Evaluating and comparing immunostaining and computational methods for spatial profiling of drug response in patient-derived explants.

Patient-derived explants (PDEs) represent the direct culture of fragments of freshly-resected tumour tissue under conditions that retain the original architecture of the tumour. PDEs have advantages over other preclinical cancer models as platforms for predicting patient-relevant drug responses in that they preserve the tumour microenvironment and tumour heterogeneity. At endpoint, PDEs may either be processed for generation of histological sections or homogenised and processed for 'omic' evaluation of biomarker expression. A significant advantage of spatial profiling is the ability to co-register drug responses with tumour pathology, tumour heterogeneity and changes in the tumour microenvironment. Spatial profiling of PDEs relies on the utilisation of robust immunostaining approaches for validated biomarkers and incorporation of appropriate image analysis methods to quantitatively and qualitatively monitor changes in biomarker expression in response to anti-cancer drugs. Automation of immunostaining and image analysis would provide a significant advantage for the drug discovery pipeline and therefore, here, we have sought to optimise digital pathology approaches. We compare three image analysis software platforms (QuPath, ImmunoRatio and VisioPharm) for evaluating Ki67 as a marker for proliferation, cleaved PARP (cPARP) as a marker for apoptosis and pan-cytokeratin (CK) as a marker for tumour areas and find that all three generate comparable data to the views of a histomorphometrist. We also show that Virtual Double Staining of sequential sections by immunohistochemistry results in imperfect section alignment such that CK-stained tumour areas are over-estimated. Finally, we demonstrate that multi-immunofluorescence combined with digital image analysis is a superior method for monitoring multiple biomarkers simultaneously in tumour and stromal areas in PDEs.

Development of a patient-derived explant model for prediction of drug responses in endometrial cancer.

OBJECTIVE: To undertake a pilot study to develop a novel Patient-Derived-Explant (PDE) model system for use in endometrial cancer (EC) that is capable of monitoring differential drug responses in a pre-clinical setting. METHODS: Fresh tumour was obtained post-hysterectomy from 27 patients with EC. Tumours were cut into 1-3 mm3 explants that were cultured at the air-liquid interface for 16-24 h in culture media. Explants were cultured in different media conditions to optimise viability. Explants were also treated with carboplatin/paclitaxel or pembrolizumab for 24 h and processed into histology slides. Multiplexed immunofluorescence for Ki67 (proliferation marker), cPARP (apoptosis marker) and CAM 5.2 (tumour mask) was performed followed by image analysis and quantitation of biomarker expression. RESULTS: EC samples are amenable to PDE culture with preserved histological architecture and PDE viability for up to 48 h, with the addition of autologous serum in culture media facilitating EC-PDE viability. Our PDE platform provides evidence of differential drug-response to conventional chemotherapeutics and immune checkpoint inhibition, and these responses can be assessed in the context of a preserved tumour microenvironment. CONCLUSIONS: Our PDE platform represents a rapid, low-cost pre-clinical model which can be easily integrated into drug development pipelines. PDE culture preserves original tumour architecture and enables evaluation of spatial relationships in the tumour microenvironment. PDE culture has the potential for personalised drug-testing in a pre-clinical setting which is increasingly important in an era of personalised medicine in the treatment of EC.

Exploration of Matrix Effects in Laser Ablation Inductively Coupled Plasma Mass Spectrometry Imaging of Cisplatin-Treated Tumors.

The use of a low aerosol dispersion ablation chamber within a laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS) setup allows for high-resolution, high-speed imaging of the distribution of elements within a sample. Here we show how this enhanced capability creates new analytical problems and solutions. We report the distribution of platinum at the cellular level in non-small cell lung cancer (NSCLC) explant models after treatment with clinically relevant doses of cisplatin. This revealed for the first time a correlation between the platinum signal and the presence of carbon deposits within lung tissue. We show how complementary ion beam analysis techniques, particle-induced X-ray emission (PIXE) and elastic backscattering spectrometry (EBS), can be used to explore potential matrix effects in LA-ICP-MS data. For these samples, it was confirmed that the enhancement was unlikely to have resulted from a matrix effect alone. Thus, the presence of carbon deposits within tissue has potential implications for the effective distribution of the cisplatin drug.

5-Hydroxymethylcytosine is an independent predictor of survival in malignant melanoma.

