El Al Research Articles

Abstract 1917: Re-thinking the concept of cancer stem cells: Polyploid giant cancer cells as mother cancer stem cells

Abstract Polyploid giant cancer cells (PGCCs) have been observed by pathologists for over a century. PGCCs contribute to solid tumor heterogeneity, but their functions are largely undefined. Little attention has been given to these cells, largely because PGCCs have been generally thought to originate from repeated mitosis/cytokinesis failure and have no capacity for long-term survival and cell proliferation. Our group has recently reported that we successfully purified and cultured PGCCs from human ovarian cancer cell lines and primary ovarian cancer. These cells are highly resistant to oxygen deprivation and could form through endoreduplication or cell fusion, generating regular-sized cancer cells quickly through budding or burst. They are positive for normal and cancer stem cell markers, divided asymmetrically and cycled slowly. They can differentiate into adipose, cartilage, and bone. A single PGCC formed cancer spheroids in vitro and generated tumors in immunodeficient mice. PGCC-derived tumor gained a mesenchymal phenotype with increased expression of cancer stem cell markers CD44 and CD133 and become more resistant to the treatment of cisplatin and also form tissue organ-like structure. Together, our results reveal that the PGCCs present a resistant form of human cancer that behaves like mother cancer stem cells that can contribute to generation of daughter cancer stem-like cells and formation of tissue organ structure. These cells can be induced in response to hypoxia and play a fundamental role in regulating tumor heterogeneity, stemness, and chemoresistance in human cancer. In contrast to the phenotype unstable cancer stem cells selected by cell surface markers, PGCCs can be stably passaged and maintained, thus these cells provide reproducible experimental system to study mechanisms involved in generation of cancer stem cells. The key references related this concept are listed below. we will provide a most updated data to support this new concept that PGCCs as mother cancer stem cells at this meeting. Zhang S et al, Oncogene 2013, March online publication; Zhang S et al, Int J Cancer. 2013 Feb 1;134(3):508-18; Zhang S el al, Cancer Lett. 2013 Jun 10;333(2):205-12. Citation Format: Jinsong Liu, Imelda Mercado-Uribe, Na Niu, Baocun Sun, Jian Kuang, Shiwu Zhang. Re-thinking the concept of cancer stem cells: Polyploid giant cancer cells as mother cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1917. doi:10.1158/1538-7445.AM2014-1917

High-Voltage, High-Frequency Electrochemical Capacitor

Electric double layer capacitors (EDLCs) with electrical performance like aluminum electrolytic capacitors have been developed.(1) They can be smaller in size and offer higher reliability. Operation of such devices relies on charge stored on vertically oriented graphene nanosheet (VOGN) carbon. Vertically oriented graphene nanosheets are prepared using RF plasma enhanced chemical deposition of a hydrocarbon feed gas.(2) Under proper growth conditions, VOGN are ohmically connected to the metal current collector on which they grow, offer high electronic conductivity, and have an “open” structure totally free of porous electrode behavior. Each of these characteristics is necessary for efficient operation at frequencies of 1 kHz and higher.EDLC cells, like battery cells, operate at low voltage. Thus cells must be series connected to operate at high-voltage. Figure 1 shows an approach devised for interconnecting the EDLC cells to create a high-voltage, high-frequency EDLC. It involves interconnecting planar EDLC cells using the substrate metallization. As shown, interdigitated gaps are created in the VOGN that has been grown on a thin metal layer covering an insulating substrate, for instance, nickel on alumina. The gap is created using a laser to cut through the VOGN and metal down to the insulating substrate. This then creates multiple electrically-isolated regions (six are shown in the figure). Two adjacent regions become a single EDLC after electrolyte is applied over the gap and the surface of that region. To make a series connection of cells, each gap must be individually covered with an electrolyte, making sure that the electrolyte band does not touch the neighboring bands. This is a “bipolar” design in two dimensions, the substrate metallization serving as the bipolar plate. Connecting M cells in series each rated at voltage Vo with capacitance Co and series resistance Ro yields a capacitor with a voltage rating M·Vo, capacitance Co/M, and series resistance M·Ro. The frequency response of this high-voltage capacitor is nearly identical to that of one of its cells because the interconnects add minimal resistance. Voltage-balance resistors can be added using, for instance, thick-film printing processes. Details of the fabrication process are described and electrical performance data on these high-voltage, high-frequency electric double layer capacitors is presented. References 1J.R. Miller el al, “Graphene Double-Layer Capacitor with ac Line-Filtering Performance”, Science 329 , 1637 (2010). 2Cai M, Outlaw RA, Butler SM, and Miller JR, “A High Density of Vertically-Oriented Graphenes for Use in Electric Double Layer Capacitors”, Carbon 50, 5481–5488 (2012)..

MP41-07 PROLIFERATIVE INFLAMMATORY ATROPHY IS ASSOCIATED TO LESS AGGRESSIVE AND INSIGNIFICANT TUMORS IN RADICAL PROSTATECTOMY SPECIMENS

