Immature Tumor Research Articles

Abstract 194: Increased PKR expression inhibits differentiation of hematopoietic stem cells

Abstract Acute myelogenous leukemia (AML) is characterized by a differentiation block at an early stage of hematopoiesis that results in the accumulation of immature myeloid cells and an impaired production of blood cells. This parallels findings in solid tumors that demonstrate differentiation stage is often associated with tumor behavior and generally an immature tumor is more aggressive than the more differentiated counterpart. Thus, new research designed to more fully understand the mechanisms of cellular differentiation may improve our understanding of neoplastic transformation and identify new therapeutic targets. Recently our laboratory has determined that increased expression and activation of the interferon-inducible, dsRNA-activated kinase (PKR) is associated with shorter remission duration and worse survival for patients with AML. PKR is a key mediator of the cellular response to inflammatory stress that has been found to be upregulated by chronic inflammation and aging. Since we have previously reported that mice expressing a PKR transgene (TgPKR) in hematopoietic cells have a reduced number of bone marrow (BM) Lin-, Sca1+, cKit+ hematopoietic stem/progenitor cells (HSPCs) and develop a dysplastic BM with an increased frequency of BM blasts, we investigated whether PKR may affect HSPC differentiation. Results indicate that BM of TgPKR mice contains a significantly reduced number of multipotent progenitor cells (MPPs) that are more frequently arrested in G0/G1 than wild type (WT) mice. In contrast, BM of PKR knockout mice (PKRKO) have an increased frequency of proliferating MPPs (S+G2/M) but a decreased frequency of both Long-term (LT) and Short-term (ST) HSCs compared to either WT or TgPKR mice. Although LT-HSCs of TgPKR mice are more frequently quiescent in vivo they remain functional in vitro as demonstrated by nearly double the colony forming capacity compared to BM of PKRKO mice after >4 weeks in long term culture initiating cell (LTC-IC) assays. In addition, when differentiation of the myeloid cell line K562 is induced by PMA treatment, cells with reduced PKR expression by siRNA knockdown display an increased rate and extent of megakaryocyte differentiation compared to control cells. These findings suggest that increased PKR expression has a previously unrecognized function to inhibit hematopoietic cell differentiation. This may account for why TgPKR mice accumulate quiescent LT-HSCs and develop BM dysplasia with age. Furthermore, these results suggest that increased PKR expression/activation as observed in high-risk MDS, AML and other hematologic malignancies may contribute to leukemogenesis, at least in part, by blocking hematopoietic cell differentiation. In addition, these new findings may be applicable to solid tumors since increased PKR signaling has also been reported to be associated with a more rapid and aggressive course of tumor progression in breast cancer, melanoma and colon cancer. Citation Format: Richard L. Bennett, Xiaodong Cheng, Michael T. Byrne, Amalin Asous, Mohini Patel, Zeel Patel, W. Stratford May. Increased PKR expression inhibits differentiation of hematopoietic stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 194.

Co-expression modules of NF1, PTEN and sprouty enable distinction of adult diffuse gliomas according to pathway activities of receptor tyrosine kinases.

Inter-individual variability causing elevated signaling of receptor tyrosine kinases (RTK) may have hampered the efficacy of targeted therapies. We developed a molecular signature for clustering adult diffuse gliomas based on the extent of RTK pathway activities. Glioma gene modules co-expressed with NF1 (NF1-M), Sprouty (SPRY-M) and PTEN (PTEN-M) were identified, their signatures enabled robust clustering of adult diffuse gliomas of WHO grades II-IV from five independent data sets into two subtypes with distinct activities of RAS-RAF-MEK-MAPK cascade and PI3K-AKT pathway (named RMPAhigh and RMPAlow subtypes) in a morphology-independent manner. The RMPAhigh gliomas were associated with poor prognosis compared to the RMPAlow gliomas. The RMPAhigh and RMPAlow glioma subtypes harbored unique sets of genomic alterations in the RTK signaling-related genes. The RMPAhigh gliomas were enriched in immature vessel cells and tumor associated macrophages, and both cell types expressed high levels of pro-angiogenic RTKs including MET, VEGFR1, KDR, EPHB4 and NRP1. In gliomas with major genomic lesions unrelated to RTK pathway, high RMPA signature was associated with short survival. Thus, the RMPA signatures capture RTK activities in both glioma cells and glioma microenvironment, and RTK signaling in the glioma microenvironment contributes to glioma progression.

