Search

Malignant melanoma results from ongoing activation of the mitogen activated protein kinases(MAPK ) pathway, commonly driven by mutations in BRAF. Several selective inhibitors of this pathway are used clinically, most notably the Vemurafenib and Dabrafenib. Different methods for BRAF mutation detection exist in the United Kingdom, including pyrosequencing, COBAS test and Sanger sequencing. However, there is significant variability in the analytical sensitivity of these tests. A highly sensitive PCR assay was developed to detect low tumor cell percentage of 0.1% in formalin fixed paraffin embedded (FFPE) tissue and plasma. The assay was developed on stable cell lines containing BRAF (codon 600) mutations. Peptide nucleic acid (PNA) Clamp PCR was used for the selective amplification of DNA target sequences. Total DNA was extracted from 48 FFPE tissues and 20 blood plasma (14 matched) stage II-IV melanoma patients and screened for BRAF mutations. Results were correlated with COBAS test data and clinical parameters. PNA Clamp PCR on FFPE tissue showed 46% (n=22) BRAF mutants. Four more BRAF mutant cases were identified using PNA clamp methodology when compared to COBAS. Sample cases were independently tested. Matched samples showed 85% (n=12) correlation for BRAF mutation status (6 + ve, 4 – ve, 2 with no residual tumor burden). There were two cases which were positive for COBAS test and PNA Clamp PCR but negative for cfDNA. This demonstrates an innovative and highly sensitive technique for the detection of the common driver mutations in melanoma using exceptionally low tumor burden samples, representing a useful tool for future research and clinical application.

To improve treatment outcomes in non–small cell lung cancer (NSCLC), preclinical models that can better predict individual patient response to novel therapies are urgently needed. Using freshly resected tumor tissue, we describe an optimized ex vivo explant culture model that enables concurrent evaluation of NSCLC response to therapy while maintaining the tumor microenvironment. We found that approximately 70% of primary NSCLC specimens were amenable to explant culture with tissue integrity intact for up to 72 hours. Variations in cisplatin sensitivity were noted with approximately 50% of cases responding ex vivo. Notably, explant responses to cisplatin correlated significantly with patient survival (P = 0.006) irrespective of tumor stage. In explant tissue, cisplatin-resistant tumors excluded platinum ions from tumor areas in contrast to cisplatin-sensitive tumors. Intact TP53 did not predict cisplatin sensitivity, but a positive correlation was observed between cisplatin sensitivity and TP53 mutation status (P = 0.003). Treatment of NSCLC explants with the targeted agent TRAIL revealed differential sensitivity with the majority of tumors resistant to single-agent or cisplatin combination therapy. Overall, our results validated a rapid, reproducible, and low-cost platform for assessing drug responses in patient tumors ex vivo, thereby enabling preclinical testing of novel drugs and helping stratify patients using biomarker evaluation. Cancer Res; 77(8); 2029–39. ©2017 AACR.

Melanoma is an aggressive cancer. Outcomes can vary significantly for lesions within the same pathological stage - a problem of increasing relevance with the promise of adjuvant treatments on the basis of immune checkpoint modulators and targeted therapies. The use of a panel of prognostic molecular biomarkers as an adjunct to stage represents a possible solution. Immunohistochemistry-based biomarkers offer greater potential for translation into clinical practice than biomarkers utilizing more complex methods. Many immunohistochemistry-based biomarkers have been identified through discovery studies, but rigorous validation of these is scarce. We take the first steps towards validating a combination of three such biomarkers in a prognostic panel - 5hmC, ki-67 and p16. Immunohistochemistry was performed on a cohort of 50 melanomas to determine the expression of 5hmC, ki-67 and p16. 5hmC and p16 showed statistically significant differences in metastasis-free survival between low-score and high-score groups, whereas the use of all three biomarkers together with stage could predict the 5-year metastasis risk more accurately than stage alone. Our results suggest that the use of multimarker panels to improve the accuracy of prognostic predictions is feasible and worthy of further study. We have shown that a small immunohistochemistry-based panel utilizing simple, inexpensive, reproducible methods can be an effective adjunct to stage in prognostic prediction. A follow-up study consisting of a large cohort of melanomas is now indicated to continue the development of the prognostic panel.

Identification of prognostic biomarkers is timely for melanoma as clinicians seek ways to stratify patients for molecular therapy. MicroRNAs are promising as tissue biomarkers because they can be assayed directly from formalin-fixed paraffin-embedded clinical samples. We previously reported that microRNA-21 (miR-21) was strongly expressed in melanoma relative to naevi and now sought to further assess the significance of this by assessing its relationship with its putative target, PTEN. Clinical melanoma samples were analysed by immunohistochemical analysis for PTEN, stem-loop qRT-PCR for miR-21 and PCR for BRAF/NRAS mutation status. Cell lines were investigated for the effect of anti-miR-21 on PTEN. A total of 81 clinical melanocytic tumour samples were investigated, with uniformly high PTEN expression in the nucleus and cytoplasm of naevi and with preferential loss of PTEN expression in the nucleus of melanoma cells. miR-21 expression was inversely associated with nuclear PTEN expression but not with cytoplasmic PTEN expression. An anti-miR-21 preferentially altered nuclear PTEN in melanoma cell lines. The presence of a BRAF or NRAS mutation had no significant effect on miR-21 expression. These data suggest miR-21 may exert an oncogenic effect in melanoma by favouring redistribution of PTEN to the nucleus.

