A retrospective comparative exploratory study on two methylentetrahydrofolate reductase (MTHFR) polymorphisms in esophagogastric cancer: the A1298C MTHFR polymorphism is an independent prognostic factor only in neoadjuvantly treated gastric cancer patients.

BACKGROUND Methylentetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism and consequently could be an important factor for the efficacy of a treatment with 5-fluorouracil. Our aim was to evaluate the prognostic and predictive value of two well characterized constitutional MTHFR gene polymorphisms for primarily resected and neoadjuvantly treated esophagogastric adenocarcinomas. METHODS 569 patients from two centers were analyzed (gastric cancer: 218, carcinoma of the esophagogastric junction (AEG II, III): 208 and esophagus (AEG I): 143). 369 patients received neoadjuvant chemotherapy followed by surgery, 200 patients were resected without preoperative treatment. The MTHFR C677T and A1298C polymorphisms were determined in DNA from peripheral blood lymphozytes. Associations with prognosis, response and clinicopathological factors were analyzed retrospectively within a prospective database (chi-square, log-rank, cox regression). RESULTS Only the MTHFR A1298C polymorphisms had prognostic relevance in neoadjuvantly treated patients but it was not a predictor for response to neoadjuvant chemotherapy. The AC genotype of the MTHFR A1298C polymorphisms was significantly associated with worse outcome (p = 0.02, HR 1.47 (1.06-2.04). If neoadjuvantly treated patients were analyzed based on their tumor localization, the AC genotype of the MTHFR A1298C polymorphisms was a significant negative prognostic factor in patients with gastric cancer according to UICC 6th edition (gastric cancer including AEG type II, III: HR 2.0, 95% CI 1.3-2.0, p = 0.001) and 7th edition (gastric cancer without AEG II, III: HR 2.8, 95% CI 1.5-5.7, p = 0.003), not for AEG I. For both definitions of gastric cancer the AC genotype was confirmed as an independent negative prognostic factor in cox regression analysis. In primarily resected patients neither the MTHFR A1298C nor the MTHFR C677T polymorphisms had prognostic impact. CONCLUSIONS The MTHFR A1298C polymorphisms was an independent prognostic factor in patients with neoadjuvantly treated gastric adenocarcinomas (according to both UICC 6th or 7th definitions for gastric cancer) but not in AEG I nor in primarily resected patients, which confirms the impact of this enzyme on chemotherapy associated outcome.

Is preoperative chemotherapy followed by surgery the appropriate treatment for signet ring cell containing adenocarcinomas of the esophagogastric junction and stomach?

BACKGROUND Recent data suggest primary resection as the preferable approach in patients with signet ring cell gastric cancer (SRC). The aim of our retrospective exploratory study was to evaluate the influence of SRC on prognosis and response in esophagogastric adenocarcinoma treated with neoadjuvant chemotherapy. METHODS A total of 723 locally advanced esophagogastric adenocarcinomas (cT3/4 N any) documented in a prospective database from two academic centers were classified according to the WHO definition for SRC (more than 50 % SRC) and analyzed for their association with response and prognosis after neoadjuvant treatment. RESULTS A total of 235 tumors (32.5 %) contained SRC. Median survival of SRC was 26.3 compared with 46.6 months (p < 0.001) for non-SRC. SRC were significantly associated with female gender, gastric localization, advanced ypT and R1/2 categories, and lower risk of surgical complications and anastomotic leakage (each p < 0.001). Clinical (21.1 vs. 33.7 %, p = 0.001) and histopathological response (less than 10 % residual tumor: 16.3 vs. 28.9 %, p < 0.001) were significantly less frequent in SRC. Clinical response (p = 0.003) and complete histopathological response (pCR) (3.4 %) (p = 0.003) were associated with improved prognosis in SRC. Clinical response, surgical complications, ypTN categories, but not SRC were independent prognostic factors in forward Cox regression analysis in R0 resected patients. Risk of peritoneal carcinomatosis was increased (p < 0.001), while local (p = 0.015) and distant metastases (p = 0.02) were less frequent than in non-SRC. CONCLUSIONS Prognosis of SRC is unfavorable. Although response to neoadjuvant chemotherapy is rare in SRC, it is associated with improved outcome. Thus, chemotherapy might not generally be abandoned in SRC. A stratification based on SRC should be included in clinical trials.

Comprehensive validation of published immunohistochemical prognostic biomarkers of prostate cancer-what has gone wrong? A blueprint for the way forward in biomarker studies

BACKGROUND Treatment planning of localised prostate cancer remains challenging. Besides conventional parameters, a wealth of prognostic biomarkers has been proposed so far. None of which, however, have successfully been implemented in a routine setting so far. The aim of our study was to systematically verify a set of published prognostic markers for prostate cancer. METHODS Following an in-depth PubMed search, 28 markers were selected that have been proposed as multivariate prognostic markers for primary prostate cancer. Their prognostic validity was examined in a radical prostatectomy cohort of 238 patients with a median follow-up of 60 months and biochemical progression as endpoint of the analysis. Immunohistochemical evaluation was performed using previously published cut-off values, but allowing for optimisation if necessary. Univariate and multivariate Cox regression were used to determine the prognostic value of biomarkers included in this study. RESULTS Despite the application of various cut-offs in the analysis, only four (14%) markers were verified as independently prognostic (AKT1, stromal AR, EZH2, and PSMA) for PSA relapse following radical prostatectomy. CONCLUSIONS Apparently, many immunohistochemistry-based studies on prognostic markers seem to be over-optimistic. Codes of best practice, such as the REMARK guidelines, may facilitate the performance of conclusive and transparent future studies.