PRMT5-PTEN molecular pathway regulates senescence and self-renewal of primary glioblastoma neurosphere cells.

Glioblastoma (GBM) represents the most common and aggressive histologic subtype among malignant astrocytoma and is associated with poor outcomes because of heterogeneous tumour cell population including mature non-stem-like cell and immature stem-like cells within the tumour. Thus, it is critical to find new target-specific therapeutic modalities. Protein arginine methyltransferase enzyme 5 (PRMT5) regulates many cellular processes through its methylation activity and its overexpression in GBM is associated with more aggressive disease. Previously, we have shown that silencing of PRMT5 expression in differentiated GBM cell lines results in apoptosis and reduced tumour growth in mice. Here, we report the critical role of PRMT5 in GBM differentiated cells (GBMDC) grown in serum and GBM neurospheres (GBMNS) grown as neurospheres in vitro. Our results uncover a very significant role for PRMT5 in GBMNS self-renewal capacity and proliferation. PRMT5 knockdown in GBMDC led to apoptosis, knockdown in GBMNS led to G1 cell cycle arrest through upregulation of p27 and hypophoshorylation of retinoblastoma protein, leading to senescence. Comparison of impact of PRMT5 on cellular signalling by the Human Phospho-Kinase Array and chromatin immunoprecipitation-PCR revealed that unlike GBMDC, PRMT5 regulates PTEN expression and controls Akt and ERk activity in GBMNS. In vivo transient depletion of PRMT5 decreased intracranial tumour size and growth rate in mice implanted with both primary tumour-derived GBMNS and GBMDC. This is the first study to identify PTEN as a potential downstream target of PRMT5 and PRMT5 is vital to support both mature and immature GBM tumour cell populations.

Shift of microRNA profile upon orthotopic xenografting of glioblastoma spheroid cultures.

Glioblastomas always recur despite surgery, radiotherapy and chemotherapy. A key player in the therapeutic resistance may be immature tumor cells with stem-like properties (TSCs) escaping conventional treatment. A group of promising molecular targets are microRNAs (miRs). miRs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. In this study we aimed to identify over-expressed TSC-related miRs potentially amenable for therapeutic targeting. We used non-differentiated glioblastoma spheroid cultures (GSCs) containing TSCs and compared these to xenografts using a NanoString nCounter platform. This revealed 19 over-expressed miRs in the non-differentiated GSCs. Additionally, non-differentiated GSCs were compared to neural stem cells (NSCs) using a microarray platform. This revealed four significantly over-expressed miRs in the non-differentiated GSCs in comparison to the NSCs. The three most over-expressed miRs in the non-differentiated GSCs compared to xenografts were miR-126, -137 and -128. KEGG pathway analysis suggested the main biological function of these over-expressed miRs to be cell-cycle arrest and diminished proliferation. To functionally validate the profiling results suggesting association of these miRs with stem-like properties, experimental over-expression of miR-128 was performed. A consecutive limiting dilution assay confirmed a significantly elevated spheroid formation in the miR-128 over-expressing cells. This may provide potential therapeutic targets for anti-miRs to identify novel treatment options for GBM patients.

Evaluation of the "steal" phenomenon on the efficacy of hypoxia activated prodrug TH-302 in pancreatic cancer.

Pancreatic ductal adenocarcinomas are desmoplastic and hypoxic, both of which are associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are specifically activated in hypoxic environments to release cytotoxic or cytostatic effectors. TH-302 is a HAP that is currently being evaluated in a Phase III clinical trial in pancreatic cancer. Using animal models, we show that tumor hypoxia can be exacerbated using a vasodilator, hydralazine, improving TH-302 efficacy. Hydralazine reduces tumor blood flow through the “steal” phenomenon, in which atonal immature tumor vasculature fails to dilate in coordination with normal vasculature. We show that MIA PaCa-2 tumors exhibit a “steal” effect in response to hydralazine, resulting in decreased tumor blood flow and subsequent tumor pH reduction. The effect is not observed in SU.86.86 tumors with mature tumor vasculature, as measured by CD31 and smooth muscle actin (SMA) immunohistochemistry staining. Combination therapy of hydralazine and TH-302 resulted in a reduction in MIA PaCa-2 tumor volume growth after 18 days of treatment. These studies support a combination mechanism of action for TH-302 with a vasodilator that transiently increases tumor hypoxia.

