Barbara Vergani

Rb and TP53 Pathway Alterations in Sporadic and NF1-Related Malignant Peripheral Nerve Sheath Tumors

Karyotypic complexities associated with frequent loss or rearrangement of a number of chromosome arms, deletions, and mutations affecting the TP53 region, and molecular alterations of the INK4A gene have been reported in sporadic and/or neurofibromatosis type I (NF1)-related malignant peripheral nerve sheath tumors (MPNSTs). However, no investigations addressing possible different pathogenetic pathways in sporadic and NF1-associated MPNSTs have been reported. This lack is unexpected because, despite similar morphologic and immunophenotypic features, NF1-related cases are, by definition, associated with NF1 gene defects. Thus, we investigated the occurrence of TP53 and p16INK4A gene deregulation and the presence of microsatellite alterations at markers located at 17p, 17q, 9p21, 22q, 11q, 1p, or 2q loci in MPNSTs and neurofibromas either related (14 cases) or unrelated (14 cases) to NF1. Our results indicate that, in MPNSTs, p16INK4A inactivation almost equally affects both groups. However, TP53 mutations and loss of heterozygosity involving the TP53 locus (43% versus 9%), and p53 wild type overexpression, related or not to mdm2 overexpression (71% versus 25%), seem to mainly be restricted to sporadic MPNSTs. In NF1-associated MPNSTs, our microsatellite results are consistent with the occurrence of somatic inactivation by loss of heterozygosity of the second NF1 allele.

Molecular abnormalities in liposarcoma: role of MDM2 and CDK4-containing amplicons at 12q13–22

It has recently been shown that mdm2 overexpression with stabilization of p53 represents a characteristic of retroperitoneal well‐differentiated‐dedifferentiated, here renamed evolved (WD‐E), liposarcomas at the immunocytochemical, molecular, and cytogenetic level. This make‐up appears to be confined to half the cases in non‐retroperitoneal well‐differentiated liposarcomas. Since in different tumours MDM2 amplification involves amplicons encompassing flanking genes, such as CDK4, the possibility was investigated that in these tumours, CDK4 could act as an alternative or additional gene involved in the transformation mechanism. Forty‐one retroperitoneal (R)/non‐retroperitoneal (NR) well‐differentiated‐dedifferentiated (WD‐DD) and 33 myxoid/round cell liposarcomas were reanalysed by immunocytochemical, molecular (nine cases) and fluorescence in situ hybridization (FISH) (one case) techniques. The results showed that all but one R WD‐E cases carried the mdm2+, p53+, cdk4+ immunophenotype. In NR‐WD liposarcomas, this immunophenotype was shared in five cases and the remainder showed mdm2+, p53−, cdk4+ in four and mdm2−, p53−, cdk4+ in one case, showing ring chromosomes by FISH analysis. TP53 mutations are confirmed to be closely correlated with NR‐DD liposarcomas and no CDK4 involvement was found in the myxoid/round cell liposarcoma group. As well as confirming the synergistic effect of MDM2 and CDK4, these results are consistent with the concept that amplicon(s) excluding MDM2 may contribute to transformation and support a role of CDK4 in opposing p53 function, particularly in NR WD liposarcoma.

TNF-Related Apoptosis-Inducing Ligand (TRAIL)–Armed Exosomes Deliver Proapoptotic Signals to Tumor Site

Purpose: Exosomes deliver signals to target cells and could thus be exploited as an innovative therapeutic tool. We investigated the ability of membrane TRAIL-armed exosomes to deliver proapoptotic signals to cancer cells and mediate growth inhibition in different tumor models. Experimental Methods and Results: K562 cells, transduced with lentiviral human membrane TRAIL, were used for the production of TRAIL+ exosomes, which were studied by nanoparticle tracking analysis, cytofluorimetry, immunoelectronmicroscopy, Western blot, and ELISA. In vitro, TRAIL+ exosomes induced more pronounced apoptosis (detected by Annexin V/propidium iodide and activated caspase-3) in TRAIL-death receptor (DR)5+ cells (SUDHL4 lymphoma and INT12 melanoma), with respect to the DR5−DR4+KMS11 multiple myeloma. Intratumor injection of TRAIL+ exosomes, but not mock exosomes, induced growth inhibition of SUDHL4 (68%) and INT12 (51%), and necrosis in KMS11 tumors. After rapid blood clearance, systemically administered TRAIL+ exosomes accumulated in the liver, lungs, and spleen and homed to the tumor site, leading to a significant reduction of tumor growth (58%) in SUDHL4-bearing mice. The treatment of INT12-bearing animals promoted tumor necrosis and a not statistically significant tumor volume reduction. In KMS11-bearing mice, despite massive perivascular necrosis, no significant tumor growth inhibition was detected. Conclusions: TRAIL-armed exosomes can induce apoptosis in cancer cells and control tumor progression in vivo. Therapeutic efficacy was particularly evident in intratumor setting, while depended on tumor model upon systemic administration. Thanks to their ability to deliver multiple signals, exosomes thus represent a promising therapeutic tool in cancer. Clin Cancer Res; 22(14); 3499–512. ©2016 AACR.

