Reduction In Ki67 Expression Research Articles

Digital image analysis of breast epithelial cells collected by random periareolar fine-needle aspirates (RPFNA) from women at high risk for breast cancer taking hormone replacement and the aromatase inhibitor, letrozole, for six months

Aromatase inhibitors are currently being evaluated as preventive agents in post-menopausal women at high risk for breast cancer. A phase II trial of 42 women on hormone replacement therapy (HRT) treated with letrozole for 6 months showed Ki-67 was reduced by 66% but showed no change in cytomorphology or Masood score. Subsequent image analytical procedures (karyometry) conducted on a subset of the samples captured subvisual information that showed reduced cellular abnormality after 6 months of letrozole. In the present study we expanded on the preliminary karyometry study to determine if the change in karyometric measurements corresponded to changes in risk biomarkers quantified in the Phase II trial; and secondly, whether these biomarkers might be used together to serve as markers of response in individual cases. Pap stained slides from the Phase II trial were used. Epithelial cell images were digitized on a CCD video-microphotometer and the nuclei were segmented from the field using a semiautomatic algorithm. Nine out of 37 cases analyzed showed a numerical decrease in all three markers, although only three of these exhibited changes substantial enough to be considered as an improvement. However, 12 cases showed improvement by cytology (a decrease in Masood score of at least 2), an additional 13 cases demonstrated a reduction in Ki-67 expression by 50% of the median baseline value, and an additional five cases exhibited a decrease of at least 10% in abnormal cells by nuclear morphometry. Thus, a total of 30 of 37 cases (81%) showed improvement in at least one marker. There was no correlation between changes in Ki-67%, karyometric abnormality, and Masood score change other than specimens that exhibited an improvement in cytology also displayed greater decreases in nuclear morphometry abnormalities. Given the heterogeneity of mechanisms leading to malignancy, the quantitative analysis of nuclear chromatin patterns may be valuable as a global, or integrating, biomarker of change in chemoprevention studies in conjunction with additional markers. Correlation with long term clinical outcome is needed to validate meaningful combinations of informative biomarkers.

Inhibition of renal cancer cell growth in vitro and in vivo with oncolytic adenovirus armed short hairpin RNA targeting Ki-67 encoding mRNA

RNA interference (RNAi) has been proved to be a powerful tool for gene knockdown purpose and holds great promise for the treatment of cancer. Our previous study demonstrated that the reduction of Ki-67 expression by means of chemically synthesized siRNAs and shRNAs expressed from plasmid resulted in proliferation inhibition in human renal carcinoma cells. In this study, we constructed a novel oncolytic adenovirus-based shRNA expression system, ZD55-Ki67, and explored ZD55-Ki67-mediated RNAi for Ki-67 gene silencing. Our results showed that ZD55-Ki67 could induce silencing of the Ki-67 gene effectively, allow for efficient tumor-specific viral replication and induce the apoptosis of tumor cells effectively in vitro and in nude mice. We conclude that combining shRNA gene therapy and oncolytic virotherapy can enhance antitumor efficacy as a result of synergism between CRAd oncolysis and shRNA antitumor responses.

Ki67 tumor expression in breast cancer post-menopausal patients following calcitriol supplementation

14612 Background: Breast cancer patients present lower 1,25(OH)2D3 or 25(OH)D3 serum levels than unaffected women. Although breast cancer cell lines are growth inhibited by to vitamin D supra-physiological concentrations, there is much uncertainty about the anti- proliferative effect of physiological concentrations of 1,25(OH)2D3, the active form of vitamin D, in human beings. Vitamin D supplementation to post-menopausal women may be indicated to reduce bone loss. Methods: We have evaluated tumor dimension, proliferation rate (Ki67 expression), 1,25(OH)2D3 serum concentration, before and after a one month calcitriol supplementation (dose to prevent osteoporosis) to post-menopausal patients. Twenty one patients with clinical stage II disease, signed the informed consent and took part in the study from Jan- Nov 2007. Median age was 56 years (50–66), all tumors were grade 2/3, ER was detected in 11/18 and HER2 in 8/18 tumors; two tumors were triple negative. The first 10 (group-GI) and the next 11 patients (group-GII) received calcitriol 0.25 and 0.50mg/day PO, respectively. Results: One third of the patients presented 25(OH)D3 insufficiency (7–20 ng/mL) and no patients presented 1,25(OH)2D3 serum concentration below the reference level (15.9 pg/mL). No differences in 1,25(OH)2D3 serum concentration (GI: 63.05±20.44 pg/mL vs 56.20±25.16, p=0.16; GII: 69.77±39.02 vs 49.33±21.01, p=0.22, paired t test) and tumor dimension evaluated by ultrasonography (GI: 27.67 ±.9.92 mm vs 26.70±8.15, p=0.57; GII: 30.82±8.21 vs 30.00±11.96, p=0,76) were detected before and after calcitriol supplementation, in both groups. There was a trend towards a reduction in Ki67 expression in GI (10.0±6.6% vs 4.5±4.7, p=0.06, paired t test) and a significant reduction in GII (9.13±1.27% vs 4.59±1.42, p=0.02). Two patients with triple negative tumors had major reductions of Ki67 expression following calcitriol supplementation (6.6–11 to 0.6–2%). Conclusions: In a small group of post-menopausal patients, a short period of calcitriol supplementation may reduce Ki67 expression in breast cancer specimens, suggesting that physiological doses of the active form of vitamin D may have antiproliferative effects in these patients. Supported by FAPESP No significant financial relationships to disclose.

