Han-Seung Yoon

Multiple Gene Expression Classifiers from Different Array Platforms Predict Poor Prognosis of Colorectal Cancer

Purpose: This study aimed to develop gene classifiers to predict colorectal cancer recurrence. We investigated whether gene classifiers derived from two tumor series using different array platforms could be independently validated by application to the alternate series of patients. Experimental Design: Colorectal tumors from New Zealand (n = 149) and Germany (n = 55) patients had a minimum follow-up of 5 years. RNA was profiled using oligonucleotide printed microarrays (New Zealand samples) and Affymetrix arrays (German samples). Classifiers based on clinical data, gene expression data, and a combination of the two were produced and used to predict recurrence. The use of gene expression information was found to improve the predictive ability in both data sets. The New Zealand and German gene classifiers were cross-validated on the German and New Zealand data sets, respectively, to validate their predictive power. Survival analyses were done to evaluate the ability of the classifiers to predict patient survival. Results: The prediction rates for the New Zealand and German gene-based classifiers were 77% and 84%, respectively. Despite significant differences in study design and technologies used, both classifiers retained prognostic power when applied to the alternate series of patients. Survival analyses showed that both classifiers gave a better stratification of patients than the traditional clinical staging. One classifier contained genes associated with cancer progression, whereas the other had a large immune response gene cluster concordant with the role of a host immune response in modulating colorectal cancer outcome. Conclusions: The successful reciprocal validation of gene-based classifiers on different patient cohorts and technology platforms supports the power of microarray technology for individualized outcome prediction of colorectal cancer patients. Furthermore, many of the genes identified have known biological functions congruent with the predicted outcomes.

A Multigene Urine Test for the Detection and Stratification of Bladder Cancer in Patients Presenting with Hematuria

PURPOSE We investigated whether the RNA assay uRNA® and its derivative Cxbladder® have greater sensitivity for the detection of bladder cancer than cytology, NMP22™ BladderChek™ and NMP22™ ELISA, and whether they are useful in risk stratification. MATERIALS AND METHODS A total of 485 patients presenting with gross hematuria but without a history of urothelial cancer were recruited prospectively from 11 urology clinics in Australasia. Voided urine samples were obtained before cystoscopy. The sensitivity and specificity of the RNA tests were compared to cytology and the NMP22 assays using cystoscopy as the reference. The ability of Cxbladder to distinguish between low grade, stage Ta urothelial carcinoma and more advanced urothelial carcinoma was also determined. RESULTS uRNA detected 41 of 66 urothelial carcinoma cases (62.1% sensitivity, 95% CI 49.3-73.8) compared with NMP22 ELISA (50.0%, 95% CI 37.4-62.6), BladderChek (37.9%, 95% CI 26.2-50.7) and cytology (56.1%, 95% CI 43.8-68.3). Cxbladder, which was developed on the study data, detected 82%, including 97% of the high grade tumors and 100% of tumors stage 1 or greater. The cutoffs for uRNA and Cxbladder were prespecified to give a specificity of 85%. The specificity of cytology was 94.5% (95% CI 91.9-96.5), NMP22 ELISA 88.0%, (95% CI 84.6-91.0) and BladderChek 96.4% (95% CI 94.2-98.0). Cxbladder distinguished between low grade Ta tumors and other detected urothelial carcinoma with a sensitivity of 91% and a specificity of 90%. CONCLUSIONS uRNA and Cxbladder showed improved sensitivity for the detection of urothelial carcinoma compared to the NMP22 assays. Stratification with Cxbladder provides a potential method to prioritize patients for the management of waiting lists.

Epigenetic silencing in non-neoplastic epithelia identifies E-cadherin (CDH1) as a target for chemoprevention of lobular neoplasia.

Invasive lobular carcinoma (ILC) of the breast is believed to develop from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). Down‐regulation of the cell–cell adhesion protein E‐cadherin is a defining feature of lobular breast cancer (LBC) and already occurs in ALH and LCIS. Apart from mutational mechanisms, epigenetic silencing of the E‐cadherin gene (CDH1) is thought to be involved in E‐cadherin down‐regulation and has been observed at a high frequency in ILC. Whether CDH1 promoter methylation is already present in in situ lesions and thus contributes to the initiation of LBC is not established. We thus examined microdissected archived tissue from 20 LBCs by methylation‐specific PCR to determine the CDH1 methylation status of lobular lesions. Nineteen of the 20 LBCs had a hypermethylated CDH1 promoter, including 13/14 ILCs and 13/13 ALHs or LCIS. Bisulphite sequencing indicated that methylation was complete within the investigated promoter fragment. Intriguingly, CDH1 methylation was likewise present in 8/8 adjacent non‐neoplastic epithelia, but not in 6/6 mammary epithelia from healthy subjects. E‐cadherin protein and mRNA were down‐regulated in in situ lesions relative to adjacent epithelia. Together, these results indicate that CDH1 promoter methylation occurs in LBC prior to E‐cadherin down‐regulation and neoplastic formation. We thus propose that epigenetic silencing represents the first of the two hits required to silence both CDH1 alleles for LBC to develop. Because promoter methylation is in principle reversible, our findings suggest that chemoprevention of LBC by epigenetic drugs should be feasible. Furthermore, the presence of CDH1 methylation in pre‐neoplastic epithelia suggests the existence of mammary regions with increased disease susceptibility, providing an explanation for the often multifocal presentation of LBC. Copyright

Slow proliferation as a biological feature of colorectal cancer metastasis.