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2014MP41-07 PROLIFERATIVE INFLAMMATORY ATROPHY IS ASSOCIATED TO LESS AGGRESSIVE AND INSIGNIFICANT TUMORS IN RADICAL PROSTATECTOMY SPECIMENS Pol Servian, Ana Celma, Jacques Planas, José Placer, Inés M. de Torres, Maria T. Quilez, Maria A. Arbos, and Juan Morote Pol ServianPol Servian More articles by this author , Ana CelmaAna Celma More articles by this author , Jacques PlanasJacques Planas More articles by this author , José PlacerJosé Placer More articles by this author , Inés M. de TorresInés M. de Torres More articles by this author , Maria T. QuilezMaria T. Quilez More articles by this author , Maria A. ArbosMaria A. Arbos More articles by this author , and Juan MoroteJuan Morote More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1224AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Proliferative inflammatory atrophy (PIA) has been proposed to be involved in prostate carcinogenesis by suggesting that it may rise to prostatic carcinoma (PCa) either directly or indirectly by first developing into high grade prostatic intraepithelial neoplasm (HGPIN)1. We have studied the PIA genetic signature 2 and more recently we have learned that PIA incidence in prostatic biopsies is around 30%, and it is associated to lower PCa detection and less aggressive tumors 3. The objectives of this study were to assess the incidence of PIA lesion in radical prostatectomy (RP) specimens and to confirm the hypothesis that PIA is associated to less aggressive tumors. Methods A retrospective study conducted in 200 consecutive patients subjected to RP was done. A single pathologist identified PIA in all specimens and this finding was related with: age, serum PSA, prostate volume, Gleason score, pathologic stage, percentage of tumor in the gland, maximal length of index tumor, perineural invasion, multifocality, bilaterality, insignificant cancer, positive margins, and HGPIN. Mann-Witney U test and Chi-square Pearson test were used to compare data. Results PIA was present in 64 specimens (32%). Age, serum PSA and prostate volume were similar in patients with and without PIA. Respectively, high Gleason grade was present in 34.4% and 49.3%, p=0.033; non-organ confine disease in 10.9% and 16.9%, p=0.187; perineural invasion in 59.4 and 74.3%, p=0.025; multifocal tumors in 59.4% and 74.3%, p=0.001; bilateral tumors in 46.9% and 70.5%, p=0.005; insignificant tumors in 17.2% and 6.6%, p=0.022; HGPIN in 92.2% and 90.4%, p=0.456. Conclusions This study confirms that the incidence of PIA in RP specimens was similar to the one observed previously in prostatic biopsies (32% versus 30%). PIA was not associated to HGPIN. PIA was associated to many pathologic characteristics of less aggressive tumors. Of great clinical interest could be the finding that PIA is associated to unilaterial, unifocal, low grade, and insignificant tumors. 1 De Marzo et al. Am J Pathol 1999; 155: 1985-92. 2 Morote et al. Annual EAU Congress 2012; P-848. 3 Morote el al. Annual AUA Meeting 2013; P-2226. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e453 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Pol Servian More articles by this author Ana Celma More articles by this author Jacques Planas More articles by this author José Placer More articles by this author Inés M. de Torres More articles by this author Maria T. Quilez More articles by this author Maria A. Arbos More articles by this author Juan Morote More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Subcellular distribution of melatonin receptors in rat parotid salivary glands: effect of melatonin intravenous infusion

The ultrastructural localization of melatonin receptors Mt1 and Mt2 in human salivary glands has previously been reported (Isola el al, 2013). In the acinar cells, the intragranular distribution of these receptors suggested to us that they may serve as a transcytotic system for melatonin. In an attempt to verify our working-hypothesis we currently exposed anaesthetized rats to the intravenous infusion of melatonin for 2 hrs. After another 30 min, the parotid gland on the right side was removed to be compared with prestimulation removed pieces of the left parotid gland serving as control specimens. Parotid gland tissues from 5 rats were cut into small pieces and fixed with a mixture of paraformaldehyde and glutaraldehyde. They were dehydrated, and embedded in Epon Resin. The ultrathin sections were collected on nickel grids and incubated overnight at 4oC with an antibody specific for Mt1 and Mt2, respectively. The grids were then incubated for 1 hr with the appropriate secondary antibody conjugated to gold particles and, finally examined using electron transmission microscopy. In all sections, the immunoreactivity to Mt2 and, with less intensity, to Mt1 was principally expressed in secretory granules of acinar cells. Occasionally, plasma membranes were also stained, although slightly. Statistical analysis showed that, in response to melatonin, the immunogold particles reactive for Mt1, but not those for Mt2, increased. To conclude, in line with our hypothesis, melatonin stimulates an acinar carrier system, which seems predominantly to involve the Mt1 receptor, in order to delivery this hormone into saliva.

MODELING MONTHLY AVERAGE DAILY DIFFUSE RADIATION FOR DHAKA, BANGLADESH

The diffuse part of solar radiation is one of the e lements necessary for the design and evaluation of energy production of a solar system. However, in most cases, when radiometric me asurements are made, only global radiation is avail able. To remedy this situation, this paper presents a model of the scatt ered radiation measured on a horizontal surface for the capital city of Bangladesh. The correlation established for the chosen site was compared to the work of Liu anf Jordan, Page, Coll ares Pereira and Rabl, Modi and Sukhatme and Gupta el al.

No Improvement Of Overall Survival After Extended Follow-Up Of Donor Versus No Donor Analysis Of Newly Diagnosed Myeloma Patients Included In The HOVON 50/54 Study

