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The humoral branch of the immune response has an important role in acute and chronic allograft dysfunction. The CD40/CD40L costimulatory pathway is crucial in B- and T- alloresponse. Our group has developed a new small interfering RNA (siRNA) molecule against CD40 that effectively inhibits its expression. The aim of the present study was to prevent rejection in an acute vascular rejection model of kidney transplant by intra-graft gene silencing with anti-CD40 siRNA (siCD40), associated or not with sub-therapeutic rapamycin. Four groups were designed: unspecific siRNA as control; sub-therapeutic rapamycin; siCD40; and combination therapy. Long-surviving rats were found only in both siCD40-treated groups. The CD40 mRNA was overexpressed in control grafts but treatment with siCD40 decreased its expression. Recipient spleen CD40+ B-lymphocytes were reduced in both siCD40-treated groups. Moreover, CD40 silencing reduced donor-specific antibodies, graft complement deposition and immune-inflammatory mediators. The characteristic histological features of humoral rejection were not found in siCD40-treated grafts, which showed a more cellular histological pattern. Therefore, the intra-renal effective blockade of the CD40/CD40L signal reduces the graft inflammation as well as the incidence of humoral vascular acute rejection, finally changing the type of rejection from humoral to cellular.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at the posttranscriptional level and are critical for many cellular pathways. The disruption of miRNAs and their processing machineries also contributes to the development of human tumors. A common scenario for miRNA expression in carcinogenesis is emerging that shows that impaired miRNA production and/or down-regulation of these transcripts occurs in many neoplasms. Several of these lost miRNAs have tumor-suppressor features, so strategies to restore their expression globally in malignancies would be a welcome addition to the current therapeutic arsenal against cancer. Herein, we show that the small molecule enoxacin, a fluoroquinolone used as an antibacterial compound, enhances the production of miRNAs with tumor suppressor functions by binding to the miRNA biosynthesis protein TAR RNA-binding protein 2 (TRBP). The use of enoxacin in human cell cultures and xenografted, orthotopic, and metastatic mouse models reveals a TRBP-dependent and cancer-specific growth-inhibitory effect of the drug. These results highlight the key role of disrupted miRNA expression patterns in tumorigenesis, and suggest a unique strategy for restoring the distorted microRNAome of cancer cells to a more physiological setting.

Degeneration of the central white matter is described in old dogs. The presence of ubiquitin-immunoreactive free granules and intracytoplasmic globules in glial cells and macrophages, together with galactocerebroside-immunoreactive precipitates and lipofuscin storage, point to the likelihood of a primary myelin degeneration with deposits of non-degraded ubiquitin-protein conjugates and complex galactolipids.

High microsatellite instability (MSI-H) allows the identification of a subset of colorectal carcinomas associated with good prognosis and a higher incidence of Lynch syndrome. The aim of this work was to assess the interobserver variability and optimize our MSI-H prediction model previously published based on phenotypic features. The validation series collected from five different hospitals included 265 primary colorectal carcinomas from the same number of patients. The eight clinicopathological parameters that integrate our original model were evaluated in the corresponding centers. Homogeneity assessment revealed significant differences between hospitals in the estimation of the growth pattern, presence of Crohn-like reaction, percentage of cribriform structures, and Ki-67 positivity. Despite this observation, our model was globally able to predict MSI-H with a negative predictive value of 97.0%. The optimization studies were carried out with 615 cases and resulted in a new prediction model RERtest8, which includes the presence of tumor infiltrating lymphocytes at the expense of the percentage of cribriform structures. This refined model achieves a negative predictive value of 97.9% that is maintained even when the immunohistochemical parameters are left out, RERtest6. The high negative predictive value achieved by our models allows the reduction of the cases to be tested for MSI to less than 10%. Furthermore, the easy evaluation of the parameters included in the model renders it a useful tool for the routine practice and can reinforce other published models and the current clinical protocols to detect the subset of colorectal cancer patients bearing hereditary nonpolyposis colorectal cancers risk and/or MSI-H phenotype.