Down-Regulation of Phosphatidylinositol 3′-Kinase/AKT/Molecular Target of Rapamycin Metabolic Pathway by Primary Letrozole-Based Therapy in Human Breast Cancer

The phosphatidylinositol 3'-kinase (PI3K)/AKT/molecular target of rapamycin (mTOR) pathway is involved in the development of tumor resistance to endocrine therapy in breast cancer cell lines and represents an attractive target for pharmacologic intervention. However, the effects of endocrine therapy with aromatase inhibitors on in vivo expression of this signaling cascade, and its relation to tumor response and patient outcome, is unknown. PI3K, phospho-AKT (pAKT) and phospho-mTOR were assessed by immunohistochemistry on tumor specimens collected at baseline and after 6 months of treatment in 113 elderly breast cancer patients consecutively enrolled in a randomized phase II trial of primary letrozole therapy and letrozole associated with metronomic cyclophosphamide. Basal expression of the pathway was not significantly correlated with response or patient outcome. Both letrozole alone and letrozole with cyclophosphamide resulted in a significant reduction of PI3K expression (P = 0.02 and P < 0.005, respectively) and phospho-mTOR expression (P = 0.0001 and P = 0.0001, respectively). pAKT showed no change in the letrozole arm, whereas it was significantly decreased in the letrozole plus cyclophosphamide arm (P < 0.005). pAKT expression reduction was associated with a greater response rate (P = 0.05) and greater reduction in Ki67 expression (P = 0.05). Phospho-mTOR expression reduction was associated with a significantly longer disease-free survival in a multivariate analysis (P = 0.02). Letrozole inhibits key molecules in the PI3K pathway that are important targets of new drugs being developed to overcome resistance. Changes in these molecules may have prognostic significance. These results should be taken into account when planning prospective trials testing up-front aromatase inhibitor with drugs targeting the PI3K/AKT/mTOR signaling pathway.

Cytotoxic and antiproliferative activity of the single agent epirubicin versus epirubicin plus tamoxifen as primary chemotherapy in human breast cancer: a single-institution phase III trial

This study was designed to address whether simultaneous primary chemo-hormonal therapy provides additional activity compared with chemotherapy alone in breast cancer patients with operable or locally advanced disease. Between January 1997 and January 2002, 211 consecutive patients with T2-4, N0-1, M0 breast cancer were randomized to receive either epirubicin alone (EPI) or epirubicin plus tamoxifen (EPI-TAM). Ki67 expression was evaluated immunohistochemically in tumor specimens obtained before chemotherapy by incision biopsy and at definitive surgery. Tumor shrinkage of >50% was obtained in 76% of patients randomized in the EPI arm and 81.9% of patients randomized in the EPI-TAM arm (not significant). The corresponding rates of clinical and pathological complete response were 20.2 and 21.9% (not significant), and 4.8 and 6.7% (not significant), respectively. Pathologically complete response was more frequently observed in estrogen receptor (ER)-negative (ER-) tumors (P=0.04) and correlated with elevated baseline Ki67 expression (P<0.01). Both EPI and EPI-TAM treatments resulted in a significant reduction in Ki67 expression, either in overall patients (P=0.000) or in patients with ER+ breast cancer (P=0.000). The reduction in Ki67 immunostaining in the EPI-TAM arm was greater than in the EPI arm, leading to a lower Ki67 expression at post-operative residual histology (P=0.0041). The addition of tamoxifen to epirubicin chemotherapy did not improve the response rate but led to a significantly higher reduction in the Ki67 expression. Baseline elevated Ki67 expression and the ER- status were both associated with a greater chance of obtaining a pathological complete response at residual histology.