BackgroundThe value of Allo-SCT in myeloma is heavily disputed. In our previous Donor versus No Donor (DvND) comparison we found no survival benefit of Allo-RIC in newly diagnosed myeloma. (Lokhorst et al:Blood 2012119: 6219-6225). However, a recent update of the EBMT-NMA 2000 trial (Gahrton el al:Blood 2013121: 5055-506) suggested that extended follow-up (> 5 years) may be necessary for a correct interpretation of a potential survival benefit for Allo-RIC. Here we present the extended follow-up of our trial, in which the median follow-up of patients now exceeds over 7.5 years since the first autologous SCT. MethodsPatients with an HLA–identical sibling donor included in the phase III HOVON-50 study, that was designed to assess the role of thalidomide in induction treatment and maintenance after high-dose therapy (HDM 200 mg/m2), could proceed to the Hovon 54 study in which an Allo-SCT was performed after conditioning with low dose TBI only, between 2-6 months after HDM. Among the 536 eligible patients randomized in the HOVON-50 trial, ultimately 260 patients were eligible to be included into the DvND analysis: 122 patients with a donor, of whom 99 patients received an Allo-RIC and 138 without a donor, of whom 115 patients started maintenance therapy with thalidomide. Groups were comparable with regard to age, myeloma stage, and prognostic factors including cytogenetics and ISS stage. Results93% of the patients in the no donor group achieved at least a PR (38% CR, 71% at least VGPR ), versus 96% of the patients in the donor group (43% CR, 73% at least VGPR). After a median follow-up of 91 months after HDM, PFS and OS were comparable between the two groups. In the intention to treat analysis median PFS was 29 months for the no donor group and 30 months the no donor group (P=0.25). Median OS was 76 for the donor group and 81 months for the no donor group (P=0.61). For the patients who actually received their allocated treatment (Allo-RIC or maintenance), PFS curves started to diverge after 3 years, however no statistical difference was observed (P=0.07). Allo-RIC improved the median overall survival from 73 to 94 months compared to patients receiving maintenance. However, due to frequent late mortality (> after 96 months) in the Allo-RIC group the benefit was not statistically significant (P=0.54). No subgroup including those achieving CR or those with high risk features (ISS, deletion of chromosome 13) did benefit from Allo-RIC. ConclusionThis analysis failed to show improvement of tandem Auto Allo-RIC as part of first line therapy in myeloma as compared to Auto-SCT followed by maintenance therapy, even after extended follow-up. Disclosures:Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria. Minnema:Janssen Cilag: Consultancy, Honoraria. Sonneveld:Janssen-Cilag: Honoraria; Celgene: Honoraria; Onyx: Honoraria; Janssen-Cilag: Research Funding; Millenium: Research Funding; Onyx: Research Funding; Celgene: Research Funding.

AB0482 Takayasu arteritis and hla-b52 allele: a single-centre analysis from japanese population

BackgroundTakayasu arteritis (TA) is a large vessel vasculitis affecting the aorta and its primary branches. The etiology of TA is still unknown, but recent studies have indicated that some genetic...

PL.95 Obstetric Practises During Childbirth: Expectations, Perceptions and Satisfaction of Women

Knowing what the woman expected to find and what she found in relation to obstetric practises, enables us to meet the needs, if we assess their satisfaction we will know...

A Case Series: Chiari Malformation and Sickle Cell Disease

AbstractAbstract 4772 Introduction:Chiari type I malformation is defined as tonsillar herniation of at least 3 to 5 mm below the foramen magnum. The prevalence of Chiari type I malformation in the general population is estimated to be 0.1 to 0.5%. In the SIT (Silent Infarct Transfusion) Trial in children with sickle cell disease, Chiari type I malformation was found as an incidental finding in 22 of 953 (2.3%) of the individuals imaged. Thus, the incidence of Chiari type I malformation in the SIT Trial appeared to be about five times greater than in the general population (Jordan et al Pediatrics 2010). Recently, four hemoglobin SS patients aged 6–17 years with and Chiari type I malformation were reported from one institution (Chonat el al J Pediatr Hematol Oncol 2011). Here we describe six sickle cell disease patients aged 17–24 years with Chiari type I malformation from two institutions.Case Series:Case123456Age221817202423GenderMaleMaleMaleMaleFemaleFemaleGenotypeHgb SSSickle/B0thalHgb SSHgb SSHgb SSHgb SSPresenting SymptomsFeeling “off balance” for 6 monthsDifficulty performing tandem walking, ataxia, and nystagmusSeveral weeks of headaches, lightheadedness, and the feeling “off balance”Global headaches aggravated by coughChronic headaches for several yearsBilateral hand numbness, headache, double visions, slurred speech, and gait imbalanceExam FindingsDifficulty performing tandem walking, ataxia, and nystagmusNoneWide-based ataxic gaitNoneNoneDiplopia on extreme lateral gaze and decreased gag reflexMRI FindingsChiari type I malformation with cerebellar tonsil elongation and herniation, deformity of the fourth ventricle, and mass effect on the brainstemLow-lying cerebellar tonsils and brainstem compatible with Chiari type I malformationChiari type I malformation of the cranio-cervical junction with the cerebellar tonsils 9 mm below the foramen magnumDownward displacement of the cerebellar tonsil and a small posterior fossa consistent with Chiari type I malformation.Chiari type I malformation with syringehydromyelia of the proximal cervical spinal cordHerniation of the cerebellar tonsils into the upper cervical spinal canal with ∼17 to 19 mm inferior displacement through the foramen magnum consistent with a Chiari type I malformation Discussion:All of the cases had severe sickling phenotype, either hemoglobin SS or sickle/B°thalassemia. The majority of the patients were experiencing neurologic symptoms that were subsequently attributed to Chiari type I malformation. Five of the six cases were eventually evaluated by neurosurgery. Neurosurgical intervention with posterior fossa decompression was recommended in four of these individuals. To date one patient has undergone surgery with relief of symptoms. Although Chiari type I malformation is thought to be primarily a congenital disorder, it has also been associated with disorders that are non-congenital. Individuals with sickle cell disease may have a thick calvarium as a result of erythroid hyperplasia and expansion of the diploe of the skull bones. Other conditions with severe anemia and thalassemia have been associated with increased thickness of the calvarium caused by diploic erythroid hyperplasia. One can question whether the increased thickness of the calvarium in some sickle cell patients results in craniocephalic disproportion with a subsequent acquired cerebellar ectopia, hence the increased Chiari type I malformation in patients with sickle cell disease. Central nervous system involvement in sickle cell disease is common. Any patient with sickle cell disease presenting with symptoms indicative of an ongoing or impending neurological event requires an in depth investigation and entails a broad differential. Chiari malformations may need to be part of this differential diagnosis. Disclosures:No relevant conflicts of interest to declare.