Recent data suggest that the phosphatidylinositol 3-kinase (PI3-K)/Akt/mammalian target of rapamycin (mTOR) pathway is important in diabetic nephropathy. The effect of mTOR blockade by sirolimus (SRL) in diabetic kidney disease in rats was investigated. Diabetes was induced by streptozotocin in male Sprague-Dawley rats. Sixteen weeks later, diabetic animals were divided into the following groups: diabetes (D; n = 8), diabetes + SRL at 1 mg/kg per d, SRL trough level 2.3 +/- 0.25 ng/ml (D+SRL; n = 7); and diabetes + normoglycemia maintained by insulin implants (D+NG; n = 5). There was an age-matched nondiabetic group (ND; n = 6). All animals were followed for 4 wk. The D group showed glomerular hypertrophy (mean glomerular volume 5.0 +/- 0.4 in D versus 3.3 +/- 0.2 10(6) mu(3) in ND; P0.05) without renal hyperplasia (calculated by reverse transcription-PCR of proliferative cell nuclear antigen) and albuminuria (29 +/- 4 in D versus 1.4 +/- 1.5 mg/24 h in ND; P0.05). Both D+NG and D+SRL groups had a significant reduction of albuminuria, although glomerular hypertrophy was still present. SRL treatment did not modify the number of infiltrating renal ED1(+) cells. Diabetic animals had greater expression of p-Akt and mTOR, unlike ND rats. NG and SRL treatment reduced p-Akt and normalized mTOR. It is interesting that D+SRL was associated with a significant reduction of renal TGF-beta1 and glomerular connective tissue growth factor. SRL treatment reduced glomerular alpha-smooth muscle actin overexpression and reduced significantly the mesangial matrix accumulation that is characteristic of diabetic nephropathy. In conclusion, mTOR blockade by low-dose SRL has a beneficial effect in diabetic kidney disease, suggesting that the mTOR pathway has an important pathogenic role in diabetic nephropathy.

The aim of the study was to characterize the role of cold ischemia in the process of acute rejection using an experimental renal transplant model. Syngeneic renal transplants were performed between Wistar Agouti rats and allogeneic grafts using Wistar-Agouti rats as recipients of Brown-Norway kidneys. For cold ischemia (CI), kidneys were preserved in Euro-Collins (4 degrees C/ 2.5 hours). Rats were bilaterally nephrectomized at the moment of renal transplant and did not receive any immunosuppressant. The groups were NoAR (n = 6): immediate syngeneic transplant; CI-NoAR (n = 6): syngeneic transplant with CI; AR (n = 13): immediate allogeneic graft; CI-AR (n = 6): allogeneic graft with CI. Allogeneic rats were followed for the survival study. Syngeneic rats, with mean survival time beyond 6 months, were sacrificed on the day 7 to compare grafts with those in the allogeneic groups. HE- and PAS-stained grafts were evaluated using the Banff criteria. Tissue INF-gamma and TNF-alpha were quantified by RT-real time-PCR on the kidney grafts. Renal insufficiency did not appear in the NoAR group, but it did from the posttransplant day 5 in both acute rejection groups. While NoAR kidneys showed well-conserved renal architecture, then AR group displayed variable degrees of tubular necrosis with scarce cellular infiltration, interstitial hemorrhage, vascular damage with fibrinoid necrosis, perivascular edema, and nuclear disruption. Cold ischemia in rejecting animals increased the mortality rate due to renal insufficiency and accelerated acute rejection. Independently of CI, the proinflammatory cytokines TNF-alpha and INF-gamma were increased in both rejection groups. In conclusion, addition of CI overactivates the acute rejection process via a humoral component.

High-frequency microsatellite instability has been reported to be associated with good prognosis in colorectal adenocarcinoma. However, methods to assess microsatellite instability (MIN) are based on genetic assays and are not ideally suited to most histopathology laboratories. The aim of the present study was to develop a model for prediction of MIN status in colorectal cancer based on phenotypic characteristics. Clinicopathological features of a cohort of 204 patients with primary colon cancer were retrospectively reviewed following predetermined criteria. Genetic assessment of MIN status was performed on DNA extracted from sections of formalin-fixed, paraffin-embedded specimens by testing a panel of 11 microsatellite markers. Logistic regression analysis generated a mathematical tool capable of identifying colorectal tumors displaying MIN status with a sensitivity of 77.8% and a specificity of 96.8%. Features associated with instability included the proximal location of the lesions, occurrence of solid and/or mucinous differentiation, absence of cribriform structures, presence of peritumoral Crohn-like reaction, expansive growth pattern, high Ki67 proliferative index, and p53-negative phenotype. This approach predicts microsatellite instability in colorectal carcinoma with an overall assigned accuracy of 95.1% and a negative predictive value of 97.8%. Implementation of this tool to routine histopathological studies could improve the management of patients with colorectal cancer, especially those presenting with stage II and III of the disease. It will also assist in identifying a subset of patients likely to benefit from adjuvant chemotherapy.

