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Expression of CCL24/eotaxin-2 in CAF supernatant-conditioned monocytes and in TDLNs.

Multivariate Cox regression analysis of the influence of the percentage of basophils in TDLNs on the survival of 35 stage IB-III pancreatic cancer patients after surgery.

In pancreatic ductal adenocarcinomas (PDAC), lymphoid infiltrates, comprised mainly of Th2 cells, predict a poor survival outcome in patients. IL4 signaling has been suggested to stabilize the Th2 phenotype in this setting, but the cellular source of IL4 in PDAC is unclear. Here, we show that basophils expressing IL4 are enriched in tumor-draining lymph nodes (TDLN) of PDAC patients. Basophils present in TDLNs correlated significantly with the Th2/Th1 cell ratio in tumors, where they served as an independent prognostic biomarker of patient survival after surgery. Investigations in mouse models of pancreatic cancer confirmed a functional role for basophils during tumor progression. The recruitment of basophils into TDLN relied partly upon the release of chemokine CCL7/MCP3 by "alternatively activated" monocytes, whereas basophil activation was induced by T-cell–derived IL3. Our results show how basophils recruited and activated in TDLNs under the influence of the tumor microenvironment regulate tumor-promoting Th2 inflammation in PDAC, helping in illuminating a key element of the immune milieu of pancreatic cancer. Cancer Res; 76(7); 1792–803. ©2016 AACR.

The incidence of well-differentiated non-functioning pancreatic neuroendocrine tumors (NF-PanNET) increased during the last decades. The risk of relapse after curative surgery, albeit low, is not negligible; moreover, adjuvant treatment is currently not an option and a reliable predictive model based on prognostic characteristics is urgently needed for tailoring a follow-up strategy. The histological classification of PanNET now relies only on the proliferative activity (mitosis and Ki67) and staging. In contrast to other endocrine neoplasms, the role of infiltrative growth pattern in NF-PanNET is not taken into consideration at present. In the current study, 247 consecutive patients who underwent surgical resection for a NF-PanNET were examined for the histological growth pattern of the tumor. Two distinct patterns (non-infiltrative vs. infiltrative) were described with the latter being further subclassified according to the type of structures invaded by the tumor (non-infiltrative: pattern 1; infiltration of adjacent pancreatic parenchyma and/or peripancreatic soft tissue: pattern 2; invasion of nearby organs and/or major vessels: pattern 3). The infiltrative growth resulted to be strongly associated with a poorer survival compared to a non-infiltrative growth (p

Assessment of HRD status is now essential for ovarian cancer patient management. A relevant percentage of high-grade serous carcinoma (HGSC) is characterized by HRD, which is caused by genetic alterations in the homologous recombination repair (HRR) pathway. Recent trials have shown that not only patients with pathogenic/likely pathogenic BRCA variants, but also BRCAwt/HRD patients, are sensitive to PARPis and platinum therapy. The most common HRD test is Myriad MyChoice CDx, but there is a pressing need to offer an alternative to outsourcing analysis, which typically requires high costs and lengthy turnaround times. In order to set up a complete in-house workflow for HRD testing, we analyzed a small cohort of HGSC patients using the CE-IVD AmoyDx HRD Focus Panel and compared our results with Myriad’s. In addition, to further deepen the mechanisms behind HRD, we analyzed the study cohort by using both a custom NGS panel that analyzed 21 HRR-related genes and FISH analysis to determine the copy numbers of PTEN and EMSY. We found complete concordance in HRD status detected by the Amoy and the Myriad assays, supporting the feasibility of internal HRD testing.