Incidence and patterns of phospho insulin growth factor receptor-1 (pIGF-1R) and matrilysin (MMP7) expression in metastatic colorectal cancer (mCRC), and correlation with KRAS status: A prospective evaluation in the PULSE trial—A GEMCAD study.

e14041 Background: MMP7 can activate IGF-1R by IGF release due to IGFBP degradation. Activation of IGF-1R can contribute to EGFR resistance by transactivation. We previously described that concomitant expression of p-IGF-1R and MMP7 (Double positive; DP), correlates with poor prognosis, in KRAS WT patients (pts) treated with anti-EGFR compounds (Horndler el al, 2011). Therefore we designed a prospective clinical trial to validate DP as a marker of resistance in KRAS WT pts treated in first-line therapy with FOLFOX-6 plus panitumumab. Methods: mCRC pts in the ongoing prospective PULSE trial (NCT0128833) were prospectively evaluated for p-IGF-1R (p-1316), MMP7 expression and KRAS mutational status. Pts defined as DP should express MMP7 (++ or +++ intensity in >66% of tumor cells) and p-IGF-1R (++ or +++ intensity in >66% of tumor cells). KRAS pts with mutations at exon 2 were excluded. The study was designed to include 40 pts in the two groups (DP vs non-DP) to detect a Hazard ratio difference in PFS of <0.5 (DP vs non-DP) with 80% power. Results: From November 2010 to December 2011, 113 consecutive pts were screened from 24 Spanish Institutions. 54 KRAS WT (40 pts non-DP and 14 DP) have been included. The non-DP arm has being recently closed for inclusion, due to pre-planned complete accrual. Among DP pts, 27% were KRAS WT and 30% KRAS mutant; p=0.63. 48% of cases were positive for p-IGF-1R. Phospho-IGF-1R positive cases had different patterns of staining: peri-nuclear in 76%, 11% nuclear and only 13% membrane-apical staining. These patterns do not differ between KRAS WT; (n=70) and KRAS mutant pts (n=43) (p=0.60). Tumors with positive p-IGF-1R expression, independently of the pattern, have higher MMP7 co-expression (59%) compared with negative cases (13%) (p<0.0001). Conclusions: MMP7 contributes to activate IGF-1R pathway in pts with mCRC. Internalization of the activated IGF-1R, could explain in part, the lack of efficacy of IGF-1R inhibitors in mCRC clinical trials.

Identifying the high-risk Brugada syndrome patient: Let us get personal

There are several reasons why risk stratification in the Brugada syndrome (BrS) continues to pose significant challenges. Prevalence of the syndrome in the general population is low, yet high-risk individuals can manifest with sudden cardiac death at a young age when the implantable cardioverter-defibrillator comes with a heavier burden of lifestyle change and cumulative long-term complications. Many patients will be asymptomatic on presentation and electrocardiographic (ECG) changes can fall in a nondiagnostic category. Despite significant and ongoing advancements in genetic discovery and technology, the yield of genetic testing in overall patients with BrS remains well below 50%. 1 The 2005 Consensus Conference established that conversion of a nondiagnostic ECG (saddle pattern type 2 or 3 ECG) to a diagnostic ECG (type 1 Brugada pattern) with a sodium-channel blocker (SCB) test indicated a positive diagnosis of the BrS. 1 This is an important issue, since increased awareness of this condition has resulted in a referral stream to electrophysiologists and cardiologists of patients with a nondiagnostic Brugada-pattern ECG who have vague or no symptoms. However, more recently, the largest BrS registry (FINGER Registry, 1029 consecutive subjects from 4 European countries) concluded that event rates in asymptomatic patients with BrS were low (0.5% per year) and that inducibility of ventricular arrhythmias and family history of sudden cardiac death were not predictive of sudden cardiac death. 2 Spontaneous type 1 ECG changes and the presence of symptoms were the only predictors of arrhythmic events in this population. In this issue of HeartRhythm, Zorzi el al 3 specifically address the utility of the SCB test among 198 patients with a nondiagnostic Brugada-pattern ECG recruited at 2 medical centers of the Veneto region of Italy. Of the total, 27 who developed spontaneous type 1 ECG changes were excluded and 18 were not available for follow-up. The remaining 153 subjects with type 2 or 3 Brugada ECGs were followed up for a mean period of 59 months. A positive SCB test result was associated with a significantly higher event rate in symptomatic patients but not in asymptomatic patients. These results suggest that the clinical utility of the SCB test in asymptomatic patients with nondiagnostic ECGs is likely to be low. This careful study incorporated placement of the right precordial leads in a superior position in order to increase sensitivity for detecting type 1 ECG changes. 4 What is the clinical message of this study and what are the caveats to keep in mind? The findings of this study provide more evidence to clinicians that SCB testing may not be indicated in asymptomatic patients with a nondiagnostic Brugada-pattern ECG. We should keep in mind that this was a relatively small study with a relatively short follow-up period given the natural history of the BrS. Also, event rates were low and the study did not provide information on pre-2005 (Consensus Conference publication) vs post-2005 clinical management. Most important, if the findings of the FINGER registry remain consistent, our clinical attention is likely to focus largely on symptomatic patients with a spontaneous type 1 BrS ECG.