The aims of this study were to investigate, in cutaneous malignant melanoma (MM), the integrity of nuclear vitamin D receptor (VDR) signalling, as implied by VDR subcellular location; to investigate the relationship between VDR and tumour progression and the inhibitory effect on VDR by mitogen-activated protein kinase (MAPK) overactivity. Archived tissue from 34 benign melanocytic naevi, 149 MMs and 44 matched metastases were stained by immunohistochemistry for VDR and a subset of primary MMs were stained for phosphorylated-extracellular signal-regulated kinase as a marker of MAPK activity. MM cell lines were investigated to show the subcellular location of VDR and cell viability in response to ligand±MAPK inhibitor. Benign melanocytic naevi showed mainly a strong nuclear VDR staining in contrast to MM where decreased nuclear and emergent cytoplasmic VDRs were associated with malignant progression in terms of dermal invasion and metastasis. MMs that retained exclusive nuclear VDR at the tumour base did not metastasize, a potentially important prognostic indicator. Decreased nuclear VDR correlated with increased cytoplasmic staining, suggesting the failure of nuclear entry as a primary cause of defective VDR signalling in MM. The histological subset analysis and MM cell line studies confirmed the inhibitory effect of MAPK activity on VDR signalling, but the pattern of VDR subcellular localization suggested failure of VDR nuclear entry as a primary effect of MAPK activity rather than direct inhibition of VDR-regulated transcription. Furthermore, high MAPK activity in tumours expressing cytoplasmic VDR was associated with worsened prognosis.

This work was funded by the Medical Research Council. Full open access supported by the Velux Foundation, the University of Zurich, and the EPFL School of Life Sciences.

Background and Aim: To study the characteristics of the inner (IM) and outer (OM) myometrium in the presence and absence of uterine adenomyosis. Methods: Case control blinded comparison carried out in a university department. Morphometric features of the myometrium were studied in uteri from pre- and postmenopausal women with and without uterine adenomyosis as the sole pathology. Uteri were also divided according to the phase of the cycle and examined using immunohistochemistry and image analysis. Results: Cell density and total nuclear area were statistically significantly greater in the IM compared to OM, in pre- and postmenopausal women, in both the adenomyosis and control uteri. The difference in nuclear size was statistically significant only in the premenopausal group. The change from the IM to the OM in cell density and total nuclear area was gradual with no distinct zonation. Examined features did not vary with cycle phase. Both the IM and OM in adenomyosis exhibited lower cell density and larger nuclei compared to controls. In adenomyosis, immunostaining for α-smooth muscle actin, desmin and Ki-67 was consistent with myometrial hyperplasia and hypertrophy. Conclusions: There are clear differences between the IM and the OM but the transition is gradual, with no distinct zonation. Adenomyosis is characterised by reduced cell density, and increased nuclear size and features of hyperplasia and hypertrophy that are not confined to the IM.

Objective To compare endometrial stromal cell invasion from women with and without adenomyosis and the effect of myometrial cells using a three-dimensional coculture. Design Case-controlled blinded comparison. Setting University department. Patient(s) Premenopausal women with and without uterine adenomyosis. Intervention(s) Human endometrial stromal and myometrial cells were grown in a three-dimensional coculture with crossover between cells from uteri with and without adenomyosis. Surface-enhanced laser desorption/ionization–time of flight–mass spectrometry proteomic analysis was performed on culture supernatants. Main Outcome Measure(s) Depth of stromal cell invasion into collagen matrix and protein expression profiles. Result(s) The depth of invasion for adenomyosis stromal cells (AS) was statistically significantly higher than controls (CS) whether grown on plain collagen, control muscle (CM), or adenomyosis muscle (AM). Coculture with AM enhanced invasion of both CS and AS. Enhanced invasion by AS was more marked in cocultures with AM than CM. Proteomic analysis identified differences that may account for the invasiveness and also many similarities between secretory products related to the disease status. Conclusion(s) Enhanced stromal invasion in adenomyosis is influenced by the myometrium in the in vitro coculture model. This suggests that adenomyosis may be a disease of both the endometrial stroma and myometrium.

The A allele of the A-1012G (rs4516035) vitamin D receptor (VDR) promoter polymorphism is associated with increased susceptibility and worsened outcome in malignant melanoma (MM). The A allele contains a GATA-3 binding site. There is a second polymorphism in the same promoter region, G-1520C (rs7139166), and there is potential for another GATA binding site in the G allele. Here, we tested the hypothesis that the G(-1520)A(-1012) haplotype might be a greater risk factor for MM than A-1012 alone. The A allele of A-1012G was preferentially linked to G of G-1520C and was more frequent in MM patients (p = 0.011) but G of G-1520C was not (p = 0.756). The CA haplotype was a very significant risk factor for MM (p = 0.0001) while the CG haplotype was protective (p = 0.014, combined model p = 0.00002). There was no effect of GA haplotype (p = 0.931), suggesting that that the difference in frequencies of the A allele between patients and controls was accounted for by the differences in frequencies of the CA haplotype. The A allele of A-1012G was more frequent in patients with metastasis (p = 0.054) than MM patients without metastasis, as was the G allele of G-1520C (p = 0.028). The GA haplotype was more frequent in patients with metastasis (p = 0.015), while frequencies of CA were similar. We suggest that the different roles of the A allele of A-1012G in susceptibility and metastasis risk may be a function of the availability of transcription factors in the differing cellular backgrounds related to susceptibility and progression of MM.

