Formamide as a denaturant for bisulfite conversion of genomic DNA: Bisulfite sequencing of the GSTPi and RARbeta2 genes of 43 formalin-fixed paraffin-embedded prostate cancer specimens.

Analysis of methylated DNA, which refers to 5-methycytosine (5mC) versus cytosine (C) at specific loci in genomic DNA (gDNA), has received increased attention in epigenomics, particularly in the area of cancer biomarkers. Many different methods for analysis of methylated DNA rely on initial reaction of gDNA with concentrated acidic sodium bisulfite to quantitatively convert C to uracil (U) via sulfonation of denatured, single-stranded gDNA under conditions where 5mC is resistant to analogous sulfonation leading to thymine (T). These methods typically employ polymerase chain reaction (PCR) amplification after bisulfite conversion, thereby leading to readily detectable amounts of amplicons where T and C are measured as surrogates for C and 5mC in the original unconverted gDNA. However, incomplete bisulfite conversion of C in gDNA has been reported to be a common source of error in analysis of methylated DNA. Incomplete conversion can be revealed during the course of bisulfite sequencing, which is the generally accepted "gold standard" for analysis of methylated DNA. Previous bisulfite sequencing investigations of conventional predenaturation of gDNA with NaOH followed by the use of bisulfite containing added urea to maintain denaturation and thus mitigate incomplete conversion of C have been reported to give conflicting results. The current study describes a new approach where conventional predenaturation of gDNA with NaOH is instead achieved with formamide and maintains denaturation during subsequent sample handling and sulfonation. This formamide-based method was applied to 46 formalin-fixed/paraffin-embedded (FFPE) biopsy tissue specimens from well-characterized patients with primary prostate cancer. These specimens were representative of difficult-to-analyze samples due to the chemically compromised nature of the gDNA, which was recovered by modifying the protocol for a commercially available total RNA/DNA extraction kit (RecoverALL). An additional novel aspect of this study was analysis of CpG-rich promoter regions of two prostate cancer-related genes: glutathione S-transferase pi (GSTPi) and retinoic acid receptor beta2 (RARbeta2). High-quality bisulfite sequencing results were obtained for both genes in 43 of 46 (93%) specimens. Detection of methylated GSTPi and RARbeta2 genes was significantly associated with primary prostate cancer as compared with the benign prostate (Fisher's exact test, P < 0.001). The sensitivity and specificity of detection of methylated GSTPi and RARbeta2 genes were 86% and 100% and 91% and 100%, respectively. Moreover, the presence of either methylated gene was detected in primary prostate cancer with sensitivity and specificity of 100% and 100%, respectively. The results demonstrated a high degree of reliability of formamide-based denaturation and bisulfite conversion that should extend, generally, to FFPE and other types of samples intended for any analytical method predicated on bisulfite conversion. This pilot study also demonstrated the efficacy of determining methylation of these two genes with high sensitivity and specificity in FFPE biopsy tissue specimens. Moreover, the results showed a highly significant association of methylated GSTPi and RARbeta2 genes with primary prostate cancer. Finally, this improved procedure for determining these two methylated genes may allow the detection of prostate cancer cells in core biopsy specimens with insufficient numbers of cells and poor morphology.

J1-31 protein expression in astrocytes and astrocytomas.

J1-31 is one of the astrocytic proteins, the expression of which has not been evaluated in astrocytomas. In the present study, we studied the expression of J1-31 protein in astrocytes and astrocytomas in comparison with GFAP, p53 and Ki-67. Materials consisted of formalin-fixed paraffin-embedded tissue specimens that included five cases of normal brain, 17 of gliosis, 15 of pilocytic astrocytoma (WHO grade I), 26 of low-grade diffuse astrocytoma (WHO grade II), four of anaplastic astrocytoma (WHO grade III), and eight of glioblastoma (WHO grade IV). GFAP was highly expressed in all specimens examined. The anti-J1-31 antibody exhibited strong cytoplasmic staining of reactive gliosis in 17/17 (100%) cases with a higher intensity of staining than that observed in the adjacent normal astrocytes. The antibody showed reactivity with tumor cells in 12/15 (80%) cases of pilocytic astrocytoma, although intensity of staining was generally weaker and more focal than observed in reactive gliosis. J1-31-positive tumor cells were detected in only 9/26 (35%) cases of the low-grade diffuse astrocytoma and none of the cases of anaplastic astrocytoma and glioblastoma. Increasing Ki-67 indices paralleled advancing tumor grades. p53 protein was expressed more commonly in infiltrating astrocytomas compared to pilocytic astrocytoma. In conclusion, down-regulation of J1-31 expression correlates with advancing grade of astrocytomas. The result suggests this protein plays some role in astrocytes that is progressively lost in malignant progression. The anti-J1-31 antibody may help further our understanding of astrocytes in disease and may be useful as an aid in the pathologic diagnosis of astrocytic lesions.

