Published by MDPI
Online ISSN: 2072-6694
The number of SLNs removed at the three institutes.
The ICG fluorescence technique for SLN biopsy (a) Subcutaneous lymphatic streams were visualized as fluorescent signals on PDE; (b) could be traced on the surface of the skin; and (c) a skin incision was made and the fluorescent basin was shown on the monitor.
All removed lymph nodes were classified in terms of fluorescent signal and/or blue color.
We investigated the feasibility of sentinel lymph node (SLN) biopsy using indocyanine green (ICG) technique in 411 patients with early breast cancer at three institutes. ICG, a fluorescence source, and blue dye were injected into the subareolar area to enable real-time image-guided surgery and identification of SLN fluorescence after meticulous dissection. The subcutaneous lymphatic channels were precisely detected in all cases. SLN identification rate was 99% (408/411) with a mean of 2.3 nodes identified per patient. Thirty-nine cases (9.5%) had SLNs involved and all of them were ICG positive. Thus, the ICG technique has a high SLN identification rate comparable with that of the radioisotope method.
Structures of (A) 1,25-dihydroxyvitamin D 3 (1,25D) and (B) its 26,26,26,27,27,27-hexadeuterated analogue (1,25-dihydroxyvitamin D 3-d 6 ; 1,25D-d 6 ). 
Cell viability in (A) MCF-7 cells; (B) SKBR-3 cells, and (C) MDA-MB-231 cells 48 h after administration of placebo (control), 100 nM 1,25D, 100 Nm 1,25D-d 6 , 500 nM etoposide, 200 nM 5-fluorouracil, and the combination of these substances at the same doses. The values represent means ± SD from three independent determinations. a = p < 0.001 vs. control cells; b = p < 0.01 vs. control cells; c = p < 0.05 vs. control cells; ns = not significant. 
(A) Flow cytometry analysis of cell cycle in MDA-MB-231 cells after 48 h of treatment with placebo (control), 100 nM 1,25D, 100 nM 1,25D-d 6 , 500 nM etoposide, and 1,25D or 100 nM 1,25D-d 6 (100 nM) + etoposide (Eto, 500 nM). The values represent means ± SD from three independent determinations.*** = p < 0.001; (B) Western blot of p53, cyclin A, cyclin B, cyclin D and GAPDH (used as loading control) from protein extracts obtained from MDA-MB-231 cells treated as in (A). Numbers show the quantification of protein expression after normalization to GAPDH. A representative experiment is shown. 
Three-dimensional growth of MDA-MB-231 cells 48 h after treatment with placebo (control), 100 nM 1,25D-d 6 , 500 nM etoposide, and 100 nM 1,25D-d 6 plus 500 nM etoposide. The values represent means ± SD from three independent determinations. a = p< 0.05 vs. control; b = p < 0.001 vs. control; c = p < 0.01 vs. etoposide. 
It has been demonstrated that 1,25-dihydroxyvitamin D3 (1,25D) and some of its analogues have antitumor activity. 1,25D labeled with deuterium (26,26,26,27,27,27-hexadeuterated 1a,25-dihydroxyvitamin D3, or 1,25D-d6) is commonly used as internal standard for 1,25D liquid chromatography-mass spectrometry (LC-MS) quantification. In the present study using human breast cancer cell lines, the biological activity of 1,25D-d6 administered alone and in combination with two commonly used antineoplastic agents, 5-fluorouracil and etoposide, was evaluated. Using an MTT assay, flow cytometry, and western blots, our data demonstrated that 1,25D-d6 has effects similar to the natural hormone on cell proliferation, cell cycle, and apoptosis. Furthermore, the combination of 1,25D-d6 and etoposide enhances the antitumoral effects of both compounds. Interestingly, the antitumoral effect is higher in the more aggressive MDA-MB-231 breast cancer cell line. Our data indicate that 1,25D-d6 administered alone or in combination with chemotherapy could be a good experimental method for accurately quantifying active 1,25D levels in cultures or in biological fluids, on both in vitro breast cancer cell lines and in vivo animal experimental models.
NSCLC cells that are VDR high and vitamin D-sensitive preferentially express 
1,25(OH) 2 D 3 supports the acquisition of an epithelial phenotype in SK-LU-1 
Correlation between VDR and EMT Signature Genes in lung cancer cell lines. The correlation between expression of VDR (Affymetrix probe 204254_s_at) and individual EMT signature genes in GEO dataset GSE4824 is presented.
1,25(OH) 2 D 3 opposes TGFβ induction of the EMT in HCC827 cells. HCC827 cells were seeded into 6-well plates at a density of 5 × 10 3 cells/well. Treatments were initiated 48 h after seeding and were repeated every other day for a total of 4 treatments. The experiment was terminated 4 h after the final treatment. (A) Representative photographs of treated cells; (B) qRT-PCR was used to measure the expression of genes associated with EMT. The expression of each gene was normalized to the level obtained for control cells, which were arbitrarily assigned a value of 1.0. Data represent the mean ± SD for 3 independent experiments. The normalized data were analyzed by ANOVA with a post-hoc Tukey's multiple comparison test (X, control vs. TGFβ p < 0.05; +,1,25(OH) 2 D 3 vs. TGFβ p < 0.05; #, TGFβ vs. combination p < 0.05). (C) Cells were seeded onto glass slides and treated as outlined in (A). Four h after the final treatment, cells were fixed and stained with PE-conjugated E-cadherin antibodies or Alexa 488-conjugated VIM antibodies. Nuclei were visualized with DAPI. Immunofluorescence controls included untreated H292 (E-cadherin positive, VIM negative) and A549 cells (E-cadherin negative, VIM positive).
1,25-dihydroxyvitamin D3 (1,25(OH)2D3) exerts anti-proliferative activity by binding to the vitamin D receptor (VDR) and regulating gene expression. We previously reported that non-small cell lung cancer (NSCLC) cells which harbor epidermal growth factor receptor (EGFR) mutations display elevated VDR expression (VDRhigh) and are vitamin D-sensitive. Conversely, those with K-ras mutations are VDRlow and vitamin D-refractory. Because EGFR mutations are found predominately in NSCLC cells with an epithelial phenotype and K-ras mutations are more common in cells with a mesenchymal phenotype, we investigated the relationship between vitamin D signaling capacity and the epithelial mesenchymal transition (EMT). Using NSCLC cell lines and publically available lung cancer cell line microarray data, we identified a relationship between VDR expression, 1,25(OH)2D3 sensitivity, and EMT phenotype. Further, we discovered that 1,25(OH)2D3 induces E-cadherin and decreases EMT-related molecules SNAIL, ZEB1, and vimentin in NSCLC cells. 1,25(OH)2D3-mediated changes in gene expression are associated with a significant decrease in cell migration and maintenance of epithelial morphology. These data indicate that 1,25(OH)2D3 opposes EMT in NSCLC cells. Because EMT is associated with increased migration, invasion, and chemoresistance, our data imply that 1,25(OH)2D3 may prevent lung cancer progression in a molecularly defined subset of NSCLC patients.
Genome- and transcriptome-wide data has significantly increased the amount of available information about primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes in cancer cell models, such as human THP-1 myelomonocytic leukemia cells. In this study, we investigated the genes G0S2, CDKN1A and MYC as master examples of primary vitamin D receptor (VDR) targets being involved in the control of cellular proliferation. The chromosomal domains of G0S2 and CDKN1A are 140-170 kb in size and contain one and three VDR binding sites, respectively. This is rather compact compared to the MYC locus that is 15 times larger and accommodates four VDR binding sites. All eight VDR binding sites were studied by chromatin immunoprecipitation in THP-1 cells. Interestingly, the site closest to the transcription start site of the down-regulated MYC gene showed 1,25(OH)2D3-dependent reduction of VDR binding and is not associated with open chromatin. Four of the other seven VDR binding regions contain a typical DR3-type VDR binding sequence, three of which are also occupied with VDR in macrophage-like cells. In conclusion, the three examples suggest that each VDR target gene has an individual regulatory scenario. However, some general components of these scenarios may be useful for the development of new therapy regimens.
Positron emission tomography (PET) with 11C-labeled 5-hydroxytryptophane (5-HTP) is a sensitive technique to visualize neuroendocrine tumours (NETs), due to high intracellular uptake of amine-precursors like L-dihydroxyphenylalanine (L-DOPA) and 5-HTP. NETs are often small and difficult to localize in spite of overt clinical symptoms due to hormonal excess. In our study, 38 consecutive NET patients underwent 11C-5-HTP-PET and morphological imaging by CT within 12 weeks prior to surgery. Surgical, histopathological and 5-HTP PET findings were correlated. 11C-5-HTP-PET corresponded to the surgical findings in 31 cases, was false negative in six, and true negative in one case resulting in 83.8% sensitivity and 100% specificity. Positive predicted value was 100%. In 11 patients 11C-5-HTP-PET was the only imaging method applied to localize the tumour. Thus, we could demonstrate that functional imaging by 11C-5-HTP-PET in many cases adds vital preoperative diagnostic information and in more than every fourth patient was the only imaging method that will guide the surgeon in finding the NET-lesion. Although the present results demonstrates that 11C-5-HTP may be used as an universal NET tracer, the sensitivity to visualize benign insulinomas and non functioning pancreatic NETs was lower.
Site-specific predicted lifetime attributable risks of SMN incidence after photon CSI at AUBMC or proton CSI at MD Anderson. Other solid cancer risks were based on H remainder , and leukemia risks were based on H redbonemarrow. These risk estimates took into account both primary and stray radiation. The ratios of the risks (proton CSI:photon CSI) are shown for each SMN site. 