Objectives Circulating cell-free tumor DNA (ctDNA) isolated from the peripheral blood of non-small-cell lung cancer (NSCLC) patients provides biomarkers for both therapeutic target selection, particularly when direct tumor biopsy is unfeasible, and also for drug resistance monitoring. This study evaluates the reliability and feasibility of ctDNA analysis in an in-house clinical molecular diagnostic workflow. Materials and methods Mutation profiling by both standard methods and Next-Generation sequencing (NGS) was carried out and compared on 2 independent lung cancer patient cohorts. Cohort 1 consisted of 50 EGFR-mutated NSCLC patients, established on tumour biopsy, for whom ctDNA was collected at disease progression after TKI-inhibitor treatment and could be used to monitor drug resistance. Cohort 2 consisted of 50 newly diagnosed lung cancer patients for whom tumour biopsy was not possible and only ctDNA was available, providing the possibility of biomarker identification. Results ctDNA analysis of Cohort 1 verified the persistence of the tumour-detected EGFR activating mutation at disease progression by both standard and NGS methods, in 84% and 92% of the cases respectively. The T790M EGFR resistance mutation was identified in 71% of the ctDNA EGFR mutated samples providing vital information for their disease management. In newly diagnosed Cohort 2 patients, EGFR activating mutations were detected in 16% of the patients by both standard and NGS analysis of ctDNA in peripheral blood, providing indication to targeted-therapy otherwise unavailable for this group of patients. Conclusion The presented study investigated lung cancer ctDNA analysis, comparing conventional methods versus NGS sequencing, and demonstrated the successful use of plasma ctDNA as a template for targeted NGS tumor gene panel in an in-house routine clinical practice. More importantly, these data underline the advantages of the clinical application of ctDNA NGS analysis for identification of therapeutic targets, real-time monitoring of therapy, and resistance mechanisms in lung cancer patients.

Background The thymic stromal lymphopoietin (TSLP), a key cytokine for development of Th2 immunity, is produced by cancer associated fibroblasts (CAFs) in pancreatic cancer where predominant tumor infiltrating Th2 over Th1 cells correlates with reduced patients’ survival. Which cells and molecules are mostly relevant in driving TSLP secretion by CAFs in pancreatic cancer is not defined. Methods We performed in vitro, in vivo and ex-vivo analyses. For in vitro studies we used pancreatic cancer cell lines, primary CAFs cultures, and THP1 cells. TSLP secretion by CAFs was used as a read-out system to identify in vitro relevant tumor-derived inflammatory cytokines and molecules. For in vivo studies human pancreatic cancer cells and CAFs were orthotopically injected in immunodeficient mice. For ex-vivo studies immunohistochemistry was performed to detect ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression in surgical samples. Bioinformatics was applied to interrogate published data sets. Results We show in vitro that IL-1α and IL-1β released by pancreatic cancer cells and tumor cell-conditioned macrophages are crucial for TSLP secretion by CAFs. Treatment of immunodeficient mice orthotopically injected with human IL-1 positive pancreatic cancer cells plus CAFs using the IL-1R antagonist anakinra significantly reduced TSLP expression in the tumor. Importantly, we found that pancreatic cancer cells release alarmins, among which ASC, able to induce IL-1β secretion in macrophages. The relevance of ASC was confirmed ex-vivo by its expression in both tumor cells and tumor associated macrophages in pancreatic cancer surgical samples and survival data analyses showing statistically significant inverse correlation between ASC expression and survival in pancreatic cancer patients. Conclusions Our findings indicate that tumor released IL-1α and IL-1β and ASC are key regulators of TSLP secretion by CAFs and their targeting should ultimately dampen Th2 inflammation and improve overall survival in pancreatic cancer. Electronic supplementary material The online version of this article (10.1186/s40425-019-0521-4) contains supplementary material, which is available to authorized users.

