and increased nuclear expression of β-catenin is associated with the progression of colorectal adenomas to carcinomas. Our results also suggest that decreased membranous expression of β-catenin may result from aberrant localisation of the protein in the cell nucleus.

Guanylyl Cyclase C Messenger RNA Is a Biomarker for Recurrent Stage II Colorectal Cancer

Stage at diagnosis is the most important prognostic determinant for patients with colorectal cancer (1-6), and it dictates the role of adjuvant chemotherapy in this disease (7-10). Given the prognostic and therapeutic importance of staging, accurate histopathologic evaluation of lymph nodes to detect invasion by tumor cells is crucial. However, conventional microscopic lymph node examination has methodologic limitations (6, 11). It can be difficult to differentiate single or even small clumps of tumor cells from other types of cells, which limits sensitivity. The standard practice of examining only a limited number of tissue sections from each lymph node can omit from review more than 99% of each specimen and can introduce sampling error. These limitations are evident when the frequency of disease recurrence in patients with stage I and stage II disease is considered. By definition, such patients do not have extraintestinal disease at the time of curative resection. However, recurrence rates of 10% to 30% have been reported for lesions confined to the mucosa (stage I disease), and rates of 30% to 50% have been reported for lesions confined to the bowel wall (stage II disease) (12, 13). Alternate methods of detecting small numbers of tumor cells have been used for staging, including intensive review of serial tissue sections, immunohistochemical analysis to detect tumor-associated antigens, polymerase chain reaction (PCR) to detect tumor-specific mutations, and reverse transcriptase (RT) PCR to detect the expression of tumor-associated biomarkers (6, 11). In some studies of colorectal cancer, staging by these sensitive methods has been correlated with disease. However, the fact that serial sectioning is labor- and cost-intensive, the lack of uniform association between mutations and neoplastic transformation, and the nonspecificity of many biomarkers limit the applicability of these methods. An easily detected biomarker that is specifically expressed by all colorectal tumors would be useful for disease staging. Guanylyl cyclase C is expressed in normal intestinal mucosal cells, adenomatous polyps, and primary and metastatic colorectal tumors but not in extraintestinal tissues or tumors (14-17). Expression of guanylyl cyclase C has been detected by RT-PCR in all of the histologically confirmed colorectal tumors and colorectal cancer cell lines that have been examined (14-17). Therefore, guanylyl cyclase C may be a specific biomarker for metastasis of extraintestinal colorectal cancer (16, 17). We examined whether expression of guanylyl cyclase C messenger RNA (mRNA) in lymph nodes was associated with disease recurrence in patients with stage II colorectal cancer who had presumably been cured by surgical resection. Methods Patients and Tissues We examined the tumor registry database at Thomas Jefferson University (Philadelphia, Pennsylvania) for patients who had been treated for colorectal cancer between 1989 and 1995, an interval that permitted adequate follow-up (Figure). The initial search was designed to exclude patients who developed recurrent disease more than 3 years after the index surgery. In this way, we avoided inadvertent inclusion of patients who had metachronous rather than recurrent cancer. Our search yielded 445 patients with invasive colon or rectal cancer and no evidence of metastases (tumor, node, metastasis [TNM] classification, N0 M0) at surgery. Of these 445 patients, 260 had surgery at Thomas Jefferson University that yielded lymph nodes. Subsequently, 167 patients were excluded because they had stage I or less severe disease (T0-T2 N0 M0), because they developed recurrent disease locally or at unspecified sites, or because they received neoadjuvant chemotherapy or radiation therapy. Fifty-six patients with no evidence of recurrence were excluded because they had less than 6 years of follow-up. Of 18 patients who had had no evidence of disease for 6 or more years after surgery and were considered clinically cured, 16 had pathologic specimens available for further analysis; these patients formed the control group. Of 19 patients who developed metastases up to 3 years after surgery, 12 had pathologic specimens available for further analysis; these patients formed the case-patient group. The remaining 9 patients were excluded from analysis. Two controls (12.5% [patients 9 and 16]) and 1 case-patient (8.3% [patient 24]) received 5-fluorouracil-based adjuvant chemotherapy after surgery. Figure. Algorithm for selecting patient biopsy samples for analysis. Reverse Transcriptase Polymerase Chain Reaction In our study, lymph nodes were obtained for analysis under an institutional review board-approved protocol that maintained patient anonymity. Preliminary tests showed that mRNA isolated from single 10-m sections of