Lorenzo D’Ambrosio

Complete remission of paraneoplastic vanishing bile duct syndrome after the successful treatment of Hodgkin’s lymphoma: a case report and review of the literature

BackgroundVanishing bile duct syndrome has been associated with different pathologic conditions (adverse drug reactions, autoimmune diseases, graft versus host disease, and cancer). Though its causes are unknown, an immune-related pathogenesis is the most likely one. Vanishing bile duct syndrome can evolve to hepatic failure and, eventually, to death. The treatment is uncertain, but it needs the resolution of the underlying pathologic condition.Case presentationWe describe the association of Hodgkin’s lymphoma with a syndrome characterized by cholestasis, aminotransferase elevation and an histological picture of bile duct loss. All other causes of hepatic function impairment were excluded (in particular, drugs, viral and autoimmune related diseases) eventually leading to the diagnosis of vanishing bile duct syndrome. Despite the fact that the dysfunction is not caused by hepatic Hodgkin’s lymphoma involvement, liver impairment can limit the optimal therapy of Hodgkin’s lymphoma. A treatment consisting of ursodeoxycholic acid, prednisone, and full dose chemotherapy restored hepatic function and achieved complete and long-lasting remission of Hodgkin’s lymphoma.ConclusionWe reviewed all case reports showing that vanishing bile duct syndrome is a dismal paraneoplastic syndrome being fatal in a high proportion of patients if not adequately treated. Indeed, this syndrome requires both an early recognition and an appropriate aggressive treatment consisting of full dose upfront chemotherapy which is the only way to achieve a resolution of the vanishing bile duct syndrome. Delayed or reduced intensity treatments unfavorably correlate with survival.

Alpha–fetoprotein elevation in NUT midline carcinoma: a case report

BackgroundNuclear protein in testis (NUT) midline carcinoma is a rarely diagnosed and potentially under-recognized type of squamous carcinoma that is considered one of the most aggressive human solid tumors. Alpha-fetoprotein elevation has been associated with chronic liver diseases and a limited number of cancers. In particular, in presence of a mediastinal mass in a young man, alpha-fetoprotein elevation is considered nearly pathognomonic of a non-seminoma germ-cell tumor.Case presentationA 22-year old man without any comorbidity was diagnosed with a large mediastinal mass with skeletal and lymph node metastases. The clinical picture was dominated by a life-threatening superior vena cava syndrome with elevated alpha-fetoprotein and lactate dehydrogenase that supported the diagnostic suspicion of mediastinal germ-cell tumor. However, a biopsy showed a poorly-differentiated and diffusely necrotic carcinoma. We eventually reached the diagnosis of the peculiar entity of NUT midline carcinoma, but the differential diagnosis was quite challenging also because alpha-fetoprotein is not reported as a marker of NUT midline carcinoma. Notably, alpha-fetoprotein levels correlated with disease course.ConclusionsThe life-threatening aggressiveness of NUT midline carcinoma mandates to reach the right diagnosis in the shortest possible time. In this regard, poorly differentiated carcinomas lacking glandular differentiation mandate testing for NUT expression by immunohistochemistry. Awareness of a potentially misleading tumor marker elevation can help to broaden the differential diagnosis and establish the most appropriate treatment.

Next generation immune-checkpoints for cancer therapy

The discovery and clinical application of immune-checkpoint inhibitors has dramatically improved the treatments, outcomes and therapeutic concepts in multiple tumor settings. This breakthrough was mainly based on monoclonal antibodies blocking the inhibitory molecule CTLA-4 and or the PD-1/PD-L1 axis, with the aim of counteracting major tumor immune evasion mechanisms. Even acknowledging these important successes, not all the patients benefit from these treatments. Translational and clinical research efforts are ongoing to explore the potentialities of a new generation of immune-modulatory molecules to extend current clinical applications and contrast the unsolved issues of resistance and disease relapse that still affects a considerable rate of patients. New immune-checkpoints, with either stimulatory or inhibitory functions are emerging with key roles in regulating T cell response but also affecting other crucial effectors belonging to the innate immune response (e.g., natural killer). Their therapeutic exploitation, either alone or in strategical combinations, is providing important preclinical results, holding promises currently explored in initial clinical trials. The first results point toward favorable safety profiles with selective hints of activity in challenging settings. Important issues regarding the dose, schedule and rational combinations remain open and data from the clinical studies are needed. Here we provide an overview of the main emerging stimulatory or inhibitory immune-checkpoints exploitable in cancer treatment, briefly reporting their biological function, preclinical activity and preliminary clinical data.