Site-specific predicted lifetime attributable risks of SMN mortality after photon CSI at AUBMC or proton CSI at MD Anderson. Other solid cancer risks were based on Hremainder, and leukemia risks were based on Hred bone marrow. These risk estimates took into account both primary and stray radiation. The ratios of the risks (proton CSI:photon CSI) are shown for each SMN site. 
Treatment field characteristics for the proton CSI, with Monitor Units listed for D Rx . "Air gap" refers to the distance between the distal edge of most downstream component of the treatment unit (range compensator) and the proximal surface of the patient along the central beam axis. 
Children receiving radiotherapy face the probability of a subsequent malignant neoplasm (SMN). In some cases, the predicted SMN risk can be reduced by proton therapy. The purpose of this study was to apply the most comprehensive dose assessment methods to estimate the reduction in SMN risk after proton therapy vs. photon therapy for a 13-year-old girl requiring craniospinal irradiation (CSI). We reconstructed the equivalent dose throughout the patient's body from therapeutic and stray radiation and applied SMN incidence and mortality risk models for each modality. Excluding skin cancer, the risk of incidence after proton CSI was a third of that of photon CSI. The predicted absolute SMN risks were high. For photon CSI, the SMN incidence rates greater than 10% were for thyroid, non-melanoma skin, lung, colon, stomach, and other solid cancers, and for proton CSI they were non-melanoma skin, lung, and other solid cancers. In each setting, lung cancer accounted for half the risk of mortality. In conclusion, the predicted SMN risk for a 13-year-old girl undergoing proton CSI was reduced vs. photon CSI. This study demonstrates the feasibility of inter-institutional whole-body dose and risk assessments and also serves as a model for including risk estimation in personalized cancer care.
Cont . 
Serum IL-4 levels in patients with benign and malignant prostate disease. Each symbol in the dot plot represents the serum level as measured by ELISA for an individual patient. Results are expressed as median (horizontal line), flanked by the inter-quartile range. Differences were calculated using the Mann-Whitney U Test and are shown in the table as 2-tailed p values. 
Serum IL-13 levels in patients with benign and malignant prostate disease. Each symbol in the dot plot represents the serum level as measured by ELISA for an individual patient. Results are expressed as median (horizontal line), flanked by the inter-quartile range. Differences were calculated using the Mann-Whitney U Test and are shown in the table as 2-tailed p values. 
Prostate cancer is the most common cancer in men, both in the USA and Europe. Although incurable, metastatic disease can often be controlled for years with anti-androgen therapy. Once the disease becomes castrate resistant, the median survival is 18 months. There is growing evidence that the immune system, and in particular cytokines, play an important role in prostate cancer immunosurveillance and progression. Here, we have undertaken a clinical investigation of the role of two closely related cytokines, IL-4 and IL-13 in prostate cancer. In the largest series studied to date, we show that serum IL-4, but not IL-13 is significantly elevated in castrate resistant, compared to androgen sensitive disease. Notably however, serum IL-4 levels are also raised in patients with benign prostatic disease. Analysis of benign and malignant prostate tissue demonstrates that the source of IL-4 is epithelial cells rather than infiltrating leukocytes. Together, our data are consistent with a dual role for IL-4 in prostate cancer development. In benign disease, our data add to the evidence that IL-4 serves a protective role. By contrast, the data support a direct role for IL-4 in the progression of prostate cancer from androgen responsive, to advanced castrate-resistant disease.
STAT3 is a transcription factor that can be activated by phosphorylation on Tyr705 by Jaks and other kinases. STAT3 then regulates the expression of genes controlling processes including proliferation, survival, invasion, and angiogenesis. STAT3 can also be phosphorylated on Ser727. This is important for its effects on mitochondrial function. STAT3 may also have effects at other cellular sites, including microtubules and focal adhesions. 
The transcription factor STAT3 regulates genes that control critical cellular processes such as proliferation, survival, pluripotency, and motility. Thus, under physiological conditions, the transcriptional function of STAT3 is tightly regulated as one part of a complex signaling matrix. When these processes are subverted through mutation or epigenetic events, STAT3 becomes highly active and drives elevated expression of genes underlying these phenotypes, leading to malignant cellular behavior. However, even in the presence of activated STAT3, other cellular modulators can have a major impact on the biological properties of a cancer cell, which is reflected in the clinical behavior of a tumor. Recent evidence has suggested that two such key modulators are the activation status of other STAT family members, particularly STAT5, and the expression of STAT3-regulated genes that are part of negative feedback circuits, including microRNAs such as miR-146b. With attention to these newly emerging areas, we will gain greater insight into the consequence of STAT3 activation in the biology of human cancers. In addition, understanding these subtleties of STAT3 signaling in cancer pathogenesis will allow the development of more rational molecular approaches to cancer therapy.
Simulation planning of three-dimensional conformal radiotherapy. Five radiation beams (arrows) were set up to irradiate the clinical target volume (CTV), defined as main tumor and portal vein tumor thrombus. Doses of the high-dose beams were limited to 38.25 Gy/18 fractions/4 weeks; three additional low-dose beams of 6.75 Gy/18 fractions/4 weeks were required to elevate the dose to the CTV, resulting in a total dose of 45 Gy. The iso-dose distribution curves indicate 40, 30, 20, and 10 Gy, from innermost to outermost. 
Computed tomography (CT; 1-A, 2-A, and 3-A) prior to three-dimensional conformal radiation treatment (3DCRT) in three cases of hepatocellular carcinoma associated with portal vein tumor thrombi. Subfigures 1-B, 2-B, and 3-B show single photon emission computed tomography (SPECT). Case 1 is classified as localized type because the filling defect is localized by a surrounding area of high accumulation; Case 2 is classified as wedge type because the area of radioisotope (RI) filling defect is in a wedge form that separates areas of high accumulation; and Case 3 is classified as extensive wedge type because the area of RI filling defect occupies almost a whole lobe. Subfigures 1-C, 2-C, and 3-C show four iso-dose lines from simulation CT during radiation planning (from innermost to outermost: 40, 30, 20, and 10 Gy). The large arrows indicate high-dose beams of 38.25 Gy; small arrows indicate low-dose beams of 6.75 Gy. Subfigures 1-D, 2-D, and 3-D show SPECT at 2 months after 3DCRT. New filling defect lesions are evident compared with Subfigures 1-B, 2-B, and 3-B; we termed these lesions radiation-induced dysfunctional liver (RIDFL). They are shown as gray areas on Subfigures 1-E, 2-E, and 3-E, and are located within the 20 Gy iso-dose curve. 
Patient characteristics.
Scatter diagram of all patients showing the correlation between clinical target volume (CTV) and functional liver volume ratio in margin volume. A significant negative correlation is seen (correlation coefficient = − 0.610, p < 0.001). The three cases of radiation-induced liver disease (arrows) are at the upper margin of patients with similar CTVs. 
Purpose To analyze the distribution of functional liver volume (FLV) in the margin volume (MV) surrounding hepatocellular carcinoma (HCC) with portal vein tumor thrombus (PVTT) before radiation therapy (RT) and to verify the safety of single photon emission computed tomography-based three-dimensional conformal radiotherapy (SPECT-B3DCRT) by exploring the relation of FLV in MV to radiation-induced liver disease (RILD). Methods and Materials Clinical target volume (CTV) included main tumor and PVTT, and planning target volume (PTV) included CTV with a 10 mm margin. MV was defined as PTV–CTV. FLV ratio in MV was calculated as FLV in MV/MV × 100 (%). The two high-dose beams were planned to irradiate FLV as little as possible. Fifty-seven cases of HCC (26/57, 46%; Child–Pugh grade B) with PVTT underwent SPECT-B3DCRT which targeted the CTV to a total dose of 45 Gy/18 fractions. The destructive ratio was defined as radiation induced dysfunctional volume/FLV × 100 (%). Results We observed a significant negative correlation between FLV ratio in MV and CTV (p < 0.001). Three cases with CTVs of 287, 587 and 1184 cm3 experienced transient RILD. The FLV ratio in MV was highest in patients with RILD: nine patients with CTV of 200–300 cm3, three with CTV of 500–600 cm3, and two with CTV of 1100–1200 cm3. The destructive ratio yielded a mean value of 24.2 ± 1.5%. Conclusions Radiation planning that takes into account the distribution of FLV appears to result in the least possible RILD.
Molecular mechanisms of HIF-1 activation. Under normoxia, HIF-1 is hydroxylated on proline residues by prolyl hydroxylase domain proteins (PHDs). Prolyl-hydroxylated HIF-1 is bound by the von Hippel-Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1  for proteasomal degradation. Under hypoxia, the prolyl hydroxylation reaction on HIF-1 is inhibited by O 2 deprivation. HIF-1 degradation is inhibited and translocated into the nucleus where it couples with HIF-1β. The heterodimer then binds together with CBP/p300 to hypoxia-response element (HRE) in the promoters of target genes. HIF-1 proteins are also stabilized by intracellular reactive oxygen species (ROS), which block PHD activities. HIF-1 can also be regulated by oxygen-independent mechanisms. It can be activated by mutations of PTEN and VHL. In addition, HIF-1 can be activated by hyperactivity of the RAS/MAPK and PI3K-AKT-mTOR pathways following growth factor protein tyrosine kinase (PTK) signaling.