Background. Ischemia added to the allogeneic background accelerates the cellular mechanisms involved in alloresponsiveness, supporting the influence of early nonspecific inflammatory injury on chronic allograft nephropathy (CAN). The authors hypothesize that reinforcing initial immunosuppressive regimens may prevent immunogenicity derived from postischemic inflammatory responses, attenuating CAN. Methods. Lewis rats engrafted with Fischer kidneys received for 15 days overimmunosuppressive doses of rapamycin, a standard cyclosporine regimen, or both, and were followed functionally for 24 weeks. Animals were grouped according to the initial immunosuppressant or cold-ischemia period. Grafts were evaluated for acute inflammatory response at 1 week and for chronic histologic damage at 24 weeks. Results. Rats under cyclosporine alone displayed the highest mortality, which was decreased in the long term by reducing cold ischemia or by strengthening immunosuppression. At 24 weeks, all rapamycin-treated groups displayed much less severe tubulointerstitial and vascular damage. The combination of both immunosuppressants offered better functional outcome and a global reduction in chronic histologic damage. After 1 week, ATN and profibrotic features appeared in all 5-hr ischemic animals, indicating that cyclosporine and rapamycin co-treatment did not induce further nephrotoxicity. Treatment with rapamycin, alone or combined with cyclosporine, greatly reduced the severe immune-inflammatory damage, including vessels, shown in cyclosporine-treated ischemic grafts. Conclusions. Strengthening initial immunosuppression attenuates the intensity and extent of the early postischemic immune-inflammatory response as well as later function and structure of renal allografts. Severe CAN may be prevented by reducing cold ischemia or strengthening immunosuppression. Because the former approach is not always possible, reinforcement of early immunosuppression constitutes an excellent alternative.

Diabetic nephropathy is the main cause of end-stage renal disease requiring dialysis in developed countries. In this study, we demonstrated the therapeutic effect of hepatocyte growth factor (HGF) on advanced rather than early diabetic nephropathy using a rat model of streptozotocin-induced diabetes. Early diabetic nephropathy (16 weeks after induction of diabetes) was characterized by albuminuria, hyperfiltration, and glomerular hypertrophy, whereas advanced diabetic nephropathy showed prominent transforming growth factor (TGF)-β1 upregulation, mesangial expansion, and glomerulosclerosis. An SP1017-formulated human HGF (hHGF) plasmid was administered by intramuscular injection combined with electroporation over a 30-day follow-up in rats with early and advanced diabetic nephropathy. hHGF gene therapy upregulated endogenous rat HGF in the diabetic kidney (rat HGF by RT-PCR was threefold higher than in diabetic rats without therapy). hHGF gene therapy did not improve functional or morphologic abnormalities in early diabetic nephropathy. hHGF gene therapy reduced albuminuria and induced strong regression of mesangial expansion and glomerulosclerosis in advanced diabetic nephropathy. These findings were associated with suppression of renal TGF-β1 and mesangial connective tissue growth factor (CTGF) upregulation, inhibition of renal tissue inhibitor of metalloproteinase (TIMP)-1 expression, and reduction of renal interstitial myofibroblasts. In conclusion, our results suggest that hHGF gene therapy may be considered as an innovative therapeutic strategy to treat advanced diabetic nephropathy.

Gastrointestinal stromal tumor (GIST) is the designation for a major subset of gastrointestinal mesenchymal tumors that histologically, immunocytochemically, and genetically differ from leiomyomas, leiomyosarcomas, and schwannomas. GISTs derive from the interstitial cells of Cajal and, in addition to variable expression of smooth muscle and neural markers, they characteristically express CD34 and CD117. The cytological appearance, including immunocytochemical and mutational analysis of c-kit gene in primary GIST has been well described. To our knowledge, only two cases of metastatic GIST diagnosed by fine-needle aspiration (FNA) have been reported. We illustrate three cases of metastatic GIST in the liver. Two cases had no prior history of gastrointestinal tumor and the third case had a 4-yr previous history of duodenal tumor. Consistent immunocytochemistry and ultrastructual studies supported the diagnosis of GIST. We emphasize that in the appropriate clinical and radiological setting, a confident diagnosis of GIST can be established by FNA of metastatic lesions.