"The CDC25A-CDK2 pathway has been proposed as critical for the oncogenic action of. human epidermal growth factor receptor 2 (HER2) in mammary epithelial cells. In. particular, transgenic expression of CDC25A cooperates with HER2 in promoting. mammary tumors, whereas CDC25A hemizygous loss attenuates the HER2-induced. tumorigenesis penetrance. On the basis of this evidence of a synergism between. HER2 and the cell cycle regulator CDC25A in a mouse model of mammary. tumorigenesis, we investigated the role of CDC25A in human HER2-positive breast. cancer and its possible implications in therapeutic response. HER2 status and. CDC25A expression were assessed in 313 breast cancer patients and we found. statistically significant correlation between HER2 and CDC25A (P = .007).. Moreover, an HER2-positive breast cancer subgroup with high levels of CDC25A and . very aggressive phenotype was identified (P = .005). Importantly, our in vitro. studies on breast cancer cell lines showed that the HER2 inhibitor efficacy on. cell growth and viability relied also on CDC25A expression and that such. inhibition induces CDC25A down-regulation through phosphatidylinositol. 3-kinase\/protein kinase B pathway and DNA damage response activation. In line. with this observation, we found a statistical significant association between. CDC25A overexpression and trastuzumab-combined therapy response rate in two. different HER2-positive cohorts of trastuzumab-treated patients in either. metastatic or neoadjuvant setting (P = .018 for the metastatic cohort and P =. .021 for the neoadjuvant cohort). Our findings highlight a link between HER2 and . CDC25A that positively modulates HER2-targeted therapy response, suggesting that,. in HER2-positive breast cancer patients, CDC25A overexpression affects. trastuzumab sensitivity."

In pancreatic ductal adenocarcinomas (PDAC), lymphoid infiltrates, comprised mainly of Th2 cells, predict a poor survival outcome in patients. IL4 signaling has been suggested to stabilize the Th2 phenotype in this setting, but the cellular source of IL4 in PDAC is unclear. Here, we show that basophils expressing IL4 are enriched in tumor-draining lymph nodes (TDLN) of PDAC patients. Basophils present in TDLNs correlated significantly with the Th2/Th1 cell ratio in tumors, where they served as an independent prognostic biomarker of patient survival after surgery. Investigations in mouse models of pancreatic cancer confirmed a functional role for basophils during tumor progression. The recruitment of basophils into TDLN relied partly upon the release of chemokine CCL7/MCP3 by "alternatively activated" monocytes, whereas basophil activation was induced by T-cell–derived IL3. Our results show how basophils recruited and activated in TDLNs under the influence of the tumor microenvironment regulate tumor-promoting Th2 inflammation in PDAC, helping in illuminating a key element of the immune milieu of pancreatic cancer. Cancer Res; 76(7); 1792–803. ©2016 AACR.

There is increased production of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) of multiple myeloma (MM) patients and these favor Th22 cell differentiation. Here, we found that the frequency of interleukin (IL)-22+IL-17-IL-13+ T cells is significantly increased in peripheral blood (PB) and BM of stage III and relapsed/refractory MM patients compared with healthy donors and patients with asymptomatic or stage I/II disease. Th22 cells cloned from the BM of MM patients were CCR6+CXCR4+CCR4+CCR10- and produced IL-22 and IL-13 but not IL-17. Furthermore, polyfunctional Th22-Th2 and Th22-Th1 clones were identified based on the co-expression of additional chemokine receptors and cytokines (CRTh2 or CXCR3 and IL-5 or interferon gamma [IFNγ], respectively). A fraction of MM cell lines and primary tumors aberrantly expressed the IL-22RA1 and IL-22 induced STAT-3 phosphorylation, cell growth, and resistance to drug-induced cell death in MM cells. IL-13 treatment of normal BM mesenchymal stromal cells (MSCs) induced STAT-6 phosphorylation, adhesion molecule upregulation, and increased IL-6 production and significantly favored MM cell growth compared with untreated BM MSCs. Collectively, our data show that increased frequency of IL-22+IL-17-IL-13+ T cells correlates with poor prognosis in MM through IL-22 and IL-13 protumor activity and suggest that interference with IL-22 and IL-13 signaling pathways could be exploited for therapeutic intervention.

