Loss of the chromatin modifier Kdm2aa causes BrafV600E-independent spontaneous melanoma in zebrafish.

KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease. Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency. Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing. In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin. Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis. These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology.

Nicolau Syndrome (Embolia Cutis Medicamentosa): A Rare and Poorly Recognized Iatrogenic Cause of Cutaneous Thrombotic Vasculopathy.

Nicolau syndrome is a rare form of iatrogenic cutaneous necrosis which affects injection sites. Although classically associated with intramuscular injections, it may develop after subcutaneous or other routes of parenteral drug administration. Clinically, it manifests as necrotic ulcers that often develop in a background of erythematous and livedoid reticular patches. The histopathologic characteristics of Nicolau syndrome are poorly documented in the dermatopathology literature and features only rarely as one of the obscure causes of cutaneous thrombotic vasculopathy. We report a case of Nicolau syndrome developing secondary to subcutaneous injection of cyclizine to familiarize the clinicians and pathologists to this unusual condition. Given that it is potentially avoidable, pathologists should alert the clinicians to the possibility of Nicolau syndrome when a skin biopsy from an injection site shows signs of extensive thrombotic vasculopathy.

Evidence for distinct alterations in the FGF axis in prostate cancer progression to an aggressive clinical phenotype.

Multiple fibroblast growth factor (FGF) axis alterations are known to occur in prostate cancer. Here we simultaneously profiled key components of this axis to determine their relevance in disease progression. An optimized immunohistochemistry protocol was used in expression analysis of FGF2, FGF8, FGFR1, FGFR4, and Sef (similar expression to FGF) in a single TMA of prostate cancer. FGF ligands and receptors were overexpressed in cancers compared to benign samples (p < 0.0001), while Sef expression was reduced (p < 0.0001). There was a positive association between higher grades and increased FGFR4 (p = 0.02), FGF2, and FGF8 (p = 0.002 and p < 0.0001). Sef expression was progressively lower with increasing grade (p = 0.005). Clinical stage was positively associated with FGF2, FGF8, and FGFR4 expression (p = 0.005, 0.03, and 0.012) but not with FGFR1 or Sef expression. Only reduced Sef was associated with bone metastasis (p = 0.02) and was also predictive of subsequent metastasis in initially localized tumours (p = 0.004). Down-regulation of Sef and increased FGFR4 were also the only independent variables associated with disease-specific survival (HR 1.73, p = 0.04 and HR 0.56, p = 0.01). In in vitro studies, silencing Sef enhanced the cell response to FGFs (p < 0.001) and substantially mitigated the effectiveness of an FGFR1 inhibitor. Conversely, increased Sef blocked the response to FGFs and had a comparable suppressive effect to the inhibitor. This study demonstrates that increased FGFR4 and reduced Sef may be critical FGF alterations associated with prostate cancer progression. Sef may also have a role in the tumour response to FGFR inhibition and warrants further investigation in this context.

Zebrafish MITF-Low Melanoma Subtype Models Reveal Transcriptional Subclusters and MITF-Independent Residual Disease.

The melanocyte-inducing transcription factor (MITF)-low melanoma transcriptional signature is predictive of poor outcomes for patients, but little is known about its biological significance, and animal models are lacking. Here, we used zebrafish genetic models with low activity of Mitfa (MITF-low) and established that the MITF-low state is causal of melanoma progression and a predictor of melanoma biological subtype. MITF-low zebrafish melanomas resembled human MITF-low melanomas and were enriched for stem and invasive (mesenchymal) gene signatures. MITF-low activity coupled with a p53 mutation was sufficient to promote superficial growth melanomas, whereas BRAFV600E accelerated MITF-low melanoma onset and further promoted the development of MITF-high nodular growth melanomas. Genetic inhibition of MITF activity led to rapid regression; recurrence occurred following reactivation of MITF. At the regression site, there was minimal residual disease that was resistant to loss of MITF activity (termed MITF-independent cells) with very low-to-no MITF activity or protein. Transcriptomic analysis of MITF-independent residual disease showed enrichment of mesenchymal and neural crest stem cell signatures similar to human therapy-resistant melanomas. Single-cell RNA sequencing revealed MITF-independent residual disease was heterogeneous depending on melanoma subtype. Further, there was a shared subpopulation of residual disease cells that was enriched for a neural crest G0-like state that preexisted in the primary tumor and remained present in recurring melanomas. These findings suggest that invasive and stem-like programs coupled with cellular heterogeneity contribute to poor outcomes for MITF-low melanoma patients and that MITF-independent subpopulations are an important therapeutic target to achieve long-term survival outcomes. SIGNIFICANCE: This study provides a useful model for MITF-low melanomas and MITF-independent cell populations that can be used to study the mechanisms that drive these tumors as well as identify potential therapeutic options.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/22/5769/F1.large.jpg.

