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.

Valutazione precoce della risposta a Sorafenib nel Carcinoma Epatocellulare mediante quantificazione dell'Ecografia con contrasto con software Sonotumor

Scopo dello studio: Stabilire se cambiamenti della perfusione di una lesione target di epatocarcinoma (HCC), valutati quantitativamente mediante ecografia con contrasto (CE-US) alla settimana 2 e 4 di terapia con sorafenib, possono predire la progressione di malattia alla settimana 8, valutata con la tomografia computerizzata o la risonanza magnetica con mezzo di contrasto (TC-RM) usando i criteri RECIST/RECIST modificati (response evaluation criteria in solid tumors). Pazienti e metodi: Il comitato etico ha approvato lo studio ed i pazienti hanno fornito un consenso informato scritto prima dell’arruolamento. Lo studio è stato effettuato su un campione di soggetti con epatocarcinoma avanzato o non suscettibile di trattamento curativo, in monoterapia con sorafenib. La valutazione della risposta tumorale è stata effettuata con TC o RM a 2 mesi usando i criteri RECIST/RECIST modificati. La CE-US è stata effettuata entro 1 settimana prima dell’inizio del trattamento con sorafenib e durante la terapia alla settimana 2, 4, 8, 16 e 32. I parametri quantitativi funzionali sono stati ottenuti impiegando un software dedicato. I cambiamenti dei valori dei parametri suddetti tra il tempo zero ed i punti temporali successivi sono stati confrontati con la risposta tumorale basata sui criteri RECIST/RECIST modificati. Risultati: La riduzione dei valori dei parametri relativi alla perfusione tumorale, in particolare di WiAUC e PE (parametri correlati con il volume ematico), al T2/T4 (settimana 2, 4), predice la risposta tumorale a 2 mesi, valutata secondo i criteri RECIST e RECIST modificati, risultata indicativa di malattia stabile (responders). Conclusione: L’ecografia con contrasto può essere impiegata per quantificare i cambiamenti della vascolarizzazione tumorale già alla settimana 2, 4 dopo la somministrazione di sorafenib nei pazienti con HCC. Questi precoci cambiamenti della perfusione tumorale possono essere predittivi della risposta tumorale a 2 mesi e possono avere un potenziale nella valutazione precoce dell'efficacia della terapia antiangiogenica nell’epatocarcinoma.

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 è 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’elasticità tissutale all’elastonografia. Studio-3:19 pazienti trattati con capecitabina metronomica dopo sorafenib. Il TTP è 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 attività anti-neoplastica ed una sicurezza accettabile.

Use of VEGFR-2 targeted microbubbles (BR55, Bracco imaging) for the early ultrasound evaluation of response to antiangiogenic treatment in a xenograft model of hepatocarcinoma

Aim: To evaluate the early response to treatment to an antiangiogenetic drug (sorafenib) in a heterotopic murine model of hepatocellular carcinoma (HCC) using ultrasonographic molecular imaging. Material and Methods: the xenographt model was established injecting a suspension of HuH7 cells subcutaneously in 19 nude mice. When tumors reached a mean diameter of 5-10 mm, they were divided in two groups (treatment and vehicle). The treatment group received sorafenib (62 mg/kg) by daily oral gavage for 14 days. Molecular imaging was performed using contrast enhanced ultrasound (CEUS), by injecting into the mouse venous circulation a suspension of VEGFR-2 targeted microbubbles (BR55, kind gift of Bracco Swiss, Geneve, Switzerland). Video clips were acquired for 6 minutes, then microbubbles (MBs) were destroyed by a high mechanical index (MI) impulse, and another minute was recorded to evaluate residual circulating MBs. The US protocol was repeated at day 0,+2,+4,+7, and +14 from the beginning of treatment administration. Video clips were analyzed using a dedicated software (Sonotumor, Bracco Swiss) to quantify the signal of the contrast agent. Time/intensity curves were obtained and the difference of the mean MBs signal before and after high MI impulse (Differential Targeted Enhancement-dTE) was calculated. dTE represents a numeric value in arbitrary units proportional to the amount of bound MBs. At day +14 mice were euthanized and the tumors analyzed for VEGFR-2, pERK, and CD31 tissue levels using western blot analysis. Results: dTE values decreased from day 0 to day +14 both in treatment and vehicle groups, and they were statistically higher in vehicle group than in treatment group at day +2, at day +7, and at day +14. With respect to the degree of tumor volume increase, measured as growth percentage delta (GPD), treatment group was divided in two sub-groups, non-responders (GPD>350%), and responders (GPD<200%). In the same way vehicle group was divided in slow growth group (GPD<400%), and fast growth group (GPD>900%). dTE values at day 0 (immediately before treatment start) were higher in non-responders than in responders group, with statistical difference at day 2. While dTE values were higher in the fast growth group than in the slow growth group only at day 0. A significant positive correlation was found between VEGFR-2 tissue levels and dTE values, confirming that level of BR55 tissue enhancement reflects the amount of tissue VEGF receptor. Conclusions: the present findings show that, at least in murine experimental models, CEUS with BR55 is feasable and appears to be a useful tool in the prediction of tumor growth and response to sorafenib treatment in xenograft HCC.