Unmethylated E-cadherin gene expression is significantly associated with metastatic human prostate cancer cells in bone.

BACKGROUND: The concurrent determination of methylation status of E-cadherin gene and E-cadherin protein expression remains scant in metastatic prostate cancer cells in bone, the most prevalent site for metastatic growth. Therefore, the study was undertaken to ascertain the methylation status of E-cadherin gene, a most frequent and known epigenetic mechanism of its regulation, and the protein expression in prostate tissue biopsy specimen. METHODS: The methylation of E-cadherin gene was determined by methylation specific-PCR and the protein expression by immunohistochemical method in the consecutive sections of each prostate tissue biopsy specimen. RESULTS: The unmethylated E-cadherin gene and homogeneous E-cadherin protein expression was significantly associated with BPH as compared to the primary prostate carcinoma (Fisher's Exact P < 0.001). A significant association was observed between the concurrent methylated gene and markedly reduced expression of the protein in the primary prostate cancer cells as compared to the BPH cells, suggesting methylation-dependent regulation of the gene expression in these cases. In contrast to the primary cancer, a highly significant increase in the frequency of metastatic prostate cancer cells in bone exhibited the concurrent expression of unmethylated gene and homogeneous protein (Fisher's Exact P < 0.001). CONCLUSIONS: The study clearly demonstrated a significant association of the concurrent expression of unmethylated E-cadherin gene and E-cadherin protein with metastatic prostate cancer cells in bone, and that its expression may have a role in the intercellular adhesion in the formation of metastatic lesions in bone.

Overexpression of E-cadherin protein in metastatic breast cancer cells in bone.

AIM: The aim of the present study was to evaluate E-cadherin, whose expression remains poorly understood in the intercellular adhesion of metastatic breast cancer cells in bone, the most prevalent site for metastatic growth. MATERIALS AND METHODS: An immunohistochemical staining method was used for the localization of E-cadherin protein in tissue biopsy specimens of normal breast (n = 9) and well- (n = 8), moderately (n = 8) or poorly (n = 14) differentiated invasive primary breast cancer and metastatic breast cancer in bone (n = 17). The expression patterns of E-cadherin were classified as homogeneous (most cells exhibiting positivity), heterogeneous (a few scattered patches of cells with positivity) or negative (cells with undetectable positivity). RESULTS: Normal breast epithelial cells showed homogeneous overexpression of E-cadherin in all cases. A progressive and statistically significant reduction of E-cadherin expression was detected in the histologically well- to moderately to poorly differentiated breast cancer cells (p < 0.001). The clumps of invasive primary breast cancer cells in CD-31-positive blood vessels exhibited E-cadherin expression. Moreover, as compared to the poorly differentiated breast cancer cells, a significantly increased frequency of the metastatic breast cancer cells in bone exhibited homogeneous expression of E-cadherin in 15 out of 17 and heterogeneous expression in the remaining 2 cases (McNemar Exact p < 0.001). This is the first demonstration of membranous overexpression of E-cadherin on metastatic breast cancer cells in bone; the high frequency of its expression may have a role in the intercellular adhesion of metastatic cells in bone.

Wnt pathway component LEF1 mediates tumor cell invasion and is expressed in human and murine breast cancers lacking ErbB2 (her-2/neu) overexpression.