A deficient HIF-1 expression increases chemosensitivity to 5-FU in the gastric cancer cells MKN-45. (A) A Western blot analysis of HIF-1 in the HIF-1-deficient gastric cancer cell line MKN45-KD in which HIF-1 siRNA plasmids were transfected. Hypoxic induction of HIF-1 was completely knocked down in MKN45-KD compared with that observed in the control cell line MKN45-SC. (B) Effects of HIF-1 knockdown on sensitivity in xenografts of nude mice. 5-FU treatment was administered three times a week starting on Day 17. The tumor size before treatment was significantly larger in the MKN45-KD tumors than in the MKN45-SC tumors. The response rate to 5-FU, which was estimated based on tumor size on Day 35, was significantly better in the KD tumors than in the SC tumors (41.7% ± 38.3% vs. 141.38 ± 48.1%). (C) A TUNEL assay showed a significant increase in the number of apoptotic cells with nuclear staining in the HIF-1 KD tumors after 5-FU treatment.  
The HIF-1 expression is an unfavorable determinant of relapse in gastric cancer patients who undergo adjuvant 5-FU chemotherapy. (A) Immunohistochemical staining of HIF-1 in gastric cancer specimens. Nuclear staining of the HIF-1 expression is shown in the left panel, while the expression is negatively observed in the right panel. (B) Kaplan-Meier estimates of disease-specific survival (DSS) of the patients according to the HIF-1 expression. Among all 91 patients, the HIF-1-negative cases showed significantly longer DSS than the HIF-1-positive cases. Among the 64 patients who underwent adjuvant chemotherapy after surgery, the DSS of the HIF-1-negative cases was also significantly longer than that of the HIF-1-positive cases. On the other hand, in the 27 patients in the surgery alone group, the HIF-1 expression did not contribute to patient survival.  
The critical impact of HIF-1 on gastric cancer biology.  
Hypoxia inducible factor-1 (HIF-1) monitors the cellular response to the oxygen levels in solid tumors. Under hypoxia conditions, HIF-1a protein is stabilized and forms a heterodimer with the HIF-1β subunit. The HIF-1 complex activates the transcription of numerous target genes in order to adapt the hypoxic environment in human cancer cells. In gastric cancer patients, HIF-1a activation following extended hypoxia strongly correlates with an aggressive tumor phenotype and a poor prognosis. HIF-1a activation has been also reported to occur via hypoxia-independent mechanisms such as PI3K/AKT/mTOR signaling and ROS production. This article argues for the critical roles of HIF-1a in glucose metabolism, carcinogenesis, angiogenesis, invasion, metastasis, cell survival and chemoresistance, focusing on gastric cancer.
Important results were shown as cell survival points in the two panels of the figure which is reproduced in this Comment letter. A curve was fitted assuming the mono-exponential recovery half-time of 17 ± 21 minutes. The wide error limits indicate that this fit is not very good, but the notable feature of both panels is that the last four points are clearly continuing to rise, above the "fitted" curve. This indicates that there is a second, slower, component of repair or recovery and this Comment explores constructively the implications of that additional discovery.
Expression of CD133 and OATP4A1 mRNA in serum-free stem cell medium. Time course of real-time qPCR of CD133 and OATP4A1 expression in COLO205 colon cancer cells cultivated in serum-free stem cell medium for the indicated period of time (mean ± SD). Expression of CD133 was significantly increased at days 2–7.
Comparison of chemosensitivities of COLO 205 cells precultivated in either standard medium or serum-free stem cell medium. Viability of COLO 205 colon cancer cells precultivated in either normal tissue culture (con) or stem cell medium (CSC-like) after exposure to a range of chemotherapeutics in normal tissue culture medium for four days in vitro (mean ± SD). All differences were statistically significant, except for satraplatin.
Assessment of gene expression levels of CD133, CYP3A4 (A) as well as ALDH1A1 and AQ3 (B) in COLO205 cells cultured in serum-free stem cell medium. Gene expression of the stem cell markers CD133 and ALDH1A1 in addition to CYP3A4 and AQ3 in COLO205 cells cultivated in either standard (con) or serum-free stem cell medium (CSClike) for three or seven days was measured by real-time qPCR (mean ± SD).
Cancer stem cells (CSCs) seem to constitute a subpopulation of tumor cells that escape from chemotherapy and cause recurrent disease. Low proliferation rates, protection in a stem cell niche and overexpression of drug resistance proteins are considered to confer chemoresistance. We established an in vitro colon CSC-like model using the COLO 205 cell line, which revealed transiently increased expression of CD133 when transferred to serum-free stem cell culture medium. Assessment of global gene expression of COLO 205 cells under these conditions identified a set of upregulated genes including cytochrome P450 3A4 (CYP3A4) and aldehyde dehydrogenase 1A1 (ALDH1A1), as confirmed by real-time qPCR. ALDH1A1 is a CSC marker for certain tumor entities and confers resistance to cyclophosphamide. CYP3A4 is expressed in liver and colon and its overexpression seems particularly relevant in colon cancer, since it inactivates irinotecan and other xenobiotics, such as taxols and vinca alkaloids. In conclusion, this COLO 205 model provides evidence for CD133 induction concomitant with overexpression of CYP3A4, which, together with ATP-binding cassette, subfamily G, member 2 (ABCG2) and others, may have a role in chemoresistant colon CSCs and a negative impact on disease-free survival in colon cancer patients.
Diastereomeric and geometric analogs of calcipotriol, PRI-2202 and PRI-2205, were synthesized as advanced intermediates from vitamin D C-22 benzothiazoyl sulfones and side-chain aldehydes using our convergent strategy. Calcitriol, calcipotriol (PRI-2201) and tacalcitol (PRI-2191) were used as the reference compounds. Among a series of tested analogs the diastereomeric analog PRI-2202 showed the strongest antiproliferative activity on the human breast cancer cell line MCF-7, whereas the geometric analog PRI-2205 was the weakest. Both analogs were less potent in antiproliferative activity against HL-60 cells compared to the reference compounds. The ability to potentiate antiproliferative effect of cisplatin or doxorubicin against HL-60 cells or that of tamoxifen against the MCF-7 cell line was observed at higher doses of PRI-2202 or PRI-2205 than those of the reference compounds. The proapoptotic activity of tamoxifen, expressed as the diminished mitochondrial membrane potential, as well as the increased phosphatidylserine expression, was partially attenuated by calcitriol, PRI-2191, PRI-2201 and PRI-2205. The treatment of the MCF-7 cells with tamoxifen alone resulted in an increase in VDR expression. Moreover, a further increase in VDR expression was observed when the analogs PRI-2201 or PRI-2205, but not PRI-2191, were used in combination with tamoxifen. This observation could partially explain the potentiation of the antiproliferative effect of tamoxifen by vitamin D analogs.
Hypoxia affects the tumor microenvironment and is considered important to metastasis progression and therapy resistance. Thus far, the majority of global analyses of tumor hypoxia responses have been limited to just a single omics level. Combining multiple omics data can broaden our understanding of tumor hypoxia. Here, we investigate the temporal change of the metabolite composition with gene expression data from literature to provide a more comprehensive insight into the system level in response to hypoxia. Nuclear magnetic resonance spectroscopy was used to perform metabolomic profiling on the MDA-MB-231 breast cancer cell line under hypoxic conditions. Multivariate statistical analysis revealed that the metabolic difference between hypoxia and normoxia was similar over 24 h, but became distinct over 48 h. Time dependent microarray data from the same cell line in the literature displayed different gene expressions under hypoxic and normoxic conditions mostly at 12 h or earlier. The direct metabolomic profiles show a large overlap with theoretical metabolic profiles deduced from previous transcriptomic studies. Consistent pathways are glycolysis/gluconeogenesis, pyruvate, purine and arginine and proline metabolism. Ten metabolic pathways revealed by metabolomics were not covered by the downstream of the known transcriptomic profiles, suggesting new metabolic phenotypes. These results confirm previous transcriptomics understanding and expand the knowledge from existing models on correlation and co-regulation between transcriptomic and metabolomics profiles, which demonstrates the power of integrated omics analysis.
( A ). A three-dimensional v-shaped helix/ribbon computer model of human alpha-fetoprotein (HAFP) is displayed. GIP-34 amino acid buried segment ( D ) is shown in the black boxed configuration. Minimal energy computer model of GIP-34 and GIP-8 and their amino acid sequences are displayed above the v-shaped HAFP model ( B and C ). 
Growth Inhibitory Peptides (GIP-34, GIP-8, and a 34-mer scrambled (SCRM) peptide were evaluated for acute effects on cell membrane potential (current) and membrane resistance in cultured human breast cancer cells. MCF-7 cells were impaled in vivo with sharp microelectrodes under inverted phase contrast microscopy. 
A flow diagram showing the proposed mechanism of growth inhibition of cancer cells by GIP-34 (left side) and GIP-8 (right side). Solid black-line arrows indicate pathways verified by direct evidence; the dash-line arrows represent hypothetical, proposed, or published pathways.