Background:We have recently reported an inverse relationship between colon cancer progression and tumour proliferative activity. Here, we extend our findings by evaluating the proliferative activity of liver metastatic lesions and primary colorectal cancers (CRC) that differ in their metastatic potential.Methods:Using an earlier established multi-gene proliferation signature (GPS), proliferative levels were analysed in 73 primary CRCs and 27 liver metastases.Results:Compared with primary CRCs, we observed a significantly lower expression of the GPS in liver metastases and confirmed their lower proliferative levels by quantitative RT–PCR and Ki-67 immunostaining. No difference could be detected in apoptotic indices as assessed by M30 immunostaining, indicating that the net growth rate is lower in metastases relative to primary tumours. Notably, relapsed primaries or those with established metastases had significantly lower proliferative activity than CRCs that were non-metastatic and did not relapse.Conclusion:Our results suggest that slow proliferation is a biological characteristic of both liver metastases and those primary tumours with the ability to metastasise. The delineation of the mechanisms underlying the inverse association between proliferation and CRC aggressiveness may be important for the development of new therapeutic strategies.

Reduced expression of a gene proliferation signature is associated with enhanced malignancy in colon cancer.

The association between cell proliferation and the malignant potential of colon cancer is not well understood. Here, we evaluated this association using a colon-specific gene proliferation signature (GPS). The GPS was derived by combining gene expression data obtained from the analysis of a cancer cell line model and a published colon crypt profile. The GPS was overexpressed in both actively cycling cells in vitro and the proliferate compartment of colon crypts. K-means clustering was used to independantly stratify two cohorts of colon tumours into two groups with high and low GPS expression. Notably, we observed a significant association between reduced GPS expression and an increased likelihood of recurrence (P<0.05), leading to shorter disease-free survival in both cohorts. This finding was not a result of methodological bias as we verified the well-established association between breast cancer malignancy and increased proliferation, by applying our GPS to public breast cancer data. In this study, we show that reduced proliferation is a biological feature characterizing the majority of aggressive colon cancers. This contrasts with many other carcinomas such as breast cancer. Investigating the reasons underlying this unusual observation may provide important insight into the biology of colon cancer progression and putative novel therapy options.

Loss of Epidermal Langerhans Cells Occurs in Human Papillomavirus α, γ, and μ but Not β Genus Infections

Human papillomaviruses (HPVs), which are contained in the alpha, beta, gamma, mu, and nu genera, differ in their oncogenic potential and their tropism for cutaneous or mucosal epidermis. Langerhans cells (LC), the only epidermal professional antigen-presenting cells, are readily detected in normal mucosal and cutaneous epithelium. The aim of this study is to determine whether LC loss, which has been reported for HPV16, occurs in other HPV genera and establish its significance in viral pathology. We found that, as for HPV16, LCs were reduced in lesions infected with high-risk mucosal (alpha7 and alpha9 species) and low-risk cutaneous (gamma and mu) types. Lesions infected with alpha10 low-risk genital types had reduced LC but contained epidermal LC patches, coincident with dermis-localized regulatory T cells (T-regs). In contrast to other genera, LCs were common in the epidermis, and T-regs occupied the dermis of the potentially high-risk cutaneous beta-HPV type infected lesions. Therefore, LC loss in the infected lesions occurred irrespective of tropism or oncogenic potential of the HPV type. LC depletion in the HPV-infected epidermis may create an environment that is permissive for viral persistence and in HPV lesions in which LCs are found, the presence of typically immunosuppressive T-regs may compensate for their continued presence.

Increased levels of active c-Src distinguish invasive from in situ lobular lesions.