Pancreatic neuroendocrine tumors (PanNETs) are the second most common malignancy of the pancreas, and the only curative option is represented by surgery. PanNETs can be classified as well- or poorly- differentiated, based on histology. Poorly-differentiated neuroendocrine carcinomas (NECs) represent less than 10% of cases, but they are highly malignant and associated with poor prognosis, and, most importantly, the inconsistency in their clinical management is due to the insufficient data on NEC genetic features. In order to widen our knowledge of PanNET genetic alterations, we performed RNASeq analysis (using Illumina NextSeq platform) of a group of 18 PanNETs and 2 NECs. TopHat-Fusion algorithm identified, among others, a fusion involving SMAD3 transcription factor and CCDC149; the alteration of SMAD3 in a poorly differentiated NEC was particularly interesting since SMAD3 is involved in TGF-b signaling, which has an oncogenic role in malignant cells. The SMAD3-CCDC149 fusion was confirmed in the index case by RT-PCR using primers spanning the breakpoint, followed by Sanger sequencing. The fusion was also detected by FISH analysis performed on both FFPE and frozen samples. By both FISH analysis and Sanger sequencing we identified two other poorly-differentiated NEC samples characterized by the presence of the SMAD3-CCDC149 translocation. Immunohistochemical (IHC) staining revealed that the 3 tumors, positive for the SMAD3-CCDC149 fusion, were characterized by nuclear localization of SMAD3. In order to test the hypothesis that the fusion generates an active form of SMAD3 that localizes exclusively within the nucleus, we transfected BON-1 cell line with plasmids containing either SMAD3 full length or SMAD3 truncated form (the latter mimics the fusion product) showing that only the truncated form of SMAD3 had an exclusively nuclear localization. Interestingly, FISH analysis performed on 3 well-differentiated PanNET samples did not detected the presence of the SMAD3-CDC149 translocation and IHC analysis showed cytoplasmic localization of SMAD3. Moreover, IHC analysis performed on a cohort of 40 cases of non-pancreatic neuroendocrine tumors (NETs) revealed that only poor-differentiated tumors showed a significant percentage of samples with nuclear localization of SMAD3. These data support the hypothesis that the translocation is a typical phenomenon of poorly-differentiated tumors and suggests that it could be involved also in non-pancreatic NETs. In this study we identified a previous unknown SMAD3 translocation in poor-differentiated NECs, which generates an active SMAD3 form. Deepening the knowledge of the fusion product will shed light on PanNET genetic alterations, opening the way to new therapeutic strategies. Citation Format: Emanuela Brunetto, Greta Grassini, Valeria De Pascali, Anna Talarico, Francesca Invernizzi, Michaela Bowden, Massimo Loda, Luca Albarello, Massimo Falconi, Lorenza Pecciarini, Maria G. Cangi, Claudio Doglioni. TGF-β pathway alteration in pancreatic neuroendocrine tumors: characterization of a novel SMAD3 translocation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3519. doi:10.1158/1538-7445.AM2017-3519

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D), herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.