The Alpha-fetoprotein (AFP) derived Growth Inhibitory Peptide (GIP) is a 34-amino acid segment of the full-length human AFP molecule that inhibits tumor growth and metastasis. The GIP-34 and its carboxy-terminal 8-mer segment, termed GIP-8, were found to be effective as anti-cancer therapeutic peptides against nine different human cancer types. Following the uptake of GIP-34 and GIP-8 into the cell cytoplasm, each follows slightly different signal transduction cascades en route to inhibitory pathways of tumor cell growth and proliferation. The parallel mechanisms of action of GIP-34 versus GIP-8 are demonstrated to involve interference of signaling transduction cascades that ultimately result in: (1) cell cycle S-phase/G2-phase arrest; (2) prevention of cyclin inhibitor degradation; (3) protection of p53 from inactivation by phosphorylation; and (4) blockage of K+ ion channels opened by estradiol and epidermal growth factor (EGF). The overall mechanisms of action of both peptides are discussed in light of their differing modes of cell attachment and uptake fortified by RNA microarray analysis and electrophysiologic measurements of cell membrane conductance and resistance. As a chemotherapeutic adjunct, the GIPs could potentially aid in alleviating the negative side effects of: (1) tamoxifen resistance, uterine hyperplasia/cancer, and blood clotting; (2) Herceptin antibody resistance and cardiac (arrest) arrhythmias; and (3) doxorubicin's bystander cell toxicity.
(a,b) Heart and lung DVHs.
Patient characteristics.
Coronal and transverse images of 3D-CRT plan (left), IMRT plan (middle), proton plan (right). 
Background. While neoadjuvant concurrent chemoradiotherapy has improved outcomes for esophageal cancer patients, surgical complication rates remain high. The most frequent perioperative complications after trimodality therapy were cardiopulmonary in nature. The radiation modality utilized can be a strong mitigating factor of perioperative complications given the location of the esophagus and its proximity to the heart and lungs. The purpose of this study is to make a dosimetric comparison of Intensity-Modulated Radiation Therapy (IMRT), proton and 3D conformal radiotherapy (3D-CRT) with regard to reducing perioperative cardiopulmonary complications in esophageal cancer patients. Materials. Ten patients with esophageal cancer treated between 2010 and 2013 were evaluated in this study. All patients were simulated with contrast-enhanced CT imaging. Separate treatment plans using proton radiotherapy, IMRT, and 3D-CRT modalities were created for each patient. Dose-volume histograms were calculated and analyzed to compare plans between the three modalities. The organs at risk (OAR) being evaluated in this study are the heart, lungs, and spinal cord. To determine statistical significance, ANOVA and two-tailed paired t-tests were performed for all data parameters. Results. The proton plans showed decreased dose to various volumes of the heart and lungs in comparison to both the IMRT and 3D-CRT plans. There was no difference between the IMRT and 3D-CRT plans in dose delivered to the lung or heart. This finding was seen consistently across the parameters analyzed in this study. Conclusions. In patients receiving radiation therapy for esophageal cancer, proton plans are technically feasible while achieving adequate coverage with lower doses delivered to the lungs and cardiac structures. This may result in decreased cardiopulmonary toxicity and less morbidity to esophageal cancer patients.
A commercially available technique named "NAVIGATOR" (Esaote, Italy) easily enables a 3-D reconstruction of a single 2-D acquisition of Contrast Enhanced Ultrasound (CEUS) imaging of the whole liver (with a volumetric correction thanks to the electromagnetic device of NAVIGATOR). Aim of the study was to evaluate this "panoramic" technique in comparison with conventional US and spiral CT in the detection of new hepatic lesions. 144 cirrhotic patients (previously treated for hepato cellular carcinoma (HCC)) in follow-up with detection of 98 new nodules (N), 28 multinodular (Nmulti), 14 loco-regional regrowth (LR) 94 efficaciously treated without new nodules (neg) and four multinodular without new nodules, were submitted to 200 examinations with this new technique from November 2008 to November 2009. 3DNavCEUS was performed using SonoVue (Bracco), as contrast agent, and a machine (Technos MPX, Esaote). Spiral CT and 3DNav CEUS were performed in the same month during follow up. Sens.,Spec.,diagn.-Acc.,PPV and NPV were evaluated; comparison and differences between the techniques were obtained with chi-square (SPSS release-15). Final diagnosis was: 98 new lesions (N) (one to three), 28 multinodular HCC (Nmulti) and 14 loco-regional regrowth (LR); in 94 no more lesions were observed during follow-up; conventional US obtained: 58 N (+18 multinodularN and 8 LR), 40 false negative (+10 Nmulti and 6 LR) (sens:59.2, spec:100%, Diagn Accur:73.6, PPV:100; NPV:70.1); spiral CT obtained: 84N (+26-multinodularN and 14-LR), 14 false-negative (+2-Nmulti), and one false-positive (sens:85.7, spec:97.9%, Diagn Accur:90.9, PPV:97.7; NPV:86.8); 3DNAV obtained: 92N (+28 multinodularN and 14LR), 6 false-negative, and two false-positives (sens:93.9, spec:97.9%, Diagn Accur:95.6, PPV:97.9; NPV:93.9). 3-DNav CEUS is significantly better than US and almost similar to spiral CT for detection of new HCC. This technique, in particular, showed the presence of lesions even in the cases not detected with spiral CT.
Baseline characteristics of 450 patients with incident melanoma shown as absolute (relative) frequencies or means ± SD. 
The present study was aimed at assessing quality of life (QoL) in a total of 450 melanoma patients who filled out the EORTC QLQ-C30 (Q1; 15 months post diagnosis) as part of the OVIS Study. Follow-up questionnaires (Q2) were administered two years after Q1. The analyses presented herein were based on the following assumptions: QoL of melanoma patients is worse than that of a German reference population. Further, both tumor location and tumor stage have an influence on self-reported QoL, with patients with tumors located on face, head, neck, and advanced tumor stage (T3/T4) reporting the worst QoL levels. Finally, patients' QoL improves over time based on the theory of disease adaptation. In contrast to the above assumptions, with the exception of global health/QoL scores, differences between OVIS and the reference population were below the minimal clinical important difference of ten points. Furthermore, no clinically meaningful differences were found between patients after stratifying our data by tumor location and tumor stage. Finally, no clinically relevant changes were seen between Q1 and Q2 across all scales of the EORTC QLQ-C30. However, when data were stratified by patients with stable disease versus those with progression, clinically relevant differences were found between Q1 and Q2 predominantly in women in the latter group regarding emotional function, insomnia, dyspnoea, and fatigue. The lack of clinically meaningful differences across strata (tumor location; tumor stage), time, and patients compared to a reference population is surprising. However, it is possible that the instrument used, a generic QoL instrument, is generally not sensitive enough to detect differences in melanoma patients. Our findings may further be explained by the fact that all patients included in our sample had been diagnosed well before Q1, i.e., main illness adaptation processes may have occurred before study entry.
Clinicopathologic data of the colorectal lesions analyzed. 
Gene mutation frequency in the colorectal lesions analyzed.
Anatomical location of the colorectal lesions with an altered Wnt or Ras-Raf- MEK-MAPK pathway. 
Anatomical location of the colorectal lesions with an altered Wnt or Ras-Raf- MEK-MAPK pathway.
In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form. CTNNB1 and B-Raf were screened by PCR-single-strand conformation polymorphism analysis, K-Ras by restriction fragment length polymorphism analysis and the APC gene mutation cluster region (codons 1243-1567) by direct DNA sequencing. APC mutations were only detected in 10% of the serrated lesions but in 34% of the adenomas. Twenty percent of the serrated lesions and 14% of the adenomas carried a mutated K-Ras. B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas. CTNNB1 was altered in 12% of the adenomas, but not in serrated lesions. By using the above gene marker panel it could be shown that 65% of the serrated lesions and 61% of the adenomas carried at least one of the four genes in a mutated form. Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer.
Percentage of growth inhibition of COLO 858, MEL HO and COLO 679 human melanoma cells at first doubling time in in vitro cultures following the addition of increasing concentrations (10 to 100 μM) of L-732,138. Level of significance: * p ≤ 0.05.
Induction of cell proliferation of COLO 858, MEL HO and COLO 679 human melanoma cells by SP at several nanomolar concentrations (5, 10, 50, 100 and 500 nM) according to the cell line studied. The NK-1 receptor antagonist L-732,138 was added in the presence (the most mitogenic exogenous SP nM concentration) or absence (none) of SP during the first doubling time. In both cases, L-732,138 inhibited cell proliferation. Using the ANOVA test, a significant difference between each group and the control group (none-none) was found. Level of significance: ** p ≤ 0.05 and # and x p ≤ 0.05. Vertical bars indicate SD.
Culture of COLO 858 melanoma cells not treated (A) or treated with the NK-1 receptor antagonist L-732,138 (B). (C) High power magnification of the region delimited by the yellow rectangle in B. Note the apoptotic figures. Chromatin condensation and nuclear fragmentation are observed.
It has been recently demonstrated that substance P (SP) and neurokinin-1 (NK-1) receptor antagonists induce cell proliferation and cell inhibition in human melanoma cells, respectively. However, the antitumor action of the NK-1 receptor antagonist L-732,138 on such cells is unknown. The aim of this study was to demonstrate an antitumor action of L-732,138 against three human melanoma cell lines (COLO 858, MEL HO, COLO 679). We found that L-732,138 elicits cell growth inhibition in a concentration dependent manner in the melanoma cells studied. Moreover, L-732,138 blocks SP mitogen stimulation. The specific antitumor action of L-732,138 occurred through the NK-1 receptor and melanoma cell death was by apoptosis. These findings indicate that the NK-1 receptor antagonist L-732,138 could be a new antitumor agent in the treatment of human melanoma.
