Changes in tumor stiffness for early prediction of tumor response to sorafenib: a proof-of-concept study with elastosonography in an animal model of Hepatocellular Carcinoma (HCC)

Background and aims: Sorafenib is the reference therapy for advanced Hepatocellular Carcinoma (HCC). No method exists to predict in the very early period subsequent individual response. Starting from the clinical experience in humans that subcutaneous metastases may rapidly change consistency under sorafenib and that elastosonography a new ultrasound based technique allows assessment of tissue stiffness, we investigated the role of elastonography in the very early prediction of tumor response to sorafenib in a HCC animal model. Methods: HCC (Huh7 cells) subcutaneous xenografting in mice was utilized. Mice were randomized to vehicle or treatment with sorafenib when tumor size was 5-10 mm. Elastosonography (Mylab 70XVG, Esaote, Genova, Italy) of the whole tumor mass on a sagittal plane with a 10 MHz linear transducer was performed at different time points from treatment start (day 0, +2, +4, +7 and +14) until mice were sacrified (day +14), with the operator blind to treatment. In order to overcome variability in absolute elasticity measurement when assessing changes over time, values were expressed in arbitrary units as relative stiffness of the tumor tissue in comparison to the stiffness of a standard reference stand-off pad lying on the skin over the tumor. Results: Sor-treated mice showed a smaller tumor size increase at day +14 in comparison to vehicle-treated (tumor volume increase +192.76% vs +747.56%, p=0.06). Among Sor-treated tumors, 6 mice showed a better response to treatment than the other 4 (increase in volume +177% vs +553%, p=0.011). At day +2, median tumor elasticity increased in Sor-treated group (+6.69%, range ���30.17-+58.51%), while decreased in the vehicle group (-3.19%, range ���53.32-+37.94%) leading to a significant difference in absolute values (p=0.034). From this time point onward, elasticity decreased in both groups, with similar speed over time, not being statistically different anymore. In Sor-treated mice all 6 best responders at day 14 showed an increase in elasticity at day +2 (ranging from +3.30% to +58.51%) in comparison to baseline, whereas 3 of the 4 poorer responders showed a decrease. Interestingly, these 3 tumours showed elasticity values higher than responder tumours at day 0. Conclusions: Elastosonography appears a promising non-invasive new technique for the early prediction of HCC tumor response to sorafenib. Indeed, we proved that responder tumours are characterized by an early increase in elasticity. The possibility to distinguish a priori between responders and non responders based on the higher elasticity of the latter needs to be validated in ad-hoc experiments as well as a confirmation of our results in humans is warranted.

Terapie sistemiche dell'epatocarcinoma (HCC): dalla preclinica alla clinica

Introduzione Attualmente i principali punti critici del trattamento dell���HCC avanzato sono: 1) la mancanza di predittori di risposta alla terapia con sorafenib, 2) lo sviluppo resistenze al sorafenib, 3) la mancanza di terapie di seconda linea codificate. Scopo della tesi 1) ricerca di predittori clinico-laboratoristici di risposta al sorafenib in pazienti ambulatoriali con HCC; 2) valutazione dell���impatto della sospensione temporanea-definitiva del sorafenib in un modello murino di HCC mediante tecniche ecografiche; 3) valutazione dell���efficacia della capecitabina metronomica come seconda linea dell���HCC non responsivo a sorafenib. Risultati Studio-1: 94 pazienti con HCC trattato con sorafenib: a presenza di metastasi e PVT-neoplastica non sembra inficiare l���efficacia del sorafenib. AFP basale <19 ng/ml �� risultata predittrice di maggiore sopravvivenza, mentre lo sviluppo di nausea di una peggiore sopravvivenza. Studio -2: 14 topi con xenografts di HCC: gruppo-1 trattato con placebo, gruppo-2 trattato con sorafenib con interruzione temporanea del farmaco e gruppo-3 trattato con sorafenib con sospensione definitiva del sorafenib. La CEUS targettata per il VEGFR2 ha mostrato al giorno 13 valori maggiori di dTE nel gruppo-3 confermato da un aumento del VEGFR2 al Western-Blot. I tumori del gruppo-2 dopo 2 giorni di ritrattamento, hanno mostrato un aumento dell���elasticit�� tissutale all���elastonografia. Studio-3:19 pazienti trattati con capecitabina metronomica dopo sorafenib. Il TTP �� stato di 5 mesi (95% CI 0-10), la PFS di 3,6 mesi (95% CI 2,8-4,3) ed la OS di 6,3 mesi (95% CI 4-8,6). Conclusioni Lo sviluppo di nausea ed astenia ed AFP basale >19, sono risultati predittivi di una minore risposta al sorafenib. La sospensione temporanea del sorafenib in un modello murino di HCC non impedisce il ripristino della risposta tumorale, mentre una interruzione definitiva tende a stimolare un ���effetto rebound��� dell���angiogenesi. La capecitabina metronomica dopo sorafenib ha mostrato una discreta attivit�� anti-neoplastica ed una sicurezza accettabile.