Outcomes for melanoma patients vary within cancer stage. Prognostic biomarkers are potential adjuncts to provide more precise prognostic information. Simple, low-cost biomarker assays, such as those based on immunohistochemistry, have strong translational potential. 5-hydroxymethylcytosine (5 hmC) shows prognostic potential in melanoma but prior studies were small. We, therefore, analysed 5 hmC in a retrospective cohort to provide external validation of its prognostic value. Two hundred primary melanomas were evaluated for 5 hmC expression using immunohistochemistry. The primary objective was to assess the effect on overall survival while controlling for important confounders. Univariable and multivariable analyses were performed. REMARK guidelines were followed. The 5 hmC immunohistochemistry scoring showed very strong inter-observer agreement (ICC 0.88) and expression was significantly related to age, site, Breslow thickness, ulceration, mitotic rate, and stage. Kaplan-Meier analysis showed 5 hmC was associated with metastasis-free, melanoma-specific, and overall survival, P<0.0001 for each. In univariable Cox proportional hazards models, 5 hmC hazard ratios were significant and remained so in a multivariable model. A two-step cox model was created using stage and 5 hmC, as stage is the gold standard for clinical practice. The addition of 5 hmC produced significant improvement in the model and 5 hmC and stage were independent significant predictors. This is the largest study of the prognostic value of 5 hmC immunohistochemistry in melanoma. The 5 hmC scoring was easily and reproducibly performed and it was an independent predictor of metastasis-free survival, melanoma-specific survival, and overall survival. This work supports further development of 5 hmC as a prognostic biomarker and suggests that it could add more precision to American Joint Committee on Cancer staging.

microRNA-10b is a prognostic biomarker for melanoma.

Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disease has been revolutionized by new agents. More therapeutic options, coupled with the desire to extend treatment to the adjuvant setting mean that prognostic biomarkers that can be assayed from formalin-fixed paraffin-embedded clinical would be valuable. microRNAs have potential to fill this need. We analyzed 377 microRNAs in 79 primary melanomas and 32 metastases using a split sample discovery strategy. From a discovery analysis using 40 thick primary melanomas (20 cases with metastasis and 20 controls without metastasis at 5 years), microRNA expression was measured by quantitative RT-PCR (QRT-PCR). MiR-10b emerged as a candidate prognostic microRNA. This was confirmed in an independent validation set of thick primary melanomas (20 cases with metastasis and 19 controls without metastasis at 5 years). In the combined discovery and validation cohorts (n=79), miR-10b expression showed a 3.7-fold increase in expression between cases and controls (P=0.005) and showed a trend of increasing expression between primary melanomas and their matched metastases (P<0.001). In situ hybridization showed expression was in melanoma cells and correlated with expression measured by QRT-PCR (P=0.0005). We used the combined discovery and validation samples to verify the prognostic value of additional candidate microRNAs identified from other studies, and proceeded to analyze miR-200b. We demonstrated that miR-10b and miR-200b showed independent prognostic value (P=0.002 and 0.047, respectively) in multivariable analysis alongside known clinico-pathological prognostic features (eg, Breslow thickness) using a Cox proportional hazards regression model. Furthermore, the addition of these microRNAs to the clinico-pathological features led to an improved regression model with better identification of aggressive thick melanomas. Taken together, these data suggest that miR-10b is a new prognostic microRNA for melanoma and that there could be a place for microRNA analysis in stratifying melanoma for therapy.

Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer.

BACKGROUND: Activating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a "liquid biopsy." METHODS: We developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-α-positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR). RESULTS: Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3%) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans. CONCLUSIONS: Early identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease.

Prognostic tissue markers in melanoma.

Prognostic tissue markers in melanoma Prognosis for patients diagnosed with cutaneous melanoma is currently based upon histopathological features alone, although tumours which are morphologically similar can behave differently. Numerous putative biomarkers have been identified in an attempt to aid prognostication for primary melanoma, using methods which include immunhistochemistry, polymerase chain reaction (PCR), array comparative genomic hybridization (CGH) and gene expression arrays. Despite this wide body of research, no biomarkers for prognosis in melanoma have been translated or are close to translation into clinical practice. In this review selected prognostic biomarkers are evaluated and the factors influencing successful biomarker translation, including phases of biomarker development and study design, are explored in an attempt to highlight the current gap between prognostic melanoma biomarker research and clinical translation.