individual lymph nodes yielded insufficient RNA for RT-PCR analysis. Consequently, at least five 10-m sections of representative lymph nodes for each patient were pooled and deparaffinized, and the total RNA was isolated (17). Reverse transcriptase polymerase chain reaction was performed by using the RNA PCR Kit, version 2 (Takara Shuzo Co., Ltd., Kyoto, Japan) (16, 17). We used only total RNA that yielded amplicons after -actin-specific RT-PCR was used (16, 17). Guanylyl cyclase C-specific RT-PCR and nested carcinoembryonic antigen-specific RT-PCR were performed as described elsewhere (16-18). Reactions from RT-PCR were separated by electrophoresis on 4% NuSieve 3:1 agarose (FMC Bioproducts, Rockland, Maine) and by amplification products visualized by ethidium bromide. We included positive control specimens, consisting of RNA isolated from human colorectal cancer cells that expressed guanylyl cyclase C and carcinoembryonic antigen (Caco2 cells [American Type Culture Collection, Rockville, Maryland]), and negative control specimens, consisting of RNA from lymph nodes without colorectal cancer and incubations in which no template was added. Amplicon identity was confirmed by sequencing (16, 17). Production of guanylyl cyclase C-specific amplicons was confirmed by Southern blot analysis, which used a 32P-labeled antisense probe that complemented a sequence found within the amplicon (19). Statistical Analysis Results are expressed as the mean SD, except for disease-free and overall survival, which are expressed as the median (range). We calculated P values by using the Fisher exact test. The odds ratios, with exact 95% CIs, were calculated by using the StatXact 4.0 statistical software package (Cytel Software Corp., Cambridge, Massachusetts). Role of the Funding Source Targeted Diagnostics and Therapeutics, Inc., which provided a portion of the grant support for this study, was not involved in the design of the study or in the collection and analysis of the data; it also had no role in the decision to submit the paper for publication. Results Characteristics of Patients Evaluated by Reverse Transcriptase Polymerase Chain Reaction Patients ranged in age from 37 to 85 years (mean, 68.1 9.5 years). Women and men were similar in age (range, 52 to 85 years [mean, 64.5 10.5 years] and 37 to 82 years [mean, 70.9 7.8 years], respectively). The ratio of men to women was 8:9 among controls and 5:7 among case-patients. One woman was African-American; all other patients were white. The ratio of cases of T3 to T4 disease was 3:13 among controls and 4:8 among case-patients. Patients were followed for 9 to 105 months (mean, 67.4 30.7 months). Controls were followed for 73 to 105 months (mean, 89.9 7.8 months), and case-patients were followed for 9 to 78 months (mean, 37.3 22.6 months). In the control group, 1 patient (6.3%) developed a new primary colonic lesion 96 months after initial diagnosis, 1 patient (6.3%) died of causes unrelated to colorectal cancer, and the remaining 14 patients (87.5%) were alive and free of disease 88 months after diagnosis (range, 73 to 97 months). In the case-patient group, 8 patients (66.7%) died of recurrent colorectal cancer after 13 months of disease-free survival (range, 3 to 35 months) and after 19 months of overall survival (range, 9 to 64 months). Four patients (33%) were alive with metastases after 12 months of disease-free survival (range, 2 to 36 months) and 52 months of overall survival (range, 17 to 78 months). Analysis by Reverse Transcriptase Polymerase Chain Reaction of RNA Expression in Lymph Nodes For all 28 patients, 524 lymph nodes (mean, 18.4 12.5 lymph nodes per patient) collected at surgery were reported to be free of tumor in the original histologic review. The number of lymph nodes obtained from each patient at the time of initial operative staging was similar in the control group (mean, 19.9 13.2 lymph nodes per patient) and the case-patient group (mean, 17.2 12.7 lymph nodes per person). Lymph nodes were omitted from RT-PCR analysis because they were not available from the pathology department (326 lymph nodes from 28 patients [62.2% of 524 lymph nodes obtained at surgery]). Of the 198 lymph nodes that were available for RT-PCR analysis, 19.7% (39 lymph nodes from 7 patients [7.4% of 524 lymph nodes obtained at surgery]) did not yield RNA. The number of lymph nodes available for RT-PCR analysis was similar in the control group (mean, 6.4 3.0 lymph nodes) and the case-patient group (mean, 8.1 6.3 lymph nodes). Twenty-one patients (75%) yielded 159 paraffin-embedded lymph nodes (mean, 7.6 5.2 lymph nodes per patient) that could be adequately evaluated by RT-PCR. In 5 case-patients (41.7%) and 2 controls (16.7%), -actin-specific amplicons (an indicator of intact RNA) were not detected in the total RNA from pooled sections of lymph nodes; these 7 patients were excluded from further analysis. Total RNA extracted from the pooled lymph node sections of the remaining 21 patients was analyzed by RT-PCR using guanylyl cyclase C-specific primers. Guanylyl cyclase C-specific amplicons were not detected in any reaction that used RNA from lymph nodes of control