Malignant mixed germ cell tumours of ovary. A Report of two cases.

Malignant germ cell tumours of ovary comprise less than 5% of all ovarian neoplasms. Malignant mixed germ cell tumours are still rare. Most common combination in mixed germ cell tumours is that of Dysgerminoma & yolk sac tumour. Mixed tumours showing three germ cell components and four or five germ cell components are still rare. Here we report two cases of Malignant mixed germ cell tumours with a histologic combination of Immature teratoma, Dysgerminoma & Yolk sac tumour. Review of literature also showed such type of combination.

Growing Teratoma Syndrome of Ovary: Avoiding A Misdiagnosis of Tumour Recurrence

Growing teratoma syndrome is characterized by conversion of immature gonadal germ cell tumour to a mature form, along with increase in size of the lesions. Being more frequently described in testicular germ cell tumours, growing teratoma syndrome in an ovarian tumour is uncommon. The exact cause of conversion and growth is unknown and is hypothesized to be induced by chemotherapy. On imaging, the diagnosis is suggested by the appearance of mature elements such as fat or calcium within these lesions and it is confirmed by normal serum markers despite the increase in its size. Knowledge of this entity is important, to avoid misdiagnosis as disease progression and continuing chemotherapy, as these lesions are refractory to chemotherapy. Though surgery is curative, they can also be safely followed-up in a majority of cases, with a favourable long term prognosis.

Pregnancy following surgery and chemotherapy for ovarian germ cell tumors.

e16550 Background: Malignant ovarian germ cell tumours(OGCT) are a rare disease, accounting for 2% of all malignant ovarian tumours diagnosed in Ireland between 1994 - 2010. They affect a younger population than epithelial ovarian cancer, with the highest number of cases in the second and third decade. In this report, we present a review of patients treated in a tertiary referral centre. Methods: We performed a retrospective review over a 26 year period in our institution. We examined clinical records, pathology, pharmacy and surgical databases to identify patients with ovarian germ cell tumours who were treated in our institution. Results: During the period 1986 – 2012, twenty patients were treated and followed at this institution for malignant ovarian germ cell tumor (OGCT). Eighteen patients are alive and well at a median follow up of 14.5 years (range 0.5 – 26). Nineteen patients received platinum based chemotherapy with a median number of four cycles (range 2 – 6) required to achieve complete remission. Twenty percent of patients had yolk sac histology; the next commonest were immature teratomas and mixed germ cell tumour (10 & 15% respectively). The commonest chemotherapy regimen used was BEP (bleomyocin, etoposide and cistplatin). Six patients (30%) underwent resection of residual disease post chemo revealing mature teratoma/necrotic tissue. During follow up, four patients had progressive or recurrent disease. Two patients at resection had mature teratomas found on histological examination. The remaining two patients (10%) had progressive disease and died within a short time period. In our patient population, nine of nine attempting pregnancy have been successful. There have been 12 pregnancies carried to term; mother and child in each case are well. At a median follow up of 14.5 years (range 0.5 – 26), there have been no second malignancies and no toxicities attributable to chemotherapy. Conclusions: We have shown that platinum based chemo in OGCT is associated with preservation of fertility in these young women. In our series there have been no adverse affects in women or their infants. Platinum based chemo highly successful in curing OGCT and this confirms that it is associated with preserved fertility in these young women.

β-Catenin Functions Pleiotropically in Differentiation and Tumorigenesis in Mouse Embryo-Derived Stem Cells

The canonical Wnt/β-catenin signaling pathway plays a crucial role in the maintenance of the balance between proliferation and differentiation throughout embryogenesis and tissue homeostasis. β-Catenin, encoded by the Ctnnb1 gene, mediates an intracellular signaling cascade activated by Wnt. It also plays an important role in the maintenance of various types of stem cells including adult stem cells and cancer stem cells. However, it is unclear if β-catenin is required for the derivation of mouse embryo-derived stem cells. Here, we established β-catenin-deficient (β-catΔ/Δ) mouse embryo-derived stem cells and showed that β-catenin is not essential for acquiring self-renewal potential in the derivation of mouse embryonic stem cells (ESCs). However, teratomas formed from embryo-derived β-catΔ/Δ ESCs were immature germ cell tumors without multilineage differentiated cell types. Re-expression of functional β-catenin eliminated their neoplastic, transformed phenotype and restored pluripotency, thereby rescuing the mutant ESCs. Our findings demonstrate that β-catenin has pleiotropic effects in ESCs; it is required for the differentiation of ESCs and prevents them from acquiring tumorigenic character. These results highlight β-catenin as the gatekeeper in differentiation and tumorigenesis in ESCs.