Purpose To report the toxicity and long-term outcomes of dose-escalated intensity-modulated radiation therapy (IMRT) for patients with localised prostate cancer. Methods and Materials From 2001 to 2005, a total of 125 patients with histologically confirmed T1-3N0M0 prostate cancer were treated with IMRT to 74Gy at the Austin Health Radiation Oncology Centre. The median follow-up was 5.5 years (range 0.5–8.9 years). Biochemical prostate specific antigen (bPSA) failure was defined according to the Phoenix consensus definition (absolute nadir + 2ng/mL). Toxicity was scored according to the RTOG/EORTC criteria. Kaplan-Meier analysis was used to calculate toxicity rates, as well as the risks of bPSA failure, distant metastases, disease-specific and overall survival, at 5 and 8-years post treatment. Results All patients completed radiotherapy without any treatment breaks. The 8-year risks of ≥ Grade 2 genitourinary (GU) and gastrointestinal (GI) toxicity were 6.4% and 5.8% respectively, and the 8-year risks of ≥ Grade 3 GU and GI toxicity were both < 0.05%. The 5 and 8-year freedom from bPSA failure were 76% and 58% respectively. Disease-specific survival at 5 and 8 years were 95% and 91%, respectively, and overall survival at 5 and 8 years were 90% and 71%, respectively. Conclusions These results confirm existing international data regarding the safety and efficacy of dose-escalated intensity-modulated radiation therapy for localised prostate cancer within an Australian setting.
The t(7;12)(q36;p13) translocation is a recurrent chromosome abnormality that involves the ETV6 gene on chromosome 12 and has been identified in 20-30% of infant patients with acute myeloid leukaemia (AML). The detection of t(7;12) rearrangements relies on the use of fluorescence in situ hybridization (FISH) because this translocation is hardly visible by chromosome banding methods. Furthermore, a fusion transcript HLXB9-ETV6 is found in approximately 50% of t(7;12) cases, making the reverse transcription PCR approach not an ideal screening method. Considering the report of few cases of variant translocations harbouring a cryptic t(7;12) rearrangement, we believe that the actual incidence of this abnormality is higher than reported to date. The clinical outcome of t(7;12) patients is believed to be poor, therefore an early and accurate diagnosis is important in the clinical management and treatment. In this study, we have designed and tested a novel three-colour FISH approach that enabled us not only to confirm the presence of the t(7;12) in a number of patients studied previously, but also to identify a cryptic t(7;12) as part of a complex rearrangement. This new approach has proven to be an efficient and reliable method to be used in the diagnostic setting.
Summary of TRR and RECIST criteria as defined by Eisenhauer, Therasse et al. 2009 [4]. 
Clinical studies of 99m Tc-HYNIC-Annexin A5 in oncology. 
Evaluation of efficacy of anti-cancer therapy is currently performed by anatomical imaging (e.g., MRI, CT). Structural changes, if present, become apparent 1-2 months after start of therapy. Cancer patients thus bear the risk to receive an ineffective treatment, whilst clinical trials take a long time to prove therapy response. Both patient and pharmaceutical industry could therefore profit from an early assessment of efficacy of therapy. Diagnostic methods providing information on a functional level, rather than a structural, could present the solution. Recent technological advances in molecular imaging enable in vivo imaging of biological processes. Since most anti-cancer therapies combat tumors by inducing apoptosis, imaging of apoptosis could offer an early assessment of efficacy of therapy. This review focuses on principles of and clinical experience with molecular imaging of apoptosis using Annexin A5, a widely accepted marker for apoptosis detection in vitro and in vivo in animal models. 99mTc-HYNIC-Annexin A5 in combination with SPECT has been probed in clinical studies to assess efficacy of chemo- and radiotherapy within 1-4 days after start of therapy. Annexin A5-based functional imaging of apoptosis shows promise to offer a personalized medicine approach, now primarily used in genome-based medicine, applicable to all cancer patients.
Clinical staging of intra-abdominal desmoids.
Summary of outcomes in patients with advanced intra-abdominal desmoid tumors.
Treatment algorithm for the surgical management of complicated intra-abdominal desmoids tumors.
Advanced intra-abdominal desmoids tumors present with severe symptoms, complications or rapid growth, which lead to adverse outcomes. Our aim was to evaluate the treatment and outcome of patients with advanced intra-abdominal desmoids tumors, and develop guidelines for surgical management of these patients. We reviewed the clinical courses of 21 adult patients with advanced stage intra-abdominal desmoid tumors who presented to an intestinal rehabilitation and transplantation program. Patients with massive intestinal resection presented in two groups. The first group had a short small intestinal remnant after resection ( < 60 cm). These patients were poor rehabilitation candidates and eventually met criteria for transplant. The second had longer intestinal remnants and were more successfully rehabilitated and have not had complications that would lead to transplantation. Advanced intra-abdominal desmoid tumors have outcomes after resection that merit aggressive resection and planned intestinal rehabilitation and intestinal transplantation as indicated.
Seventy-one years old, female with desmoid (arrows) in left iliopsoas muscle. ( A ) T2-weighed axial image of MRI before meloxicam treatment. ( B ) MRI of the same patient after 54 months’ treatment with meloxicam. 
Clinical data of patients with desmoid.
Twenty years old female with desmoid (arrows) in abdominal rectus muscle. ( A ) T2-weighed sagittal image of MRI before meloxicam treatment. ( B ) MRI of the same patient after 8 months’ treatment with meloxicam . The patient was subjected to the surgical treatment. 
Treatment modalities for desmoid tumors have been changed because of the high recurrence rate, even after wide resection, and some cases experience spontaneous self-regression during clinical course. The treatment modality in our institutions before 2003 was surgical resection with wide surgical margin, however, meloxicam, which is a NSAID and a selective COX-2 inhibitor has been applied consecutively since 2003. We reviewed the previously reported outcomes of surgical and conservative treatment in our institutions. Among 30 patients receiving surgical treatment, 16 (53%) recurred. Younger age ( p < 0.05) was a significant poor factor. According to RECIST for meloxicam treatment, CR was in one, PR in 10, SD in eight, PD in one evaluated at 2011. Older age ( p < 0.01) was significantly associated with good outcome for meloxicam treatment. Results of the previous study indicated that surgical treatment alone could not control desmoid tumors, even with negative surgical margin. Considering the functional impairment resulting from surgery with negative surgical margin, a conservative and effective treatment modality with fewer complications is desired. Conservative treatment with meloxicam is a promising novel modality for patients with extra-abdominal desmoid tumors.
Demographic information for patients in the present study who received chemotherapy for treatment of fibromatosis.
Cytotoxic chemotherapy response rates of prior studies evaluating the effects of chemotherapy on fibromatosis.
Histological appearance of fibromatosis.
T2 sagittal MRI images before and after treatment demonstrate significant reduction in lesion size after treatment.
Fibromatosis, or extra-abdominal desmoid tumor, is a benign disease which often has an aggressive clinical course that can be difficult to treat. We performed a retrospective review of 16 patients (12 females and four males) with a mean age of 34.2 years treated with methotrexate and vinblastine for newly diagnosed or recurrent extra-abdominal desmoid tumor. The mean age of our patient cohort was 34.2 years (range 11-70), and the mean tumor size was 11.5 cm (range 2.5-21.2 cm). The mean duration of therapy was 12 months with an average follow-up of 43 months (range 1-149 months). Fourteen of 16 patients demonstrated a clinical response to treatment. Eight of 14 patients demonstrated a radiologic decrease in tumor size. Only one patient progressed on therapy. Six patients developed recurrent symptoms after discontinuation of treatment. Chemotherapy-related symptoms including neutropenia, nausea, and vomiting were common and observed in most patients, however these side effects were mild and transient. Five patients developed peripheral neuropathy that prompted a change from vinblastine to vinorelbine during treatment. One potentially life-threatening complication (pneumocystis pneumonia) occurred which was diagnosed early and successfully treated. The use of methotrexate and vinblastine/vinorelbine in the management of fibromatosis appears to be an effective treatment with minimal treatment-related side effects.
Abdominal Non-Hodgkin lymphomas (NHL) are the most common extra nodal presentation of pediatric NHL. Our aim is to assess the role of surgery as a risk factor and to evaluate the impact of risk-adjusted systemic chemotherapy on survival of patients with stages II and III disease. This study included 35 pediatric patients with abdominal NHL treated over five years at South Egypt Cancer Institute (SECI), Assiut University, between January 2005 and January 2010. The data of every patient included: Age, sex, and presentation, staging work up to determine extent of the disease and the type of resection performed, histopathological examination, details of chemotherapy, disease free survival and overall survival. The study included 25 boys and 10 girls with a median age of six years (range: 2.5:15). Thirty patients (86%) presented with abdominal pain, 23 patients (66%) presented with abdominal mass and distention, 13 patients (34%) presented with weight loss, and intestinal obstruction occurred in six patients (17%). The ileo-cecal region and abdominal lymph nodes were the commonest sites (48.5%, 21% respectively). Burkitt's lymphoma was the most common histological type in 29 patients (83%). Ten (28.5%) stage II (group A) and 25 (71.5%) stage III (group B). Complete resections were performed in 10 (28.5%), debulking in 6 (17%) and imaging guided biopsy in 19 (54%). A11 patients received systemic chemotherapy. The median follow up duration was 63 months (range 51-78 months). The parameters that significantly affect the overall survival were stage at presentation complete resection for localized disease. In conclusion, the extent of disease at presentation is the most important prognostic factor in pediatric abdominal NHL. Surgery is restricted to defined situations such as; abdominal emergencies, diagnostic biopsy and total tumor extirpation in localized disease. Chemotherapy is the cornerstone in the management of pediatric abdominal NHL.