IntroductionMounting molecular evidence suggests that invasive lobular carcinoma (ILC) is developing from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). However, little is known about the mechanisms promoting the progression of lobular breast cancer (LBC) to invasive disease. Here, we investigated whether c-Src kinase, an established inducer of invasive states, contributes to the progression from ALH/LCIS to ILC.MethodsImmunochemistry for c-Src and other cancer-related molecules was performed on archived tissue specimens from 57 LBC patients. Relative c-Src activity was estimated by comparing fluorescence intensity of ILC with that of adjacent ALH/LCIS and nonneoplastic epithelia after staining with an antibody against active c-Src. Expression of active c-Src was correlated with markers of invasion and malignancy and with relapse among LBC patients.ResultsLevels of activated c-Src were increased in ILC relative to ALH/LCIS (1.63-fold ± 0.24 SD) and nonneoplastic epithelia (1.47 ± 0.18 SD). Increased c-Src levels correlated with the activation of c-Src downstream targets (Fak, Stat-3) and the expression of mesenchymal markers. ILC cells with activated c-Src co-expressed metastatic markers (Opn, Cxcr4) and included cells positive for the cancer stem cell marker Aldh1. A tendency for high c-Src levels (P = 0.072) was observed among the seven LBC patients with relapsed disease.ConclusionsOur data indicate elevated c-Src activity in ILC relative to noninvasive neoplastic tissue. The associated molecular changes suggest that c-Src promotes LBC invasiveness by inducing an epithelial-mesenchymal transition. Therefore, c-Src antagonists might counteract the acquisition of invasiveness during LBC progression. Inhibition of c-Src may also affect ILC cells thought to have a high metastatic potential and to be capable of initiating/maintaining tumor growth. Together with the possible association between high c-Src levels and disease recurrence, our findings encourage the evaluation of c-Src antagonists for the treatment of LBC.

Prognostic Association of YB-1 Expression in Breast Cancers: A Matter of Antibody

The literature concerning the subcellular location of Y-box binding protein 1 (YB-1), its abundance in normal and cancer tissues, and its prognostic significance is replete with inconsistencies. An explanation for this could be due in part to the use of different antibodies in immunohistochemical and immunofluorescent labeling of cells and tissues. The inconsistencies could also be due to poor resolution of immunohistochemical data. We analyzed two cohorts of breast tumours for both abundance and subcellular location of YB-1 using three different antibodies; two targeting N-terminal epitopes (AB- a and AB- b) and another (AB- c) targeting a C-terminal epitope. We also investigated stress-induced nuclear translocation of YB-1 in cell culture. We report that both AB- a and AB- c detected increased YB-1 in the cytoplasm of high-grade breast cancers, and in those lacking estrogen and progesterone receptors; however the amount of YB-1 detected by AB- a in these cancers is significantly greater than that detected by AB- c. We confirm our previously published findings that AB- b is also detecting hnRNP A1, and cannot therefore be used to reliably detect YB-1 by immunohistochemistry. We also report that AB- a detected nuclear YB-1 in some tumour tissues and stress treated cells, whereas AB- c did not. To understand this, cancer cell lines were analyzed using native gel electrophoresis, which revealed that the antibodies detect different complexes in which YB-1 is a component. Our data suggest that different YB-1 antibodies show different staining patterns that are determined by the accessibility of epitopes, and this depends on the nature of the YB-1 complexes. It is important therefore to standardize the protocols if YB-1 is to be used reproducibly as a prognostic guide for different cancers.

Deregulation of E-cadherin by human papillomavirus is not confined to high-risk, cancer-causing types.

Background  E‐cadherin is a tumour suppressor protein, which is normally expressed on keratinocytes and antigen‐presenting Langerhans cells (LCs) in the epidermis. We have previously shown that E‐cadherin is lost from tissues infected with the high‐risk cancer‐causing human papillomavirus (HPV) type 16.

MITF and PAX3 Play Distinct Roles in Melanoma Cell Migration; Outline of a "Genetic Switch" Theory Involving MITF and PAX3 in Proliferative and Invasive Phenotypes of Melanoma.

Melanoma is a very aggressive neoplasm with a propensity to undergo progression and invasion early in its evolution. The molecular pathways underpinning invasion in melanoma are now just beginning to be elucidated, but a clear understanding of the transition from non-invasive to invasive melanoma cells remains elusive. Microphthalmia-associated transcription factor (MITF), is thought to be a central player in melanoma biology, and it controls many aspects of the phenotypic expression of the melanocytic lineage. However, recently the paired box transcription factor PAX3 was shown to transcriptionally activate POU3F2/BRN2, leading to direct repression of MITF expression. Here we present a theory to explain melanoma phenotype switching and discuss the predictions that this theory makes. One prediction is that independent and opposing roles for MITF and PAX3 in melanoma would be expected, and we present empirical evidence supporting this: in melanoma tissues PAX3 expression occurs independently of MITF, and PAX3 does not play a key role in melanoma cell proliferation. Furthermore, we show that knockdown of PAX3 inhibits cell migration in a group of “lower MITF” melanoma cell lines, while knockdown of MITF promotes cell migration in a complementary “higher MITF” group of melanoma cell lines. Moreover, the morphological effects of knocking down PAX3 versus MITF in melanoma cells were found to differ. While these data support the notion of independent roles for MITF and PAX3, additional experiments are required to provide robust examination of the proposed genetic switch theory. Only upon clear delineation of the mechanisms associated with progression and invasion of melanoma cells will successful treatments for invasive melanoma be developed.