Trends and predictors of non-AIDS-defining cancers in men and women with HIV infection: a single-institution retrospective study before and after the introduction of HAART.

Background:The incidence of non–AIDS-defining cancers (NADCs) in HIV-positive patients has increased over recent years. Most studies of the risk and spectrum of NADCs are primarily based on male populations, and only a few have provided specific information regarding females. Methods:We retrospectively analyzed all incident NADCs occurring in a cohort of HIV-positive patients followed up between 1985 and 2011. Incidence rates before and after the introduction of highly active antiretroviral therapy (HAART) were examined using Poisson regression models. Standardized incidence ratios (SIRs) were used to compare the cancer risk of HIV-infected subjects with that of the age- and gender-matched general population as estimated by the Milan Cancer Registry. Results:Five thousand nine hundred twenty-four patients (4382 men and 1542 women) contributed 50,990 person-years to the follow-up. Among them, 144 had new NADC diagnosis. The overall incidence increased from 1.0 case/1000 person-years in the pre-HAART period to 4.5 cases/1000 person-years in the HAART period (P < 0.01). In women, the risks were higher than expected in the case of cancer of the vulva (SIR = 69.2), Hodgkin lymphoma (SIR = 7.5), anal cancer (SIR = 41.2), and lung cancer (SIR = 4.8). In men, the risks were higher than expected in the case of anal cancer (SIR = 91.5), Hodgkin lymphoma (SIR = 13.0), tonsil cancer (SIR = 10.9), lung cancer (SIR = 2.1), and liver cancer (SIR = 7.1). Conclusions:The spectrum and incidence of NADCs in our cohort increased over time. The incidence of NADCs, especially virus- and smoking-associated cancers, was significantly higher than expected in HIV-positive men and women.

Transcriptional profiling of melanoma sentinel nodes identify patients with poor outcome and reveal an association of CD30(+) T lymphocytes with progression.

Sentinel lymph nodes set the stance of the immune system to a localized tumor and are often the first site to be colonized by neoplastic cells that metastasize. To investigate how the presence of neoplastic cells in sentinel lymph nodes may trigger pathways associated with metastatic progression, we analyzed the transcriptional profiles of archival sentinel node biopsy specimens obtained from melanoma patients. Biopsies from positive nodes were selected for comparable tumor infiltration, presence or absence of further regional node metastases, and relapse at 5-year follow-up. Unsupervised analysis of gene expression profiles revealed immune response to be a major gene ontogeny represented. Among genes upregulated in patients with progressing disease, the TNF receptor family member CD30/TNFRSF8 was confirmed in biopsy specimens from an independent group of patients. Immunohistochemical analysis revealed higher numbers of CD30(+) lymphocytes in nodes from progressing patients compared with nonprogressing patients. Phenotypic profiling demonstrated that CD30(+) lymphocytes comprised a broad population of suppressive or exhausted immune cells, such as CD4(+)Foxp3(+) or PD1(+) subpopulations and CD4(-)CD8(-) T cells. CD30(+) T lymphocytes were increased in peripheral blood lymphocytes of melanoma patients at advanced disease stages. Our findings reinforce the concept that sentinel nodes act as pivotal sites for determining progression patterns, revealing that the presence of CD30(+) lymphocytes at those sites associate positively with melanoma progression.

Overcoming melanoma resistance to vemurafenib by targeting CCL2-induced miR-34a, miR-100 and miR-125b

In melanoma, the adaptative cell response to BRAF inhibitors includes altered patterns of cytokine production contributing to tumor progression and drug resistance. Among the factors produced by PLX4032-resistant melanoma cell lines, CCL2 was higher compared to the sensitive parental cell lines and increased upon drug treatment. CCL2 acted as an autocrine growth factor for melanoma cells, stimulating the proliferation and resistance to apoptosis. In patients, CCL2 is detected in melanoma cells in tumors and in plasma at levels that correlate with tumor burden and lactate dehydrogenase. Vemurafenib treatment increased the CCL2 levels in plasma, whereas the long-term clinical response was associated with low CCL2 levels. Increased CCL2 production was associated with miRNA deregulation in the resistant cells. miR-34a, miR-100 and miR-125b showed high expression in both resistant cells and in tumor biopsies that were obtained from treated patients, and they were involved in the control of cell proliferation and apoptosis. Inhibition of CCL2 and of the selected miRNAs restored both the cell apoptosis and the drug efficacy in resistant melanoma cells. Therefore, CCL2 and miRNAs are potential prognostic factors and attractive targets for counteracting treatment resistance in metastatic melanoma.

Morphologic-cytogenetic analysis of dedifferentiated liposarcomas with an extensive misleading leiomyosarcomatous component.