Prozessanalytische Technologien "PAT" zur Optimierung von Wirbelschichtgranulationsprozessen

In der Herstellung fester Darreichungsformen umfasst die Granulierung einen komplexen Teilprozess mit hoher Relevanz f��r die Qualit��t des pharmazeutischen Produktes. Die Wirbelschichtgranulierung ist ein spezielles Granulierverfahren, welches die Teilprozesse Mischen, Agglomerieren und Trocknen in einem Ger��t vereint. Durch die Kombination mehrerer Prozessstufen unterliegt gerade dieses Verfahren besonderen Anforderungen an ein umfassendes Prozessverst��ndnis. Durch die konsequente Verfolgung des PAT- Ansatzes, welcher im Jahre 2004 durch die amerikanische Zulassungsbeh��rde (FDA) als Guideline ver��ffentlicht wurde, wurde der Grundstein f��r eine kontinuierliche Prozessverbesserung durch erh��htes Prozessverst��ndnis, f��r Qualit��tserh��hung und Kostenreduktion gegeben. Die vorliegende Arbeit befasste sich mit der Optimierung der Wirbelschicht-Granulationsprozesse von zwei prozesssensiblen Arzneistoffformulierungen, unter Verwendung von PAT. rnF��r die Enalapril- Formulierung, einer niedrig dosierten und hochaktiven Arzneistoffrezeptur, wurde herausgefunden, dass durch eine feinere Zerst��ubung der Granulierfl��ssigkeit deutlich gr����ere Granulatk��rnchen erhalten werden. Eine Erh��hung der MassRatio verringert die Tr��pfchengr����e, dies f��hrt zu gr����eren Granulaten. Sollen Enalapril- Granulate mit einem gew��nschten D50-Kornverteilung zwischen 100 und 140 um hergestellt werden, dann muss die MassRatio auf hohem Niveau eingestellt werden. Sollen Enalapril- Granulate mit einem D50- Wert zwischen 80 und 120��m erhalten werden, so muss die MassRatio auf niedrigem Niveau eingestellt sein. Anhand der durchgef��hrten Untersuchungen konnte gezeigt werden, dass die MassRatio ein wichtiger Parameter ist und zur Steuerung der Partikelgr����e der Enalapril- Granulate eingesetzt werden kann; unter der Voraussetzung dass alle anderen Prozessparameter konstant gehalten werden.rnDie Betrachtung der Schnittmengenplots gibt die M��glichkeit geeignete Einstellungen der Prozessparameter bzw. Einflussgr����en zu bestimmen, welche dann zu den gew��nschten Granulat- und Tabletteneigenschaften f��hren. Anhand der Lage und der Gr����e der Schnittmenge k��nnen die Grenzen der Prozessparameter zur Herstellung der Enalapril- Granulate bestimmt werden. Werden die Grenzen bzw. der ���Design Space��� der Prozessparameter eingehalten, kann eine hochwertige Produktqualit��t garantiert werden. rnUm qualitativ hochwertige Enalapril Tabletten mit der gew��hlten Formulierung herzustellen, sollte die Enalapril- Granulation mit folgenden Prozessparametern durchgef��hrt werden: niedrige Spr��hrate, hoher MassRatio, einer Zulufttemperatur von mindestens > 50 ��C und einer effektiven Zuluftmenge < 180 Nm��/h. Wird hingegen eine Spr��hrate von 45 g/min und eine mittlere MassRatio von 4.54 eingestellt, so muss die effektive Zuluftmenge mindestens 200 Nm��/h und die Zulufttemperatur mindestens 60 ��C betragen, um eine vorhersagbar hohe Tablettenqualit��t zu erhalten. Qualit��t wird in das Arzneimittel bereits w��hrend der Herstellung implementiert, indem die Prozessparameter bei der Enalapril- Granulierung innerhalb des ���Design Space��� gehalten werden.rnF��r die Metformin- Formulierung, einer hoch dosierten aber wenig aktiven Arzneistoffrezeptur wurde herausgefunden, dass sich der Wachstumsmechanismus des Feinanteils der Metformin- Granulate