MicroRNA expression profiling of male breast cancer.

IntroductionMicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. To test the hypothesis that there is a specific miRNA expression signature which characterizes male breast cancers, we performed miRNA microarray analysis in a series of male breast cancers and compared them with cases of male gynecomastia and female breast cancers.MethodsParaffin blocks were obtained at the Department of Pathology of Thomas Jefferson University from 28 male patients including 23 breast cancers and five cases of male gynecomastia, and from 10 female ductal breast carcinomas. The RNA harvested was hybridized to miRNA microarrays (~1,100 miRNA probes, including 326 human and 249 mouse miRNA genes, spotted in duplicate). To further support the microarray data, an immunohistochemical analysis for two specific miRNA gene targets (HOXD10 and VEGF) was performed in a small series of male breast carcinoma and gynecomastia samples.ResultsWe identified a male breast cancer miRNA signature composed of a large portion of underexpressed miRNAs. In particular, 17 miRNAs with increased expression and 26 miRNAs with decreased expression were identified in male breast cancer compared with gynecomastia. Among these miRNAs, some had well-characterized cancer development association and some showed a deregulation in cancer specimens similar to the one previously observed in the published signatures of female breast cancer. Comparing male with female breast cancer miRNA expression signatures, 17 significantly deregulated miRNAs were observed (four overexpressed and 13 underexpressed in male breast cancers). The HOXD10 and VEGF gene immunohistochemical expression significantly follows the corresponding miRNA deregulation.ConclusionsOur results suggest that specific miRNAs may be directly involved in male breast cancer development and that they may represent a novel diagnostic tool in the characterization of specific cancer gene targets.

Use of guanylyl cyclase C for detecting micrometastases in lymph nodes of patients with colon cancer