Pancreatic Ductal AdenoCarcinoma (PDAC) is a very aggressive disease, characterized by desmoplasia and Th2-inflammation. Th2-inflammation correlates with poor prognosis and it is driven by the release of the Thymic Stromal LymphoPoietin (TSLP) from Cancer Associated Fibroblasts (CAFs) activated by tumor-derived pro-inflammatory cytokines. The aims of our study are: i) to verify which tumor-derived cytokines are responsible for CAFs activation, ii) to understand the stimuli/mechanisms of tumor-derived cytokine production in pancreatic cancer cells. Analyzing a series of 9 PDAC cell lines by both Real-time PCR and immunofluorescence, we found heterogeneous expression of IL-1α, IL-1β and TNF-α. Furthermore, ELISA tests revealed that some PDAC cell lines could either actively secrete or release during necrosis these pro-inflammatory cytokines. Next, we treated CAFs obtained from surgical patient samples with rhIL-1α, rhIL-1β, rhTNF-α and PDAC cell supernatants. Interestingly, we observed an induction in TSLP secretion that was significantly reduced by anti-IL1α and Anakinra (the recombinant form of IL-1Ra), suggesting a predominant role of the IL-1/IL-1R axis. It is known that IL-1α and TLRs ligands can activate NF-kB that in turn induces the synthesis of proIL-1β that is cleaved to matureIL-1β by caspase-1, upon inflammasome activation. In line with this, we performed Western Blot analysis and we observed constitutive activation of both NF-kB and inflammasome. We also demonstrated that IL-1α is able to increase IL-1β production in PDAC cells, with an autocrine loop. Furthermore, treating THP1 cells (a monocytic cell line that secrete IL-1β only upon stimulation) with PDAC cell supernatants we found that both IL-1-expressing and non-IL-1-expressing cells can release inflammasome-activating factors. Since pancreatic cancer is highly hypoxic, we cultured PDAC cell lines in hypoxic conditions and we demonstrated that prolonged severe hypoxia, in combination with starvation, induces an increase in both IL-1α and IL-1β secretion. In conclusion, our study demonstrates that PDAC cells can constitutively express IL-1α, IL-1β and TNF-α, thus inducing TSLP secretion from CAFs and eventually Th2-inflammation. PDAC cells secrete inflammasome-activating factors that sustain cytokine production with an autocrine loop, and that could induce cytokine release from infiltrating macrophages. Finally, the hypoxic environment of pancreatic cancer can further increase IL-1α and IL-1β secretion. Our findings support the development of clinical studies combining the use of Anakinra with conventional chemotherapy, in order to interfere with the IL-1/IL-1R axis and therefore Th2-inflammation, thus improving the prognosis of pancreatic cancer patients. Citation Format: Emanuela Brunetto, Lucia De Monte, Silvia Heltai, Maria Pia Protti. Mechanisms of IL-1 production and release in pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 443. doi:10.1158/1538-7445.AM2015-443

HER2 gene amplification/overexpression has been identified in 10-34% of invasive breast cancers and correlates with reduced disease-free and overall survival. The HER2-targeted therapy with trastuzumab decreases the risk of relapse and prolongs survival. Resistance to trastuzumab is a significant barrier to the effective HER2-positive breast cancer treatment: response rate ranges from 20-35%, underlining that HER2-positive breast cancer is hardly a homogeneous disease and, more importantly, that other HER2-related pathways need to be unveiled. Recent studies provided evidence that HER2 overexpression increases skin tumorigenesis through CDC25A maintenance: HER2 suppresses DNA damage-induced checkpoint activation through lack of CDC25A degradation. CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. CDC25A misregulation contributes to genomic instability and cancer development. Oncogene activation during early stages of tumor development causes DNA replication stress resulting in the DNA damage response (DDR) induction and the selection of cells defecting in their DDR could lead to malignant progression. Accordingly, CDC25A overexpression in primary human breast cells promotes the DDR alteration, increasing the chances of tumorigenic conversion by enhancing genomic instability at common fragile sites. The aim of our work was to verify a relation between HER2 and CDC25A in human breast cancer. To this end HER2 amplification and CDC25A overexpression were determined in 220 breast cancer patients. We found a statistically significant correlation between HER2 gene status and CDC25A expression (p=0,000): 64,4% of HER2 amplified tumours show CDC25A overexpression and 75,5% of tumours with no HER2 amplification do not overexpress CDC25A. Furthermore HER2 status and CDC25A expression significantly correlate with prognosis (p=0,003 and p=0,047 respectively). A mortality risk stratification was identified when four different combinations of HER2 status and CDC25A expression were evaluated for their relative effect on survival: the group with the highest mortality was the one in which breast carcinomas showed both HER2 amplification and CDC25A overexpression (p=0,019). Our in vitro studies demonstrate that HER2 can stabilize CDC25A protein levels in breast cancer cell lines, through PI3K-AKT pathway: decreased HER2-mediated signaling by HER2 inhibitors resulted in CDC25A decreased expression; remarkably CDC25A inactivation by specific inhibitor can restore response to IR-induced DNA damage in HER2-positive, both trastuzumab sensitive and resistant, breast cancer cell lines. These findings suggest a CDC25A role for a more specific characterization of HER2-positive breast cancers, warranting further study of its potential as therapeutic target in combination with HER2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2011-3147