Background MicroRNAs (miRNAs) are small non-coding RNA molecules. Reduced or increased levels of specific miRNAs are observed in colon and other cancers, supporting their role in carcinogenesis. Detection of colorectal polyps is the cornerstone of the Bowel Cancer Screening Programme in the UK. However, uptake of screening nationally remains under 60%. We aimed to see whether circulating plasma miRNAs can be used to screen for patients with colorectal polyps, adenomas, or both. Methods Blood samples were taken from patients from the Bowel Cancer Screening Programme (asymptomatic but faecal occult blood testing [FOBt] positive). Plasma RNA was extracted, target miRNAs (19a, 98, 146b, 186, 191, 222*, 331-5p, 452, 625, 664, 1247) were identified on pooled case miRNA assay cards, and miRNA fraction was quantified by quantitative RT-PCR assay. Results were compared with endoscopy reports and with histology of any polyps identified and removed. Analysis was done with Excel (2011) and SPSS (version 20) software. Findings 210 patients were included (117 with polyps, 12 with cancer, 81 healthy controls [FOBt positive]). The miRNA panel showed significant differences in expression (on t testing) for patients compared with controls for those with polyps, cancer, or both (miR-19a, p=0·0184; miR-98, p=0·0206; miR-146b, p=0·0029; miR-186, p=0·0006; miR-62,5 p=0·0008), polyps (miR-19a, p=0·0233; miR-98, p=0·0224; miR-146b, p=0·003; miR-186, p=0·0004; miR-625, p=0·001), adenomas (miR-19a, p=0·0339; miR-98, p=0·0266; miR-146b, p=0·0045; miR-186, p=0·0008; miR-625, p=0·0049), multiple adenomas (both sides of colon; miR-146b, p=0·0194; miR-186, p=0·0226; miR-625, p=0·0013), and right-sided adenomas (miR-98, p=0·031; miR-146b, p=0·0076; miR-186, p=0·0041; miR-331-5p, p=0·0142; miR-625, p=0·0049). Receiver operating characteristic analysis showed sensitivity of 60% or more, and specificity of 86% or more for men with polyps, men with adenomas, all patients with haemorrhoids or diverticulosis and polyps, and all patients with haemorrhoids or diverticulosis and adenomas. Interpretation The target miRNAs that we identified showed significant differences in expression levels for patients with polyps and patients with adenomas from controls. Use of this panel has potential as a screening test. Funding Bowel Disease Research Foundation.

Matrix metalloproteinases (MMP) are considered to be of critical importance in the initiation of menstruation where MMP protein levels are reciprocally modulated by the actions of the gonadal steroid hormones, estradiol (E(2)) and progesterone (P4), with P4 being considered the principal suppressor of endometrial MMP expression. Trimegestone (T) is a novel progestagen that tightly controls menstruation timing and duration through mechanisms that might involve MMP suppression. Endometrial stromal cells treated with 10(-6) M E(2), P4 or T in the presence and absence of 10(-6)M RU486 showed that both T and P4 suppressed the expression of MMP-1 and MMP-3 transcripts and secreted protein, whereas MMP-9 was not produced in culture. The suppressive effect of T or P4 on MMP-1 and MMP-3 transcript levels was enhanced in the presence of E(2) and attenuated in the presence of RU486, although MMP-1 proteins were unaffected by the presence of RU486, which alone acted as a partial progesterone agonist in these cultures. Immunohistochemistry with MMP-1, MMP-3 and MMP-9-specific antibodies performed on endometrial biopsies obtained from non-treated, LH-dated, normally cycling women and endometrial biopsies obtained from postmenopausal women treated with T-based HRT showed that immunoreactive MMP-1 and MMP-3 was higher in the menstrual phase, whilst MMP-9 expression was higher in the late luteal phase (P = 0.03) and T significantly inhibited the presence of MMP-9(+) cells. These data suggest that T acts in a similar manner to P4, but causes subtle differences in expression patterns of MMPs that may explain the different clinical effect that this progestagen has on endometrial behaviour compared to P4.

The uterine endometrium responds to blastocyst implantation with extensive proliferation and differentiation of stromal cells into decidual cells, forming the antimesometrial and mesometrial decidua. These undergo regression by apoptosis but as this process occurs at different time periods suggest that there is spatially dependent temporal control of apoptosis in these specific regions. To elucidate the role of the mitochondrion-dependent signalling pathway in tissue regression, we investigated the spatial and temporal pattern of expression of the Bcl-2 family members in uterine tissues of the implantation site, from the post-implantation period to parturition. Furthermore, the activities of the initiator caspases-8 and -9, and of the executioner caspase-3 were determined. Overall Bax and Bcl-2 were expressed from day 8 till day 19, whilst Bcl-x(L) was extinguished by day 16. In the antimesometrial and in the mesometrial decidua both Bcl-2 and Bax declined from days 10 to 12. In the latter Bcl-2 immunoreactivity decreased till the end of pregnancy, whilst for Bax a constant level remained thereafter. The pattern of variation of enzymatic activities throughout pregnancy for all the enzymes was similar, increasing from days 10 to 14 and decreasing towards the end of pregnancy. The increased levels of active caspase-9 correlated with Bax/Bcl-2 and Bcl-x(L) expression suggesting that the apoptotic mitochondrion-dependent pathway is involved in decidual regression during pregnancy progression.

Psoriasis is a genetically determined disease characterized by hyperproliferation and disordered maturation of the epidermis. Th1 lymphocytes are implicated in its pathogenesis. The vitamin D receptor (VDR) is a candidate modifying gene, having immunosuppressive effects and being involved in anti-proliferative and pro-differentiation pathways in keratinocytes. There is suggestive evidence that the A allele of the A-1012G polymorphism is associated with down-regulation of the Th1 response, via GATA-3. The F and T alleles of Fok1 and Taq1 have been associated with increased VDR activity. The present study aimed to test the hypothesis that the A allele of A-1012G is protective for occurrence and severity of psoriasis and enhances therapeutic response to vitamin D analogues and that these effects would be additive to those of Fok1 and Taq1. The study group comprised 206 psoriasis patients who had received topical calcipotriol treatment and 80 controls. There was no significant linkage disequilibrium between any pair of the three polymorphic sites (P=0.3-0.8). The A, F and T alleles were positively associated with calcipotriol response: AA genotype (compared to AG/GG), odds ratio (OR)=2.18 (P=0.04); TT, OR=1.97 (P=0.03); AAFF genotype combination, OR=4.11 (P=0.03); AATT, OR=5.64 (P=0.005); and FFTT, OR=3.22 (P=0.01). Comparing patients without, to patients with, a family history of psoriasis, the A allele was under represented (P=0.01) and the AAFF genotype combination even more so (compared to residual genotypes) (OR=0.24; P=0.005). AAFF was also under-represented in patients without a family history compared to controls (OR=0.31; P=0.04). There were no associations of family history with Fok1 and Taq1. There were no associations of severity of psoriasis with any polymorphism. In conclusion, the A-1012G, Fok1 and Taq1 VDR polymorphisms were associated with response to calcipotriol. A-1012G and Fok1 were associated with susceptibility to non-familial psoriasis.