To study angiogenesis in early steps of melanoma progression, 113 cutaneous melanomas 1 mm or less in thickness were stained with Ulex europaeus lectin. Vascular density was determined in the areas of greatest vascularization. To avoid the effect of anatomic location, the quotient between vascular density at the tumor base and in normal skin (vascular ratio) was obtained in each case. Of these melanomas, 46 were immunohistochemically stained for the presence of vascular endothelial growth factor (VEGF). Positivity was scored from 1-4 by comparing staining of melanoma cells with keratinocytes. Vascular ratio values in vertical growth phase melanomas were higher than those in radial growth phase when counting per 200 or 400 magnification (2.29 +/- 1.3 and 2.48 +/- 1.5 for vertical growth phase and 1.34 +/- 0.62 and 1.41 +/- 0.83 for radial growth phase melanomas, respectively). This difference was statistically significant (p < 0.0001 and p < 0.001 at x200 and x400, respectively). Also, VEGF staining was stronger in vertical growth phase melanomas when compared with radial growth phase melanomas (Chi square, p < 0.025). In conclusion, our findings suggest that angiogenesis and VEGF expression are associated with the development of vertical growth phase.

HSP-70 was induced in the gerbil following 20 min of forebrain ischemia. The induction, as revealed with immunohistochemistry, is stronger and longer-lasting in CA3 and dentate gyrus than in CA1. Most neurons in this region, except GABAergic interneurons containing the calcium-binding protein parvalbumin, eventually cease to live as a result of delayed cell death. Double-labeling of inducible HSP-70 and parvalbumin has shown that no co-localization occurs in the hippocampus and neocortex of the gerbil in this model of transient forebrain ischemia. These results show that different thresholds of sensitivity and vulnerability exist for different subpopulations of neurons in the ischemic hippocampus, and suggest that HSP-70 protein induction is probably not essential for the survival of particular neuronal subpopulations subjected to transient ischemia.

427 The Major Vault Protein (MVP) Mediates Starvation Resistance of Human Glioblastoma Cells Via Deregulation of the PI3kinase Pathway D. Lotsch, S. Spiegl-Kreinecker, C. Pirker, J. Hlavaty, H. Petznek, M. Grusch, W. Berger. Medical University of Vienna, Internal Medicine I / Institute of Cancer Research, Vienna, Austria, Wagner-Jauregg Hospital, Department of Neurosurgery, Linz, Austria, University of Veterinary Medicine, Institute of Virology / Department of Pathobiology, Vienna, Austria

Background: Epithelial ovarian cancer (EOC) is the leading cause of deaths in women diagnosed with gynecologic malignancies. The low survival rate is due to its advanced-stage diagnosis and either intrinsic or acquired resistance to standard platinum-based chemotherapy. As such, the development of effective innovative therapeutic strategies to overcoming cisplatin resistance remains a high priority. PM01183 is a new alkaloid that binds to the minor groove of DNA and induces delayed progression of S phase and cycle arrest in G2/M. Currently, PM01183 clinical development includes Phase II single agent trials (pancreas, breast and platinum-resistant/refractory ovarian) as well as Phase I trials in combination (with doxorubicin and gemcitabine) and in advanced acute leukemia. Here, we report that PM01183 is effective in the treatment of new developed orthotopic subject-derived epithelial serous ovarian tumor grafts Cisplatin-sensitive and-resitan models. Material and Methods: Athymic nude female mice were orthotopically engrafted with derived-fragments of OVAX1 (Cisplatin-Sensitive) or OVAX1R (Cisplatin-Resistant) tumors, and when tumors reached homogeneous palpable size were randomly allocated into the treatment groups (N = 8-12/group): i) Placebo; ii) PM01183 (0.18 mg/kg); iii) Cisplatin (3.5 mg/kg); and, iv) PM01183 plus Cisplatin (0.18+3.5 mg/kg). Drugs were intravenously administered once per week for 3 consecutive weeks (days 0, 7 and 14) and seven days after the last dose (Day 21), animals were sacrificed, their ovaries dissected-free and weighed. Statistical differences between groups were determined using two-tailed Mann-Whitney U test. Results: On day 21, OVA1X tumors experienced reductions of 95.3, 88.3 and 87.2% following the treatment with Cisplatin, PM01183 and PM01183+Cisplatin, respectively. The results obtained for OVA1XR tumor demonstrated tumor weight reductions of 48.2, 93.6 and 96.7% for Cisplatin, PM01183 and PM01183+Cisplatin treatments, respectively. Single therapy with PM01183 induced a statistically significant better response than Cisplatin (P=0.003). Also, PM01183+Cisplatin treatment increased the activity following Cisplatin (P=0.002) or PM01183 (P=0.022) administration as single agents, suggesting thus a synergistic effect of both compounds. Conclusions: PM01183 demonstrated strong in vivo antitumor activity in pure patient-derived Cisplatin-sensitive and Cisplatin-resistant serous epithelial ovarian orthotopic tumor models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1778. doi:1538-7445.AM2012-1778