COBRA methylation analyses of RASSF2, RASSF5A, RASSF5C and RASSF10 in tumor tissue. Methylation analysis by COBRA for RASSF2, RASSF5A, RASSF5C and RASSF10 was performed and representative results are shown for different tumor samples (numbers are indicated above each gel). DNA was bisulfite treated and COBRA PCR with according primers was performed. Mock ( − ) and TaqI (+) digested PCR products are resolved in 2% TBE agarose gel together with 100 bp marker. An in vitro methylated (ivm) DNA was used as positive control. Methylated samples (m) are indicated below and PCR product sizes are shown beside picture. 
Merkel cell carcinoma (MCC) is one of the most aggressive cancers of the skin. RASSFs are a family of tumor suppressors that are frequently inactivated by promoter hypermethylation in various cancers. We studied CpG island promoter hypermethylation in MCC of RASSF2, RASSF5A, RASSF5C and RASSF10 by combined bisulfite restriction analysis (COBRA) in MCC samples and control tissue. We found RASSF2 to be methylated in three out of 43 (7%), RASSF5A in 17 out of 39 (44%, but also 43% in normal tissue), RASSF5C in two out of 26 (8%) and RASSF10 in 19 out of 84 (23%) of the cancer samples. No correlation between the methylation status of the analyzed RASSFs or between RASSF methylation and MCC characteristics (primary versus metastatic, Merkel cell polyoma virus infection, age, sex) was found. Our results show that RASSF2, RASSF5C and RASSF10 are aberrantly hypermethylated in MCC to a varying degree and this might contribute to Merkel cell carcinogenesis.
Glioblastoma multiforme (GBM), a WHO grade IV malignant glioma, is the most common and lethal primary brain tumor in adults; few treatments are available. Median survival rates range from 12-15 months. The biological characteristics of this tumor are exemplified by prominent proliferation, active invasiveness, and rich angiogenesis. This is mainly due to highly deregulated signaling pathways in the tumor. Studies of these signaling pathways have greatly increased our understanding of the biology and clinical behavior of GBM. An integrated view of signal transduction will provide a more useful approach in designing novel therapies for this devastating disease. In this review, we summarize the current understanding of GBM signaling pathways with a focus on potential molecular targets for anti-signaling molecular therapies.
Aberrant expression and activation of oncogenes in somatic cells has been associated with cancer initiation. Required for reacquisition of pluripotency in the developing germ cell, PRDM14 initiates lymphoblastic leukemia when misexpressed in murine bone marrow. Activation of pluripotency in somatic cells can lead to aneuploidy and copy number alterations during iPS cell generation, and we hypothesized that PRDM14-induced lymphoblastic leukemias would demonstrate significant chromosomal damage. High-resolution oligo array comparative genomic hybridization demonstrated infrequent aneuploidy but frequent amplification and deletion, with amplifications occurring in a 5:1 ratio with deletions. Many deletions (i.e., Cdkn2a, Ebf1, Pax5, Ikzf1) involved B-cell development genes and tumor suppressor genes, recapitulating deletions occurring in human leukemia. Pathways opposing senescence were frequently deactivated via Cdkn2a deletion or Tbx2 amplification, with corollary gene expression. Additionally, gene expression studies of abnormal pre-leukemic B-precursors showed downregulation of genes involved in chromosomal stability (i.e., Xrcc6) and failure to upregulate DNA repair pathways. We propose a model of leukemogenesis, triggered by pluripotency genes like Prdm14, which involves ongoing DNA damage and failure to activate non-homologous end-joining secondary to aberrant gene expression.
―Chronological‖ steps in tumor angiogenesis (marked in green). Causative mechanisms are marked in yellow and pro-angiogenic factors are marked in red. 
Newly formed microvessels in most solid tumors show an abnormal morphology and thus do not fulfil the metabolic demands of the growing tumor mass. Due to the chaotic and heterogeneous tumor microcirculation, a hostile tumor microenvironment develops, that is characterized inter alia by local hypoxia, which in turn can stimulate the HIF-system. The latter can lead to tumor progression and may be involved in hypoxia-mediated radioresistance of tumor cells. Herein, cellular and molecular mechanisms in tumor angiogenesis are discussed that, among others, might impact hypoxia-related radioresistance.
Molecular characteristics of ACFs.
Three ACF types and possible pathways in colon carcinogenesis. The inset shows the speculated pattern of proliferation zone in the crypt of subtypes of ACF. Hypothetical scheme for the transition of crypts from normal to dysplastic ACF showing nuclear morphometry (dark blue is ACF; purple is stratified nucleus). Three types of dysplastic ACF reported in the literature: mucin depleted foci (MDF, goblet cells in pink are lost and replaced by stratified nuclei in purple; brown represent β-catenin which can accumulate in MDF), β-catenin accumulated crypts (BCAC, brown denoted nuclear β-catenin within the crypts) and flat-dysplastic ACF (flat ACF, brown is β-catenin within highly dysplastic and pleiomorphic crypts).
Aberrant crypt foci (ACF) are one of the earliest histopathological manifestations of colon cancer. In this review, we critically present the molecular, cellular, histopathological, and chemopreventive evidence that ACF are relevant biomarkers for colon cancer. The laboratory and clinical evidence are highly suggestive that ACF are in the pathway leading to colon cancer, but not all ACF will do so. The possible fate and outcome of ACF in the progression toward colon cancer may be dependent on a number of features that define their predictive power for the prevention or progression of cancer.
Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5 is predominantly present in the cytoplasm and the survival of the Jak2(V617F)+ HEL cells is impeded through the inhibition of the cytoplasmic functions of Stat5.
The management and diagnosis of renal tumors have changed significantly over the last decade. Due to advances in imaging techniques, more than 50% of kidney tumors are discovered incidentally and many of them represent an early stage lesion. This has stimulated the development of nephron-sparing surgery and of the minimally invasive treatment options including ablative techniques, i.e., radiofrequency ablation (RFA) and cryoablation. The objective of the minimally invasive approach is to preserve the renal function and to lower the perioperative morbidity. RFA involves inducing the coagulative necrosis of tumor tissue. Being probably one of the least invasive procedures in kidney tumor management, RFA may be performed percutaneously under ultrasound (US), computed tomography (CT) or magnetic resonance (MR) guidance. Most of the studies show that the RFA procedure is efficient, safe and has a low complication rate. Due to the still limited data on the oncological outcome of RFA, the indication for this intervention remains limited to selected patients with small organ-confined renal tumors and contraindication to surgery or who have a solitary kidney. The aim of our study is to review the literature on RFA of kidney tumors.
Demographics and clinical data of 106 patients who underwent radiofrequency ablation for pancreatic ductal adenocarcinoma. Data are reported as absolute number, frequency, median and range. NR = not reported.
Radiofrequency ablation (RFA) and ultrasonographic (US) evaluation of the technique in a patient with locally advanced pancreatic head carcinoma. Panel A. Intraoperative US showing the pancreatic adenocarcinoma; Panel B. Intraoperative RFA; Panel C. Intraoperative US showing the ablated carcinoma.
Type and frequency of postoperative complications in 30 patients who received radiofrequency ablation for pancreatic adenocarcinoma. One or more complication may be present in the same patient.
Advanced ductal pancreatic carcinoma (PC) remains a challenge for current surgical and medical approaches. It has recently been claimed that radiofrequency ablation (RFA) may be beneficial for patients with locally advanced or metastatic PC. Using the MEDLINE database, we found seven studies involving 106 patients in which PC was treated using RFA. The PC was mainly located in the pancreatic head (66.9%) with a median size of 4.6 cm. RFA was carried out in 85 patients (80.1%) with locally advanced PC and in 21 (19.9%) with metastatic disease. Palliative surgical procedures were carried out in 41.5% of the patients. The average temperature used was 90 °C (with a temperature range of 30-105 °C) and the ratio between the number of passes of the probe and the size of the tumor in centimeters was 0.5 (range of 0.36-1). The median postoperative morbidity and mortality were 28.3% and 7.5%, respectively; the median survival was 6.5 months (range of 1-33 months). In conclusion, RFA is a feasible technique: however, its safety and long-term results are disappointing; Thus, the RFA procedure should not be recommended in clinical practice for a PC patient.
Some comments about the role of ablation techniques in the management of advanced pancreatic cancer as palliative procedure.
Radiofrequency ablation in the management of advanced pancreatic cancer should be no longer utilized in patients with locally advanced or metastatic pancreatic adenocarcinoma.
Genetic and epigenetic molecular pathological mechanism of onset and progression of mucosa associated lymphoid tissue (MALT) lymphoma. Arrows indicate genetic or epigenetic events during onset and/or progression of MALT lymphoma.
Schematic illustration of development and progression of gastric lymphoma in terms of specific gene methylation. Genes associated with orange arrows indicate significant increase of methylation frequency from arrow start point status to end point status. On the other hand, genes associated with green arrow show statistically significant decrease of methylation frequency from start-point to end-point status (p < 0.05). Left panel: schematic illustration of H. pylori (-) L-MALT-related diseases in terms of CpG hypermethylation; right panel: schematic illustration of H. pylori (+) L-MALT related diseases in terms of CpG hypermethylation.
Natural course from the infection of human T lymphotropic virus type-I (HTLV-I) to onset and progression of adult T-cell leukemia/lymphoma (ATLL). Accumulation of genetic and epigenetic changes in host and virus genome during long latent period induce onset of ATLL.