Statins mediate anti- and pro-tumourigenic functions by remodelling the tumour microenvironment.

Anti-cancer properties of statins are controversial and possibly context dependent. Recent pathology/epidemiology studies of human lung adenocarcinoma showed reduced pro-tumourigenic macrophages associated with a shift to lower-grade tumours amongst statin users but, paradoxically, worse survival compared with that of non-users. To investigate the mechanisms involved, we have characterised mouse lung adenoma/adenocarcinoma models treated with atorvastatin. Here, we show that atorvastatin suppresses premalignant disease by inhibiting the recruitment of pro-tumourigenic macrophages to the tumour microenvironment, manifested in part by suppression of Rac-mediated CCR1 ligand secretion. However, prolonged atorvastatin treatment leads to drug resistance and progression of lung adenomas into invasive disease. Pathological progression is not driven by acquisition of additional driver mutations or immunoediting/evasion but is associated with stromal changes including the development of desmoplastic stroma containing Gr1+ myeloid cells and tertiary lymphoid structures. These findings show that any chemopreventive functions of atorvastatin in lung adenocarcinoma are overridden by stromal remodelling in the long term, thus providing mechanistic insight into the poor survival of lung adenocarcinoma patients with statin use.

Expression of tenascin-C and its isoforms in the breast.

Tenascin-C (TNC) is an extracellular matrix glycoprotein which is frequently up-regulated in a variety of pathological conditions including chronic inflammation and cancer. TNC has been implicated in the modulation of cell migration, proliferation, invasion and angiogenesis. Multiple isoforms of TNC can be generated through the alternative splicing of nine exons located in the fibronectin type III region of the molecule. The profile of isoforms expressed differs between cancers and normal breast, with the fully truncated TNC isoform being predominant in normal and benign tissues and higher molecular weight isoforms induced predominantly in cancer. The addition of extra domains within the fibronectin type III repeat domain greatly affects TNC function with multiple exon combinations available for splicing. Exons 14 and 16 are considered to be tumour-associated and have been shown to affect breast cell line invasion and growth in vitro to a greater extent than the full-length TNC isoform. This mini review will provide a summary of the literature to date regarding the expression of TNC isoforms in the breast and also discuss more recent developments in the field regarding exon AD1.

Association of invasion-promoting tenascin-C additional domains with breast cancers in young women.

INTRODUCTION: Tenascin-C (TNC) is a large extracellular matrix glycoprotein that shows prominent stromal expression in many solid tumours. The profile of isoforms expressed differs between cancers and normal breast, with the two additional domains AD1 and AD2 considered to be tumour associated. The aim of the present study was to investigate expression of AD1 and AD2 in normal, benign and malignant breast tissue to determine their relationship with tumour characteristics and to perform in vitro functional assays to investigate the role of AD1 in tumour cell invasion and growth. METHODS: Expression of AD1 and AD2 was related to hypoxanthine phosphoribosyltransferase 1 as a housekeeping gene in breast tissue using quantitative RT-PCR, and the results were related to clinicopathological features of the tumours. Constructs overexpressing an AD1-containing isoform (TNC-14/AD1/16) were transiently transfected into breast carcinoma cell lines (MCF-7, T-47 D, ZR-75-1, MDA-MB-231 and GI-101) to assess the effect in vitro on invasion and growth. Statistical analysis was performed using a nonparametric Mann-Whitney test for comparison of clinicopathological features with levels of TNC expression and using Jonckheere-Terpstra trend analysis for association of expression with tumour grade. RESULTS: Quantitative RT-PCR detected AD1 and AD2 mRNA expression in 34.9% and 23.1% of 134 invasive breast carcinomas, respectively. AD1 mRNA was localised by in situ hybridisation to tumour epithelial cells, and more predominantly to myoepithelium around associated normal breast ducts. Although not tumour specific, AD1 and AD2 expression was significantly more frequent in carcinomas in younger women (age ≤40 years; P < 0.001) and AD1 expression was also associated with oestrogen receptor-negative and grade 3 tumours (P < 0.05). AD1 was found to be incorporated into a tumour-specific isoform, not detected in normal tissues. Overexpression of the TNC-14/AD1/16 isoform significantly enhanced tumour cell invasion (P < 0.01) and growth (P < 0.01) over base levels. CONCLUSIONS: Together these data suggest a highly significant association between AD-containing TNC isoforms and breast cancers in younger women (age ≤40 years), which may have important functional significance in vivo.

Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms.

INTRODUCTION: The stromal microenvironment has a profound influence on tumour cell behaviour. In tumours, the extracellular matrix (ECM) composition differs from normal tissue and allows novel interactions to influence tumour cell function. The ECM protein tenascin-C (TNC) is frequently up-regulated in breast cancer and we have previously identified two novel isoforms - one containing exon 16 (TNC-16) and one containing exons 14 plus 16 (TNC-14/16). METHODS: The present study has analysed the functional significance of this altered TNC isoform profile in breast cancer. TNC-16 and TNC-14/16 splice variants were generated using PCR-ligation and over-expressed in breast cancer cells (MCF-7, T47D, MDA-MD-231, MDA-MB-468, GI101) and human fibroblasts. The effects of these variants on tumour cell invasion and proliferation were measured and compared with the effects of the large (TNC-L) and fully spliced small (TNC-S) isoforms. RESULTS: TNC-16 and TNC-14/16 significantly enhanced tumour cell proliferation (P < 0.05) and invasion, both directly (P < 0.01) and as a response to transfected fibroblast expression (P < 0.05) with this effect being dependent on tumour cell interaction with TNC, because TNC-blocking antibodies abrogated these responses. An analysis of 19 matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases 1 to 4 (TIMP 1 to 4) revealed that TNC up-regulated expression of MMP-13 and TIMP-3 two to four fold relative to vector, and invasion was reduced in the presence of MMP inhibitor GM6001. However, this effect was not isoform-specific but was elicited equally by all TNC isoforms. CONCLUSIONS: These results demonstrate a dual requirement for TNC and MMP in enhancing breast cancer cell invasion, and identify a significant role for the tumour-associated TNC-16 and TNC-14/16 in promoting tumour invasion, although these isoform-specific effects appear to be mediated through MMP-independent mechanisms.

WNT5A expression increases during melanoma progression and correlates with outcome.

PURPOSE: Wnt ligands play a major role in development and are important in cancer. Expression microarray analysis correlates one member of this family, WNT5A, to a subclass of melanomas with increased motility and invasion. There are no large studies of clinical samples primarily addressing the importance of WNT5A in melanoma progression or outcome. Therefore, this study aimed to assess the protein expression of WNT5A during melanoma progression and its effect on outcome. EXPERIMENTAL DESIGN: Expression of WNT5A was determined in a series of 59 primary melanomas with matched metastases. To provide a benchmark of progression against which to assess WNT5A, expression of p16(ink4a) was analyzed, as this has been previously well documented in melanoma. The effect of WNT5A protein expression on outcome was assessed in 102 melanomas. RESULTS: Cytoplasmic WNT5A showed a trend of increasing expression with melanoma progression (P = 0.013), whereas there was diminishing p16(ink4a) expression (P = 0.006). Nevi showed relatively strong WNT5A expression. Strong cytoplasmic WNT5A was an independent risk factor for reduced metastasis-free and overall survival in multivariate analysis (P = 0.001 and 0.003, respectively). CONCLUSION: Cytoplasmic WNT5A increases with melanoma progression and strong expression is associated with poor outcome.

Cutaneous melanoma subtypes show different BRAF and NRAS mutation frequencies.

PURPOSE: BRAF mutations are present in two thirds of cutaneous melanomas and many of the rest have NRAS mutations. However, cutaneous melanoma is a heterogeneous disease with many clinicopathologic subtypes. Of these, the majority fits into four categories: superficial spreading, nodular, lentigo maligna, and acral lentiginous melanoma (ALM). Thus far, there is very limited data combining BRAF and NRAS mutation analysis to explore differences between cutaneous melanoma subtypes. The aim of this study was to address this issue. EXPERIMENTAL DESIGN: The frequency of BRAF and NRAS hotspot mutations, in exons 15 and 2, respectively, was assessed in 59 cutaneous melanomas comprising superficial spreading, nodular, lentigo maligna, and ALM using single-strand conformational polymorphism and RFLP-PCR analysis. RESULTS: Only 2 of 21 (9.5%) ALM showed BRAF exon 15 mutation compared with 9 of 14 (64.3%) superficial spreading malignant melanomas, 4 of 11 (36.4%) nodular melanomas, and 7 of 13 (53.4%) lentigo maligna melanomas (P < 0.01). However, our key finding is that the combined analysis of BRAF exon 15 and NRAS exon 2 showed that there were no significant differences in the overall mutation frequency between subtypes. In particular, 9 of 19 (47.4%) ALM without BRAF exon 15 mutation had an NRAS exon 2 mutation. CONCLUSIONS: We show that the overall BRAF/NRAS frequency in mutation hotspots is not significantly different among cutaneous melanoma subtypes. These data show that mitogen-activated protein kinase pathway activation may be important in all major subtypes of cutaneous melanoma, although the mechanism by which this is achieved varies.