The association of Taq 1 and Fok 1 restriction fragment length polymorphisms of the vitamin D receptor with occurrence and outcome of malignant melanoma (MM), as predicted by tumour (Breslow) thickness, has been reported previously. We now report a novel adenine–guanine substitution −1012 bp relative to the exon 1a transcription start site (A-1012G), found following screening by single-stranded conformational polymorphism of this promoter region. There was a total of 191 MM cases , which were stratified according to conventional Breslow thickness groups, cases being randomly selected from each group to form a distribution corresponding to the known distribution of Breslow thickness in our area, and this population (n=176) was compared to 80 controls. The A allele was over-represented in MM patients and, with GG as reference, odds ratio (OR) for AG was 2.5, 95% confidence interval (CI) 1.1–5.7, (P=0.03) and AA 3.3, CI 1.4–8.1, (P=0.007). The outcome was known in 171 of 191 patients and the A allele was related to the development of metastasis, the Kaplan–Meier estimates of the probability of metastasis at 5 years being: GG 0%; AG 9%, CI 4–16%; AA 21%, CI 12–36%; (P=0.008), and to thicker Breslow thickness groups (P=0.04). The effect on metastasis was independent of tumour thickness and A-1012G may have predictive potential, additional to Breslow thickness. Neither the Fok 1 nor Taq 1 variants (f and t) were significantly related to the development of metastasis, although there was a strong relationship of fftt with the thickest Breslow thickness group (P=0.005). There was an interaction between the A-1012G and Fok 1 polymorphisms (P=0.025) and the Fok 1 variant enhanced the effect of the A allele of the A-1012G polymorphism on metastasis, the probability of metastasis for AAff at 5 years follow-up being 57%, CI 24–92%.

Introduction : Colorectal cancer is the third most common neoplasm worldwide. The sequential progression of colorectal cancer from adenoma to carcinoma highlights that opportunities exist to alter the natural course of disease progression. The aim of this study was to characterize the expression levels of microRNAs linked to development and progression of colorectal neoplasia. Patient, Design, Patients & Methods : MicroRNA expression signature was developed for RNA extracted from freshly frozen tumour and adjacent normal tissue ( n = 5). Based on differential expression and literature search, hsa-miR-135b was selected for further characterisation in different types of colonic polyps and cancer tissue. Formalin Fixed Paraffin Embedded tissue were studied for miRNA expression, KRAS, BRAF, PIK3CA mutations, and immuno-histochemistry for APC and p53 proteins for normal colon ( n = 11), hyperplastic polyps ( n = 11), high grade adenomas ( n = 10), low grade adenomas ( n = 34) and adenocarcinoma ( n = 13). Results : CRC tissue had significantly higher expression levels of hsa-miR-135b ( p = 0.0017) than their adjacent paired normal tissues (mean increase = 8.90 fold, 95% CI = 2.98–26.50). Linear trend analysis showed a progressive increase in expression level of hsa-miR-135b across normal epithelium, low grade adenomas, high grade adenomas and carcinomas ( p = 0.0007, R squared 0.16, slope −0.35). KRAS mutant colonic polyps and cancer tissue had significantly higher (3.04 fold, 95% CI = 1.23–7.46) expression levels of hsa-miR-135b compared to polyps and cancers with non mutant KRAS gene ( p = 0.001). Whereas, hsa-miR-135b expression levels were significantly lower in colonic polyp and cancer tissue stained positively for APC proteins ( p Conclusion : There is a progressive increase in expression levels of hsa-miR-135b correlating with the sequential progression of normal epithelium to adenoma and carcinoma. Expression levels of hsa-miR-135b correlate with expression of APC proteins in colorectal tissue suggesting its role in tumour initiation. Highlights : This study highlights the role of tissue microRNAs for their role in the development of colorectal carcinoma. Identification of tissue specific microRNAs will help is designing microRNAs based gene therapies to control the development and progression of colonic neoplasia.

Aim MicroRNAs (miRNAs) from tumour tissue and common gene mutations were studied to determine whether they predict the development of metastasis in patients with Dukes B colorectal cancer. Method Patients who underwent curative resection for Dukes B colorectal cancer who subsequently developed distant metastatic disease at some stage in the following 5 years (‘high-risk B’) were compared with case-matched controls of Dukes A, Dukes B (no metastases, ‘low-risk B’) and Dukes C patients without any detectable metastasis at 5 years of follow-up. MiRNAs from tumour and adjacent normal tissue and common gene mutations (KRAS, BRAF, PIK3CA) in primary cancer tissue were analysed to identify prognostic tissue markers for the development of metastasis in patients with Dukes B colorectal cancer. Results Expression of miR-15b and miR-135b was significantly downregulated (P

Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCR–based assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. The reproducibility and accuracy of the DRTs were demonstrated and appropriate correction factors for DNA quality calculated for each assay, based on the results of 108 diploid samples. As a panel, seven DRTs were able to differentiate unambiguous cases of melanoma and nevi with a sensitivity of 87% (95% CI, 83%–91%) and a specificity of 88% (95% CI, 84%–92%) in a series of 145 melanomas and 123 nevi. The DRT scores for 20 nonmetastasizing primary melanomas and 20 metastasizing primary melanomas revealed that DRTs had a marginal benefit as prognostic markers. DRTs have early potential to act as molecular biomarkers of melanoma on FFPE specimens pending validation, and DRTs may have applicability as prognostic markers in melanoma or other tumor types if new DRTs to relevant loci are developed.