A SEVERE shortage of human transplant donors has triggered interest in the use of animals as a source of organs and tissues for transplantation. Immune xenoreactions in the hamster-to-rat Lewis transplantation model are completely inhibited by the combination of tacrolimus and mycophenolate mofetil. When immunosuppression is withdrawn, grafts are rejected by a process called late xenograft rejection (LXR). Some authors have reported that under immunosuppression there is a T-cell–independent B-cell response, but when immunosuppression is withdrawn the immunologic B-cell response now becomes T-cell dependent. It has also been proposed that in the LXR phase, the natural antibodies may be less specific for the antigen and may activate smaller quantities of the complement system. The aim of this study is to describe and compare the histologic and immunohistochemical features in heart and liver LXR; the histopathologic pattern of this type of rejection has not been described exhaustively.

4652 Background: Germ cell tumors (GCTs) are the most common malignancy in young men. Recently, our group has reported the development of human nonseminoma (NSE) after orthotopic nude mice implanta...

Parvalbumin-immunoreactive dystrophic neurites and aberrant terminal sprouts associated with senile plaques, together with preserved density of parvalbumin-immunoreactive neurons, were found in the cerebral cortex of two patients with Alzheimer's disease, one of them familiar, in which a biopsy of the left frontal lobe was carried out for diagnostic and counselling purposes. These findings suggest that, although parvalbumin-immunoreactive cells are relatively resistant to degeneration in Alzheimer's disease, parvalbumin-immunoreactive neuronal processes can degenerate in some cases of Alzheimer's disease.

Anti-Hu autoantibodies in high titres, as revealed with immunocytochemistry and Western blot, were present in a patient with gastrointestinal pseudo-obstruction and small-cell lung cancer (SCLC) bearing the Hu antigen. Marked neuron and nerve fibre loss were found in the myenteric plexus at postmortem. These findings show that neuronopathic Hu-associated gastrointestinal pseudo-obstruction can occur as the only paraneoplastic neurological symptom in patients with SCLC.

e16143 Testicular germ cell tumors (TGCTs) are the most common malignancy in young men. Recently, our group has reported the development of a model of human nonseminoma (NSE) after orthotopic nude mice implantation (Piulats et al, Amer Assoc Cancer Res. 2006). Pure and mix NSE anatomies were represented and they reproduce the main histological, genetic and epigenetic characteristics of paired primary tumors. Xenografts mimic distal dissemination patterns and cisplatin (CDDP) tumor behavior responses. This model has been useful for the study of antiangiogenic therapies (Piulats et al, ASCO. 2007), furthermore we want to demonstrate it is an useful tool to study the genetic mechanisms related with CDDP adquired resitance. We have generated in vivo five tumors showing increased resistance to CDDP by exposition to repetitive cycles and increasing the dose applied through different passages (1 yolk-sac; 1 choriocarcinoma; 2 embrional carcinoma; 1 mix tumor). A shortness time elipse between pasajes was observed for each tumor through CDDP treatments. To confirm increasin resistance, a parallel assay of chemotherapy response was performed between nontreated and CDDP resistant tumors. Whole genome analysis of tour xenografted tumors and their paired CDDP resistant tumor (#3 and #5 passage) were analyzed by CGH NimbeGen arrays using 60 Kb average windows. Few differential genomic changes were identified some of them were consistent across resistant tumors including gain of 9q21.11–9q33.3, 15q23–15q24.1, and 15q26.3 regions and loss of Xp22.33. In one tumour showing strong CDDP resistance compared with its sensitive counterpart it occur in absence of new genomic changes. No changes in the MSI or mutational TP53 status were observed in resisant tumors. Our data suggest that acquisition of tumor resistance to CDDP in TGCTs may proably depend of a combination of different mechanisms, including cromosomal imbalances. No significant financial relationships to disclose.