Primary letrozole therapy versus the combination of letrozole plus oral cyclophosphamide in elderly breast cancer patients. A single Institution randomized phase II trial

8108 Background: Letrozole has repeatedly found to be active as primary endocrine therapy in breast cancer patients. The aim of the study was to explore the activity of single agent Letrozole or Letrozole plus oral cyclophosphamide in a elderly patient population. Methods: From November 2001 to June 2004 114 elderly patients with T2-T4N0–1 estrogen receptor positive primary breast cancer were randomized to receive primary letrozole (arm L) (1 tablet/daily) or letrozole plus low dose protracted administration of oral cyclophosphamide (arm LC) (1 tablet 50 mg daily). Six month treatment was planned in both arms before definitive surgery. Paired tumor specimens were obtained before chemotherapy by incisional biopsy and at surgery. Results: Median age (range) was 79 years (64–89) in the 57 patients randomised in L arm and 75 years (62–94) in the 57 randomized in LC arm. Tumor characteristics were well balanced across the two treatment groups. Three patients were not available because too early. Among the 55 assessable patients in arm L, 22 attained a clinical complete response cCR (40.1%), 18 a partial response PR (32.7%), 11 stable disease SD (20.0%) and 2 progressive disease PD (3.6%); 2 patients were not evaluable. The corresponding disease response in the 56 assessable cases in the LC arm were: 25 cCR (44.6%), 25 PR (44.6%), 3 SD (5.4%) and 3 PD (5.4%). Three patients attained a pathological complete response, 2 in the L arm, 1 in the LC arm; 2 patients had residual in situ carcinoma (cis), 1 in each arm. Baseline Ki67 did not differ between the two arms [mean (95% confidence interval): 15.2% (12.8–17.6) in L arm and 19.5% (15.0–23.9) in LC arm, respectively]. Both treatments led to a significative reduction in Ki67 expression, but at residual tumor histology Ki67 immunostaining was lower in LC patients as opposed to L ones: mean 5.9% (95% CI 2.4–9.4) and 9.9% (95% CI 6.1–13.6). Conclusions: Primary hormone therapy with letrozole confirms to be very active in breast cancer patients. The addition of cyclophosphamide may lead to a higher response rate and greater reduction in proliferative activity. Statistics will be provided at the meeting. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis

Effect of chronic intermittent restraint stress on hippocampal expression of marker proteins for synaptic plasticity and progenitor cell proliferation in rats

Chronic restraint stress may change hippocampal mRNA levels of markers for synaptic plasticity such as synaptophysin, growth-associated protein 43 (GAP-43), and brain-derived neurotrophic factor (BDNF). In order to examine the relation between that stressor and those biochemical markers on protein level as well as the Ki-67 protein, a marker of progenitor cell proliferation, we subjected rats to chronic intermittent restraint stress for 6 h per day for 14 days excluding the weekends. This stress intensity caused a significant increase in adrenal gland weight and decrease in body weight gain. However, we did not find significant alteration of protein expression levels for synaptophysin, GAP-43, and BDNF by using Western blot analysis. Unlike these findings, the hippocampal protein expression of Ki-67 was significantly reduced by using both Western blot and immunohistochemical analyses. This reduction of Ki-67 expression in chronically stressed rats was correlated with increased adrenal gland weight and decreased body weight gain. All marker proteins used did not show any changes of hippocampal expression level after a single restraint stress session of 3 h. In conclusion, chronic intermittent restraint stress caused changes in the physiological stress response in rats, and a decrease of hippocampal progenitor cells using the Ki-67 protein as marker which indicates a suppression of adult neurogenesis. The results might contribute to understand the relationship between stress and cellular neurobiology of depression, since chronic antidepressant treatment have been shown to increase adult neurogenesis in the rat hippocampus.

Prognostic and predictive value of changes in tumour cell proliferation in locally advanced breast cancer primarily treated with doxorubicin

We previously reported that high tumour cell proliferation evaluated by Ki-67 expression, high mitotic frequency and high histological grade were associated with resistance to primary doxorubicin monotherapy in locally advanced breast cancer harbouring wild-type (wt) TP53. The aim of our present study was to evaluate the predictive and prognostic impact of proliferation parameters assessed in tumour tissue obtained after chemotherapy, and alterations induced in tumour cell proliferation. While we found a significant reduction in Ki-67 expression and mitotic frequency in tumours with wtTP53 (p=0.001 and p=0.008, respectively), no significant change was recorded in tumours expressing mutant TP53. For histological grade there was no significant change in either group. There was a direct correlation between pre- and post-treatment values for Ki-67 and mitotic frequency in tumours harbouring wtTP53 (p=0.0001 for both), but no correlation in tumours harbouring mutated TP53. High post-treatment Ki-67 expression and mitotic frequency were found to predict doxorubicin resistance only in patients with wtTP53 (p=0.04 and p=0.03, respectively). The prognostic importance of proliferation markers and histological grade was found to be similar whether they were determined in the pre- or post-treatment samples (Ki-67; pre: p=0.02; post: p=0.03; mitotic frequency; p=0.002 and p=0.01, respectively; histological grade; p=0.0001 and p=0.002, respectively). While the reduction in mitotic frequency was associated with improved survival (p=0.03), no significant associations between changes in other parameters and outcome were recorded.

