Development of a Ki-67-based clinical trial assay for neoadjuvant endocrine therapy response monitoring in breast cancer

Purpose

The recent publication of the ACOSOG Z1031 trial results demonstrated that Ki-67 proliferation marker-based neoadjuvant endocrine therapy response monitoring could be used for tailoring the use of adjuvant chemotherapy in ER+HER2-negative breast cancer patients. In this paper, we describe the development of the Ki-67 clinical trial assay used for this study.

Methods

Ki-67 assay assessment focused on reproducing a 2.7% Ki-67 cut-point (CP) required for calculating the Preoperative Endocrine Prognostic Index and a 10% CP for poor endocrine therapy response identification within the first month of neoadjuvant endocrine treatment. Image analysis was assessed to increase the efficiency of the scoring process. Clinical outcome concordance for two independent Ki-67 scores was the primary performance metric.

Results

Discordant scores led to a triage approach where cases with complex histological features that software algorithms could not resolve were flagged for visual point counting (17%). The final Ki-67 scoring approach was run on T1/2 N0 cases from the P024 and POL trials (N = 58). The percent positive agreement for the 2.7% CP was 87.5% (95% CI 61.7–98.5%); percent negative agreement 88.9% (95% CI: 65.3–98.6%). Minor discordance did not affect the ability to predict similar relapse-free outcomes (Log-Rank P = 0.044 and P = 0.055). The data for the 10% early triage CP in the POL trial were similar (N = 66), the percentage positive agreement was 100%, and percent negative agreement 93.55% (95% CI: 78.58–99.21%). The independent survival predictions were concordant (Log-rank P = 0.0001 and P = 0.01).

Conclusions

We have developed an efficient and reproducible Ki-67 scoring system that was approved by the Clinical Trials Evaluation Program for NCI-supported neoadjuvant endocrine therapy trials. Using the methodology described here, investigators are able to identify a subgroup of patients with ER+HER2-negative breast cancer that can be safely managed without the need of adjuvant chemotherapy.

     

MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer

MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2'-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer.     

DICKKOPF-4 activates the noncanonical c-Jun-NH2 kinase signaling pathway while inhibiting the Wnt-canonical pathway in human renal cell carcinoma

BACKGROUND:

To the authors' knowledge, the functional significance of the Wnt antagonist dickkopf homolog 4 (DKK4) has not been investigated previously in renal cancer.

METHODS:

The authors initially observed that the expression of DKK4 was significantly higher in renal cancer tissues compared with adjacent normal kidney tissues. To assess the function of DKK4, stable DKK4‐transfected cells were established, and functional analyses were performed, including a T‐cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay and tests for cell viability, colony formation, apoptosis, cell cycle, invasive capability, wound‐healing capability, and in vivo tumor growth.

RESULTS:

The relative TCF/LEF activity was significantly lower in DKK4‐transfected cells compared with empty vector, and nuclear β‐catenin expression was decreased in DKK4 transfectants. In addition, expression levels of the β‐catenin downstream effector proteins cyclin D1 and c‐Myc were decreased in DKK4 transfectants. However, greater invasiveness and migration were observed in stably transfected DKK4 cells. Increased growth of DKK4‐transfected tumors also was observed in nude mice. Members of the Wnt noncanonical/c‐Jun‐NH2 kinase (JNK) signaling pathway also were effected, such as c‐Jun, which had significantly increased expression and phosphorylation in DKK4‐stable transfectants, and matrix metalloproteinase‐2, which had significantly increased expression in DKK4‐stable transfectants.

CONCLUSIONS:

This is the first study to indicate that DKK4 expression is increased in renal cancer tissues and that DKK4 activates the noncanonical JNK signaling pathway while inhibiting the Wnt‐canonical pathway. Cancer 2011. © 2010 American Cancer Society.     

Tumour suppressor microRNA-584 directly targets oncogene Rock-1 and decreases invasion ability in human clear cell renal cell carcinoma

Background:

The purpose of this study was to identify new tumour suppressor microRNAs (miRs) in clear cell renal cell carcinoma (ccRCC), carry out functional analysis of their suppressive role and identify their specific target genes.

Methods:

To explore suppressor miRs in RCC, miR microarray and real-time PCR were performed using HK-2 and A-498 cells. Cell viability, invasion and wound healing assays were carried out for functional analysis after miR transfection. To determine target genes of miR, we used messenger RNA (mRNA) microarray and target scan algorithms to identify target oncogenes. A 3′UTR luciferase assay was also performed. Protein expression of target genes in ccRCC tissues was confirmed by immunohistochemistry and was compared with miR-584 expression in ccRCC tissues.