16070 Background: Germ cell tumors (GCTs) of the testis are highly curable, but patients refractory to cisplatin (CDDP) based chemotherapy have a poor prognosis. Several studies support an important role of angiogenesis in GCT, suggesting anti-angiogenic treatment as a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor with antiangiogenic and antitumoral activities. We evaluate the effect of sunitinib, CDDP or the combination of both compounds on tumoral growth using an orthotopic model of human testicular GCTs (Piulats et al., AACR 2006; abstract 2760). Methods: Mice were implanted with 4 different tumors: a yolk sac without prior exposure to CDDP; a choriocarcinoma from a CDDP-resistant patient; and finally both a choriocarcinoma without prior exposure to CDDP and its CDDP refractory variant induced in mice by a continous CDDP exposure. Mice were treated with sunitinib (40 mg/kg, daily for 15 days), CDDP (2 mg/kg three times at 5-day interval) or combination of both. Animals wer...

The aim of this article is to propose guidelines and recommendations in problematic areas in pathologic reporting of endometrial carcinoma (EC) regarding special techniques and ancillary studies. An organizing committee designed a comprehensive survey with different questions related to pathologic features, diagnosis, and prognosis of EC that was sent to all members of the International Society of Gynecological Pathologists. The special techniques/ancillary studies group received 4 different questions to be addressed. Five members of the group reviewed the literature and came up with recommendations and an accompanying text which were discussed and agreed upon by all members of the group. Twelve different recommendations are made. They address the value of immunohistochemistry, ploidy, and molecular analysis for assessing prognosis in EC, the value of steroid hormone receptor analysis to predict response to hormone therapy, and parameters regarding applying immunohistochemistry and molecular tests for assessing mismatch deficiency in EC.

Recent efforts to sequence human cancer genomes have highlighted that point mutations in genes involved in the epigenetic setting occur in tumor cells. Small cell lung cancer (SCLC) is an aggressive tumor with poor prognosis, where little is known about the genetic events related to its development. Herein, we have identified the presence of homozygous deletions of the candidate histone acetyltransferase KAT6B, and the loss of the corresponding transcript, in SCLC cell lines and primary tumors. Furthermore, we show, in vitro and in vivo, that the depletion of KAT6B expression enhances cancer growth, while its restoration induces tumor suppressor–like features. Most importantly, we demonstrate that KAT6B exerts its tumor-inhibitory role through a newly defined type of histone H3 Lys23 acetyltransferase activity. Cancer Res; 75(18); 3936–45. ©2015 AACR.

Altres ajuts: The research leading to these results received funding from the Olga Torres Foundation. AlV is supported by the Fondo de Investigaciones Sanitarias FIS (PI13-01339), Fundación Mutua Madrileña AP150932014 and the Asociación Española Contra el Cáncer (AECC)-Barcelona. HH is a Miguel Servet (CP14/00229) researcher funded by the Spanish Institute of Health Carlos III (ISCIII). Core funding is from the ISCIII and the Generalitat de Catalunya. A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-beta 3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM. This work was funded by the Government of Catalonia grant SGR 2014-364, the "Red Tematica de Investigacion Colaborativa en Cancer" grant RD12/0036/0008, the Telemaraton 2014 "Todos Somos Raros, Todos Somos Unicos" grant P35, the Spanish Ministry of Health "Instituto de Salud Carlos III Fondo de Investigacion Sanitaria" grant PI12/01528, and a Spanish Society of Pneumology and Thoracic Surgery (Sociedad Espanola de Neumologia y Cirugia Toracica, SEPAR) grant for 2012. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Chromosome 18q allelic loss has been reported to have prognostic significance in stage II colorectal carcinoma. We have developed a fluorescent multiplex polymerase chain reaction assay to analyze five microsatellite markers (D18S55, D18S58, D18S61, D18S64, and D18S69) for allelic loss at the long arm of chromosome 18. Amplicon detection and evaluation was accomplished by capillary electrophoresis using an ABI 310 genetic analyzer. Robustness of the assay when performed on DNA extracted from formalin-fixed, paraffin-embedded tissue sections was confirmed by analyzing its repeatability and reproducibility. Allelic loss was assessed in 61 stage II colorectal tumors and was detected in 58% (31 of 53) of tumors not showing instability. As part of the study, results of 207 previous polymerase chain reaction/polyacrylamide-based assays were re-evaluated by two independent observers to determine the degree of concordance of visual evaluation. In the case of stage II colorectal tumors, when electropherogram results were compared with those obtained from visual evaluation of the same markers after polyacrylamide gel electrophoresis, discrepancies between observers were detected in 16.4% of determinations. In conclusion, we have developed a robust and reliable assay for multiplexed loss of heterozygosity determination that improves assessment of chromosome 18q allelic loss in colorectal tumors processed as routine formalin-fixed, paraffin-embedded specimens.