This report describes two cases of recurrent retroperitoneal dedifferentiated liposarcoma characterized by an extensive leiomyomatous component that prevented the correct diagnosis before the last recurrence. Strong immunoreactivity with smooth muscle and desmin antibodies and ultrastructural features consistent with leiomyosarcoma were observed in the spindle-cell and/or myxoid-like components in all four recurrences in case 1, and in the spindle-cell component of the primary tumor and the first recurrence in case 2. In case 1, the correct diagnosis was suggested by the cytogenetic evidence of ring markers, a hallmark of well-differentiated/dedifferentiated liposarcoma. In case 2, tumor type was yielded mainly by the morphology of the second recurrence, which consisted entirely of a well-differentiated liposarcoma, a sclerosing inflammatory variant, as confirmed by the karyotype. Reevaluation of the first two surgical specimens of each case revealed small areas consistent with well-differentiated liposarcoma that had been previously overlooked. Despite the smooth-muscle antigen profile, both cases retained an mdm2+/p53+/cdk4+ immunophenotype consistent with the genotype.

Adaptive immune contexture at the tumour site and downmodulation of circulating myeloid-derived suppressor cells in the response of solitary fibrous tumour patients to anti-angiogenic therapy.

Background:Host immunity is emerging as a key player in the prognosis and response to treatment of cancer patients. However, the impact of the immune system and its modulation by therapies are unknown in rare soft tissue sarcomas such as solitary fibrous tumours (SFTs), whose management in the advanced forms includes anti-angiogenic therapy. Here, we studied the in situ and systemic immune status of advanced SFT patients and the effects of sunitinib malate (SM) in association with the clinical efficacy.Methods:Immune contexture of SFTs was assessed by immunohistochemistry in lesions from untreated or SM-treated patients. Frequency of circulating myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and T-cell functions was assessed ex vivo in SFT patients prior and during anti-angiogenic therapy. Patients with long-term tumour control were included to correlate immune profiles and clinical responses.Results:Anti-angiogenic naïve SFT lesions were heavily infiltrated by CD163+CD14+CD68− and CD163+CD14−CD68− myeloid cells but devoid of T cells. Conversely, post-SM tumours acquired a new subset of CD68+CD14+ myeloid cells and displayed traits of an on-going adaptive immunity, strongly enriched in activated CD8+ and CD4+ T cells. These changes at the tumour site paralleled the alleviation of systemic immunosuppression and the drop in the frequency of circulating monocytic MDSCs (mMDSCs) and granulocytic MDSCs (gMDSCs). Rebound in the number of mMDSCs, but not of gMDSCs occurred at disease progression, and a reduced percentages of mMDSCs, comparable to those found in healthy donors (HDs), endured only in the SM-responsive patients.Conclusions:The immune contexture of SFT patients is heavily involved in anti-angiogenic therapy and it could be exploited to achieve more durable disease control through immune-based combination strategies.

Structured myeloid cells and anti-angiogenic therapy in alveolar soft part sarcoma

Alveolar soft part sarcoma (ASPS) is a rare soft tissue sarcoma and the clinical management of patients with unresectable, metastatic disease is still challenging. ASPS expresses an array of potentially therapeutically targetable, angiogenesis-related molecules and, importantly, it has a distinctive angiogenic phenotype marked by a peculiar tumor-associated vasculature. Several studies, conducted in transgenic mouse models and in a large variety of human tumors of different histotype, clearly proved the substantial contribution of tumor-infiltrating myeloid cells, such as myeloid derived suppressor cells, monocytes and macrophages, in the formation and maintenance of abnormal blood vessels in tumors. By immunohistochemistry we thus explored the presence and the distribution of cells expressing myeloid markers in the inflammatory infiltrate of surgical treated metastatic ASPS. Indeed, we found that myeloid cells expressing CD14 and CD163 markers constitute the prominent cells in the inflammatory infiltrate of ASPS. These macrophage-like cells form a network surrounding the endothelial cells, or, interspersed in the tumor nest, they keep deep contact with tumor cells. In this commentary, we discussed our findings in relation to the recently published paper by Kummar and colleagues reporting the clinical and molecular results of a phase II clinical trial in patients with unresectable, metastatic ASPS treated with the anti-angiogenic drug cediranib, targeting the VEGFR-1,-2,-3 tyrosine kinases.

Pulmonary resections: cytostructural effects of different-wavelength lasers versus electrocautery

AIMS AND BACKGROUND There are few papers on the cytostructural effects of surgical instruments used during pulmonary resections. The aim of the present study was to evaluate the parenchymal damage caused by different surgical instruments: a new generation electrosurgical scalpel and two different-wavelength lasers. METHODS Six surgical procedures of pulmonary resection for nodules were performed using a new generation electrosurgical scalpel, a 1318 nm neodymium (Nd:YAG) laser or a 2010 nm thulium laser (two procedures for each instrument). Specimens were analyzed using optical microscopy and scansion electronic microscopy. RESULTS Severe cytostructural damage was found to be present in an average of 1.25 mm in depth from the cutting surface in the patients treated using electrosurgical cautery. The depth of this zone dropped to less than 1 mm in patients treated by laser, being as small as 0.2 mm using the laser with a 2010 nm-wavelength and 0.6 mm with the 1318 nm-wavelength laser. DISCUSSION These preliminary findings support the use of laser to perform conservative pulmonary resections (i.e., metastasectomies), since it is more likely to avoid damage to surrounding structures. Controlled randomized trials are needed to support the clinical usefulness and feasibility of new types of lasers for pulmonary resections.