This study examines the role of LEF1, a component of the Wnt signaling pathway, in human breast and murine mammary carcinoma and its relationship to ErbB2 (her-2/neu) expression. Mammary tissue and tumors from 5 different Wnt pathway-activated transgenic mouse strains and 5 different ErbB2 pathway-activated transgenic mouse strains were studied for the amount and distribution of expression of beta-catenin and LEF1. Fourteen samples of human infiltrating ductal breast cancer arising from a background of ductal carcinoma in situ (DCIS) were analyzed for LEF1, estrogen and progesterone receptor (ER and PR) and her-2/neu expression. in vitro, the effect of estradiol on LEF1 protein expression was examined in several breast cancer cell lines. The functional role of LEF1 was analyzed by a Matrigel invasion assay following transfection of breast cancer cell lines with either an LEF1 expression construct or a dominant-negative LEF1 construct. A significant (p=0.023) negative correlation between the expression of LEF1 and her-2/neu was observed in human breast cancer. LEF1 was strongly expressed, and beta-catenin had nuclear localization, in mammary tumors derived from Wnt pathway transgenic mice but not in ErbB2 pathway transgenic mice. In estrogen-receptor-positive breast cancer cell lines, LEF1 protein expression increased significantly following estradiol incubation (>200% of baseline). Following transient transfection, overexpression of LEF1 promoted and dominant-negative LEF1 inhibited tumor cell invasion. LEF1, a downstream component of the Wnt signaling pathway, defines a distinct, her-2/neu negative (non-overexpressing) subset of breast/mammary cancers in both humans and mice, mediates breast cancer cell invasion, and may be regulated in part by estradiol.

LEA.135 expression: identifies low-risk patients with breast ductal carcinoma in situ.

The expression of a cell surface-associated sialoglycoprotein (LEA.135), which has been shown to be significantly associated with decreased incidence of recurrence and increased overall survival of patients with primary invasive breast carcinoma, was evaluated in a retrospective study to identify subsets of patients with breast ductal carcinoma in situ (DCIS) who are at high risk of subsequently developing invasive breast carcinoma. The study was carried out by an immuno-histochemical method on formalin-fixed and paraffin-embedded tissue sections from 63 patients initially with DCIS. Pathological parameters such as DCIS histological type, nuclear grade, as well as time and type of recurrence (either a second DCIS or the diagnosis of locally invasive breast carcinoma) together with follow-up in years were available for the cohort of patients. A comparison of recurrence was made of patients whose tumor cells exhibited LEA.135 expression (24 +/- 8% recurring by 7 years), compared with those patients whose specimens showed the absence of LEA.135 expression (41 +/- 10% recurring by 7 years). A statistically significant univariant association between LEA.135 expression and the absence of recurrence of DCIS or development of locally invasive breast carcinoma was observed, suggesting a favorable prognostic significance of LEA. 135 expression (log-rank p = 0.032). It is worthy of mention that none of the LEA. 135-positive patients developed recurrence as DCIS or locally invasive breast carcinoma (0.24 +/- 0.08) after 5 years of the initial diagnosis of DCIS, whereas those from LEA. 135-negative progressively increased their recurrence at 5 years (0.30 +/- 0.09), 7 years (0.41 +/- 0.10) and 10 years (0.63 +/- 0.12). The results of this pilot study show that LEA.135 expression is significantly associated with a favorable prognosis of patients with DCIS, leading to a decreased incidence of recurrence.

LEA-135 expression: its association with a lower risk of recurrence and increased overall survival of patients with lymph node-positive primary invasive breast cancer.