Results:

Expression of miR-584 in RCC (A-498 and 769-P) cells was downregulated compared with HK-2 cells. Transfection of miR-584 dramatically decreased cell motility. The ROCK-1 mRNA was inhibited by miR-584 and predicted to be target gene. The miR-584 decreased 3′UTR luciferase activity of ROCK-1 and ROCK-1 protein expression. Low expression of miR-584 in ccRCC tissues was correlated with high expression of ROCK-1 protein. The knockdown of ROCK-1 by siRNA inhibited cell motility.

Conclusion:

miR-584 is a new tumour suppressor miR in ccRCC and inhibits cell motility through downregulation of ROCK-1.     

Coordinated regulation of polycomb group complexes through microRNAs in cancer

Polycomb Repressive Complexes (PRC1 and PRC2)-mediated epigenetic regulation is critical for maintaining cellular homeostasis. Members of Polycomb Group (PcG) proteins including EZH2, a PRC2 component, are upregulated in various cancer types, implicating their role in tumorigenesis. Here, we have identified several microRNAs (miRNAs) that are repressed by EZH2. These miRNAs, in turn, regulate the expression of PRC1 proteins BMI1 and RING2. We found that ectopic overexpression of EZH2-regulated miRNAs attenuated cancer cell growth and invasiveness, and abrogated cancer stem cell properties. Importantly, expression analysis revealed an inverse correlation between miRNA and PRC protein levels in cell culture and prostate cancer tissues. Taken together, our data have uncovered a coordinate regulation of PRC1 and PRC2 activities that is mediated by miRNAs.     

Catechol-O-methyltransferase-mediated metabolism of 4-hydroxyestradiol inhibits the growth of human renal cancer cells through the apoptotic pathway

Long-term exposure to estrogen and its metabolites may play an important role in renal cell carcinogenesis. Catechol-O-methyltransferase (COMT) participates in the estrogen metabolism pathway by neutralizing toxic substances. Although reduced COMT activity has been suggested to be a risk factor for estrogen-associated cancers, no studies have investigated the biological significance of COMT in the pathogenesis of human renal cell cancers (RCCs). We initially found that COMT levels are significantly decreased in human RCC tissues and cells suggesting it plays a suppressive role in tumor development. However, transient overexpression of COMT has no functional effect on RCC cell lines. In contrast, when cells overexpressing COMT are treated with its substrate 4-hydroxyestradiol (4-OHE(2)), growth is inhibited by apoptotic cell death. We also found that COMT overexpression combined with 4-OHE(2) induces upregulation of growth arrest- and DNA damage-inducible protein α (GADD45α). We further show that downregulation of GADD45α by a small interfering RNA-mediated approach inhibits cell death, indicating the essential role of GADD45α in the underlying mechanism of COMT action in response to 4-OHE(2). Finally, 4-methoxyestradiol fully reproduces the antiproliferative function of COMT with 4-OHE(2) by promoting GADD45α induction. Together, these findings show that COMT in the presence of 4-OHE(2) prevents RCC cell proliferation by enhancing apoptosis and that GADD45α plays a critical role in the COMT-mediated inhibition of RCC.     

Pityriasis rubra pilaris exacerbation with topical use of imiquimod

The role of immune response modifiers is increasing in the treatment of dermatologic diseases. Imiquimod, a toll-like receptor agonist, results in up-regulation of proinflammatory cytokines for improved immune surveillance. Although topical use is generally well-tolerated, imiquimod can potentially result in systemic effects and exacerbate generalized inflammatory papulosquamous diseases of the skin. We report the case of a 67-year-old man who was treated with imiquimod for actinic keratosis and developed fever and a progressive erythematous papulosquamous eruption that was histologically consistent with pityriasis rubra pilaris.     

Updated overview of current biomarkers in head and neck carcinoma

Squamous cell cancer is the most common type of malignancy arising from the epithelial cells of the head and neck region. Head and neck squamous cell carcinoma (HNSCC) is one of the predominant causes of cancer related casualties worldwide. Overall prognosis in this disease has improved to some extent with the advancements in therapeutic modalities but detection of primary tumor at its initial stage and prevention of relapse are the major targets to be achieved for further improvement in terms of survival rate of patients. Latest achievements in basic research regarding molecular characterization of the disease has helped in better perception of the molecular mechanisms involved in HNSCC progression and also in recognizing and targeting various molecular biomarkers associated with HNSCC. In the present article, we review the information regarding latest and potential biomarkers for the early detection of HNSCC. A detailed molecular characterization, ultimately, is likely to improve the development of new therapeutic strategies, potentially relevant to diagnosis and prognosis of head and neck cancers. The need for more accurate and timely disease prediction has generated enormous research interests in this field.