Carcinoma-associated fibroblasts (CAFs) are important contributors of microenvironment in determining the tumor’s fate. This study aimed to compare the influence of liver microenvironment and primary tumor microenvironment on the behavior of colorectal carcinoma. Conditioned medium (CM) from normal colonic fibroblasts (NCFs), CAFs from primary tumor (CAF-PT) or liver metastasis (CAF-LM) were obtained. We performed functional assays to test the influence of each CM on colorectal cell lines. Microarray and gene set enrichment analysis (GSEA) were performed in DLD1 cells cultured in matched CM. In DLD1 cells, CAF-LM CM compared with CAF-PT CM and NCF led to a more aggressive phenotype, induced the features of an epithelial-to-mesenchymal transition more efficiently, and stimulated migration and invasion to a greater extent. Sustained stimulation with CAF-LM CM evoked a transient G2/M cell cycle arrest accompanied by a reduction of apoptosis, inhibition of proliferation, and decreased viability of SW1116, SW620, SW480, DLD1, HT-29, and Caco-2 cells and provoked nonapoptotic cell death in those cells carrying KRAS mutations. Cells resistant to CAF-LM CM completely changed their morphology in an extracellular signal-regulated protein kinase-dependent process and depicted an increased stemness capacity alongside the Wnt pathway stimulation. The transcriptomic profile of DLD1 cells treated with CAF-LM CM was associated with Wnt and mitogen-activated protein kinase pathways activation in GSEA. Therefore, the liver microenvironment induces more efficiently the aggressiveness of colorectal cancer cells than other matched microenvironments do but secondarily evokes cell death. Resistant cells displayed higher stemness capacity.

The generation of B cells is a complex process requiring several cellular transitions, including cell commitment and differentiation. Proper transcriptional control to establish the genetic programs characteristic of each cellular stage is essential for the correct development of B lymphocytes. Deregulation of these particular transcriptional programs may result in a block in B-cell maturation, contributing to the development of hematological malignancies such as leukemia and lymphoma. However, very little is currently known about the role of transcriptional repressors in normal and aberrant B lymphopoiesis. Here we report that histone deacetylase 7 (HDAC7) is underexpressed in pro-B acute lymphoblastic leukemia (pro-B-ALL) and Burkitt lymphoma. Ectopic expression of HDAC7 induces apoptosis, leads to the downregulation of c-Myc and inhibits the oncogenic potential of cells in vivo, in a xenograft model. Most significantly, we have observed low levels of HDAC7 expression in B-ALL patient samples, which is correlated with the increased levels of c-Myc. From a mechanistic angle, we show that ectopically expressed HDAC7 localizes to the nucleus and interacts with the transcription factor myocyte enhancer factor C (MEF2C) and the corepressors HDAC3 and SMRT. Accordingly, both the HDAC7-MEF2C interaction domain as well as its catalytic domain are involved in the reduced cell viability induced by HDAC7. We conclude that HDAC7 has a potent anti-oncogenic effect on specific B-cell malignancies, indicating that its deregulation may contribute to the pathogenesis of the disease. This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2011-28290; to MP). MP was supported by a Ramón y Cajal contract from the Spanish Ministry of Science and Innovation (MICIIN). LR-G was supported by an IDIBELL PhD fellowship. NL-B acknowledges the funding from MICIIN (SAF2012-36199).