Patterns of Myeloma (MM) Progression After Autologous Transplant (AHCT) – Biochemical Progression Vs. Clinical Relapse

Abstract 5097 Background:The uniform response criteria for MM suggest that in clinical practice early biochemical progression (EBP) of MM and clinical relapse (clinREL) be distinguished to identify symptomatic patients requiring therapy. The interval between EBP and clinREL in MM is not well characterized. Biochemical progression in those with intact immunoglobulin MM (IIM) could involve serum free light chain (SFLC) progression or monoclonal component (M spike) progression by serum protein electrophoresis (SPE) or both. ClinREL could precede, be concurrent with or follow tumor marker elevation. Methods:We studied EBP and clinREL in 221 consecutive AHCT recipients for MM between 2005 and 2010. Patients with IIM and measurable M spike and SFLC elevation at diagnosis were analyzed. Patients were uniformly monitored at a single institution with SFLC and SPE every 8–12 weeks. Biochemical progression and clinREL were defined by standard IMWG criteria (Durie el al, Leukemia 2006). ClinREL was defined by new/progressive bone lesion or plasmacytoma, hypercalcemia (>11.5 mg/dl), decrease in hemoglobin ≥ 2 g/dl, rise in creatinine ≥ 2 mg/dl, or indication for new myeloma therapy. Results:76 patients had clinREL after AHCT, 24 were excluded due to light chain predominant or oligosecretory disease, subsequent AlloHCT or other preemptive treatment. Among 52 evaluable patients, median age was 57 years (42–74), 72% had DSS stage III, 40% ISS stage III, 40% high-risk (HR) MM based on t(4;14), t(14;16), 17q del or karyotypic 13 q abnormality. Median time from AHCT to relapse was 484 days (76–1631).Three temporal patterns of MM recurrence were identified: EBP where SFLC/M spike elevation (or both) preceded clinREL (n=28, 54%); concurrent clinREL and SFLC/M spike elevation (n=22, 42%) or isolated clinREL (4%). The proportion of HR MM was similar among groups. The EBP cohort had superior OS compared to concurrent relapse cohort (log rank p <0.0001).Additionally in the EBP cohort, SFLC elevation alone (39%), M spike progression alone (22%) or concurrent SFLC and M spike (39%) progression could be the initial relapse marker. After isolated SFLC progression, median time to M spike progression was 112 days and to clinREL was 200 days. After isolated M spike progression median time to clinREL was 254 days. After simultaneous SFLC and M spike progression median time to clinREL was 234 days. In these 3 groups, overall time to clinREL after AHCT was longest for patients with M spike progression alone (17.8, 20.5 and 36.8 months, respectively; p=0.05). This was due to HR MM being more common in the first 2 groups (45% for isolated SFLC and 37% in the simultaneous SFLC/M spike group). Discussion:In this cohort of relapsed MM after transplant, 54% had isolated early biochemical progression prior to clinREL and this pattern predicted for superior survival. The category of clinREL may not be detected in advance in about one half of patients and predicts for more aggressive relapse. Present guidelines do not recommend SFLC monitoring for relapse in IIM patients with measurable serum M spike. Based on the above data early SFLC progression was seen in 20% of relapses and identified a higher risk subgroup within those with biochemical progression preceding clinREL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Genome-Wide DNA Methylation Profiling of Hematopoietic Stem and Progenitor Cells Reveals Over-Representation of ETS Transcrition Factor Binding Sites

Abstract 211The hematopoietic system is ideal for the study of epigenetic changes in primary cells because hematopoietic cells representing distinct stages of hematopoiesis can be enriched and isolated by differences in surface marker expression. DNA methylation is an essential epigenetic mark that is required for normal development. Conditional knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment have revealed that methylation is critical for long-term renewal and lineage differentiation of hematopoietic stem cells (Broske et al 2009, Trowbridge el al 2009). To better understand the role of DNA methylation in self-renewal and differentiation of hematopoietic cells, we characterized genome-wide DNA methylation in primary cells representing three distinct stages of hematopoiesis. We isolated mouse hematopoietic stem cells (HSC; Lin- Sca-1+ c-kit+), common myeloid progenitor cells (CMP; Lin- Sca-1- c-kit+), and erythroblasts (ERY; CD71+ Ter119+). Methyl Binding Domain Protein 2 (MBD2) is an endogenous reader of DNA methylation that recognizes DNA with a high concentration of methylated CpG residues. Recombinant MBD2 enrichment of DNA followed by massively-parallel sequencing was used to map and compare genome-wide DNA methylation patterns in HSC, CMP and ERY. Two biological replicates were sequenced for each cell type with total read counts ranging from 32,309,435–46,763,977. Model-based analysis of ChIP Seq (MACS) with a significance cutoff of p<10−5 was used to determine statistically significant peaks of methylation in each replicate. Globally, the number of methylation peaks was highest in HSC (85,797peaks), lower in CMP (50,638 peaks), and lowest in ERY (27,839 peaks). Comparison of the peaks in HSC, CMP and ERY revealed that only 2% of the peaks in CMP or ERY are absent in HSC indicating that the vast majority of methylation in HSC is lost during differentiation. Comparison of methylation with genomic features revealed that CpG islands associated with promoters are hypomethylated, while many non-promoter CpG islands are methylated. Furthermore, methylation of non-promoter associated CpG islands occurs infrequently in cell-type specific peaks but is more abundant in common methylation peaks. When the DNA methylation patterns were compared to mRNA expression, we found that as expected, proximal promoter sequences of expressed genes were hypomethylated in all three cell types, while methylation in the gene body positively correlated with gene expression in HSC and CMP. Utilizing de novo motif discovery we found a subset of transcription factor consensus binding motifs that were overrepresented in methylated sequences. Motifs for several ETS transcription factors, including GABPalpha and ELF1 were found to be overrepresented in cell-type specific as well as common methylated regions. Other transcription factor consensus sites, such as the NFAT factors involved in T-cell activation, were specifically overrepresented in the methylated promoter regions of CMP and ERY. Comparison of our methylation data with the occupancy of hematopoietic transcription factors in the HPC7 cell line, which is similar to CMP (Wilson et al 2010), revealed a significant anti-correlation between DNA methylation and the binding of Fli1, Lmo2, Lyl1, Runx1, and Scl.Our genome-wide survey provides new insights into the role of DNA methylation in hematopoiesis. Firstly, the methylation of CpG islands is associated with the most primitive hematopoietic cells and is unlikely to drive hematopoietic differentiation. We feel that the elevated genome-wide DNA methylation in HSC compared to CMP and ERY, combined with the positive association between gene body methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSC. Finally, the finding that transcription factor binding sites are over represented in the methylated sequences of the genome leads us to conclude that DNA methylation modulates key hematopoietic transcription factor programs that regulate hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Complex networks from lévy noise