Discovery of a highly potent anti-inflammatory epoxyisoprostane-derived lactone.

Epoxyisoprostanes EI (1) and EC (2) are effective inhibitors of the secretion of proinflammatory cytokines IL-6 and IL-12. In detailed studies toward the investigation of the molecular mode of action of these structures, a highly potent lactone (3) derived from 1 was identified. The known isoprostanoids 1 and 2 are most likely precursors of 3, the product of facile intramolecular reaction between the epoxide with the carboxylic acid in 2.

Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia.

BACKGROUND It is unknown why patients with extensive ulcerative colitis (UC) have a higher risk of colorectal cancer compared with patients with left-sided UC. This study characterizes the inflammatory processes in left-sided UC, pancolitis, and UC-associated dysplasia at the transcriptional level to identify potential biomarkers and transcripts of importance for the carcinogenic behavior of chronic inflammation. METHODS The Affymetrix GeneChip Human Genome U133 Plus 2.0 was applied on colonic biopsies from UC patients with left-sided UC, pancolitis, dysplasia, and controls. Reverse transcription polymerase chain reaction and immunohistochemistry were performed for validating selected transcripts in the initial cohort and in 2 independent cohorts of patients with UC. Microarray data were analyzed by principal component analysis, and reverse transcription polymerase chain reaction and immunohistochemistry data by the Wilcoxon's rank-sum test. RESULTS The principal component analysis results revealed separate clusters for left-sided UC, pancolitis, dysplasia, and controls. Close clustering of dysplastic and pancolitic samples indicated similarities in gene expression. Indeed, 101 and 656 parallel upregulated and downregulated transcripts, respectively, were identified in specimens from dysplasia and pancolitis. Validation of selected transcripts hereof identified insulin receptor alpha (INSRA) and MAP kinase interacting serine/threonine kinase 2 (MKNK2) with an enhanced expression in dysplasia compared with left-sided UC and controls, whereas laminin γ2 (LAMC2) was found with a lower expression in dysplasia compared with the remaining 3 groups. CONCLUSIONS This study demonstrates pancolitis and left-sided UC as distinct inflammatory processes at the transcriptional level, and identifies INSRA, MKNK2, and LAMC2 as potential critical transcripts in the inflammation-driven preneoplastic process of UC.

Coping with esophageal cancer approaches worldwide.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on approaches to the epidemiology, diagnosis, and treatment of esophageal cancer in Europe, South Africa, Kenya, Australia, and China; the molecular classification of esophageal cancers (including cancers at the gastroesophageal junction); the Japanese classification; the scope of the Human Variome Project; and the topographic-anatomic subclassification of adenocarcinomas of the gastroesophageal junction.

The search for the primary tumor in metastasized gastroenteropancreatic neuroendocrine neoplasm.

Gastroenteropancreatic neuroendocrine tumors (NETs) often present as liver metastasis from a carcinoma of unknown primary. We recently showed that primary NETs from the pancreas, small intestine and stomach as well as their respective liver metastases differ from each other by the expression profile of the three genes CD302, PPWD1 and ABHB14B. The gene and protein expression of CD302, PPWD1, and ABHB14B was studied in abdominal NET metastases to identify the site of the respective primary tumors. Cryopreserved tissue from NET metastases collected in different institutions (group A: 29, group B: 50, group C: 132 specimens) were examined by comparative genomic hybridization (Agilent 105 K), gene expression analysis (Agilent 44 K) (groups A and B) and immunohistochemistry (group C). The data were blindly evaluated, i.e. without knowing the site of the primary. Gene expression analysis correctly revealed the primary in the ileum in 94 % of the cases of group A and in 58 % of group B. A pancreatic primary was predicted in 83 % (group A) and 20 % (group B), respectively. The combined sensitivity of group A and B was 75 % for ileal NETs and 38 % for pancreatic NETs. Immunohistochemical analysis of group C revealed an overall sensitivity of 80 %. Gene and protein expression analysis of CD302 and PPWD1 in NET metastases correctly identifies the primary in the pancreas or the ileum in 80 % of the cases, provided that the tissue is well preserved. Immunohistochemical profiling revealed CD302 as the best marker for ileal and PPWD1 for pancreatic detection.

Multi-colour FISH in oesophageal adenocarcinoma-predictors of prognosis independent of stage and grade.