von dem Wachstumsmechanismus der D50- und D90- Kornverteilung unterscheidet. Der Wachstumsmechanismus der Granulate ist abh��ngig von der Partikelbenetzung durch die verspr��hten Fl��ssigkeitstr��pfchen und vom Gr����enverh��ltnis von Partikel zu Spr��htr��pfchen. Der Einfluss der MassRatio ist f��r die D10- Kornverteilung der Granulate vernachl��ssigbar klein. rnMit Hilfe der St��rgr����en- Untersuchungen konnte eine Regeleffizienz der Prozessparameter f��r eine niedrig dosierte (Enalapril)- und eine hoch dosierte (Metformin) Arzneistoffformulierung erarbeitet werden, wodurch eine weitgehende Automatisierung zur Verringerung von Fehlerquellen durch Nachregelung der St��rgr����en erm��glicht wird. Es ergibt sich f��r die gesamte Prozesskette ein in sich geschlossener PAT- Ansatz. Die Prozessparameter Spr��hrate und Zuluftmenge erwiesen sich als am besten geeignet. Die Nachregelung mit dem Parameter Zulufttemperatur erwies sich als tr��ge. rnFerner wurden in der Arbeit Herstellverfahren f��r Granulate und Tabletten f��r zwei prozesssensible Wirkstoffe entwickelt. Die Robustheit der Herstellverfahren gegen��ber St��rgr����en konnte demonstriert werden, wodurch die Voraussetzungen f��r eine Echtzeitfreigabe gem���� dem PAT- Gedanken geschaffen sind. Die Kontrolle der Qualit��t des Produkts findet nicht am Ende der Produktions- Prozesskette statt, sondern die Kontrolle wird bereits w��hrend des Prozesses durchgef��hrt und basiert auf einem besseren Verst��ndnis des Produktes und des Prozesses. Au��erdem wurde durch die konsequente Verfolgung des PAT- Ansatzes die M��glichkeit zur kontinuierlichen Prozessverbesserung, zur Qualit��tserh��hung und Kostenreduktion gegeben und damit das ganzheitliche Ziel des PAT- Gedankens erreicht und verwirklicht.rn

La traduzione dei racconti di Pat Mora: muoversi tra due lingue e due culture

This dissertation deals with the translations of seven books for children written by the Chicano author Pat Mora. I started to be interested in the Chicano world, a world suspended between Mexico and the United States, after reading a book by Sandra Cisneros. I decided to deepen my curiosity and for this reason, I discovered a hybrid reality full of history, culture and traditions. In this context, the language used is characterized by a continuous code switching between Spanish and English and I thought it was an interesting phenomenon from the literary and translation point of view. During my research in the Chicano culture, I ran across Pat Mora. Her books for children fascinated me because of their actual themes (the cultural diversity and the defense of identity) and their beautiful illustrations. For this reason, I chose to translate seven of her books because I believe they could be an enrichment for children literature in Italy. The work consists of five chapters. The first one deals with the identity of Chicano people, their history, their literature and their language. In the second chapter, I outline Pat Mora���s profile. I talk about her biography and I analyze her most famous works. In the third chapter, I introduce the seven books for children to be translated and I point out their plots and main themes. In the fourth chapter, I present the translation of the books. The fifth chapter is the translation comment. I deal with the linguistic analysis of the source texts and the analysis of the target texts focusing on the choices made during the translation process.

