Insulin-like growth factor 1 (IGF-1) and its receptor mRNA levels in breast cancer and adjacent non-neoplastic tissue.

UNLABELLED: Previous studies investigating the insulin-like growth factor 1 receptor (IGF-1R) expression in breast cancer tissue and adjacent non-neoplastic breast tissue (ANCT) have produced conflicting results. The IGF-1 and IGF-1R expression in pairs of breast cancer tissue and ANCT were investigated using RT-PCR and immunohistochemistry. The results of both methods were compared. MATERIALS AND METHODS: IGF-1 and IGF-1R mRNA from 31 specimen pairs were estimated using RT-PCR. Immunohistochemistry for IGF-1R was carried out on 20 specimen pairs and the strength of staining was scored. RESULTS: The mean relative IGF-1 mRNA level was lower in the cancerous tissue (mean 0.450 +/- 0.206) than in the ANCT (mean 0.632 +/- 0.384) (paired t-test, p = 0.001). There was no measurable difference in relative IGF-1R mRNA levels in the cancerous tissue (mean 0.146 +/- 0.08) and the ANCT (mean 0.14608 +/- 0.108) (paired t-test, p = 0.807). Using immunohistochemistry, there was no statistical difference (paired t-test, p = 0.910) in IGF-1R staining scores between cancer (mean 1.93) and ANCT (mean 1.90). The comparison between the two methodologies showed no correlation (Pearson's Correlation Coefficient = -0.393). DISCUSSION: It can be concluded that IGF-1 expression is lower in cancerous tissue, thus supporting a paracrine relationship between cancerous tissue and ANCT, which may be useful in the prevention, diagnosis and treatment of breast cancer. There was no difference in the expression of the IGF-1 receptor in both types of tissue, as proven by RT-PCR and immunohistochemistty. Conflicting results in previous studies may be due to the different methods used to measure IGF-1R expression.

hTERT mRNA expression is associated with a poor clinical outcome in human breast cancer.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase) gene is the rate-limiting determinant of telomerase reactivation. The present study aims to quantitatively measure the expression of hTERT mRNA in human breast cancer, examine the association between hTERT and the clinicopathological characteristics of the cancer specimens including the Nottingham Prognostic Index (NPI) and to explore the relationship between hTERT expression and clinical outcome. MATERIALS AND METHODS: RNA was extracted from 116 breast carcinomas and 31 matched adjacent non-cancerous tissue (ANCT). hTERT mRNA expression was estimated by reverse transcriptase-PCR (RT-PCR) and Taqman methodology. RESULTS: hTERT mRNA was present in all of the cancerous specimens (mean=0.1701, median=0.0205) and most ANCT specimens with levels being 2.6 times higher in the cancerous tissue than in ANCT (mean=0.156 vs. 0.68, p=0.18). The mean mRNA levels increased with NPI scores (0.0816 for NPI 1, 0.1186 for NPI 2 and 0.68 for NPI 3), however this failed to reach statistical significance (P-values= 0.33 for NPI 1 vs. 2, 0.27for NPI2 vs. 3 and 0.24 for NPI 1 vs. 3). hTERT levels also increased with increasing tumour's grade (mean= 0.0459 for grade 1, 0.111for grade 2, and 0.27 for grade 3) but this trend did not reach a statistical significance. Low levels of hTERT were associated with mucinous carcinoma compared with ductal (p=0.023) and lobular (p=0.021) types. hTERT mRNA levels were higher in patients who had recurrent disease or died from breast cancer compared with those who remained alive without disease after a median follow up of 6 years (p=0.0026). CONCLUSION: High hTERT mRNA levels are associated with a poor clinical outcome in human breast cancer and should be included as a prognostic marker in future validation studies.

hTERT protein expression is independent of clinicopathological parameters and c-Myc protein expression in human breast cancer.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. The hTERT (human telomerase reverse transcriptase) subunit seems to be the rate-limiting determinant of telomerase and knowledge of factors controlling hTERT transcription may be useful in therapeutic strategies. The hTERT promoter contains binding sites for c-Myc and there is some experimental and in vitro evidence that c-Myc may increase hTERT expression. We previously reported no correlation between c-Myc mRNA expression and hTERT mRNA or telomerase activity in human breast cancer. This study aims to examine the correlation between hTERT expression as determined by immunohistochemistry and c-Myc expression, lymph node status, and tumour size and grade in human breast cancer. MATERIALS AND METHODS: The immunohistochemical expression of hTERT and c-Myc was investigated in 38 malignant breast tumours. The expression of hTERT was then correlated with the lymph node status, c-Myc expression and other clinicopathological parameters of the tumours. RESULTS: hTERT expression was positive in 27 (71%) of the 38 tumours. 15 (79%) of 19 node positive tumours were hTERT positive compared with 11 (63%) of 19 node negative tumours. The expression was higher in node positive tumours but this failed to reach statistical significance (p = 0.388). There was no significant association with tumour size, tumour grade or c-Myc expression. However, hTERT expression correlated positively with patients' age (correlation coefficient = 0.415, p = 0.0097). CONCLUSION: hTERT protein expression is independent of lymph node status, tumour size and grade and c-Myc protein expression in human breast cancer.