Changes in tenascin-C isoform expression in invasive and preinvasive breast disease.

Tenascin-C (TN) is an extracellular matrix protein that is expressed at low levels in normal adult tissue but is highly expressed around many tumors including breast carcinoma. TN exists as multiple isoforms generated through alternative splicing, and these isoforms have different effects on cell growth and migration. This study has analyzed in detail the pattern of TN isoform expression in benign, preinvasive, and invasive breast lesions using reverse transcription-PCR and Southern blotting. Significant differences in the profile of TN isoforms were identified. Although all tissues expressed the fully truncated TN, expression of two additional isoforms, one containing exon 16 (TN16) and one containing both exons 14 and 16 (TN14/16), were significantly associated with the invasive phenotype (P < 0.001). A subset of ductal carcinoma in situ (DCIS) cases were also found to express these isoforms, which may be indicative of a high risk of invasion in these lesions. Expression of these isoforms correlated with the presence of TN protein in the stroma in place of or in addition to basement membrane TN. Immunohistochemistry and in situ hybridization confirmed the production of exon 14-containing higher molecular weight isoforms by stromal fibroblasts in malignant tissue and both periductal fibroblasts and residual myoepithelial cells in DCIS. Although no evidence of tumor cell synthesis of TN was detected in the tissues, two highly invasive breast cancer cell lines (MDA-MB 231 and MDA-MB 468) were found to produce TN in contrast with tumor cells with a lower invasive capacity (MCF-7 and T47D). These results demonstrate for the first time that specific TN isoforms are expressed in invasive breast carcinomas and that these isoforms are identified in a subset of DCIS and suggest that detection of TN16 and/or TN14/16 may be used as a predictor for invasion. Functional studies are now essential to establish the effect of these isoforms on tumor behavior and evaluate whether they will provide appropriate targets for therapeutic intervention.

Mechanisms of resistance to TRAIL-induced apoptosis in primary B cell chronic lymphocytic leukaemia.

Primary B cells from B cell chronic lymphocytic leukaemia (B-CLL) were resistant to the novel selective cytotoxic agent, TNF-related apoptosis-inducing ligand (TRAIL). Low levels of the death-inducing TRAIL receptors, TRAIL-R1 and TRAIL-R2 but not the putative 'decoy' receptors, TRAIL-R3 and TRAIL-R4, were expressed on the surface of B-CLL cells. Resistance to TRAIL was upstream of caspase-8 activation, as little or no caspase-8 was processed in TRAIL-treated B-CLL cells. Low levels of a TRAIL death-inducing signalling complex (DISC) were formed in these cells, accompanied by the recruitment of endogenous FADD, caspase-8 and c-FLIP(L) but not c-FLIP(S). Both caspase-8 and c-FLIP(L) were cleaved to form two stable intermediates of approximately 43 kDa, which remained associated with the DISC. Caspase-8 was not further processed to its active heterotetramer. Thus the resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP(L) to caspase-8 within the DISC, which would prevent further activation of caspase-8. Our results highlight the possibility of sensitising B-CLL cells to TRAIL by modulation of c-FLIP levels or by upregulation of surface expression of death receptors.

Nuclear immunostaining in rat neuronal cells using two anti-Kir2.2 ion channel polyclonal antibodies.

The inwardly rectifying potassium ion channel Kir2.2 has recently been demonstrated to have nuclear and plasma membrane subcellular localization. Nuclear expression of Kir2.2 is controversial, as a functional role for Kir2.0 potassium channels in the nucleus has not been investigated. However, in this report we have demonstrated Kir2.2 nuclear localization in sections of rat hindbrain and dorsal root ganglia tissue, using two anti- Kir2.2 polyclonal antisera with different epitope specificities. These data confirm nuclear localization and are suggestive of new functions of Kir2.0 potassium ion channels in the nucleus.