Fetal membranes overlying the cervix in patients prior to and during labour, and within the rupture tear after spontaneous delivery at term, exhibit altered morphology. In this study we report that in comparison to mid-zone fetal membranes biopsies, these regions are characterized by increased expression of the matricellular protein osteonectin or SPARC (Secreted Protein Acidic and Rich in Cysteine). In the reticular layer, the percentage of vimentin positive mesenchymal cells immunoreactive for osteonectin increased in these regions from 3-4% to 25-33% and represented a fraction of the alpha-smooth muscle actin positive myofibroblasts elevated in the same regions. In the fibroblastic layer, the percentage of osteonectin positive cells increased from 1-5% to 8-13%; however, these did not exhibit the same relationship to the alpha-smooth muscle actin positive myofibroblasts in this layer. In the cytotrophoblastic layer the percentage of cytotrophoblastic cells immunoreactive for osteonectin increased from 1% to 6-12%. Elevation of in-situ detectable mRNA was also observed in the same cellular populations in this region. The incidence of cells positive for osteonectin mRNA or protein in the reticular layer correlated with morphological changes. Osteonectin has been implicated in the regulation of extracellular matrix turnover, and its pattern of expression suggests a role in the regional connective tissue and cytotrophoblastic changes proposed to be involved in the cleavage and rupture of fetal membranes.

Remodelling of the extracellular matrix requires tight control not only of matrix synthesis, but also of matrix degradation. Control of matrix degradation is achieved mainly through the matrix metalloproteinase (MMP) enzymes. In the glomerulus, MMP-2 and MMP-9 are believed to be particularly important, as they have activity against type IV collagen. This study has demonstrated by immuno-electron microscopy that most of the immunoreactivity for MMP-2 in the normal glomerulus is located within the glomerular basement membranes and mesangial matrix. mRNA for MMP-2 is also detectable in normal glomeruli, but the other main gelatinase, MMP-9, could not be localized by immuno-electron microscopy. In the normal glomerulus, it seemed likely that MMP-2 is present in an inactive form. To confirm this, in situ zymography was carried out using frozen sections of normal kidney. Baseline activity of normal kidney was relatively weak, but this was dramatically increased by chemical activation of metalloproteinases. The results imply that MMP-2, in an inactive form, is a normal constituent of the extracellular matrix and glomerular basement membranes. Activation would presumably render the matrix 'self-degrading'; membrane-bound MMPs (MT-MMPs) seem particularly likely to be involved in leukocyte penetration of basement membranes in inflammation.

Rabbit polyclonal antibodies were raised to rat Kir2.0 (Kir2.1, Kir2.2 and Kir2.3) inwardly rectifying potassium ion channel proteins. The antibody specificities were confirmed by immunoprecipitation of [35S]-methionine-labelled in vitro translated channel proteins and western blotting. Immunohistochemistry revealed a different patterns of expression of Kir2.0 subfamily proteins in the rat hind-brain (cerebellum and medulla) and fore-brain (hippocampus). Notably, only Kir2.2 protein was detected in the cerebellum and medulla, Kir2.1, Kir2.2 and Kir2.3 proteins were expressed in the hippocampus and immunostaining was not limited to neuronal cell types. Anti-Kir2.1 (fore-brain only) and anti-Kir2.2 (fore- and hind-brain) antibodies showed positive staining in macroglia, endothelia, ependyma and vascular smooth muscle cells. In contrast, anti-Kir2.3 (fore-brain only) immunostaining was limited to neurons, macroglia and vascular smooth muscle. These results indicate that specific regions within the rat fore- and hind-brain have differential distributions of inwardly rectifying potassium ion channel proteins.

The uterus and the placenta synthesize insulin-like growth factors (IGFs) and insulin-like binding proteins (IGFBPs). These growth factors are implicated in processes of proliferation and differentiation that occur in the uterus. To determine the patterns of expression of IGFs during rat pregnancy we used in situ hybridization with digoxigenin labeled probes on uterus from day 7 to day 16 of pregnancy. In early gestation days (7-8) both IGF mRNAs showed similar tissue distribution with relative abundance in the stroma and circular muscle layer. On days 11 and 12 expression for IGF-I mRNA was found in the mesometrial decidua and metrial gland and in the ectoplacental cone while clear expression of IGF-II mRNA could only be found in the latter. On days 13 and 14, expression for IGF-I mRNA could be detected in the mesometrial decidua and metrial gland but no expression was observed for IGF-II mRNA. A gradient of IGF-I mRNA expression could be observed in the placenta on day 16, with the trophoblastic cells of the basal zone expressing the signal with stronger intensity than in the labyrinthine zone. For IGF-II mRNA the highest expression was associated with the labyrinthine zone. Endovascular trophoblast was positive for both mRNAs. The spatial and temporal patterns of expression suggests a role for IGFs in the process of decidualization as well as in the establishment, growth and differentiation of the various trophoblast cells of the placenta.

It is not clear whether the rare combination of Hodgkin's diseases with non-Hodgkin lymphomas are true composite lymphomas or differentiation stages of one tumour cell clone. We used in situ hybridization and immunohistochemistry for the demonstration of immunoglobulin light chains in order to investigate the relationship between the two lymphoma components. In three cases of nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma the Hodgkin cells, as well as the tumour cells in the diffuse large B-cell lymphoma, showed the same messenger RNA for one light chain. Thus, using in situ hybridization in nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma in a small number of cases a possible genetic relationship between the two components could be shown. In nodular sclerosis combined with diffuse large B-cell lymphoma, in situ hybridization did not support a common clonal origin of both tumour parts. However, a unique clonal derivation cannot be excluded by the techniques applied.

We present evidence for a novel histological and embryological relationship at the human materno-fetal interface. Here an epi- endo- thelium forms an integrated unicellular layer lining the intervillus space in between the anchoring villi that attach the placenta to the uterus. This layer appears to be derived from two different germ layers (mesoderm and ectoderm). The data presented here reveals that when a probe for the Y-chromosome is used to test the gender of placental cells following the birth of male or female babies, the cell-sheet is a genetic mosaic derived from two individuals (mother and baby). The endothelium is maternally derived; the epithelium is fetal derived. This new allo- epi- endothelium model is relevant to theories of germ layer separation in development, reproductive immunology and the endocrinology of implantation and placentation. It demonstrates cooperative intercellular interactions that are fundamental to achieving a major goal of human interstitial implantation the establishment of a blood sinus for haematotrophic nutrition. Poor implantation is a fundamental cause of pregnancy pathology and this knowledge will be useful in development of our understanding of pregnancy diseases.