hTERT expression in human breast cancer and non-cancerous breast tissue: correlation with tumour stage and c-Myc expression.

Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase) gene is the rate-limiting determinant of telomerase reactivation. The present study aims to quantitatively measure the expression of hTERT mRNA in human breast cancer, adjacent non-cancerous tissue (ANCT) and benign breast lesions, examine the association between hTERT and the clinicopathological characteristics of the cancer specimens and to explore the relationship between c-Myc and hTERT expressions. RNA was extracted from 49 breast carcinomas, 46 matched ANCT, and eight fibroadenomas. hTERT and c-Myc mRNA expressions were estimated by reverse transcriptase-PCR (RT-PCR) and Taqman methodology. hTERT mRNA was present in all of the cancerous and most of ANCT specimens with levels being much higher in the cancerous tissue than in ANCT. The ratio of hTERT mRNA in tumour to that in ANCT was 2011 (95% confidence interval 373-10,853, P < 0.0001). There was no significant association between tumour hTERT expression and patient's age, tumour size, grade, nodal metastasis, estrogen receptor (ER) positivity, lymphovascular (LVI) or c-Myc expression. However, there was a weak but significant negative correlation between hTERT expression and progesterone receptor (PR) status (p = 0.04) in tumours. hTERT mRNA expression was also significantly higher in carcinomas (median = 2.61 x 10(6)) than in fibroadenomas (median = 424).We conclude that hTERT mRNA expression is significantly higher in human breast cancer than in non-cancerous breast tissue suggesting that hTERT has a potential role in breast cancer diagnosis. The hTERT mRNA levels in tumour do not seem to be associated with the patient's age or advanced tumour stage. Furthermore, hTERT mRNA expression does not correlate with c-Myc mRNA expression in breast cancer.

The mRNA expression of cyclo-oxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in human breast cancer.

AIMS: There is a growing body of evidence that cyclo-oxygenase 2 (COX-2) plays an important role in carcinogenesis and angiogenesis of human tumours. The present study aims to compare COX-2 expression in human breast cancer and adjacent non-cancerous tissue (ANCT), and to identify any correlation between COX-2 and VEGF expression. METHODS: Total cellular RNA was extracted from frozen breast tissue samples according to standard methodology. The mRNA copy numbers for COX-2 and vascular endothelial growth factor 189 (VEGF-189) were determined 40 infiltrating carcinomas and 40 matched ANCT specimens using quantitative RT-PCR and TaqMan methodology. RESULTS: The COX-2 mRNA copy number per microg of RNA was two-fold higher in ANCT compared with the cancerous tissue (p = 0.01). Median mRNA copy number was 5.44 x 10(6) for ANCT and 2.30 x 10(6) for tumour, (ANCT range: 1 x 10(6) to 4.12x 10(7)) (tumour range: 1.29 x 10(5) to 1.07 x 10(7)). There was a significant correlation between COX-2 and VEGF-189 mRNA copy numbers in the cancer specimens (correlation coefficient = 0.5528, p = 0.0076). CONCLUSIONS: COX-2 mRNA is overexpressed in both human breast cancer and ANCT. We found higher levels in the matched ANCT which suggests that paracrine effects may be important in the role of COX-2 in mammary carcinogenesis. Furthermore, our results indicate that in human breast cancers COX-2 overexpression is linked to VEGF-189 overexpression and therefore tumour angiogenesis.

Telomerase activity and Bcl-2 expression in human breast cancer.