Although cancers have been thought to be predominantly driven by acquired genetic changes, it is becoming clear that microenvironment-mediated epigenetic alterations play important roles. Aberrant promoter hypermethylation is a prevalent phenomenon in human cancers as well as malignant lymphoma/leukemia. Tumor suppressor genes become frequent targets of aberrant hypermethylation in the course of gene-silencing due to the increased and deregulated DNA methyltransferases (DNMTs). The purpose of this article is to review the current status of knowledge about the contribution of cumulative epigenetic abnormalities of the host genes after microbial and virus infection to the crisis and progression of malignant lymphoma/leukemia. In addition, the relevance of this knowledge to malignant lymphoma/leukemia assessment, prevention and early detection will be discussed.
Among the most challenging of clinical targets for cancer immunotherapy are Tumor Associated Carbohydrate Antigens (TACAs). To augment immune responses to TACA we are developing carbohydrate mimetic peptides (CMPs) that are sufficiently potent to activate broad-spectrum anti-tumor reactivity. However, the activation of immune responses against terminal mono- and disaccharide constituents of TACA raises concerns regarding the balance between "tumor destruction" and "tissue damage", as mono- and disaccharides are also expressed on normal tissue. To support the development of CMPs for clinical trial testing, we demonstrate in preclinical safety assessment studies in mice that vaccination with CMPs can enhance responses to TACAs without mediating tissue damage to normal cells expressing TACA. BALB/c mice were immunized with CMPs that mimic TACAs reactive with Griffonia simplicifolia lectin 1 (GS-I), and tissue reactivity of serum antibodies were compared with the tissue staining profile of GS-I. Tissues from CMP immunized mice were analyzed using hematoxylin and eosin stain, and Luxol-fast blue staining for myelination. Western blots of membranes from murine mammary 4T1 cells, syngeneic with BALB/c mice, were also compared using GS-I, immunized serum antibodies, and naive serum antibodies. CMP immunization enhanced glycan reactivities with no evidence of pathological autoimmunity in any immunized mice demonstrating that tissue damage is not an inevitable consequence of TACA reactive responses.
Molecular and Cellular Consequences Associated with p53 Acetylation a . 
Cellular stress leads to p53 transcriptional activation of downstream targets. Normally, p53 levels are kept low by its major antagonist, Mdm2, an E3 ubiquitin ligase that is itself a transcriptional target of p53. Stress signals, such as DNA damage, oncogene activation and hypoxia, promote p53 stability and activity by inducing posttranslational modifications (PTMs) and tetramerization of p53. p53 functions as a transcription factor that binds to specific p53 response elements upstream of its target genes. p53 affects many important cellular processes linked to tumor suppression, including the induction (green) of senescence, apoptosis, and DNA repair as well as inhibition (red) of metabolism, angiogenesis, and cell migration. These functions are largely mediated through transcriptional regulation of its targets (examples given). 
The p53 signaling pathway is altered in the majority of human cancers. The TP53 gene is deleted or mutated in approximately 55% of human cancers while its signaling is disrupted by alterations to its many regulators (listed to the right) and/or targets (not listed) in the remaining tumors. 
p53 acetyltransferases are targeted by multiple proteins that regulate their function. Schematic of p53 highlighting its acetylation by six different histone acetyltransferases, p300/CBP, PCAF, Tip60, MOF, and MOZ. The ability of these acetyltransferases to regulate p53 is influenced both positively (arrows) and negatively (perpendicular bars) by many types of proteins, including the indicated viral proteins, ubiquitin ligases, kinases, and other cellular factors. TAD, transactivation domain; TET, tetramerization domain, REG, regulatory domain. 
Acetylation-dependent mechanisms of p53 activation. Two primary mechanisms exist by which p53 function is enhanced by its acetyltransferases. (I) C-terminal acetylation of p53 by p300/CBP and PCAF promotes an open conformation of p53 by preventing the occlusion of the DNA binding domain by the C-terminal tail. This enhances p53 transcriptional activity, leading to growth arrest and/or apoptosis; (II) Interactions of MYST HATs with the central domain of p53 containing K120. Tip60 is an essential component of p53 signaling that activates p53 through direct association on target promoters as well as acetylation of p53 at K120. p21 expression is induced by Tip60-p53 complexes that bind to and promote histone H4 acetylation at the p21 promoter, which leads to a potent growth arrest. K120 acetylation mediated by Tip60 and MOF selectively increases p53 binding to apoptotic gene promoters, thus inducing cell death. Unlike Tip60 and MOF, MOZ-mediated K120 acetylation of p53 specifically induces senescence by promoting transactivation of the p21 promoter. For simplicity, other concomitant p53 acetylation events and association with p300/CBP and/or PCAF are not shown. 
Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.
The epithelial to mesenchymal transition (EMT) enables epithelial cells with a migratory mesenchymal phenotype. It is activated in cancer cells and is involved in invasion, metastasis and stem-like properties. ZEB1, an E-box binding transcription factor, is a major suppressor of epithelial genes in lung cancer. In the present study, we show that in H358 non-small cell lung cancer cells, ZEB1 downregulates EpCAM (coding for an epithelial cell adhesion molecule), ESRP1 (epithelial splicing regulatory protein), ST14 (a membrane associated serine protease involved in HGF processing) and RAB25 (a small G-protein) by direct binding to these genes. Following ZEB1 induction, acetylation of histone H4 and histone H3 on lysine 9 (H3K9) and 27 (H3K27) was decreased on ZEB1 binding sites on these genes as demonstrated by chromatin immunoprecipitation. Of note, decreased H3K27 acetylation could be also detected by western blot and immunocytochemistry in ZEB1 induced cells. In lung cancers, H3K27 acetylation level was higher in the tumor compartment than in the corresponding stroma where ZEB1 was more often expressed. Since HDAC and DNA methylation inhibitors increased expression of ZEB1 target genes, targeting these epigenetic modifications would be expected to reduce metastasis.
Definition of relative biological effect (RBE). 
Relationship between LET and RBE, for variable low LET α values (Gy −1 ), as shown by the colour codes. The low LET β value is 0.035 Gy −2 , increasing with LET. The change in the RBE rank order at lower values of α is due to the higher preponderance of β related cell kill at low α values. 
The proposal to randomise between a tumour RBE of 1 against a tumour RBE of 1.1 in radiosensitive tumours. Also, and in other studies of tumours of normal or reduced radiosensitivity, randomisation between an RBE of 1.2 (or higher) in late reacting normal tissues compared with 1.1 should also be considered. 
Dose status for each ICRU volume and likely clinical outcome changes produced by CPT compared with MXT, where ↑ is an increased dose, = is the "equivalent" dose and ↓ is a reduced dose for the CPT. OTV is the volume of the body outside the PTV. 
Despite increasing use of proton therapy (PBT), several systematic literature reviews show limited gains in clinical outcomes, with publications mostly devoted to recent technical developments. The lack of randomised control studies has also hampered progress in the acceptance of PBT by many oncologists and policy makers. There remain two important uncertainties associated with PBT, namely: (1) accuracy and reproducibility of Bragg peak position (BPP); and (2) imprecise knowledge of the relative biological effect (RBE) for different tissues and tumours, and at different doses. Incorrect BPP will change dose, linear energy transfer (LET) and RBE, with risks of reduced tumour control and enhanced toxicity. These interrelationships are discussed qualitatively with respect to the ICRU target volume definitions. The internationally accepted proton RBE of 1.1 was based on assays and dose ranges unlikely to reveal the complete range of RBE in the human body. RBE values are not known for human (or animal) brain, spine, kidney, liver, intestine, etc. A simple efficiency model for estimating proton RBE values is described, based on data of Belli et al. and other authors, which allows linear increases in α and β with LET, with a gradient estimated using a saturation model from the low LET α and β radiosensitivity parameter input values, and decreasing RBE with increasing dose. To improve outcomes, 3-D dose-LET-RBE and bio-effectiveness maps are required. Validation experiments are indicated in relevant tissues. Randomised clinical studies that test the invariant 1.1 RBE allocation against higher values in late reacting tissues, and lower tumour RBE values in the case of radiosensitive tumours, are also indicated.
A comparison between the interaction spaces presented by the spite models proposed by Hamilton (left panel) and Wilson (right panel). Mutualism could be represented through the support of tumor growth by nonmalignant cells [30]. Moreover, some cancer cells become addicted to InterLeukin-3 to survive (primed cell for death) and so benefit from the immune system, and this is a kind of selfishness [31]. Yet, microenvironment acidity-induced cancer spite (MAICS) results in excessive cell death and eventually might prevent its spreadability and so it lies under the altruism umbrella because it results in an encapsulated tumor [32]. Cannibalism at the end results in death of both normal and malignant cells under the context of organismal selection so it is compatible with spite. Therefore, it would be very interesting if further studies carry on for determining how tumors handle the thresholds of the four quadrants.
Here we propose a hypothetical model in which the environment where the player interactions are carried out (i.e., the acidic extracellular space; blue background) can play an important role in determining the choice and dynamics of spite. At this point, it is not possible to determine if there is a space-time quadrant where the environment has a greater or lesser effect and this will be an interesting point for future research. (a) Because microenvironment acidity-induced cancer spite (MAICS) blunts the immune system [33], interference with MAICS would probably create a shifting to Wilsonian spite. Thus, the third party reappears (Z-axis in (b) (Wilsonian spite) (b) This kind of shifting does not misconstrue to malignant and normal cells only but a spite process could happen also between secondary tumor cells (cannibal cells) and primary tumor cells.