INTRODUCTION: Guanylyl cyclase C appears to be expressed only in colorectal cancer cells in extraintestinal tissues. Thus, guanylyl cyclase C may be useful as a marker to detect colorectal cancer micrometastases not detectable by histopathology in lymph nodes of patients. METHODS: Twelve patients with colon adenocarcinoma, Dukes Stages A through C2, and one patient with a tubulovillous adenoma were included in this study. Forty-two lymph nodes were collected from fresh surgical specimens, and each was examined by histopathology and reverse transcription followed by polymerase chain reaction using guanylyl cyclase C-specific primers. Histopathology identified colon cancer cells in 6 of 16 lymph nodes from five Dukes Stage C patients but not in lymph nodes from the patient with a tubulovillous adenoma, the Dukes Stage A patient, or six Dukes Stage B patients. Reverse transcription followed by polymerase chain reaction using guanylyl cyclase C-specific primers was performed on all 42 lymph nodes. RESULTS: Guanylyl cyclase C messenger RNA was not detected by reverse transcription followed by polymerase chain reaction in lymph nodes from the patient with the tubulovillous adenoma or the patient with Dukes Stage A colon carcinoma. Seven lymph nodes from Dukes Stage C patients revealed guanylyl cyclase C messenger RNA including six lymph nodes containing histopathologically confirmed metastases. Of significance, guanylyl cyclase C messenger RNA was detected in 6 of 21 lymph nodes from Dukes Stage B patients. Indeed, clinical staging of two patients could be upgraded from B to C using reverse transcription followed by polymerase chain reaction and guanylyl cyclase C-specific primers. CONCLUSION: Reverse transcription followed by polymerase chain reaction using guanylyl cyclase C-specific primers might be useful to more accurately assess micrometastases in lymph nodes of colorectal cancer patients undergoing disease staging.

Proceedings of the International Consensus Conference on Breast Cancer Risk, Genetics, & Risk Management, April, 2007

A consensus conference including 30 experts was held in April, 2007, to discuss risk factors for breast cancer and their management. Four categories of risk were outlined, from “average” through “very high” risk, the latter including individuals with high penetrance BRCA1/2 gene mutations. Guidelines for management of patients in each of these categories were discussed, with the major portion of the conference devoted to individuals with BRCA1/2 mutations. Prevalence of these mutations in the general population was estimated to be 1 in 250‐500 individuals, with an increased prevalence in Ashkenazi Jews and other founder groups. Risk‐reduction strategies for these individuals included surveillance, with or without chemoprevention drugs, or surgical procedures to remove the organs at risk, ie, bilateral mastectomy and/or bilateral salpingo‐oophorectomy. These risk reduction strategies were evaluated fully, and recommendations were made for the care of patients in each risk category. These guidelines for patient care were approved by the entire group of experts. Cancer 2008.

A Validated Quantitative Assay to Detect Occult Micrometastases by Reverse Transcriptase-Polymerase Chain Reaction of Guanylyl Cyclase C in Patients with Colorectal Cancer

Purpose: Guanylyl cyclase C (GCC), a receptor for bacterial diarrheagenic enterotoxins, may be a prognostic and predictive marker to detect occult micrometastases in patients undergoing staging for colorectal cancer. However, quantification of GCC expression in tissues by the quantitative reverse transcription-PCR (qRT-PCR) has not undergone analytic and clinicopathologic validation. Experimental Design: A technique to quantify GCC mRNA in tissues employing RT-PCR was developed and validated employing external calibration standards of RNA complementary to GCC. Results: GCC qRT-PCR exhibited reaction efficiencies >92%, coefficients of variations <5%, linearity >6 orders of magnitude, and a limit of quantification of >25 copies of GCC cRNA. This assay confirmed that GCC mRNA was overexpressed by colorectal tumors from 41 patients, which correlated with increased GCC protein quantified by immunohistochemistry. Analyses obtained with 164 lymph nodes from patients free of cancer and 15 nodes harboring metastases established a threshold for metastatic disease of ∼200 GCC mRNA copies/μg total RNA, with a sensitivity of 93% and specificity of 97%. GCC mRNA above that threshold was detected in 76 of 367 (∼21%) nodes free of disease by histopathology from 6 of 23 (26%) patients, suggesting the presence of occult micrometastases. Conclusions: Quantifying GCC mRNA in tissues by RT-PCR employing external calibration standards is analytically robust and reproducible, with high clinicopathologic sensitivity and specificity. This validated assay is being applied to ∼10,000 lymph nodes in a prospective trial to define the sensitivity of GCC qRT-PCR for staging patients with colorectal cancer.