Application of transcript profiling in formalin-fixed paraffin-embedded diagnostic prostate cancer needle biopsies.

OBJECTIVE: To investigate the feasibility of transcript profiling in diagnostic formalin-fixed and paraffin-embedded (FFPE) biopsies for prostate cancer. MATERIALS AND METHODS: Laser-capture microdissection (LCM) was used to microdissect glandular epithelium as well as stromal tissue in archival prostate needle biopsies. Optimized RNA extraction, reverse transcription and real-time PCR (QPCR) protocols were used to detect transcript expression. RNA degradation effects were assessed using hydrolysed cell line RNA and matched xenograft FFPE and frozen tumours. RESULTS: LCM and RNA extraction was achieved in all biopsies from a pilot cohort of five patients. cDNA produced was successfully used to detect expression of glyceraldehyde-3-phosphate dehydrogenase, RPL13, prostate-specific antigen, vimentin, inhibitor of differentiation/DNA binding 1 (Id-1) and polycomb group protein enhancer of zeste homolog 2 (EZH2) transcripts. In the cell line and xenograft models, we investigated the effect of RNA degradation on transcript quantification by QPCR. In both models normalization of transcript quantity with a housekeeping gene resulted in restored expression in all degraded samples to within a 50% difference of control samples. Using an extended cohort of 29 biopsies, we tested application in detecting differences in EZH2 and Id-1 expression between malignant and benign epithelium. The results confirmed that our technique was capable of quantifying significant differences in expression between malignant and benign epithelium consistent with the reported trends. CONCLUSION: This study reports the use of standard FFPE needle biopsies for transcript profiling and supports the concept of molecular prognostic studies in tissue acquired at diagnosis in prostate cancer.

ALDH1 Bio-activates Nifuroxazide to Eradicate ALDHHigh Melanoma-Initiating Cells.

5-Nitrofurans are antibiotic pro-drugs that have potential as cancer therapeutics. Here, we show that 5-nitrofurans can be bio-activated by aldehyde dehydrogenase (ALDH) 1A1/1A3 enzymes that are highly expressed in a subpopulation of cancer-initiating (stem) cells. We discover that the 5-nitrofuran, nifuroxazide, is selective for bio-activation by ALDH1 isoforms over ALDH2, whereby it both oxidizes ALDH1 and is converted to cytotoxic metabolites in a two-hit pro-drug mechanism. We show that ALDH1High melanoma cells are sensitive to nifuroxazide, while ALDH1A3 loss-of-function mutations confer drug resistance. In tumors, nifuroxazide targets ALDH1High melanoma subpopulations with the subsequent loss of melanoma-initiating cell potential. BRAF and MEK inhibitor therapy increases ALDH1 expression in patient melanomas, and effectively combines with nifuroxazide in melanoma cell models. The selective eradication of ALDH1High cells by nifuroxazide-ALDH1 activation goes beyond current strategies based on inhibiting ALDH1 and provides a rational basis for the nifuroxazide mechanism of action in cancer.

A conditional zebrafish MITF mutation reveals MITF levels are critical for melanoma promotion vs. regression in vivo.