Dissecting the role of IGF2BP3 in the stress-adaptive response and in intercellular communication in Ewing Sarcoma.

Tumor microenvironment has emerged as key factor influencing tumor progression and metastatization. In this context, small vesicles produced by cancer cells can influence the fate of their surroundings via the horizontal transfer of specific molecular cargos. Ewing Sarcoma, the second most common bone tumor in young patients, presents early metastasis associated to worse prognosis. The RNA binding protein Insulin-like Growth Factor 2 mRNA Binding Protein 3 (IGF2BP3) exerts a pro-oncogenic role associated with metastasis formation and worse prognosis in Ewing Sarcoma. Our aim was to investigate the still unexplored role of IGF2BP3 in the stress-adaptive response to tumor microenvironment and in the interactions between Ewing Sarcoma cells. Hypoxia is a major feature of Ewing Sarcoma microenvironment and we demonstrated that IGF2BP3 can direct the CXCR4-mediated migratory response to CXCL12 in Ewing Sarcoma cells subjected to oxygen deprivation. We also discovered that the interaction between IGF2BP3 and CXCR4 is regulated through CD164 and which colocalize at plasma membrane level, upon CXCL12 exposure. Interestingly, high IGF2BP3 levels in Ewing Sarcoma metastatic lesions positively correlated with the expression of both CD164 and CXCR4, indicating the IGF2BP3/CD164/CXCR4 oncogenic axis as a critical modulator of Ewing Sarcoma metastatic progression. We demonstrated for the first time that IGF2BP3 is loaded into Ewing Sarcoma derived exosomes, accordingly to its cellular levels. We discovered that IGF2BP3+ exosomes carry high levels of IGF2BP3-client mRNAs involved in cellular migration, CD164 and IGF1R, and, by transferring this cargo, sustain the migratory abilities of receiving cells, induce a sharp up-regulation of CD164, CXCR4 and IGF1R and enhance the activation of AKT/mTOR and ERK down-stream signalling pathways. We demostrated that the pro-tumorigenic role of IGF2BP3 is not only exerted at cellular level, but that intercellular communication is crucial in the context of Ewing Sarcoma microenvironment.

Bright-field multiplex immunohistochemistry for tumor. Microenvironment evaluation in mucosal melanoma of the head and neck region: technical aspects, results, and correlation with clinicopathologic features and molecular landscape

Mucosal melanoma of the head and neck region (MM-H&N) is a rare disease, characterized by a poor prognosis and limited therapeutic strategies, especially regarding targeted therapy (lower rate of targetable mutations compared to cutaneous melanoma) and immunotherapy (lack of diagnostic tools able to predict the response). Meanwhile, bright-field multiplex immunohistochemistry (BF-mIHC) is emerging as a promising tool for characterizing tumor microenvironment (TME) and predicting response to immunotherapy in several tumors, including melanoma. This PhD project aims to develop a BF-mIHC protocol to evaluate the TME in MM-H&N, analyze the correlation between immune markers/immune profiles and MM-H&N features (clinicopathologic and molecular), and find new biomarkers useful for prognostic-therapeutic stratification of these patients. Specific aims are: (I) describe the clinicopathological features of MM-H&N; (II) analyze the molecular status of MM-H&N and correlate it with the clinicopathological features; (III) analyze the molecular status of multiple specimens from the same patient to verify whether molecular heterogeneity of MM-H&N could affect the results with relevant prognostic-therapeutic implications; (IV) develop a BF-mIHC protocol to study TME in MM-H&N; (V) analyze the correlation between immune markers/immune profiles and MM-H&N features (clinicopathologic and molecular) to test whether BF-mIHC could be a promising tool for prognostic-therapeutic characterization of these patients.