Personalization of regorafenib treatment in metastatic gastrointestinal stromal tumours in real-life clinical practice:

Background: Regorafenib (REG) has now been approved as the standard third-line therapy in metastatic gastrointestinal stromal tumour (GIST) patients at the recommended dose and schedule of 160 mg once daily for the first 3 weeks of each 4-week cycle. However, it has a relevant toxicity profile that mainly occurs within the first cycles of therapy, and dose and schedule adjustments are often required to reduce the frequency or severity of adverse events and to avoid early treatment discontinuation. To date, large amounts of data on the use of REG in metastatic GIST patients in daily clinical practice are not available, and we lack information about how this treatment personalization really affects the quality of life (QoL) of patients. The aim of the present retrospective study is to build a comprehensive picture of all alternative REG strategies adopted in daily clinical practice for use in metastatic GIST patients. Methods: Metastatic GIST patients treated with dose adjustment or alternative schedules of REG at seven reference Italian centres were retrospectively included. Results: For a total of 62 metastatic GIST patients, we confirmed that REG treatment adjustment is common in clinical practice and that it is very heterogeneous, with approximately 20 different strategies being adopted. Independent of which strategy is chosen, treatment personalization has led to a clinical benefit defined as complete or partial resolution of side effects in almost all patients, affecting the duration of REG treatment. Conclusions: The personalization of REG, even if it is heterogeneous, seems to be crucial to maximize the overall treatment duration.

How a Clinical Trial Unit can improve independent clinical research in rare tumors: the Italian Sarcoma Group experience

BackgroundThe Italian Sarcoma Group (ISG) is a nonprofit group of professionals established in 1997 aimed to improve the quality of care and promote the independent research in sarcomas. The increased regulatory requirements, the chance to increase the number of trials with other cooperative groups and an interest from pharmaceutical companies in supporting independent research, generated the need of an internal service for research management.Methods and resultsIn 2010, ISG implemented in its organization a Clinical Trial Unit (CTU). The CTU was appointed to fully manage Clinical Trial Operations, to guarantee regulation compliance and provide a central support to the investigators, fostering a collaboration both at national and international level. In 2016 ISG promoted 25 studies in about 120 centers, with a fivefold increase in the last 5xa0years: 68% were interventional and 32% observational. Nine of the 17 interventional studies (52%) were supported by pharmaceutical companies, while 4 (24%) were funded by European Commission within specific projects on sarcomas and 4 (24%) were supported by the ISG itself.ConclusionThe contribution of ISG researchers to the international community was striking from the earliest years of the ISG creation. The challenges of the regulatory clinical research scenario, which imposes solid and hard-fast methodology with deep knowledge and expertise, highlighted the need to identify qualified and dedicated experts able to run and follow the multifaceted aspects of trials. Our analysis demonstrated how this model has led to a growth in competitiveness of the group. The collaboration between clinicians and CTU made possible to support the research with high scientific and ethical standards and to increase the number of trials, sites and overall enrolled patients. The reduced time for approvals, the continuous support to sites, the increased speed in data collection and analysis make the ISG research attractive for pharmaceutical industries, despite the problems that have characterized the independent research in the last years. The ability to fully manage and oversight Clinical Operations and the high quality of delivered services, have led the ISG to be recognized as a reliable partner and coordinator within the international sarcoma networks.

Imatinib rechallenge in patients with advanced gastrointestinal stromal tumors following progression with imatinib, sunitinib and regorafenib

Background: Rechallenge with imatinib is an option in advanced gastrointestinal stromal tumor (GIST) patients following progression with standard tyrosine-kinase inhibitors (TKIs), imatinib, sunitinib and regorafenib. We retrospectively collected data from metastatic Italian GIST patients treated with imatinib resumption after progression to conventional TKIs. Methods: A total of 104 eligible advanced GIST patients, previously treated with imatinib, sunitinib and regorafenib, were collected from six referral Italian institutions. Mutational analysis was recorded and correlated with survival and response according to RECIST 1.1 or CHOI criteria. Results: Overall, 71 patients treated with imatinib 400 mg as rechallenge were included. Mutational status was available in all patients. The median follow up was 13u2009months. In patients who received a rechallenge therapy, the median time to progression (TTP) was 5.4u2009months [95% confidence interval (CI) 1.9–13.5] and overall survival (OS) was 10.6u2009months (95% CI 2.8–26.9). A correlation between mutational status, response rate, TTP and OS was not found but comparing deleted versus nondeleted KIT exon 11 patients, a significant difference was identified in terms of TTP and OS (pu2009=u20090.04 and pu2009=u20090.02, respectively). Conclusions: Our retrospective data confirm that imatinib rechallenge is a reasonable option in advanced GIST. The prognostic value of the specific KIT mutations was confirmed in our series.