Abstract 692: Prognostic indicators associated with breast cancer molecular subtypes in African-American women

Abstract Background: Overexpression of Fascin, Endoglin (CD105), and Beta-Catenin has been found to be associated with unfavorable prognosis and progression in hormone-receptor negative breast carcinomas. Specifically, fascin promotes cell motility, while Beta-catenin is involved in cancer invasion, cellular transformation, and metastasis. CD105 is selectively upregulated in small, immature tumor vessels in malignant tumors. The objective of this study was to correlate fascin, CD105, and Beta-Catenin protein expression with clinicopathological factors including age, grade, tumor size, stage, regional node status, survival, and with the four major molecular breast cancer subtypes (luminal A, luminal B, HER2 positive, and Triple Negative [TN]). Methods: Tissue microarrays (TMAs) were constructed from optimally-fixed formalin-fixed, paraffin-embedded tumor blocks from primary breast carcinomas in 203 African-American females. Sections were stained with antitibodies against fascin, CD105, and beta-catenin. The sections were evaluated for the intensity of reactivity (0-3) and the percentage of reactive cells. Cases were categorized as having negative/weak (score <10) or moderate/strong (score >10) fascin expression, negative (score=0) or positive (score>0) CD105 (in tumor cells only) and beta-catenin (cytoplasmic and nuclear) expression. Bivariate analysis was done via χ2 analysis and survivability data was calculated via the generation of Kaplan-Meier curves (SPSS v19). Statistical significance was assumed if P < 0.05. Results: On the TMA, there were 67 triple negatives, 88 Luminal As, 29 Luminal Bs, and 18 Her2 overexpressing cancers. The mean age for the patients in the study was 57.65. Fascin expression was significantly linked to the triple negative subtype (p<0.0001), ER negativity (p<0.0001), PR negativity (0.0001), and Grade III differentiation (p=0.03). CD105 cytoplasmic expression significantly correlated with HER2 positive subtype (p<0.0001), Luminal B subtype (0.01), HER2 positivity (p<0.0001), tumor size (p<0.01) and was associated with decreased disease-free survival (p=0.038). Cytoplasmic beta-catenin expression was significantly associated with triple negative subtype (p<0.03), HER2+ subtype (p<0.001), ER negativity (p<0.001), PR negativity (p<0.001), and HER2 negativity (p=0.03). Conclusions: Our results indicate that fascin, CD105, and Beta-catenin may be potential markers of aggressive breast cancer subtypes and predictors of recurrence and survival. These data also support pharmacological targets for these aggressive subtypes. 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 692. doi:1538-7445.AM2012-692

Abstract 408: Intermittent hypoxia effect on osteoclastogenesis stimulated by neuroblastoma cells

Abstract Neuroblastoma is the most common extracranial pediatric solid tumor that is derived from the developing sympathetic nervous system and results from the improper differentiation of neural crest cells. Neuroblastomas show a tremendous clinical heterogeneity ranging from benign ganglioneuromas to highly aggressive immature tumors. Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. Intermittent hypoxia (IH) which is characterized by cyclic periods of hypoxia and reoxygenation, occurs in tumor cells that are dependent on tumor blood vessels having intermittent perfusion fluctuations in blood flow. The occurrence of IH episodes varies significantly in rapidly growing malignant tumors. Bone is one of the target organs of metastasis in advanced neuroblastoma. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by the osteoclast. The present study focuses on how IH-conditioned neuroblastoma cells modulate the differentiation of osteoclasts. IH-conditioned cells were derived by exposing tumor cells to 10 repeated cycles of hypoxia followed by reoxygenation. In order to assess the role of HIF-1α overexpression in osteolysis, we stably transfected human neuroblastoma cells with an expression vector containing a HIF-1α cDNA. Additionally, cells were stably transfected with a plasmid expressing HIF-1α shRNA or luciferase shRNA and selected in neomycin. The expression of HIF-1α protein was analyzed by western blotting in IH-conditioned and stable transfectants of neuroblastoma cells. Changes in gene expression were determined by real-time PCR for IL-8, MCP-1α, PTHrP, CXCR-4 and RANKL and the expression of osteoclastogenic factors was found increased in IH-conditioned and HIF-1α protein overexpressing cells. Conditioned medium collected from IH-conditioned and HIF-1α cDNA overexpressing neuroblastoma cells has shown enhanced osteoclast formation capabilities in the tartrate-resistant acid phosphatase (TRAP) assay. IH-conditioned and HIF-1α protein overexpressing neuroblastoma cells secrete increased amounts of VEGF protein, which has been found to promote osteoclast activity. Calcium-sensing receptor (CaR) plays a pivotal role in osteoclast differentiation. We found enhanced expression of CaR in RAW 264.7 (osteoclast precursors) cells treated with conditioned medium collected from IH-conditioned and HIF-1α protein overexpressing neuroblastoma cells compared with the control. Thus, IH was found to enhance osteolytic capabilities of neuroblastoma cells in vitro in part through HIF-1α protein stabilization. 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 408. doi:1538-7445.AM2012-408