Relationship between tumour shrinkage and reduction in Ki67 expression after primary chemotherapy in human breast cancer.

The association between tumour shrinkage and reduction in kinetic cell activity after primary chemotherapy in human breast cancer is still a matter of investigation. 157 patients with T2-4, N0-1, M0 breast cancer received primary chemotherapy consisting of either the CMF regimen + tamoxifen (the first consecutive 76 cases) or the single agent epirubicin (the subsequent 81). Ki67, p53, bcl2, c-erbB2 and steroid hormone receptors were evaluated immunohistochemically in tumour specimens obtained before chemotherapy and at surgery. Tumour shrinkage of >50% occurred in 72.4% of patients. Ki67 expression significantly decreased after chemotherapy; the reduction correlated with tumour response in both univariate (P < 0.005) and multivariate analysis (P = 0.02). p53, bcl-2, steroid hormone receptor and c-erbB2 immunostaining were scarcely affected. Baseline bcl2 (P = 0.04) and c-erbB2 (P = 0.02) were directly and inversely associated with the reduction in Ki67 immunostaining, respectively. Baseline p53 expression (P < 0.01) was directly related with Ki67 expression at residual tumour, whereas oestrogen receptor expression (P < 0.001) was inversely related. Ki67 at residual tumour was a better predictor for relapse-free survival (RFS) than baseline Ki67. Clinical response (P < 0.03), but not reduction in Ki67, was a significant independent predictor for disease recurrence. Chemotherapy was found to induce tumour shrinkage and to reduce the number of cells in the cell cycle, but its effect on tumour biology/aggressiveness was minimal. Reduction in Ki67 immunostaining correlated with clinical response but failed to be related to RFS. Ki67 expression at surgery rather than at baseline appears to be a better predictor for disease relapse. © 2001 Cancer Research Campaign http://www.bjcancer.com

Cytotoxic and antiproliferative activity of the CMF regimen administered in association with tamoxifen as primary chemotherapy in breast cancer patients.

Seventy six consecutive patients with T2-4, N0-1, M0 primary breast cancer (BC) received a median of 3 cycles of CMF (cyclophosphamide, methotrexate, 5-fluorouracil) regimen. Tamoxifen was concomitantly administered in patients with estrogen receptor positive (ER+) BC. Ki67 antigen was evaluated immunohistochemically in tumor specimens obtained before chemotherapy and at mastectomy. At post chemotherapy evaluation, tumor shrinkage greater than 50% was obtained in 60 patients (78.9%), 21 of them being complete responders (27.6%). As a whole, primary chemotherapy significantly decreased the number of Ki67 positive cells. More than 50% decrease in Ki67 expression was observed in 78.9% of patients attaining a clinical complete response (CR), in 44.7% of patients with partial remission (PR) and in 50.0% of non-responders, while an increase (>25%) in Ki67 expression was found in 5.3%, 18.4% and 18.7% of patients with CR, PR and non-response, respectively. Both CR and PR rates were superimposable in patients with ER+ and ER- primary BC, while the reduction in Ki67 expression was mainly found in ER+ cases. Patients with increased Ki67 expression from baseline, at the end of primary chemotherapy, had a shorter disease-free interval (70 months) with respect to patients with no change (88+ months) or decrease (87+ months), p<0. 05. To conclude, the activity of CMF + tamoxifen in primary BC does not seem superior to that expected administering CMF alone. The reduction in Ki67 expression, as a whole, correlated with clinical CR, but some individual discrepancies between tumor shrinkage and Ki67 pattern have been observed. The Ki67 reduction mainly confined to the ER+ primary BC suggests that tumor response in this subset may be linked to the reduction in proliferation activity, whereas other mechanisms such as apoptosis might be responsible for the tumor shrinkage in ER- tumors. Since the increase in proliferation activity after primary chemotherapy was associated with a greater recurrence rate and lower disease free interval, irrespective of tumor response, changes in proliferation activity after primary chemotherapy may represent a potentially available parameter that, in addition to the tumor response, can discriminate patients who would benefit from the cytotoxic treatment from patients who would not.