The microphthalmia-associated transcription factor (MITF) is the "master melanocyte transcription factor" with a complex role in melanoma. MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma. How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model. Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity. We show that low levels of endogenous MITF activity are oncogenic with BRAF(V600E) to promote melanoma that reflects the pathology of the human disease. Remarkably, abrogating MITF activity in BRAF(V600E)mitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis. These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.

Generating and analyzing fish models of melanoma.

Melanoma is the most deadly form of skin cancer and incidence continues to rise rapidly (Gray-Schopfer et al., 2007). Melanoma develops from melanocytes, the pigmented cells that color our skin, hair, and eyes. Fish also have melanocytes, among other pigment cell types, and the fish and human developmental programme are highly conserved (Kelsh, 2004). The first fish models of melanoma were established in Xiphophorus, and more recently, transgenic melanoma models in zebrafish and medaka have been developed (Meierjohann and Schartl, 2006; Patton et al., 2010; Schartl et al., 2010). In this Chapter, we describe the basic techniques to generate genetic, environmental, and transgenic models of melanoma, discuss diagnoses, and describe standard molecular analysis techniques.

Twenty-three years of disease-free survival following cutaneous metastasis from a primary bladder transitional cell carcinoma.

We present a case of cutaneous metastases from a primary bladder transitional cell carcinoma (TCC), with a prolonged survival of 23 years. Cutaneous metastases from primary bladder TCC are uncommon and, like all metastases, have a poor prognosis. The common modality of treatment of cutaneous metastases from a primary bladder cancer is wide local excision of the metastases followed by combination chemotherapy. Here, we present a case of a solitary cutaneous metastasis from a primary bladder TCC, which was treated with wide local excision and single agent chemotherapy. Twenty-three years on, the patient remains disease and recurrence free.

How predictive is a cervical smear suggesting glandular neoplasia?

The prevalence of endocervical adenocarcinoma and its precursors has increased, in part due to increased diagnostic awareness of these lesions. To date, limited information has been published regarding the predictive value of glandular abnormalities in cervical smears. This study details the histological follow up of 418 cervical smears showing glandular abnormality, reported in our department over a six year period from 1993 to 1998. Histological follow up was available for 395 of the 418 smears (94.50%). The overall positive predictive value (PPV) for this group of smears was 72.66% for either significant glandular or squamous pathology (at least low grade cervical glandular intraepithelial neoplasia or CIN2 on follow up biopsy), and 55.70% for significant glandular pathology alone. Examination of subcategories of abnormal glandular smear showed that the PPV increased with the degree of abnormality reported within the smears.

Cytokeratin 7: a useful adjunct in the diagnosis of chromophobe renal cell carcinoma.

AIMS: The histopathological diagnosis of chromophobe renal cell carcinoma can present a diagnostic challenge, as these tumours can resemble either conventional renal cell carcinoma or oncocytoma. The aim of this study was to determine whether cytokeratin 7 expression is of practical use in the distinction of these three entities. METHODS AND RESULTS: A total of 40 cases previously diagnosed as either chromophobe renal cell carcinoma, conventional renal cell carcinoma or oncocytoma were identified. A representative section of each was stained with H&E and cytokeratin 7. Following independent review of the cases by three pathologists, a consensus diagnosis for each case was reached and the pattern of cytokeratin 7 staining was assessed. There were 12 cases of chromophobe renal cell carcinoma in the study, all of which showed a characteristic peripheral membrane pattern of staining for cytokeratin 7. Seventeen of the 18 cases of conventional renal cell carcinoma studied were negative for cytokeratin 7, while one case showed weak focal staining of <5% of the cells. The 10 cases of oncocytoma showed patchy weak to moderate cytoplasmic expression of cytokeratin 7, without the characteristic peripheral membrane accentuation seen in the chromophobe carcinomas. CONCLUSIONS: Immunohistochemical staining for cytokeratin 7 appears to be a useful adjunct in the diagnosis of chromophobe renal cell carcinoma, and in distinguishing this tumour from both oncocytoma and conventional renal cell carcinoma.