Previous studies using immunohistochemistry have shown variable hormone production by carcinoid tumours of the appendix. In order to confirm the existence of a specific subset of these tumours, in situ hybridization using synthetic oligonucleotide probes to detect pre-proglucagon and pre-proinsulin mRNA was performed in formalin-fixed, paraffin-embedded material from eight tubular carcinoids, 12 insulin carcinoids, and two mucinous carcinoids. The results were correlated with standard silver and mucin stains. All tubular carcinoids but none of the insular or mucinous carcinoids contained proglucagon mRNA. Proinsulin mRNA was not detected in any of the tumours. Tubular carcinoids of the appendix constitute a definable subset of appendiceal carcinoids which have a similar distribution and prognosis to typical insular carcinoids and can be diagnosed on haematoxylin and eosin-stained sections confirmed by routine special stains. The main need for recognition is to avoid confusion with mucinous carcinoids, which have a worse prognosis and may require more aggressive treatment.

Summary Background The relationships between so-called spitzoid tumours have proven difficult to understand. Objectives To address three questions: does spitzoid tumour morphological similarity reflect molecular similarity? Does Spitz naevus progress into spitzoid melanoma? Are ambiguous spitzoid tumours genuine entities? Methods BRAF, NRAS and HRAS mutations were analysed using single-strand conformational polymorphism analysis and sequencing. Results Both Spitz naevi and spitzoid melanoma had a lower combined BRAF and NRAS mutation frequency compared with common acquired naevi (P = 0·0001) and common forms of melanoma (P = 0·0072), respectively. To look for evidence of progression from Spitz naevi to spitzoid melanoma, HRAS was analysed in 21 spitzoid melanomas, with no mutations identified. The binomial probability of this was 0·03 based on an assumption of a 15% mutation frequency in Spitz naevi with unbiased progression. Under these assumptions, HRAS mutations must be rare/absent in spitzoid melanoma. Thus, Spitz naevi seem unlikely to progress into spitzoid melanoma, implying that ambiguous spitzoid tumours cannot be intermediate degrees of progression. In addition, the data suggest that HRAS mutation is a potential marker of benign behaviour, in support of which none of three HRAS mutant spitzoid cases metastasized. Conclusions First, the morphological similarity of spitzoid tumours reflects an underlying molecular similarity, namely a relative lack of dependence on BRAF/NRAS mutations. Second, Spitz naevi do not appear to progress into spitzoid melanoma, and consequently ambiguous spitzoid tumours are likely to be unclassifiable Spitz naevi or spitzoid melanoma rather than genuine entities. Third, HRAS mutation may be a marker of Spitz naevus, raising the possibility that other molecular markers for discriminating Spitz naevi from spitzoid melanoma can be discovered.

P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential “field effect” of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia. [Cancer Res 2008;68(19):7760–8]

Objective To study the ultrastructure of the inner and outer myometrium, in the presence and absence of uterine adenomyosis. Design Case control blinded comparison. Setting University departments. Patient(s) Four premenopausal women with and six without uterine adenomyosis as the sole pathology. Intervention(s) Multiple samples were studied using transmission electron microscopy. Main Outcome Measure(s) Ultrastructure feature of the myometrium. Result(s) In uteri with adenomyosis, the myocytes exhibited cellular hypertrophy. The cytoplasmic myofilaments were less abundant. Abundant intermediate filaments formed cytoplasmic aggregates. The nuclei had a smooth outline with a clear ground substance, prominent nucleoli and peripherally arranged nuclear chromatin. There was occasional infolding of the nuclear envelope with entrapment of cytoplasmic organelles. The sarcolemmal bands were significantly longer and there were fewer caveolae. The perinuclear cell organelles were more distinct. The rough endoplasmic reticulum and Golgi apparatus were more prominent, denoting active protein synthesis, consistent with the observed cellular hypertrophy. All features were more prominent at the junctional zone. Conclusion(s) Smooth muscle cells from uteri with adenomyosis are ultrastructurally different from smooth muscle cells of normal uteri. These distinct features suggest a possible effect on myometrial contractility, together with hypertrophy.

Spitzoid tumours are a morphologically diverse group of lesions that share histological similarity to the Spitz naevus, a benign melanocytic skin tumour. Distinguishing classic Spitz naevi from cutaneous malignant melanoma is usually achievable on standard histology sections, but occasionally equivocal lesions are encountered that show features intermediate between these two entities and consequently generate considerable clinical and histopathological concern. The nomenclature and diagnostic criteria for spitzoid lesions are not standardized and this article begins by considering the adverse effect this has on our understanding of spitzoid tumour biology. Investigations of some of the hallmark features of cancer and neoplasia in spitzoid tumours are described, and the contribution of these studies to our understanding of spitzoid tumour biology is considered, along with their potential diagnostic utility. These studies compare spitzoid tumours with better-characterized melanocytic lesions, and from such comparisons assumptions concerning the biological nature of different spitzoid tumours can be made. In contrast, investigations of the mitogen-activated protein kinase (MAPK) pathway and DNA gains and losses have suggested that Spitz naevi may be genetically distinct from other melanocytic tumours. The studies that led to this conclusion are reviewed, as well as subsequent work examining whether the same applies to all spitzoid tumours. Possible explanations for the considerable inconsistencies within some of these data are explored. Finally, potential pathways of tumour progression within spitzoid lesions are considered, with an emphasis placed upon insights gained from investigations of MAPK genes and DNA gains and losses.