A retrospective study was undertaken to determine and compare the prognostic significance of LEA-135 protein expression by immunohistochemistry with other prognostic pathological parameters, with respect to recurrence and overall survival. This study was conducted in freshly-frozen tissue sections from a cohort of 367 patients having primary invasive breast cancer, with axillary lymph node metastasis. The association of LEA-135 expression was compared with estrogen and progesterone receptor status, segmentectomy or radical mastectomy and hormonal therapy or chemotherapy in terms of recurrence or disease-free survival. Pathologic parameters including tumor size, histological tumor type and histological grade, as well as age of patients at the time of initial diagnosis, and the treatments, together with a median follow-up of 8.8 years were contemplated for the study. Among these parameters, tumor size and histological grade were individually and significantly associated with an increased probability of recurrence (log rank p<0.001 in both cases) and short survival (log ranks p<0.001 and p=0.002, respectively), whereas age was only significantly associated with an increased probability of recurrence (log rank p=0.002) by univariate analysis. By multivariate analysis, both tumor size and histological grade remained statistically significant for recurrence (log rank p<0.001 and p=0.013, respectively) and overall survival (log ranks p<0.001 and p=0.016, respectively). Among the prognostic biomarkers, both ER and PR expression were associated with a decreased rate of recurrence (log ranks p<0.001 and p=0.008, respectively) and overall survival (log ranks p<0.001 and p=0.002, respectively) by univariate analysis. By multivariate analysis, only the ER expression remained significantly associated with a decreased recurrence and increased overall survival (log ranks p=0.023 and p=0.002, respectively). Patients with high (>50% positive cells) or moderate (5-50% positive cells) number of LEA-135-positive cells had a lower probability (46%) of recurrence at 10 years after surgery compared to 76% in LEA-135-negative patients (log rank p<0.001) by univariate analysis. Moreover, the probability of overall survival was higher in patients with high or moderate expression of LEA-135 (46% and 47%, respectively) compared to LEA-135-negative patients (24%) by univariate analysis (log rank p=0.009). By multivariate analysis, the association remained statistically significant for recurrence (log rank p<0.001) and survival (log rank p=0.002). However, there was no significant association between LEA-135 and any of the pathological parameters, age, hormone receptor status, the mode of surgery or the form of therapy (chemo- and/or hormonal) received by this cohort of patients. The results show that an improved prognosis was directly associated with the density of LEA-135-positive cancer cells, while loss of LEA-135 expression was associated with an aggressive phenotype of cancer cells during breast cancer progression. Thus, LEA-135 expression can be implicated as a significant and independent biomarker to identify and distinguish high- from low-risk patients with lymph node-positive invasive breast cancer for an aggressive treatment. Moreover, according to the present results, LEA-135 expression appears to be associated with the tumor cells that have retained certain normal biological characteristics, leading to their lack of aggressiveness and hence a better prognosis.

Stromal response to prostate cancer: nanotechnology-based detection of thioredoxin-interacting protein partners distinguishes prostate cancer associated stroma from that of benign prostatic hyperplasia.

Histological staining of reactive stroma has been shown to be a predictor of biochemical recurrence in prostate cancer, however, molecular markers of the stromal response to prostate cancer have not yet been fully delineated. The objective of this study was to determine whether or not the stromal biomarkers detected with a thioredoxin-targeted nanodevice could be used to distinguish the stroma associated with benign prostatic hyperplasia from that associated with PCA. In this regard, we recently demonstrated that a thioredoxin-targeted nanodevice selectively binds to reactive stroma in frozen prostate tumor tissue sections. To accomplish this, random frozen prostate tissue sections from each of 35 patients who underwent resection were incubated with the nanodevice and graded for fluorescent intensity. An adjacent section from each case was stained with Hematoxylin & Eosin to confirm the diagnosis. Select cases were stained with Masson's Trichrome or immunohistochemically using antibodies to thioredoxin reductase 1, thioredoxin reductase 2 or peroxiredoxin 1. Our results demonstrate that the graded intensity of nanodevice binding to the stroma associated with PCA was significantly higher (p = 0.0127) than that of benign prostatic hyperplasia using the t-test. Immunohistochemical staining of adjacent sections in representative cases showed that none of the two commonly studied thioredoxin interacting protein partners mirrored the fluorescence pattern seen with the nanodevice. However, thioredoxin reductase 2 protein was clearly shown to be a biomarker of prostate cancer-associated reactive stroma whose presence distinguishes the stroma associated with benign prostatic hyperplasia from that associated with prostate cancer. We conclude that the signal detected by the nanodevice, in contrast to individual targets detected with antibodies used in this study, originates from multiple thioredoxin interacting protein partners that distinguish the M2 neutrophil and macrophage associated inflammatory response in prostate cancer-associated stroma from the CD4+ T-Lymphocyte linked inflammation in benign prostatic hyperplasia.

Biomarker identification with ligand-targeted nucleoprotein assemblies.

AIMS: Since many biomarkers of both the tumor and its microenvironment are expected to involve differential expression of divalent proteins capable of protein or peptide ligand interaction, we are developing multivalent nanodevices for the identification of biomarkers in prostate cancer. PATIENTS & METHODS: We compared a multivalent thioredoxin-targeted nanodevice with monovalent thioredoxin in binding to human prostate cell line(s) and freshly frozen tissue specimens obtained after resection from patients with biopsy-proven prostate cancer. CONCLUSION: The nanodevice binds specifically with enhanced avidity to tumor microenvironment-associated stromal cells in prostate cancer tissue specimens. Cells that bind the nanodevice also reacted with antibodies to dimeric thioredoxin reductases 1 and 2, suggesting the utility of the nanodevice as a potentially specific and functional marker of tumor stromal cells.