CD44v6 as innovative sarcoma target for CAR-redirected CIK cells

ABSTRACT Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR+.CIK). We set a patient-derived experimental platform. CAR+.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR+.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR+.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR+.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p<0.0001). Specificity of tumor killing was confirmed by blocking with anti-CD44v6 antibody. CAR+.CIK produced higher amounts of IL6 and IFN-γ compared to control CIK. CAR+.CIK were highly active in mice bearing subcutaneous STS xenografts, with significant delay of tumor growth (p<0.0001) without toxicities. We report first evidence of CAR+.CIKs activity against high grade STS and propose CD44v6 as an innovative target in this setting. CIK are a valuable platform for the translation of CAR-based strategies to challenging field of solid tumors. Our findings support the exploration of CAR+.CIK in clinical trials against high grade STS.

Cytokine Induced Killer cells are effective against sarcoma cancer stem cells spared by chemotherapy and target therapy.

ABSTRACT Metastatic bone and soft tissue sarcomas often relapse after chemotherapy (CHT) and molecular targeted therapy (mTT), maintaining a severe prognosis. A subset of sarcoma cancer stem cells (sCSC) is hypothesized to resist conventional drugs and sustain disease relapses. We investigated the immunotherapy activity of cytokine induced killer cells (CIK) against autologous sCSC that survived CHT and mTT. The experimental platform included two aggressive bone and soft tissue sarcoma models: osteosarcoma (OS) and undifferentiated-pleomorphic sarcoma (UPS). To visualize putative sCSC we engineered patient-derived sarcoma cultures (2 OS and 3 UPS) with a lentiviral sCSC-detector wherein the promoter of stem-gene Oct4 controls the expression of eGFP. We visualized a fraction of sCSC (mean 24.2 ± 5.2%) and confirmed their tumorigenicity in vivo. sCSC resulted relatively resistant to both CHT and mTT in vitro. Therapeutic doses of doxorubicin significantly enriched viable eGFP+sCSC in both OS (2.6 fold, n = 16) and UPS (2.3 fold, n = 29) compared to untreated controls. Treatment with sorafenib (for OS) and pazopanib (for UPS) also determined enrichment (1.3 fold) of viable eGFP+sCSC, even if less intense than what observed after CHT. Sarcoma cells surviving CHT and mTT were efficiently killed in vitro by autologous CIK even at minimal effector/target ratios (40:1 = 82%, 1:4 = 29%, n = 13). CIK immunotherapy did not spare sCSC that were killed as efficiently as whole sarcoma cell population. The relative chemo-resistance of sCSC and sensitivity to CIK immunotherapy was confirmed in vivo. Our findings support CIK as an innovative, clinically explorable, approach to eradicate chemo-resistant sCSC implicated in tumor relapse.

Abstract 3709: PARP1 expression (PARP1expr) drives synergy between PARP1 inhibitors (PARP1-Is) and trabectedin (TR)

Purpose of study. An attractive strategy to improve antitumor treatments is to inflict cytotoxic DNA damage with chemotherapy, and then impede DNA repair by molecular targeting. TR is a new drug characterized by a peculiar mechanism of action: TR traps DNA repair machinery leading to DNA damage, particularly in BRCA1/2-deficient tumors. We speculated that TR might activate PARP1, a key player in DNA-repair, and that subsequent PARP1 inhibition perpetuates TR-induced DNA damage leading to cell death. Experimental procedures and results. We developed a preclinical platform of 31 cell lines from different histotypes to explore the potential synergy between TR and the PARP1-Is olaparib (OL) and veliparib. We demonstrated that, regardless of BRCA1/2 status, PARP1-Is significantly increased TR activity, but a 15-fold range of sensitivity to the combination was observed. OL was proven the best PARP-I to combine with TR, probably due to its PARP1 trapping activity. In selected experiments, whole-genome expression profiling and GSEA analysis comparing cells displaying high vs. low synergism of the combination (HS-C vs. LS-C) revealed that DDR, G2/M cell cycle checkpoint, and DNA repair pathways were mechanistically involved in TR+OL synergy. TR induced PARP1 activation in 3/6 cell lines and PARP1-Is completely blocked both basal and TR-induced PARP1 activation. OL enhanced DNA damage response in 6/6 cell lines, but unrepairable DNA fragmentation was obtained in cells with high PARP1expr only. In two independent cell panels TR+OL synergism was directly related to PARP1expr both at mRNA (Pearson score r: 0.70, p = 0.00079) and protein level (r: 0.71, p = 0.015). Silencing and overexpression experiments validated the functional role of PARP1expr in determining TR+OL synergism: in HS-C the downmodulation of PARP1expr reduced sensitivity to TR+OL while the overexpression of PARP1 in LS-C rose TR+OL activity to levels observed in HS-C. Subcutaneous, intravenous and orthotopic xenografts of one HS-C (DMR) and one LS-C (SJSA-1) in NOD/SCID mice revealed OL significantly increased antitumor and antimetastatic activity of TR in cells with high PARP1expr only. Finally, we demonstrated that basal PARP1expr PARP1 activation by other cytotoxics with a stronger PARP1 activation observed in cells with high vs. low PARP1expr, regardless of the considered drug. Conclusions. OL enhances and potentially broaden TR cytotoxicity. TR+OL combination is particularly attractive in tumors harboring high PARP1expr and specific DDR-R gene signatures that might become predictive biomarkers of response. Future clinical validation of TR+OL combination may extend the use of PARP1-Is beyond BRCA1/2 defective tumors. Indeed, the crucial role of PARP1expr is confirmed regardless of tumor histotype and BRCA1/2 status. Further studies of combination between PARP1-Is and other cytotoxics should consider basal PARP1expr and activation after drug exposure. Citation Format: Ymera Pignochino, Federica Capozzi, Lorenzo D’ambrosio, Carmine Dell’aglio, Marco Basirico, Paola Boccone, Erica Palesandro, Loretta Gammaitoni, Dario Sangiolo, Maria Serena Benassi, Massimo Aglietta, Giovanni Grignani. PARP1 expression (PARP1expr) drives synergy between PARP1 inhibitors (PARP1-Is) and trabectedin (TR). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3709.