We construct complex networks from levy noise (LN) using visibility algorithm proposed by Lucas lacasa el al. It is found that as the stability index α of the symmetric LN decreases, the corresponding complex network will transit from exponential network to long-tailed-degree-distribution one, and then to Gaussian one. The associated network for symmetric LN is the high clustering, hierarchy, and 18 community network. The properties of the associated networks for asymmetric LN except the skewness parameter β = −1 are similar with that for symmetric one. The associated network for the asymmetric LN with the skewness parameter β = −1 is always the exponential, high clustering, and hierarchy one with small k-clique communities.

Aggregate Homotopy Method for Min-Max-Min Programming Satisfying a Weak-Normal Cone Condition

Min-max-min programming is an important but difficult nonsmooth programming. An aggregate homotopy method was given for solving min-max-min programming by Bo Yu el al. However, the method requires a difficult to verify weak-normal cone condition. Moreover, the method is only theoretically with no algorithmic implementation. In this paper, the weak normal cone condition is discussed first. A class of min-max-min programming satisfying the condition is introduced. A detailed algorithm to implement the method is presented. Models arising from some applications such as support vector machine for multiple-instance classification in data mining, can be included in the problem. In the end, the aggregate homotopy method is given to solve the multiple-instance support vector machine model.

PTEN Promoter Silencing and Cowden Syndrome

THE PHOSPHATASE AND TENSIN HOMOLOG GENE (PTEN) (RefSeq NM_000314) is a tumor suppressor located on the human chromosome 10q arm and is an important mediator of carcinogenesis in a variety of human malignancies. By the strictest definition, a tumor suppressor is a gene whose loss confers an increased lifetime risk of developing tumors. The most illustrative examples of genes that fulfill this criterion are those associated with familial cancer syndromes whereby heritable inactivation of 1 allele and subsequent increased tumor risk is passed along to each generation in an autosomaldominant fashion. Using this as a framework, PTEN is a bona fide tumor suppressor gene in that heritable germline mutations have been described in Cowden syndrome (CS), giving rise to a number of human tumors and cancers, most notably thyroid and breast cancers. As is the paradigm of tumor suppressor genes, affected patients with CS inherit 1 mutant inactive copy of PTEN from either parent, and the ensuing loss of the second allele results in tumor formation with subsequent genetic events that eventually lead to cancer. Although there are notable exceptions to this model, most heritable cancer syndromes are believed to adhere to this pattern. Although anomalies within a specific gene often account for the majority of a given familial cancer syndrome, there are striking examples in which a pedigree matches the syndrome, yet no mutations are found in the affected individuals. Such perplexing cases have led to the identification of additional genes that can account for a significant fraction in these cases. For example, germline mutations of the TP53 gene account for the majority of families with LiFraumeni syndrome, a heritable cancer syndrome hallmarked by a number of different cancers, including breast cancers, sarcomas, and leukemias. However, in families diagnosed with Li-Fraumeni syndrome without a TP53 mutation, germline mutations in the cell cycle checkpoint gene CHK2 can account for a considerable proportion of these familial cases. Similarly, patients with CS generally have been found to have germline mutations in PTEN, but in a substantial number of these families no PTEN mutations have been found. Along the same lines of CHK2 mutations and Li-Fraumeni syndrome, germline mutations in the succinate dehydrogenase subunits SDHB-D have been previously described in patients with PTEN wild-type CS as well as individuals who have features of CS but do not meet diagnostic criteria: patients who are said to have Cowdenlike syndrome (CLS). Despite the discovery of germline mutations in these genes, there remains a sizeable number of patients with CS and CLS who lack mutations in known genes. The study by Bennett et al in this issue of JAMA offers an intriguing explanation for some of these families with PTEN wild-type CS and CLS. It has previously been described that epigenetic inactivation of genes via hypermethylation of their promoters, of other DNA modifications, such as histone deacetylation, or both, can silence tumor suppressor genes. Indeed, silencing of tumor suppressor genes in sporadic cancers by epigenetic regulation is a welldocumented event, and in the case of specific tumor suppressor genes appears to be the more common mechanism of inactivation rather than gene mutation. In some cases, this can occur in the germline, and therefore these epigenetic changes, or “epimutations,” can then be passed to subsequent generations and predispose those individuals to an increased risk of certain cancers. This was the basis of the starting point for Bennett et al wherein they examined the peripheral lymphocytes derived from patients with CS and CLS for hypermethylation of the PTEN promoter. Perhaps not unexpectedly, the investigators did discover, in a significant fraction of these patients, PTEN promoter hypermethylation. However, rather unexpectedly, gene silencing of PTEN was not found, suggesting that methylation of the PTEN promoter did not alter or regulate expression of this tumor suppressor gene. Further study by Bennett el al led to the analysis of a relatively new gene called KILLIN. KILLIN has only recently been identified, and relatively little is known regarding its function or role in human cancers. KILLIN was originally identified as a TP53 regulated inhibitor of DNA synthesis, and

IGF-Induced Gene Profiles Are Dependent on IRS Expression and Convey Prognostic Value in Human Breast Cancer.