Human breast cancer histoid: an in vitro 3-dimensional co-culture model that mimics breast cancer tissue.

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 µm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.

Overexpression of E-cadherin and beta-catenin proteins in metastatic prostate cancer cells in bone.

BACKGROUND: The expression of E-cadherin in the intercellular adhesion of metastatic prostate cancer cells in bone, which is the most prevalent site of metastatic growth, remains elusive. METHODS: The aim of the study was to compare the concurrent membranous expression of E-cadherin and beta-catenin proteins, the state which is known to be associated with the cellular adhesion function of E-cadherin, in prostate biopsy tissue specimens by immunohistochemical staining method. The expression patterns of E-cadherin or beta-catenin were classified as homogeneous (most cells exhibiting positively), heterogeneous (a few scattered patches of cells with positivity) or negative. RESULTS: Benign prostate hyperplasia cells exhibited homogeneous expression of both E-cadherin and beta-catenin in 9 of 11 (82%), whereas the primary prostate cancer cells were homogeneously positive for both proteins only in 4 of 22 (18%) of the cases. The results are similar to those reported in literature. However, in contrast to the primary cancer, a significantly increased frequency of the metastatic prostate cancer cells in bone exhibited homogeneous expression of E-cadherin and beta-catenin in 12 of 17 (71%) of the cases. A statistically significant association was observed between the overexpression of both proteins and the metastatic prostate cancer cells in bone (Fisher's exact P < 0.001). CONCLUSIONS: The result of the study demonstrated for the first time that the membranous overexpression of E-cadherin and beta-catenin are significantly associated with the metastatic prostate cancer cells in bone and that the high frequency of expression suggest their involvement in the intercellular adhesion of the metastatic cells in bone.

Telomerase and markers of cellular proliferation are associated with the progression of cervical intraepithelial neoplasia lesions.

The expression of the catalytic subunit of telomerase protein (human telomerase reverse transcriptase [hTERT]), which is associated with telomerase activity, was evaluated as a potential marker of the high-grade premalignant cervical intraepithelial neoplasia (CIN 2/3) lesions. For comparison, cases of normal cervical squamous mucosa, low-grade CIN1 lesion, and cervical squamous cell carcinoma were included. The hTERT expression was also compared with Ki-67 and topoisomerase II-alpha (TPII-alpha) to determine the proliferative activity of the hTERT-positive dysplastic cells by a quantitative immunohistochemical staining method and was classified as follows: negative, 5% or less; moderate, 6% to 50%; or high, greater than 50% of the positive cells. The hTERT-positive cells were detected in a patchy pattern in the lower parabasal layers and in much of the basal layer in normal squamous mucosa. A similar frequency of Ki-67- or TPII-alpha-positive cells was observed, with the exception of the basal layer cells that were mostly negative. It is worthy to note that the recognizable intact basal layer cells in cases of CIN lesions were also consistently positive for the expression of hTERT, but rarely for Ki-67 or TPII-alpha. The expression of hTERT was detected in a less patchy pattern at a high or moderate percentage of the dysplastic epithelial cells each in 28.5% of cases of CIN1 lesions. A similar frequency, high and moderate percentage combined, of the TPII-alpha-positive dysplastic cell was also observed. In contrast, a high percentage of the hTERT-positive dysplastic cells were detected as diffuse basal or full-length thickness in 87.5% or 95% of cases of CIN2 or CIN3, respectively. A similar frequency of Ki-67 or TPII-alpha expression was observed in the dysplastic cells of CIN3 lesions. The pattern of hTERT-positive malignant cells in squamous cell carcinoma and dysplastic cells in the high-grade CIN lesions, to a greater extent, and dysplastic cells in the low-grade CIN lesion, to a lesser extent, was distinct from that of the normal cervical squamous mucosa. The results suggest that the progressive increase in the hTERT expression, together with the proliferative activity of the dysplastic epithelial cells of the high-grade CIN lesions, represents an early genetic abnormality in cervical pathogenesis.