AIMS: Telomerase is a ribonucleoprotein that synthesizes telomeres and plays an important role in cellular immortalization. Bcl-2 gene encodes for a mitochondrial protein thought to prevent apoptosis of normal cells. We previously reported telomerase activity in 74% of human invasive breast cancers and detected a significant association between telomerase activity and prognostic parameters such as nodal status, tumour size and cellular proliferation. We hypothesized that telomerase reactivation in human breast cancer was associated with increased immunohistochemical expression of Bcl-2. METHODS: Bcl-2 immunohistochemical expression was determined in 25 infiltrating breast carcinomas with known telomerase activity (17 telomerase-positive and 8 telomerase-negative). The percentage of strongly and moderately stained tumour cells for Bcl-2 was determined by a breast pathologist who was blinded to telomerase data. Fisher's exact test was used to examine the association between telomerase activity and Bcl-2 expression. RESULTS: The median percentage of strongly stained tumour cells was 50% for telomerase-positive tumours (range, 0--100%) and 45% for telomerase-negative tumours (range, 0--100%). Twelve (70%) of 17 telomerase-positive tumours expressed strong or moderate Bcl-2 staining in >50% of tumour cells compared with six (75%) of eight telomerase-negative tumours (P=1.0). CONCLUSION: Telomerase reactivation seems to be independent of Bcl-2 protein expression in human breast cancer.

Pure antiestrogens and breast cancer.

Tamoxifen, which is the most commonly used drug for treatment of breast cancer, has both estrogen agonist and antagonist actions. Pure antiestrogens are devoid of any estrogen agonist effects. ICI 182,780 (fulvestrant) (Faslodex) and ICI 164,384 are competitive inhibitors of estrogen by binding to the estrogen receptor (ER). Preclinical and clinical studies show that fulvestrant and ICI 164,384 are more potent than tamoxifen in inhibiting the growth of breast cancer cells. They are devoid of any estrogen-agonist action on the uterus and vagina but lack the beneficial effects of tamoxifen on the bone and serum lipid profile. Fulvestrant is the first pure antiestrogen to complete phase III clinical trials. Such studies have shown that fulvestrant is at least as good as anastrozole in the treatment of post-menopausal women with advanced breast cancer who had relapsed or progressed on prior endocrine therapy. The drug was well tolerated and only minor side-effects were reported. Its potential role in the adjuvant setting will be determined by its adverse effects on bone mass and serum lipids. EM-800 and EM-652 are the most potent pure antiestrogens and EM-652 has the highest affinity of all antiestrogens to ER. They have no stimulatory effects on the uterus or vagina. It seems reasonable to expect that pure antiestrogens will be good alternatives to tamoxifen and aromatase inhibitors in the treatment of breast cancer.

Recent advances in breast cancer (the 37th ASCO meeting, May 2001).

Data from phase III clinical trials suggest that high dose chemotherapy (HDC) is currently not indicated for any stage of breast cancer. Therefore HDC should only be considered within the context of clinical trials. Furthermore, there is no significant evidence to support the routine use of taxanes in women with metastatic breast cancer (MBC) and further research is required to address this issue. A well-designed randomised controlled trial has shown that expressive support psychosocial therapy does not improve survival of women with MBC. Her2 overexpression seems to be a significant predictor of response to taxanes and anthracyclines, and FISH testing for Her2 seems to be superior to IHC in predicting response to Herceptin. Recent evidence confirms the independent prognostic value of VEGF, UPA and PAI-1 in women with early breast cancer and suggests that such parameters may have a role in selecting systemic therapy. Biological therapy using inhibitors/antagonists of angiogenesis and EGFR seems to be safe and well tolerated. Although the response rates are currently unimpressive, further research using survival as an endpoint is required.

Zonal variations of types II, IX and XI collagen mRNAs in rat epiphyseal cartilage chondrocytes: quantitative evaluation of in situ hybridization by image analysis of radioautography.

The spatial-temporal distribution of the mRNAs for type IX and type XI collagens were compared to that of type II collagen mRNA in the tibial epiphyseal plate cartilage of normal growing rats. The mRNAs were detected by in situ hybridization with radio-labelled specific probes and visualized by radioautography. The areas covered by the resulting silver grains were quantified by computer assisted image analysis. The areas in chondrocytes of each zone of the epiphyseal plate cartilage, which correspond to the stages of chondrocyte development and function were determined. Types II, IX and XI mRNAs were present to some extent in chondrocytes of all zones. The distributions of type II and type IX collagen mRNAs were similar with the highest concentrations in the proliferative zone, and the lowest in the resting and calcifying zones chondrocytes. In contrast, type XI collagen mRNA had a different distribution, with the lowest concentration in the resting zone chondrocytes and a significant decrease in the calcifying zone chondrocytes. These patterns correlates with the changes in chondrocyte function, and may reflect the roles of the type IX and type XI collagens. The data show that computer assisted image analysis of in situ hybridization radioautographic images is a precise, rapid tool for analysing differences in gene expression.