Most cancer cells shift their metabolic pathway from a metabolism reflecting the Pasteur-effect into one reflecting the Warburg-effect. This shift creates an acidic microenvironment around the tumor and becomes the driving force for a positive carcinogenesis feedback loop. As a consequence of tumor acidity, the tumor microenvironment encourages a selection of certain cell phenotypes that are able to survive in this caustic environment to the detriment of other cell types. This selection can be described by a process which can be modeled upon spite: the tumor cells reduce their own fitness by making an acidic environment, but this reduces the fitness of their competitors to an even greater extent. Moreover, the environment is an important dimension that further drives this spite process. Thus, diminishing the selective environment most probably interferes with the spite process. Such interference has been recently utilized in cancer treatment.
Summary of the patients.
(A) Patient 4: black to brown colored pigmented patch on the left ring finger (B) Patient 7: irregularly pigmented patch on the right heel.
(A) Patient 5: retiform epidermal hyperplasia with somewhat broad rete pegs (H&E, ×40); (B) Patient 5: diffuse lentiginous pattern of melanocytic proliferation along the dermo-epidermal junction with hyperplastic epidermis compatible with ALM in situ (H&E, ×100); (C) Patient 7: irregular epidermal hyperplasia with compact hyperkeratosis (H&E, ×40); (D) Patient 7: large atypical melanocytes with irregular shapes and hyperchoramatic nuclei (H&E, ×200).
Early stage recognition of acral lentiginous melanoma (ALM) is important for a better prognosis, but in-depth understanding and proper management of ALM in situ is complicated, because there are only a few reports, probably due to its rarity and diagnostic difficulty. We have reviewed our experience with seven patients who were diagnosed as having ALM in situ and discuss how to accurately diagnose and properly manage these rare lesions. Clinically the lesions showed black to brown discoloration of the nail with Hutchinson's sign and hyperpigmented macules on the heel with color variegation. All the lesions showed a diffuse lentiginous pattern of melanocytic proliferation with variable level of atypism along the dermoepidermal junction. Dermoscopic findings were available in three and revealed parallel ridge patterns. Confrontation of clinical and histopathologic findings was observed in three, and the lesions were not recognized or diagnosed as ALM in situ in the first place. Excision of the primary lesion with variable operative margin was done as an initial treatment. Recurrence was observed in three patients and one developed invasive ALM and lymph node metastasis. Integration of all available information concerning the clinical presentation, histopathology, and dermoscopic findings is very important and can lead to the best classification for correct diagnosis. Lack of knowledge upon clinical course and optimal margin to control ALM in situ provokes the need for further studies with longer follow up and larger number of cases.
It is well-established that the actin cytoskeleton plays an important role in tumor development yet the contribution made by nuclear actin is ill-defined. In a recent study, nuclear actin was identified as a key mediator through which laminin type III (LN1) acts to control epithelial cell growth. In the breast, epithelial tumors are surrounded by an environment which lacks LN1. These findings point to actin as a potential mediator of tumor development. Here our current understanding of the roles of cytoplasmic and nuclear actin in normal and tumor cell growth is reviewed, relating these functions to cell phenotype in a tissue context.
Phylogenetic analysis of MsuE and other experimentally validated bacterial nitroreductase families. MsuE (dark red) and two SsuE orthologues from E. coli and Pseudomonas syringae pv. tomato DC3000 (orange) were genetically aligned against members of the NfsA (red), NfsB (dark blue), MdaB (black), YieF (black), NemA (pink), YwrO (light blue) and AzoR (green) nitroreductase families, and a human NQO1 out-group (grey), using the online tool ClustalW2. With the exception of the msuE and ssuE genes, nitroreductase sequences are as previously presented by Prosser et al. [14]. Following the nomenclature of that paper, the two letter suffix following the enzyme name indicates the bacterial strain of origin: Ba, B. amyloliquefaciens ; Bs, Bacillus subtilis ; Ck, Citrobacter koseri ; Es, Enterobacter sakazakii ; Ec, Escherichia coli ; Kp, Klebsiella pneumonia ; Li, Listeria innocua ; Pa, Pseudomonas aeruginosa ; Pp, Pseudomonas putida ; Ps, Pseudomonas syringae ; St, Salmonella typhi ; Vf, Vibrio fischeri ; Vh, Vibrio harveyi ; Vv, Vibrio vulnificus . The phylogenetic tree was constructed using the online tool MrBayes 3.2.1 [27] and bootstrap confidence values are indicated at major branchpoints. The final figure was generated using Geneious version 6.1 [28]. 
Fold-induction of SOS response in SOS-R2 strains over-expressing NfsA_Ec, NfsB_Ec or MsuE upon challenge with CB1954, nitro-CBI-DEI or PR-104A. SOS-R2 reporter strains over-expressing nfsA_Ec, nfsB_Ec or msuE, or containing an empty pUCX plasmid control, were incubated for 3 h in the presence of (A) 20 µM CB1954; (B) 5 µM nitro-CBI-DEI; or (C) 40 µM PR-104A (structures inset); after which relative induction of the SOS response in each strain was measured by β-galactosidase assay as previously described [13]. Data are the mean Miller units from three independent experiments, each performed in duplicate; and error bars are ± 1 standard deviation. The black dotted line indicates the upper error limit of basal SOS activity in the empty plasmid control. * indicates p < 0.05, ** indicates p < 0.005, *** indicates p < 0.001 (one-way ANOVA with Dunnett comparison of test to control).
Bacteria-delivered enzyme prodrug cytotoxicity assay. Calculated IC 50 of HCT-116 cells post-incubation with E. coli 6KO cells (over-expressing either NfsA_Ec, NfsB_Ec, MsuE, or an empty pUCX control) across a 2-fold dilution series of (A) CB1954 (25 M to 400 M); or (B) nitro-CBI-DEI (0.06 M to 15 M). Percentage cell survival at each prodrug concentration was calculated relative to an unchallenged control by CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Data are the mean of three independent experiments, each performed in duplicate; and error bars are ±1 standard deviation. ** indicates p < 0.005 and *** p < 0.001 by one-way ANOVA with Dunnett comparison of test to control. A B
Bacterial nitroreductase enzymes that can efficiently catalyse the oxygen-independent reduction of prodrugs originally developed to target tumour hypoxia offer great potential for expanding the therapeutic range of these molecules to aerobic tumour regions, via the emerging cancer strategy of gene-directed enzyme prodrug therapy (GDEPT). Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI. We describe here use of a nitro-quenched fluorogenic probe to identify MsuE from Pseudomonas aeruginosa as a novel nitroreductase candidate for GDEPT. In SOS and bacteria-delivered enzyme prodrug cytotoxicity assays MsuE was less effective at activating CB1954 (a first-generation GDEPT prodrug) than the "gold standard" nitroreductases NfsA and NfsB from Escherichia coli. However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so. These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT.
Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.
Successful cancer immunotherapy is confounded by the magnitude of the tumor burden and the presence of immunoregulatory elements that suppress an immune response. To approach these issues, 26 patients with advanced treatment refractory cancer were enrolled in a safety/feasibility study wherein a conventional treatment modality, intensity modulated radiotherapy (IMRT), was combined with dendritic cell-based immunotherapy. We hypothesized that radiation would lower the tumor burdens, decrease the number/function of regulatory cells in the tumor environment, and release products of tumor cells that could be acquired by intratumoral injected immature dendritic cells (iDC). Metastatic lesions identified by CT (computed tomography) were injected with autologous iDC combined with a cytokine-based adjuvant and KLH (keyhole limpet hemocyanin), followed 24 h later by IV-infused T-cells expanded with anti-CD3 and IL-2 (AT). After three to five days, each of the injected lesions was treated with fractionated doses of IMRT followed by another injection of intratumoral iDC and IV-infused AT. No toxicity was observed with cell infusion while radiation-related toxicity was observed in seven patients. Five patients had progressive disease, eight demonstrated complete resolution at treated sites but developed recurrent disease at other sites, and 13 showed complete response at various follow-up times with an overall estimated Kaplan-Meier disease-free survival of 345 days. Most patients developed KLH antibodies supporting our hypothesis that the co-injected iDC are functional with the capacity to acquire antigens from their environment and generate an adaptive immune response. These results demonstrate the safety and effectiveness of this multimodality strategy combining immunotherapy and IMRT in patients with advanced malignancies.
STAT3 as convergence point in the oncogenic pathway. 
Sites of potential STAT3 signal disruption.
Signal transducer and activator of transcription 3 (STAT3) is a potent regulator of gliomagenesis through its induction of angiogenesis, host immunosuppression, and tumor invasion. Gain of function mutations result in constitutive activation of STAT3 in glioma cells, making STAT3 an attractive target for inhibition in cancer therapy. Nevertheless, some studies show that STAT3 also participates in terminal differentiation and apoptosis of various cell lines and in glioma with phosphatase and tensin homolog (PTEN)-deficient genetic backgrounds. In light of these findings, the utility of STAT3 as a prognostic indicator and as a target of drug therapies will be contingent on a more nuanced understanding of its pro- and anti-tumorigenic effects.
The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation.
Top-cited authors
Dietrich Büsselberg
  • Weill Cornell Medicine in Qatar
Sarah Heerboth
  • Boston University
Karolina Lapinska
  • Boston University
Nicole Snyder
  • Boston University
Mckenna Longacre
  • Boston University