Abstract 1385: Molecular changes in breast tumors following bevacizumab-based treatment: Final analysis of a randomized neoadjuvant study of bevacizumab or placebo, followed by chemotherapy with or without bevacizumab, in patients with stage II or III breast cancer

Abstract Background: Bevacizumab (bev) has been widely studied in breast cancer (BC), yet no randomized trial in BC has reported in vivo molecular effects of bev on human tumor tissue. We conducted a trial to evaluate the safety, clinical & molecular effects of neoadjuvant chemotherapy plus bev for locally advanced BC. Methods: 90 pts were randomized (2:1:2:1) to 1 of 4 arms: Arm A: TAC (docetaxel, T: 75 mg/m2, doxorubicin, A: 50 mg/m2, cyclophosphamide, C: 500 mg/m2) + low dose bev (7.5 mg/kg); Arm B: TAC + low dose placebo (P); Arm C: TAC + standard dose bev (15 mg/kg); Arm D: TAC + Std-P. A run-in cycle of bev or P was followed by 6 cycles of TAC plus P or bev. Tumor biopsies pre- and 7-10 days post-run-in with bev or P were taken. Unblinding occurred post surgery (Sx): Arms A/C received maintenance bev to complete 52 wks, Arms B/D received no further P. Eligible patients were females with >3 cm, HER2(-) BC. Endpoints included safety and pathologic complete response (pCR) in breast & lymph nodes. To assess the effects of VEGF pathway inhibition on tumor vasculature, PCR analysis using the fluidigm array platform was performed on RNA from the pre- and post-run in samples to evaluate expression of 67 genes known to play a defined role in VEGF signaling. Results: 28 pts were assigned to Arm A, 30 to Arm C, and 32 to Arms B/D. 12 pts came off Tx before surgery (Arm A:2, Arm C:6, Arms B/D:4) and 78 received all Tx, underwent Sx and are evaluable for pCR. Two pts (6%) in Arms B/D, 5 pts (18%) in Arm A and 10 pts (33%) in Arm C had wound healing complications. 17% of pts in Arm C had Gr 3 (N=4) or Gr 4 (N=1) heart failure, none in Arms A/B/D. The pCR rate in evaluable patients was 18% (14/78): 5 (19%) Arm A, 3 (13%) Arm C, 6 (21%) Arms B/D. 45 samples (20 from Arms B/D and 25 from bev arms) were included in the pair-wise analysis to identify 6 genes that were differentially expressed. Bev resulted in a decrease in expression of Dll4, Cox2, Fibronectin (FN_EIIIB), angiopoietin 2 (Angpt2) and ESM1. In addition, upregulation of the cytokine, stromal derived growth factor (SDF1) was observed. Notably, genes such as CD31 or VE-cadherin were not appreciably differentially expressed. Of the genes that were downregulated, Dll4 and Angpt2 represent genes enriched in endothelial tip cells, which guide the migration of newly formed blood vessels. The decrease in these genes likely represents the effect of bev on reducing immature, growing vasculature in the tumor. Conclusions: This trial enabled the clinical and molecular evaluation of breast cancer tumor tissue pre and post-bev. Clinically, bev was associated with more wound healing and heart failure events and similar pCR rates compared to P. Tumor expression analysis for genes in the angiogenesis pathway supports the preclinical hypothesis that bev may primarily target immature tumor vasculature. 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 1385. doi:1538-7445.AM2012-1385

Presence of Tumour-infiltrating FOXP3+Lymphocytes Correlates with Immature Tumour Angiogenesis in Renal Cell Carcinomas