Inhibition of endometrial carcinoma cell growth using antisense estrogen receptor oligodeoxyribonucleotides.

BACKGROUND: The sex hormone 17 beta-estradiol acts as a mitogen in a number of tissues, including the endometrium, through direct interaction with the estrogen receptor (ER alpha). In the protection of the female breast and endometrium from cancer progression it would be advantageous to inhibit estrogenic action, therefore many estrogen receptor antagonists have been made. However, the most clinically relevant anti-estrogens for breast cancer have a detrimental effect on the endometrium and induce or exacerbate existing endometrial oncogenesis. Specific anti-estrogenic potential that is directed against the endometrial ER alpha theoretically could be achieved using antisense oligodeoxyribonucleotide transfection techniques. MATERIALS AND METHODS: To discover the most effective antisense oligodeoxyribonucleotides against the human ER alpha, a series of oligodeoxyribonucleotides were synthesised and tested in a human endometrial cancer cell line that expresses a functional ER alpha. RESULTS: Transfection with antisense oligodeoxyribonucleotides, directed against different regions of the human ER alpha, significantly inhibited ER alpha protein production without affecting ER beta protein levels. Antisense oligodeoxyribonucleotides directed against the translational start site demonstrated reduced binding of radiolabeled 17 beta-estradiol and a complete inhibition of estrogen-dependent endometrial cancer cell proliferation. The inhibitory effect of the antisense oligodeoxyribonucleotides on ER alpha production and ligand binding was enhanced in the presence of exogenous 17 beta-estradiol. CONCLUSION: The data suggest that antisense oligodeoxyribonucleotides against the human ER alpha have the potential of acting as anti-proliferative agents for estrogen-dependent endometrial cancers and may help in elucidating the relative roles of the two estrogen receptors, ER alpha and ER beta, in cell processes other than proliferation.

Accurate detection of copy number changes in DNA extracted from formalin-fixed, paraffin-embedded melanoma tissue using duplex ratio tests.

A minority of melanocytic lesions cannot confidently be classified as benign or malignant on histopathological examination, causing diagnostic uncertainty. DNA copy number changes can be used to distinguish nevi from melanoma, although the use of FFPE tissue can pose technical challenges. DNA copy number assays, called duplex ratio tests, have been developed with duplex real-time PCR, using a simple method with potential for high throughput. Five duplex ratio test assays targeting loci with common DNA copy number changes in melanoma were designed and tested using DNA extracted from FFPE samples microdissected from melanoma, common nevi, benign tonsil (10 each), and two melanoma cell lines. The assays proved accurate when DNA extracted from fresh and FFPE melanoma cell lines were compared (intraclass correlation coefficient, 0.99) and gave precise results when repeated on DNA from FFPE tissue (intraclass correlation coefficient range, 0.90 to 0.96). In combination, duplex ratio test values from three of the assays distinguished between the nevi and melanomas with 100% sensitivity (95% CI, 69.1% to 100%) and 100% specificity (95% CI, 69.1% to 100%). Duplex ratio test assays have been shown to be accurate and precise and can distinguish melanomas from common nevi using DNA from FFPE tissue. Appropriately designed assays could have value in assessment of other cancers.

Influence of plasma processing on recovery and analysis of circulating nucleic acids.

Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp(®) DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.

A switch in the expression of embryonic EMT-inducers drives the development of malignant melanoma.

Aberrant expression of embryonic epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) in epithelial cells triggers EMT, neoplastic transformation, stemness, and metastatic dissemination. We found that regulation and functions of EMT-TFs are different in malignant melanoma. SNAIL2 and ZEB2 transcription factors are expressed in normal melanocytes and behave as tumor-suppressor proteins by activating an MITF-dependent melanocyte differentiation program. In response to NRAS/BRAF activation, EMT-TF network undergoes a profound reorganization in favor of TWIST1 and ZEB1. This reversible switch cooperates with BRAF in promoting dedifferentiation and neoplastic transformation of melanocytes. We detected EMT-TF reprogramming in late-stage melanoma in association with enhanced phospho-ERK levels. This switch results in E-cadherin loss, enhanced invasion, and constitutes an independent factor of poor prognosis in melanoma patients.