Introduction In the UK BSCP, patients aged 60–75 are screened using biennial faecal occult blood testing (FOBt), FOBt+ patients undergo colonoscopy. At screening colonoscopy, yield for adenomas is 46.5%, adenocarcinomas in 6%. 1 Whilst effective, FOBt lacks high sensitivity, specificity and accuracy – half of screening colonoscopies are normal or reveal haemorrhoids/diverticular disease (DD). 1 One off sigmoidoscopy screening (“bowel scope”) is being introduced for patients aged 55 after a large trial showed 23% reduction in CRC incidence. 2 However, endoscopy based screening is invasive, resource intensive and can cause harm. Uptake of FOBt screening is 1 Early reports suggest this is lower for bowel scope. A blood based biomarker screening test is appealing. Method We investigated microRNAs (miRs) – short non-coding RNA molecules as potential biomarkers. 181 FOBt+ patients undergoing BCSP colonoscopy and 29 patients undergoing planned polypectomy were recruited (blood samples taken pre-procedure). 210 patients – 128 male, 82 female. 117 with adenomas, 12 with CRC, 81 controls (normal or non-adenoma/adenocarcinoma). RNA was extracted from plasma and processed. Pooled groups of patients with adenomas and controls were analysed using array cards. MiRs 19a, 98, 146b, 186, 331–5p, 452 and 625 were identified as candidate biomarkers. Cases were analysed for candidate miRs using quantative polymerase chain reaction. Results Candidate miRs showed significant levels of expression in patients with adenomas compared to controls. MiRs 19a, 331–5p, 452 p = Modelling of miR panels were performed, ROC curve analysis showed: Polyps (excl hyperplastic) in males, panel; miRs 98, 186, 452, sensitivity 0.600/specificity 0.872. Adenomas in males, panel; miRs 98, 186, 452, sens 0.606/spec 0.875 or sens 0.591/spec 0.900. Polyps (excl hyperplastic) in pts with DD/haemorrhoids, panel; miRs 186, 452, 331–5p, sens 0.600/spec 0.889 or sens 0.633/spec 0.867. Adenomas in pts with DD/haemorrhoids, panel; miRs 625, 452, 331–5p, sens 0.714/spec 0.864. Conclusion This study suggest plasma microRNAs are potential screening biomarkers for male patients with colorectal polyps/adenomas and patients with adenomas and background DD/ haemorrhoids. Further study is needed to validate these exciting findings. Disclosure of interest None Declared. References Lee TJW, Rutter MD, Blanks RG, et al . Colonoscopy quality measures: experience from the NHS Bowel Cancer Screening Programme. Gut 2012;61:1050–1057 Atkin WS, Edwards R, Kralk-Hans I, et al . Once-only flexible sigmoidoscopy screening in prevention of colorectal cancer: a multicentre randomised controlled trial. Lancet 2010;375:1624–1633

During gestation, the balance between cell proliferation and death is crucial for successful embryo implantation and maintenance of pregnancy. The uterine endometrium responds to blastocyst implantation with extensive proliferation and differentiation of stromal cells into decidual cells, forming the antimesometrial and mesometrial decidua, which regress by apoptosis. In the latter region it is also observed the growth of metrial gland. To elucidate the events underlying this tissue remodelling we investigated the spatial and temporal pattern of expression of the proliferating cell nuclear antigen (PCNA) and localized the apoptotic cells, by the TUNEL assay and by the expression of active caspase-3. We found that PCNA is expressed at high levels during decidualization until day 12 of gestation declining thereafter abruptly. On the contrary, the appearance of apoptotic cells was detected, by the TUNEL and active caspase-3 expression, in the mesometrial decidua on day 12, increasing from days 14 to 16 in the decidua and metrial gland. In the antimesometrial decidua apoptosis was observed from early to day 12 of pregnancy. However, on day 13 only cell debris and neutrophils were observed, indicating also the presence of necrosis. These results suggest that decidual cells undergo, in distinct regions and at different stages of pregnancy, cell death by apoptosis and secondary necrosis.

Embryonic transcription factors (ETFs) of the Snail, Zeb and Twist families are essential mediators of EMT programs (Epithelial to Mesenchymal Transition) and play central roles in cell determination and cell plasticity. The aberrant expression of ETFs is frequently observed in various cancer types, particularly in carcinomas, and is associated with poor prognosis and high risk of metastasis. We and others have demonstrated that their oncogenic potential relies on their ability to enable escape from oncogene-induced failsafe programs, senescence and apoptosis. Therefore, EMT-resembling processes initiated by ETFs contribute to neoplastic transformation in vitro and accelerate tumor progression in vivo in an epithelial context. We now unveil a completely different scenario in melanoma, a highly metastatic neural crest-derived cancer. Consistent with their role in melanoblasts determination, SNAIL2 and ZEB2 are already expressed in melanocytes and naevi, but their expression is down-regulated in primary melanoma. In contrast, TWIST1 and ZEB1 are aberrantly reactivated in primary and metastatic melanoma. Interestingly, this switch in ETFs expression represents a novel independent factor of poor prognosis in patients with malignant melanoma. We further demonstrate that BRAF activation, the most frequent driving event in melanoma, induces a drastic upregulation of TWIST1 and ZEB1 at the expense of SNAIL2 and ZEB2 in murine and human melanocytes. This reversible switch, orchestrated by the FRA1 transcription factor, is determinant for BRAF-driven melanocyte transformation in vitro and in a xenograft model. As a whole, while TWIST1 and ZEB1 cooperate with oncogenic BRAF to induce neoplastic transformation, SNAIL2 and ZEB2 rather behave as oncosuppressive proteins in melanoma. Gene expression profiles analyses further show that this oncogenic reprogramming of ETFs regulates the expression of the MITF master regulator, with an inhibition of MITF-regulated differentiation program and an activation of TGFβ- and invasion-associated gene expression signatures. Collectively, these data highlight yet unexpected antagonistic properties of ETFs in tumor progression, and place the ETF network as a master regulator of MITF-dependent phenotype switching during melanomagenesis. Citation Format: Julie Caramel, Eftychios Papadogeorgakis, Louise Hill, Gareth J. Browne, Geoffrey Richard, Anne Wierinckx, Gerald Saldanha, Peter Hutchinson, James H. Pringle, Alain Puisieux, Eugene Tulchinsky, Stephane Ansieau. Antagonistic functions of EMT-inducing transcription factors during melanoma development. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 285. doi:10.1158/1538-7445.AM2013-285

We describe a case report of a 21-year-old male with fatal Epstein-Barr virus-associated haemophagocytic syndrome. Virus is detected in multiple organs by polymerase chain reaction and in the tissue-specific cells of those organs by in situ hybridisation. It is suggested that organ failure may be a direct response to infection.