BACKGROUND Oesophageal adenocarcinoma or Barrett's adenocarcinoma (EAC) is increasing in incidence and stratification of prognosis might improve disease management. Multi-colour fluorescence in situ hybridisation (FISH) investigating ERBB2, MYC, CDKN2A and ZNF217 has recently shown promising results for the diagnosis of dysplasia and cancer using cytological samples. METHODS To identify markers of prognosis we targeted four selected gene loci using multi-colour FISH applied to a tissue microarray containing 130 EAC samples. Prognostic predictors (P1, P2, P3) based on genomic copy numbers of the four loci were statistically assessed to stratify patients according to overall survival in combination with clinical data. RESULTS The best stratification into favourable and unfavourable prognoses was shown by P1, percentage of cells with less than two ZNF217 signals; P2, percentage of cells with fewer ERBB2- than ZNF217 signals; and P3, overall ratio of ERBB2-/ZNF217 signals. Median survival times for P1 were 32 vs 73 months, 28 vs 73 months for P2; and 27 vs 65 months for P3. Regarding each tumour grade P2 subdivided patients into distinct prognostic groups independently within each grade, with different median survival times of at least 35 months. CONCLUSIONS Cell signal number of the ERBB2 and ZNF217 loci showed independence from tumour stage and differentiation grade. The prognostic value of multi-colour FISH-assays is applicable to EAC and is superior to single markers.

Prognostic value of histopathological regression in 850 neoadjuvantly treated oesophagogastric adenocarcinomas.

BACKGROUND Recently, histopathological tumour regression, prevalence of signet ring cells, and localisation were reported as prognostic factors in neoadjuvantly treated oesophagogastric (junctional and gastric) cancer. This exploratory retrospective study analyses independent prognostic factors within a large patient cohort after preoperative chemotherapy including clinical and histopathological factors. METHODS In all, 850 patients presenting with oesophagogastric cancer staged cT3/4 Nany cM0/x were treated with neoadjuvant chemotherapy followed by resection in two academic centres. Patient data were documented in a prospective database and retrospectively analysed. RESULTS Of all factors prognostic on univariate analysis, only clinical response, complications, ypTNM stage, and R category were independently prognostic (P<0.01) on multivariate analysis. Tumour localisation and signet ring cells were independently prognostic only when investigator-dependent clinical response evaluation was excluded from the multivariate model. Histopathological tumour regression correlates with tumour grading, Laurén classification, clinical response, ypT, ypN, and R categories but was not identified as an independent prognostic factor. Within R0-resected patients only surgical complications and ypTNM stage were independent prognostic factors. CONCLUSIONS Only established prognostic factors like ypTNM stage, R category, and complications were identified as independent prognostic factors in resected patients after neoadjuvant chemotherapy. In contrast, histopathological tumour regression was not found as an independent prognostic marker.

Supraorbital cutaneous fetal rhabdomyoma of intermediate type: a case report.

A 7-year-old boy was presented with a long-standing slowly growing mass of the left supraorbital area. A biopsy specimen revealed a bland spindle cell proliferation with scattered polygonal cells with acidophilic cytoplasm and cross-striations. Our differential diagnosis included rhabdomyoma of fetal type, leiomyoma with trapping of regenerating skeletal muscle elements, and rhabdomyomatous mesenchymal hamartoma of the skin. Immunohistochemistry demonstrated strong positivity of myoglobin and desmin as well as negativity of caldesmon, suggesting skeletal muscle lineage. The excisional specimen confirmed our diagnosis of cutaneous fetal rhabdomyoma of intermediate type. Additional immunostaining performed on the excisional specimen showed strong Wilms Tumor 1 but only a very faint and focal p63 expression.

Memory regulatory T cells reside in human skin.

Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.

Angiomatous kaposi sarcoma: a variant that mimics hemangiomas.

We describe 14 cases of angiomatous Kaposi sarcoma (KS), a distinct histological variant of KS first mentioned by Gottlieb and Ackerman in 1988 that can easily be mistaken for a hemangioma. Intriguingly, this variant of KS has not attracted much attention and has not been studied in detail. Immunohistochemistry showed prominent staining of podoplanin (D2-40) of the neoplastic vasculature but not the preexisting vessels, suggesting lymphatic differentiation, despite the erythrocyte-filled round lumens. To test whether D2-40 staining of round vessels with erythrocytes was distinctive, we stained sinusoidal hemangiomas and cellular angiolipomas, both of which have these structures. In contrast to angiomatous KS, the vessels in both entities were podoplanin (D2-40) negative. The finding of round erythrocyte-filled vessels with podoplanin (D2-40) positivity may be distinctive for this form of KS.

miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas.

Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O(6)-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor κB (NF-κB)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-κB is in part owing to downregulation of negative regulators of NF-κB activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-κB inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-κB activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNFα- and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-κB confirming that miR-125b is implicated in NF-κB signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy.

Relative positioning of classical benzodiazepines to the γ2-subunit of GABAA receptors.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABAA receptor isoforms might help to separate these divergent effects. As a first step, we characterized the interaction between diazepam and the major GABAA receptor isoform α1β2γ2. We mutated several amino acid residues on the γ2-subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ2Tyr58, γ2Asn60, and γ2Val190 were located relative to classical benzodiazepines in their binding pocket on GABAA receptors.