Abstract The insulin-like growth factor (IGF) pathway mediates aberrant function during the initiation and progression of primary tumors and secondary metastases in cancer. As a result, a host of tyrosine kinase inhibitors and monoclonal antibodies directed against the type 1 IGF receptor (IGF-1R) have entered clinical trials with early positive results. However, no predictive biomarkers have yet emerged from these initial studies. We propose that expanding IGF biomarkers beyond IGF-1R alone may identify the most appropriate candidates for anti-IGF therapy. Previous work has shown that the insulin receptor substrate (IRS) proteins serve as the functional link between IGF-I-induced IGF-1R phosphorylation and downstream signaling linked to cellular behavior. Our work has demonstrated that IRS isoforms differentially mediate IGF-I action, whereby IRS-1 drives proliferation and IRS-2 triggers motility. Here we employed the T47D-YA (IRS null) breast cancer cell line and T47D-YA/IRS clones stably transfected with IRS-1 or IRS-2. In response to acute (4h) and chronic (24h) IGF-I stimulation, global gene expression patterns were assessed by Affymetrix U133 Plus 2.0 microarray analysis. Analysis revealed that IGF-1R activation alone was insufficient to affect gene expression as no genes were regulated by IGF-I in T47D-YA cells. Conversely, ligand stimulation of IRS-1 and IRS-2 clones induced or repressed hundreds of transcripts in both overlapping and distinct fashions. Direct comparison of IRS-1 to IRS-2 clones revealed a number of early (4h) IRS-2 genes linked to metastasis and late (24h) IRS-1 proliferative genes. Interestingly, a 10-fold upregulation in the transforming growth factor beta 2 (TGFβ2) gene by IGF-I in IRS-2 cells suggested a link between the IRS-derived gene signatures and the TGFβ pathway known to regulate breast cancer metastasis. We then compared our arrays with published IGF-I (MCF-7) and TGFβ-derived (MCF10A, MDA-231, HaCaT, HPL1) microarrays to find a list of commonly regulated genes and performed cluster analysis to reveal consistent patterns of gene expression (Creighton el al 2008 &amp; Padua et al 2008). We found 75 genes that were regulated in common between these signatures. To explore the clinical relevance of the signatures we developed, we examined the NKI-295 dataset used to establish the 70-gene profile of prognosis and found 71 genes regulated in common between all four datasets (van de Vijver et al 2002). Strikingly, we discovered that patient survival was heavily influenced by the degree to which tumor expression correlated to the conserved signatures. A high degree of correlation resulted in the poorest disease free survival and an inverse correlation resulted in an improved disease free survival. Our data suggest that IGF stimulation of breast cancer cells results in distinct profiles of gene expression that are dependent on IRS adaptor protein expression. In addition, some of the “IRS-regulated” genes are shared in common with other gene signatures of poor prognosis. With the use of anti-IGF therapies in breast cancer, attention should focus on the use of these profiles as prognostic and predictive biomarkers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3031.

Adjuvant Chemotherapy for Elderly Patients With Colon Cancer: A Vanishing Paradigm?

At least two thirds of all diagnosed colon cancers occur in patients who are at least 65 years of age, and half of these patients are over 75 years of age. The clinical evidence generated to date suggests that elderly patients with colorectal cancer (CRC) are understaged, undertreated, and underrepresented in clinical trials.1 With this in mind, Folprecht el al recently analyzed the effects of treatment of metastatic CRC in elderly patients. Among 2692 patients (22% ≥ aged 70 years) treated in 4 clinical trials with irinotecan-based therapy, a significantly improved progression-free survival and a trend toward improved overall survival (OS) for elderly patients (aged ≥ 70 years) was achieved with the addition of irinotecan to 5-fluorouracil (5-FU)/leucovorin (LV) chemotherapy.2 In addition, Goldberg et al investigated the pooled clinical data from 4 oxaliplatin-based trials, which included a total of 3740 patients (16% were aged ≥ 70 years), and they observed similar clinical efficacy as well as incidence of grade 3/4 toxicity in both young and old patients (aged ≥ 70 years).3 With respect to adjuvant therapy, a metaanalysis was performed by Sargent and colleagues that included 7 randomized trials of 5-FU plus levamisole/LV versus surgery alone. This analysis included a total of 3351 patients, of which 15% were aged ≥ 70 years, and showed no significant interaction between age and efficacy of treatment.4 In sharp contrast to these studies is the presentation by Meyerhardt et al at this year’s meeting of the American Society of Clinical Oncology. In this report, Meyenhardt presented updated results from the ACCENT database, which includes > 12,500 patients with stage II/III colon cancer from 6 recent adjuvant trials (MOSAIC, NSABP C-07, CALGB 89803, PETACC 03, X-ACT, and NSABP C-06; Table 1). As outlined in Table 2, patients aged ≥ 70 years did not derive a clear benefit from combination chemotherapy with irinotecan or oxaliplatin added to modulated 5-FU when compared with 5-FU/LV chemotherapy in terms of disease-free survival (DFS), OS, or time to recurrence.5 Even when considering the lack of efficacy of irinotecan-based adjuvant trials and looking at only the 2 oxaliplatin-based studies (MOSAIC and NSABP C-07), the results remain dismal in the older population (Table 3). The lack of clinical benefit from combination chemotherapy was present in both stage II and III older patients. It should be noted that this analysis of the ACCENT database has several limitations, which include lack of toxicity data as well as specific details relating to dose intensity and presence of comorbidities. The absence of this important clinical information might have confounded the interaction between age and efficacy of the newer chemotherapy adjuvant regimens. These data do not demonstrate any superiority for the experimental regimens (namely FOLFOX4 [oxaliplatin/5-FU/LV] and FLOX [5-FU/LV/oxaliplatin) over 5-FU/LV alone in older patients. What might be the reason for this? Even admitting that tumor biology could be different in older versus younger patients with related implications in sensitivity to chemotherapy,6 it would be probably more important to look at disease-specific OS (when such data will be available), given that oxaliplatin-based chemotherapy might reduce the risk of early recurrence without achieving a DFS or OS benefit because of deaths from other causes, especially in the subpopulation of older patients. In the ACCENT analysis, oxaliplatin-based regimens were associated with a higher rate of death within 6 months of treatment in the entire treatment population (2.57% vs. 1.37% for 5-FU/LV). One of the immediate consequences of this presentation was that the ongoing PETACC 08 study comparing FOLFOX4 with or without cetuximab in the adjuvant setting for early-stage colon cancer was recently amended so as to not allow the further enrollment of patients > 70 years of age. However, what is the take-home message from the results of these new and quite unexpected data on adjuvant therapy for older patients with colon cancer? Even keeping in mind that these data do not contradict earlier studies showing a benefit of adjuvant chemotherapy with modulated 5-FU over surgery alone in elderly patients,4 it should be emphasized that contradictory results about the efficacy of this simple adjuvant treatment for elderly patients was published more recently with the final results of the Quasar study, in which there was no reduction