A well-recognized complication of Sjogren's syndrome (SS) is the development of malignant lymphoma, with a risk 44 times that of the general population. Although a few clinical signs may indicate the onset of lymphoma, there are few reliable laboratory markers which predict the development of neoplasia. A non-isotopic in situ hybridization technique has been applied to routinely processed labial salivary gland (LSG) biopsies of patients under investigation for SS. Serial section of 70 LSGs were examined for a kappa and gamma immunoglobulin light chain mRNA using digoxigenin-labelled oligonucleotide probes. As controls, 39 biopsies from non-SS-associated sialadenitis were also examined. Sections were analysed using computer-assisted quantification to determine the percentage of kappa-expressing cells in each case. The range of kappa expression in the SS group was 24.1-93.4 percent and in the non-SS group 48.3-75.4 per cent. Light chain restriction was found in 13/70 (18.6 percent) cases from the SS group but in no cases of the control group. Of the SS cases showing restriction, 4/13 (30.7 percent) have subsequently developed extrasalivary gland lymphoma. Two patients not showing light chain restriction in LSG have subsequently developed lymphoma. The positive predictive value of this test to identify patients at risk of lymphoma was 30.7 percent with a detection rate (sensitivity) of 66.7 percent and a false-positive rate of 14.1 per cent(specificity 85.9 percent). This study has identified a high prevalence of light chain restriction in labial gland biopsies of patients with SS and provides objective quantitative criteria to identify those patients at greater risk of lymphoma development.

To distinguish reactive versus neoplastic monocytoid B-cell (MBC) proliferations, the clonality of MBC was examined in paraffin-embedded tissues by in situ hybridization (ISH) of immunoglobulin (Ig) light chain messenger RNA (mRNA) with sensitive oligonucleotide probes in 26 cases. They included 13 cases of lymphadenitis with MBC reaction and 13 cases of nodal (n = 8) and extranodal (n = 5) monocytoid B-cell lymphoma (MBCL). Two cases represented a composite lymphoma showing a centroblastic-centrocytic and MBCL component. The clonality of MBC infiltrates could be demonstrated in 16 of 26 (61.5%) cases by immunostaining for Ig light chains and in all (100%) cases by ISH. Neoplastic MBC usually expressed a faint-to-moderate light chain restriction of mRNA, whereas some MBC (10% to 30% of total MBCL population) showed a strong positivity irrespective of plasmacytoid differentiation as indicated by Ig immunostaining (present in 9 of 13 cases). Reactive MBC expressed a faint kappa and lambda light-chain mRNA positivity. Five percent to 20% of total reactive MBC showed also a strong positivity for both Ig light chain mRNA, although only a minor part of these cells (7 of 13 cases) expressed polyclonal Ig by immunohistochemistry. These results indicate that (1) both reactive and neoplastic MBC can differentiate into plasma cells; and (2) a relatively high percentage of reactive and neoplastic MBC show a detectable mRNA transcription, but not a corresponding Ig synthesis. Either the Ig detection is not sensitive enough or these cells might be in an early differentiation phase, where the Ig production has not yet started.

A 47-year-old man with persistent severe oropharyngeal ulceration developed a high-grade T-cell lymphoma soon after commencing treatment with cyclosporin A. Using Southern blotting to identify T-cell beta-chain gene rearrangements, evidence of clonal restriction was found both in blood and lymph node DNA samples. Two BamH1 rearranged bands were demonstrated in both samples. In the blood a 16 Kb band predominated, with a weaker 28 kb band. In the lymph node sample this pattern was reversed. The findings suggest that a bi-clonal population of T-lymphocytes or clonal evolution of an existing T-cell monoclone had developed, and that cyclosporin contributed to the emergence of a high-grade T-cell lymphoma.

Computer-assisted learning combined with interactive video provides an excellent teaching method for a visual subject such as pathology. A system, based on the BBC microcomputer, is described. A videodisc provides illustrative material and the output from both the computer and the videodisc player are mixed to a single screen. Using the mouse as a pointing device, the students can interact directly with the video picture and so improve their understanding of what they see. A program listing for this type of interaction is given. Interactive methods are based on the pre-identification of features on the video frame by the creation of “maps” in text coordinates with storage to a data file. The use of “maps” and overlay diagrams to provide feedback is discussed and the other features of a tutorial are described. The outline of a program to design maps and diagrams is also included. These methods should prove useful in teaching any subject with an important visual content.

In this study, the phenotype of psoriatic keratinocytes and fibroblasts in reconstructed skin models was compared to those constructed from normal cells. Characterization of this model by immunohistochemistry showed that classical markers of keratinocyte differentiation exhibited similar patterns of distribution in the psoriatic models to those derived from normal cells and generally reflected in vivo observations. Some crucial differences, however, were observed between normal and psoriatic models when pro-inflammatory gene expression and keratinocyte proliferation were investigated. Notably, the chemokine receptor CXCR2 was overexpressed in the psoriatic models, and, moreover, was localized to the granular layer of keratinocytes as seen in psoriasis in vivo. Pro-inflammatory genes (tumor necrosis factor alpha [TNF-alpha], interferon gamma [IFN-gamma], and interleukin 8 [IL-8]) were expressed at high levels in the psoriatic models, but were only minimally expressed in the normal models. Models derived from uninvolved psoriatic skin showed the same gene expression profile as those derived from involved skin along with an increased proliferation rate when compared to normal models. These results suggest that psoriatic individuals possess an inherent predisposition to develop the disease phenotype even in the absence of T cells. This study represents a comprehensive characterization of psoriatic human skin reconstructed in vitro, and demonstrates the potential of this model as a valuable tool in drug discovery.