Anti-angiogenic therapy for HCC

Hepatocellular carcinoma is an insidious disease that grows without eliciting pain. In the absence of surveillance, the diagnosis of hepatocellular carcinoma is usually made at a late stage, which excludes curative treatments and leaves patients with few therapeutic options. For years, conventional chemotherapy was administered but yielded poor results. This is not surprising since hepatocytes are well equipped to survive exposure to chemotherapeutics. Hepatocytes posses an extensive repertoire of enzymes and pumps capable of degrading and exporting these drugs. Bypassing hepatocytic tumor cells in favour of supportive cells represents an alternative treatment target that has achieved modest success. The supportive cells in the hepatic vasculature comprise endothelial cells and pericytes. Thanks to a concerted effort from fundamental and pharmacological researchers, several drugs targeted to the vasculature are reaching the clinic. This manuscript reviews the rationale for targeting the vascular cells to treat hepatocellular carcinoma, the signalling pathways underlying angiogenesis and the most promising drugs.

Met-thodology

Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype. Kaposi-Novak P, Lee JS, Gomez-Quiroz L, Coulouarn C, Factor VM, Thorgeirsson SS. Identification of specific gene expression signatures characteristic of oncogenic pathways is an important step toward molecular classification of human malignancies. Aberrant activation of the Met signaling pathway is frequently associated with tumour progression and metastasis. In this study, we defined the Met-dependent gene expression signature using global gene expression profiling of WT and Met-deficient primary mouse hepatocytes. Newly identified transcriptional targets of the Met pathway included genes involved in the regulation of oxidative stress responses as well as cell motility, cytoskeletal organization, and angiogenesis. To assess the importance of a Met-regulated gene expression signature, a comparative functional genomic approach was applied to 242 human hepatocellular carcinomas (HCCs) and 7 metastatic liver lesions. Cluster analysis revealed that a subset of human HCCs and all liver metastases shared the Met-induced expression signature. Furthermore, the presence of the Met signature showed significant correlation with increased vascular invasion rate and microvessel density as well as with decreased mean survival time of HCC patients. We conclude that the genetically defined gene expression signatures in combination with comparative functional genomics constitute an attractive paradigm for defining both the function of oncogenic pathways and the clinically relevant subgroups of human cancers. [Abstract reproduced by permission of J Clin Invest 2006;116:1582-1595].

The Other Side of the Doors: Butte & Beyond -- Congressman Pat Williams ���In the Crucible of Change���

Pat Williams emerged from the Mining City of Butte, Montana with a sense of grassroots, people-oriented politics. His inherent belief in the power of ordinary citizens carried him through the Montana Legislature and into Congress for a record-setting period. The accomplishments of his long career partially obscured his innate progressive and populist instinct that is reflective of the period of ���in the Crucible of Change.��� This film addresses Pat���s early years when his progressive instincts and activities resulted in pushback from the giant Anaconda Company which had held Montana hostage for 75 years. Pat is joined for part of the film by former campaign staffer, and now prominent media consultant, Michael Fenenbock for reflections on Pat���s 1978 ���Door-to-Door to Congress��� campaign, which demonstrated the power of his belief in the people on the other side of the doors. Pat Williams (b. 1937) rose from teaching grade school in his hometown of Butte, MT, to serving for the longest number of consecutive terms (9 terms, 18 years) in the US House of Representatives of anyone in Montana history. Pat was a member of the National Guard and attended UM in Missoula and William Jewel College, graduating from the University of Denver. Pat also served in the Montana legislature for 2 terms (1966 & 1968 elections). In 1969. Pat helped his legislative seat-mate John Melcher get elected as Montana���s Eastern District Congressman in the Special Election that June. Pat went to Washington DC as Melcher���s Executive Assistant. Upon returning to Montana, Pat headed up the Montana offices of the innovative Mountain Plains Family Education Program. In 1974, Pat ran unsuccessfully for Montana���s Western District Congressional seat in a three-way race with former Congressman Arnold Olsen and state Legislator Max Baucus. After the drafting and passage of the 1972 Montana Constitution, Pat was named a member of Montana���s first-ever Reapportionment Commission. In 1978 he successfully ran for Congress, conducting a massive grass-roots door-to-door campaign of 1�� years, reaching 50,000 doors. In a hotly contested 6-way Democratic primary, Pat won going away and also handily won the general election. Pat served in Congress from January, 1979 until January of 1997, 14 years representing the Western District and 4 years representing the entire state. Upon his retirement from Congress, in 1997 Williams returned to Montana where has been an instructor at the University of Montana and Senior Fellow and Regional Policy Associate at the Center for the Rocky Mountain West. He is a former member of the Montana Board of Regents and serves on a number of national education-related boards. In Congress Pat was a Deputy Whip of the U.S. House of Representatives and sat on committees on: Budget, Natural Resources, Education and Labor, and Agriculture. Pat���s leadership helped pass trailblazing legislation to assist hard-working middle-class families and ensure opportunities for every child. Pat���s fingerprints are on many pieces of important legislation, including the College Middle Income Assistance Act, the Family and Medical Leave Act, the Toddlers and Childhood Disability Act, the Library Services and Construction Act, and the Museum Services Act. Pat successfully sponsored the Lee Metcalf Wilderness Area and the Rattlesnake Wilderness area, helped save the Bob Marshall Wilderness from oil and gas exploration, and helped ban geothermal energy drilling near the borders of Yellowstone National Park. As Chairman of The Post-Secondary Education Committee, he protected the National Endowment for the Arts from elimination, a remarkable undertaking during a very trying time for the Agency. Pat worked tirelessly with Tribal College Leaders to build Montana���s seven Tribal Colleges. He was also responsible for the legislation that created The American Conservation Corps, which became the Corporation for National Service, giving thousands of America���s young people a chance to serve their country and pursue higher education. Pat lives in Missoula with his wife Carol Griffith Williams, former Montana Senate Majority Leader. They have three children and five grandchildren.

Antitumor effect of SIRT1 inhibition in human HCC tumor models in vitro and in vivo

Sirtuins (SIRT1-7) are a highly conserved family of NAD(+)-dependent enzymes that control the activity of histone and nonhistone regulatory proteins. SIRT1 is purposed to promote longevity and to suppress the initiation of some cancers. Nevertheless, SIRT1 is reported to function as a tumor suppressor as well as an oncogenic protein. Our data show that compared with normal liver or surrounding tumor tissue, SIRT1 is strongly overexpressed in human hepatocellular carcinoma (HCC). In addition, human HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for the expression of the sirtuin family members and only SIRT1 was consistently overexpressed compared with normal hepatocytes. To determine its effect on HCC growth, SIRT1 activity was inhibited either with lentiviruses expressing short hairpin RNAs or with the small molecule inhibitor, cambinol. Knockdown or inhibition of SIRT1 activity had a cytostatic effect, characterized by an altered morphology, impaired proliferation, an increased expression of differentiation markers, and cellular senescence. In an orthotopic xenograft model, knockdown of SIRT1 resulted in 50% fewer animals developing tumors and cambinol treatment resulted in an overall lower tumor burden. Taken together, our data show that inhibition of SIRT1 in HCC cells impairs their proliferation in vitro and tumor formation in vivo. These data suggest that SIRT1 expression positively influences the growth of HCC and support further studies aimed to block its activity alone or in combination as a novel treatment strategy.

PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.