FOXP3+ regulatory T cells (Tregs) inhibit effector T cell functions and are implicated in tumour progression. However, together with microvessel density (MVD) they remain controversial prognostic predictors for renal cell carcinoma (RCC), and potential associations have yet to be determined. The objective of this study was to determine the prognostic significance of Tregs and MVD and their potential relationship in RCCs. Paraffin-embedded tissues from 62 RCC patients were analysed using immunohistochemistry to detect FOXP3+ lymphocytes, and double immunohistochemistry to detect different microvessel types in the tumour interior, rim and normal kidney tissue, and their correlation with clinicopathological characteristics. Survival analysis was also performed. The presence of FOXP3+ cells in the tumour interior or the rim showed no correlation with death from RCC and other pathological characteristics. Negative correlations were noted between the immature MVD in the tumour interior or the rim and tumour size, tumour stage and overall survival; however, there was no correlation with the nuclear grade or pathological type. A positive correlation between FOXP3+ Tregs and immature MVD (r=0.363, P=0.014) and mature MVD (r=0.383, P=0.009) was confirmed in the tumour interior. However, there was no correlation between FOXP3+ Tregs and mature MVD (r=0.281, P=0.076) or immature MVD (r=0.064, P=0.692) in the tumour rim. In this study, a positive correlation between the presence of FOXP3+ Tregs and immature and mature MVD in RCC was confirmed, which suggests a link between suppression of immunity, tumour angiogenesis and poor prognosis.

Fibroblast Growth Factor-2 Is an Important Factor that Maintains Cellular Immaturity and Contributes to Aggressiveness of Osteosarcoma

Osteosarcoma is the most frequent, nonhematopoietic, primary malignant tumor of bone. Histopathologically, osteosarcoma is characterized by complex mixtures of different cell types with bone formation. The role of environmental factors in the formation of such a complicated tissue structure as osteosarcoma remains to be elucidated. Here, a newly established murine osteosarcoma model was used to clarify the roles of environmental factors such as fibroblast growth factor-2 (Fgf2) or leukemia-inhibitory factor (Lif) in the maintenance of osteosarcoma cells in an immature state. These factors were highly expressed in tumor environmental stromal cells, rather than in osteosarcoma cells, and they potently suppressed osteogenic differentiation of osteosarcoma cells in vitro and in vivo. Further investigation revealed that the hyperactivation of extracellular signal-regulated kinase (Erk)1/2 induced by these factors affected in the process of osteosarcoma differentiation. In addition, Fgf2 enhanced both proliferation and migratory activity of osteosarcoma cells and modulated the sensitivity of cells to an anticancer drug. The results of the present study suggest that the histology of osteosarcoma tumors which consist of immature tumor cells and pathologic bone formations could be generated dependent on the distribution of such environmental factors. The combined blockade of the signaling pathways of several growth factors, including Fgf2, might be useful in controlling the aggressiveness of osteosarcoma.

Cancer cell differentiation heterogeneity and aggressive behavior in solid tumors

The differentiation stage of tumors is a central aspect in the histopathological classification of solid malignancies. The differentiation stage is strongly associated with tumor behavior, and generally an immature tumor is more aggressive than the more differentiated counterpart. While this is common knowledge in surgical pathology, the contribution of differentiation-related gene expression and functions to tumor behavior is often overlooked in the experimental, tumor biological setting. The mechanisms by which tumor cell differentiation stages are perturbed or affected are poorly explored but have recently come into focus with the introduction.of the tumor stem cell concept. While developmental biologists view the differentiation as a unidirectional event, pathologists and tumor biologists have introduced the concept of dedifferentiation to explain phenotypic changes occurring in solid tumors. In this review we discuss the impact of the tumor cell differentiation stage as used in surgical pathology. We further discuss knowledge gained from exploring the molecular basis of the differentiation and dedifferentiation processes in neuroblastoma and breast cancer, two tumor forms where the tumor cell differentiation concept is used in the clinical diagnostic work and where the tumor stem cell theory has been applied.

Gastric teratoma in a 6-month-old boy

Gastric teratomas are very rare embryonal neoplasms, accounting for 2.6% of all perinatal diagnosed germ cell tumours. About 85% are well-differentiated mature lesions and about 15% are immature tumours with the potential of malignant transformation. The recommended therapy for gastric teratomas is surgical excision. We present the case of a 6-month-old boy with an incidentally detected epigastric mass. The histological examination revealed a mature gastric teratoma. The diagnostic imaging, therapy and postoperative follow-up are discussed.