Purpose: We examined whether PI3K–AKT or extracellular signal–regulated kinase (ERK) signaling pathways could play a role in the development of cisplatin (CDDP) resistance in testicular germ cell tumor (TGT) cells. Experimental Design: We compared AKT and ERK activation levels in CDDP-sensitive testicular tumor cells and in their corresponding CDDP-resistant–derived cells. We also analyzed these pathways in orthotopic testicular tumors and human patient samples. Results: Our results indicated that there was overactivation of AKT in CDDP-resistant cells compared with sensitive cells, but no effect on activated ERK levels. We observed an increase in mRNA and protein levels for platelet-derived growth factor (PDGF) receptor β and PDGF-B ligand. These were responsible for AKT overactivation in CDDP-resistant cells. When PDGFRβ levels were decreased by short hairpin RNA (shRNA) treatment or its activation was blocked by pazopanib, CDDP-resistant cells behaved like sensitive cells. Moreover, CDDP-resistant cells were more sensitive to incubation with PDGFRβ inhibitors such as pazopanib or sunitinib than sensitive cells, a finding consistent with these cells being dependent on this signaling pathway. We also found overexpression of PDGFRβ and pAKT in CDDP-resistant choriocarcinoma orthotopic tumor versus their CDDP-sensitive counterparts. Finally, we found high PDGFRβ levels in human testicular tumors, and overexpression in CDDP-resistant testicular choriocarcinomas compared with the CDDP-sensitive and nontreated tumors. Conclusions: The PDGFRβ–AKT pathway plays a critical role in the development of CDDP resistance in testicular tumoral cells. Clin Cancer Res; 20(3); 658–67. ©2013 AACR.

e15092 Background: Germ cell tumors (GCTs) are the most common malignancy in young men. Recently have been proposed Mismatch repair (MMR) deficiency, microsatellite instability (MSI) and BRAF mutat...

e16560 Background: To provide useful material for discussion, we report an analysis of a retrospective accrued, single-institution population with BOTs. Methods: 99 consecutive patients treated between April 1989 and July 2008, were reviewed for clinical characteristics, histology, surgical types, recurrence, and prognosis. Results: Median age was 46 years (19–77). 90% of patients were diagnosed by vaginal ultrasound as complex adnexal mass. CA-125 and CA19.9 was elevated in 60% and 30% of patients respectively. Primary treatment was surgery: laparotomy and laparoscopy (LT/LC) approach in 78% and 22% of patients, respectively. 33 patients (34%) underwent comprehensive staging operation; fertility-sparing surgery was performed in 18 patients (18%), and an incomplete staging in 47 patients (48%). The pathology results were: clear cell BOTs 1 case (1%); serous 42 cases (42.4%); mucinous in 56 women (56.6%) (46 intestinal/10 mullerian). 2 out of 56 mucinous BOTs were associated with pseudomixoma peritonei. Cyst-rupture was found in 22.5% and 38.9% of patients with LT/LC approach. 91women (93.8%) had stage I; 2 (2%) had stage II; and 6 (4.2%) had stage III disease. We identified 6 patients (7.8%) with noninvasive implants involving abdominal peritoneum (1), pelvic peritoneum (1), omentum (2) and both pelvic peritoneum and omentum (2). With a median follow-up of 3.6 years (0–18.4), we observed 5 recurrences (2 in contralateral ovary, 1 ipsilateral, 1 invasive peritoneal implant, and 1 para-aortic node). All were stage I disease at diagnosis. 2 out of 5 had incomplete staging, 3 had conservative surgery.3 cases were serous type. Histology recurrences were: 2 borderline, 2 invasive ovarian cancer, and 1 unknown. All patients underwent secondary cytoreduction achieving an optimal debulking in 4. Only one patient received chemotherapy. Two women died of intercurrent disease and 1 woman died of disease. The 5-year overall survival rate for entire cohort was 98.5% (95.6%-100%; CI95%). Conclusions: Our results confirm published data. Overall BOTs have an excellent prognosis. Proper staging is the cornerstone of treatment. Laparoscopy approach is associated with higher cyst rupture. Recurrences are more frequent after incomplete or conservative surgery. No significant financial relationships to disclose.

Chronic cerebral hypoperfusion was produced in adult gerbils aged 3-6 months following bilateral stenosis of the carotid artery lasting 8 weeks. Animals with no evidence of cerebral infarction were used in the present study. Ubiquitin-immunoreactive free granules in the subcortical cerebral white matter and corpus callosum were observed in five of 12 animals. No similar lesions were found in sham-operated animals, age-matched controls, and gerbils subjected to transient forebrain ischaemia for 20 min and killed at different intervals. These results indicate that diffuse damage of the subcortical white matter may be encountered as the only neuropathological change following chronic hypoperfusion.