Abstract 2298: Cytokine-induced killer cells redirected with anti-CD44v6 chimeric antigen receptor against soft tissue sarcomas

Purpose of our study is to explore the anti-sarcoma activity of cytokine-induced killer (CIK) cells engineered with a chimeric antigen receptor (CAR) against the isoform variant 6 of adhesive receptor CD44 (CD44v6). CD44v6 may be an ideal target for immunotherapy as it is a tumor-promoting antigen, associated with the metastatic process and tumor initiating cells. Advanced and metastatic soft tissue sarcomas (STS) are currently incurable and in great need for new therapeutic strategies. CIK cells are ex vivo expanded T lymphocytes endowed with MHC-independent antitumor activity. CIK cells are active against STS (Sangiolo et al. Cancer Research 2014) but their function decreases at low effector/target (E/T) ratios with limitations in clinical perspective. We hypothesized that CIK cells may be effective candidates for CAR-based strategies, considering their intense ex vivo expansibility and innate antitumor activity. Experimental procedures and results. CIK cells were expanded from 9 STS patients and engineered with a lentiviral vector encoding for anti-CD44v6 CAR containing a CD28 signaling domain (Casucci et al, Blood 2013) and the HSV-TK suicide switch. Tumor killing was assessed in vitro against 10 STS (undifferentiated pleomorphic n = 5; Liposarcoma n = 2; Fibrosarcoma n = 1; GIST n = 2), in 2 cases STS targets were autologous. All 10 STS expressed CD44v6 (Relative Fluorescence Intensity = 4, SE = 1). Mean transduction efficiency was 60% (SE = 6%). Expansion rates and phenotype of CAR-CIK were comparable with unmodified controls (CD3+CD56+ = 50%; CD8 = 66%; NKG2D = 85%). Anti-CD44v6 CAR-CIK cells efficiently killed STS in vitro. Mean tumor-specific killing, at low E/T ratios, was significantly higher compared with unmodified CIK cells: 98% vs 84% (E/T = 10:1, p>0.05), 89% vs 41% (E/T = 1:1, p = 0.0001), 65% vs 26% (E/T = 1:4 p = 0.0001). In vitro treatment with Ganciclovir (10 μM) significantly inhibited tumor killing activity of CAR-CIK from 70% to 10% (E/T 1:1, n = 3, p = 0.008). Blocking experiments (n = 2) with anti-CD44v6 antibody against the same epitope recognized by the CAR (VFF-18) decreased tumor-specific killing from 96% to 43% (E/T = 1:1) and 27% (E/T = 1:2). Adoptive infusion of anti-CD44v6 CAR-CIK cells significantly delayed tumor growth (p = 0.01) and reduced proliferative index (p = 0.001) of established subcutaneous fibrosarcoma xenografts in NOD/SCID mice (n = 3) compared to untreated controls (n = 3) without any sign of toxicity. Conclusions. Ours is the first report of CAR-engineered CIK cells against solid tumors. This approach significantly potentiates the innate tumor killing ability of CIK cells with a new redirected antitumor specificity. CIK cells may be appealing alternative candidates to conventional T cells for future CAR-based strategies against solid tumors. Our findings support CD44v6 as a valuable target for adoptive cell therapies against currently incurable sarcomas. Citation Format: Valeria Leuci, Monica Casucci, Giovanni Grignani, Ramona Rotolo, Elisa Vigna, Loretta Gammaitoni, Giulia Mesiano, Lorenzo D’Ambrosio, Massimo Aglietta, Attilio Bondanza, Dario Sangiolo. Cytokine-induced killer cells redirected with anti-CD44v6 chimeric antigen receptor against soft tissue sarcomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2298.