SU‐EE‐A1‐03: The Neutron Dose Evaluation and Shielding at the Maze Entrance of the Varian Clinac 2300EX

Purpose: After the installation of a Varian Clinac 2300EX accelerator, neutron and photon doses at the outer maze entrance were measured and compared with several empirical calculations. The measurements were taken before and after borated polyethylene (BPE) boards were installed on the maze wall as neutron absorption material. Method and Materials: The accelerator delivered an 18 MV photon beam at 600 cGy/min dose rate. An NRC NP‐2 type neutron REM‐meter and a Ludlum 14C survey meter with a 44‐6 type of probe were used to measure the neutron dose and photon dose, respectively. The measurements were taken at the center axis of the maze, 0.8 m above the floor, 0.3 m away from the maze door. Results: With the gantry head tilted close to the inner maze entrance with closed jaws, both neutron dose and photon dose reach their maximum. The measurement data were compared with empirical calculation results obtained by Kersey's method, modified Kersey's method and a newly proposed method by R.C. Falcão el al. The estimation from Kersey's method is about 2 – 4 times of the measurement (Ratio ≈ 2.4–3.8). Falcão's method has estimation about 22 times of the measurement (Ratio ≈ 21.9). The modified Kersey's method has the best prediction of the dose (Ratio ≈ 1.0). The McGinley's photon dose equation gives estimation about 80% of the measurement. After applying borated polyethylene boards as lining on maze wall, the neutron dose and the photon dose at maze entrance were decreased by 41% and 58%, respectively. Conclusion: This work indicates that Kersey's method overestimates the neutron dose by about 2 to 4 times for this study. Falcão's method largely overestimates the neutron dose. The modified Kersey's method is recommended to be used in neutron shielding calculation for the 2300EX accelerator.

Over-Expression of IRF4 (MUM1) Protein Predicts Poorer Outcome in Myeloma Patients

IRF4 (also known as MUM1) is a member of the interferon regulatory factor family of transcription factors whose expression is critical for the transition from pre B-cell to immature B-cell and for differentiation of mature B-cell to plasma cell. It has previously been reported by Heintel el al (Leukemia (2008) 22, 441–445) that over-expression of IRF4 at the mRNA level is associated with a poor outcome in myeloma. Measurement of expression at the protein level using flow cytometry would remove the need for plasma cell purification and also allow evaluation of IRF4 in other cell populations. We have developed a multi-colour flow cytometry assay incorporating an indirect intracellular staining using the goat polyclonal antibody IRF4 (Santa Cruz). This permits simultaneous analysis of IRF4 in combination with five cell surface markers, allowing quantitation of the IRF4 levels present in specific B-cell and plasma cell populations. The aim of this study was to assess the expression profile of IRF4 in neoplastic and normal B-cells and plasma cells and evaluate the relationship between IRF4 protein expression and outcome in multiple myeloma patients. Leucocytes prepared from whole bone marrow using ammonium chloride lysis were incubated with surface markers CD52 PE, CD38 PerCPCy5.5, CD19 Pe-Cy7, CD138 APC and CD20 APC-Cy7. Cells were then fixed and permeabilised before incubation with unconjugated IRF4, washing and incubation with an Alexa488 secondary antibody. IRF4 expression showed the expected profile in samples with normal B-lineage cells. Low levels of expression were found in normal mature B-cells (mean MFI 1033, range 395–2265), higher levels in progenitor B-cells (mean 4.1-fold higher than mature B-cells, mean MFI 4252, range 612–22958) and strong uniform expression in normal BM plasma cells (mean 13.8-fold higher than mature B-cells, mean MFI 14250, range 7391–21914). Plasma cells from untreated myeloma patients showed a mean 1.9-fold higher IRF4 expression level than normal plasma cells (P=<0.001, normal n=16, neoplastic n = 19, mean MFI 27510, range 6807–53662). The myeloma patients were then separated into two groups; those with IRF4 expression in the normal range (n=6, mean MFI 12858, range 4182–21028) and those with high IRF4 expression (n=13, mean MFI 34501, range 24892–53662). After a median follow-up of 19 months, 6 patients had died in the high expression group (46.2%) compared with no deaths in the low expression group; this difference was statistically significant (Cox regression analysis, p=0.036). The assay developed can detect quantitative differences in IRF4 expression between B-progenitors, mature B-cells, normal plasma cells and neoplastic plasma cells. The ability to simultaneously analyse IRF4 expression with other cell surface markers allows rapid enumeration of IRF4 protein levels without plasma cell purification and greatly enhances the potential for IRF4 to be used as a prognostic marker. Neoplastic plasma cells can be categorised as having low or high levels of IRF4 expression with over-expression predicting a significantly shorter overall survival.