A case of growing teratoma syndrome in mixed germ cell tumors of the ovary

Growing teratoma syndrome (GTS) is an increase in tumor size containing only mature teratoma component, during or after chemotherapy for germ cell tumors. A surgical resection is important to confirm diagnosis and is considered as the most appropriate therapeutic management. GTS is rare event in association with ovarian germ cell tumors. In this report, we present a case of GTS which occurred following the treatment of mixed ovarian germ cell tumor with immature teratoma and endodermal sinus tumor. The patient was given adjuvant chemotherapy consisting of six cycles of etoposide and cisplatin. Abdomen pelvis computed tomography performed after chemotherapy demonstrated no evidence of recurrence. She is on regular follow up and remained disease-free for 8 months following second surgery.

Spontaneous Nephroblastoma in a Japanese Giant Salamander (<i>Andrias japonicus</i>)

An adult male Japanese giant salamander (Andrias japonicus) died accidentally, and necropsy showed a white mass (23 × 15 mm) in the left kidney and hepatorrhexis with hemoperitoneum. Histologically, the renal mass was mainly composed of immature nephroblastic tumor cells. In the tumor tissue, a trabecular pattern lined by oval to polygonal tumor cells with a rich interstitium, solid growth and a few tubular structures was observed. Nephroblastic tumor cells were strongly positive for vimentin and weakly positive, and epithelium-like tumor cells were strongly positive for cytokeratin. However, antibody for Wilms' tumor protein 1 did not react with the salamander's cells. On electron microscopy, a desmosome junction was observed between tumor cells. This is the first report of nephroblastoma in a Japanese giant salamander.

Dopamine stabilizes tumor blood vessels by up-regulating angiopoietin 1 expression in pericytes and Krüppel-like factor-2 expression in tumor endothelial cells

Impaired blood flow in the tumor vascular bed caused by structurally and functionally abnormal blood vessels not only hinders the delivery of chemotherapeutic agents but also aggravates tumor hypoxia, making the tumor cells further resistant to antineoplastic drugs. Therefore, normalization of tumor blood vessels may be an important approach to increase therapeutic efficacy in the treatment of cancer patients. As blood vessels are supplied by sympathetic nerves containing dopamine (DA), and DA regulates functions of normal blood vessels through its receptors present in these vessels, we investigated the effect of DA on tumor vasculature. Here we report loss of sympathetic innervation and endogenous DA in abnormal and immature tumor blood vessels in malignant colon and prostate tumor tissues. In contrast, exogenous administration of DA normalizes the morphology and improves the functions of these vessels by acting on pericytes and endothelial cells, the two major cellular components of blood vessels. DA acts through its D(2) receptors present in these cells to up-regulate directly the expression of angiopoietin 1 (Ang1) in pericytes and the expression of the zinc finger transcriptional factor, Krüppel-like factor-2 (KLF2) in tumor endothelial cells. Importantly, this vessel stabilization by DA also significantly increases the concentration of anticancer drug in tumor tissues. These results show a relationship between vascular stabilization and a neurotransmitter and indicate that DA or its D(2) receptor-specific agonists can be an option for the treatment of cancer and disorders in which normalization of blood vessels may have therapeutic benefits.

The apelin/APJ system induces maturation of the tumor vasculature and improves the efficiency of immune therapy

Immature and unstable tumor vasculature provides an aberrant tumor microenvironment and leads to resistance of tumors to conventional therapy. Hence, normalization of tumor vessels has been reported to improve the effect of immuno-, chemo- and radiation therapy. However, the humoral factors, which can effectively induce maturation of tumor vasculature, have not been elucidated. In this study, we found that the novel peptide apelin and its receptor APJ can induce the morphological and functional maturation of blood vessels in tumors. This apelin-induced tumor vascular maturation enhances the efficacy of cancer dendritic cell-based immunotherapy and significantly suppresses tumor growth by promoting the infiltration of invariant natural killer T cells into the central region of the tumor and thereby robustly inducing apoptosis of tumor cells. Additionally, we showed APJ expression to be enhanced in the tumor endothelium in comparison with normal-state endothelial cells. These findings provide a new target for tumor vascular-specific maturation, which is expected to improve the efficacy of conventional cancer therapies.