Maria Mihaela Mina

Chromosomal gains in the sarcomatoid transformation of chromophobe renal cell carcinoma.

The hallmark of chromophobe renal cell carcinoma is multiple chromosomal losses from among chromosomes 1, 2, 6, 10 and 17. Chromophobe renal cell carcinoma with distant metastases or sarcomatoid transformation are uncommon and little is known about their chromosomal abnormalities. We collected six sarcomatoid chromophobe renal cell carcinomas and three primary chromophobe renal cell carcinomas with distant metastases. A cytogenetic analysis by fluorescent in situ hybridization on paraffin-embedded tissue was performed using centromeric probes for chromosomes 1, 2, 6, 10 and 17. We found more than one signal in four of six (66%) sarcomatoid chromophobe renal cell carcinomas, in both sarcomatoid and adjacent epithelial components. Both primary chromophobe renal cell carcinomas and matched metastases showed single signals for all chromosomes studied in two cases and no abnormalities in the remaining case. We concluded that: (1) both epithelial and sarcomatoid components of sarcomatoid chromophobe renal cell carcinoma show different genetic abnormalities from those characteristic of chromophobe renal cell carcinoma; (2) sarcomatoid chromophobe renal cell carcinomas frequently have multiple gains (polysomy) of chromosomes 1, 2, 6, 10 and 17; (3) distant metastases show the same genetic patterns, usually chromosomal losses (monosomy), found in the primary tumors.

Diagnostic utility of S100A1 expression in renal cell neoplasms: an immunohistochemical and quantitative RT-PCR study.

S100A1 is a calcium-binding protein, which has been recently found in renal cell neoplasms. We evaluated the diagnostic utility of immunohistochemical detection of S100A1 in 164 renal cell neoplasms. Forty-one clear cell, 32 papillary, and 51 chromophobe renal cell carcinomas, and 40 oncocytomas, 164 samples of normal renal parenchyma adjacent to the tumors and 13 fetal kidneys were analyzed. The levels of S100A1 mRNA detected by quantitative RT-PCR analysis of frozen tissues from seven clear cell, five papillary, and six chromophobe renal cell carcinomas, four oncocytomas, and nine samples of normal renal tissues adjacent to neoplasms were compared with the immunohistochemical detection of protein expression. Clear cell and papillary renal cell carcinomas showed positive reactions for S100A1 in 30 out of 41 tumors (73%) and in 30 out of 32 (94%) tumors, respectively. Thirty-seven renal oncocytomas out of 40 (93%) were positive for S100A1, whereas 48 of 51 (94%) chromophobe renal cell carcinomas were negative. S100A1 protein was detected in all samples of unaffected and fetal kidneys. S100A1 mRNA was detected by RT-PCR in all normal kidneys and renal cell neoplasms, although at very different levels. Statistical analyses comparing the different expression of S100A1 in clear cell and chromophobe renal cell carcinomas observed by immunohistochemical and RT-PCR methods showed significant values (P<0.001), such as when comparing by both techniques the different levels of S100A1 expression in chromophobe renal cell carcinomas and oncocytomas (P<0.001). Our study shows that S100A1 protein is expressed in oncocytomas, clear cell and papillary renal cell carcinomas but not in chromophobe renal cell carcinomas. Its immunodetection is potentially useful for the differential diagnosis between chromophobe renal cell carcinoma and oncocytoma. Further, S100A1 protein expression is constantly detected in the normal parenchyma of the adult and fetal kidney.

An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients

Purpose: The majority of gastric cancer patients who achieve an initial response to trastuzumab-based regimens develop resistance within 1 year of treatment. This study was aimed at identifying the molecular mechanisms responsible for resistance. Experimental Design: A HER2+-trastuzumab sensitive NCI-N87 gastric cancer orthotopic nude mouse model was treated with trastuzumab until resistance emerged. Differentially expressed transcripts between trastuzumab-resistant and sensitive gastric cancer cell lines were annotated for functional interrelatedness by Ingenuity Pathway Analysis software. Immunohistochemical analyses were performed in pretreatment versus posttreatment biopsies from gastric cancer patients receiving trastuzumab-based treatments. All statistical tests were two-sided. Results: Four NCI-N87 trastuzumab-resistant (N87-TR) cell lines were established. Microarray analysis showed HER2 downregulation, induction of epithelial-to-mesenchymal transition, and indicated fibroblast growth factor receptor 3 (FGFR3) as one of the top upregulated genes in N87-TR cell lines. In vitro, N87-TR cell lines demonstrated a higher sensitivity than did trastuzumab-sensitive parental cells to the FGFR3 inhibitor dovitinib, which reduced expression of pAKT, ZEB1, and cell migration. Oral dovitinib significantly (P = 0.0006) reduced tumor burden and prolonged mice survival duration in N87-TR mouse models. A higher expression of FGFR3, phosphorylated AKT, and ZEB1 were observed in biopsies from patients progressing under trastuzumab-based therapies if compared with matched pretreatment biopsies. Conclusions: This study identified the FGFR3/AKT axis as an escape pathway responsible for trastuzumab resistance in gastric cancer, thus indicating the inhibition of FGFR3 as a potential strategy to modulate this resistance. Clin Cancer Res; 22(24); 6164–75. ©2016 AACR.

Combined inhibition of IL1, CXCR1/2, and TGFβ signaling pathways modulates in-vivo resistance to anti-VEGF treatment.

Resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. We recently identified interleukin-1 (IL1), CXC receptors (CXCR)1/2 ligands, and transforming growth factor &bgr; (TGF&bgr;) among the proinflammatory factors that were expressed at higher levels in murine models resistant to the antivascular endothelial growth factor (anti-VEGF) antibody bevacizumab. Here, we hypothesized that the combined inhibition of these proinflammatory signaling pathways might reverse this anti-VEGF resistance. Bevacizumab-resistant FGBR pancreatic cancer cells were treated in vitro with bevacizumab, the recombinant human IL1 receptor antagonist anakinra, the monoclonal antibody against TGF&bgr; receptor type II TR1, and a novel recombinant antibody binding CXCR1/2 ligands. The FGBR cells treated with these agents in combination had significantly higher levels of E-cadherin and lower levels of vimentin, IL6, phosphorylated p65, and SMAD2, and showed significantly lower migration rates than did their controls treated with the same agents without bevacizumab or with a single agent bevacizumab as a control. Consistently, the combination of these agents with bevacizumab reduced the FGBR tumor burden and significantly prolonged mice survival compared with bevacizumab in monotherapy. Tumors from mice receiving the combination treatment showed significantly lower expression of IL6 and phosphorylated SMAD2, higher expression of E-cadherin and lower levels of vimentin, and a significantly lower infiltration by CD11b+ cells compared with bevacizumab-treated controls. This study suggests that inhibition of IL1, CXCR1/2, and TGF&bgr; signaling pathways is a potential therapeutic approach to modulate the acquired resistance to anti-VEGF treatment by reversing epithelial–mesenchymal transition and inhibiting CD11b+ proangiogenic myeloid cells’ tumor infiltration.

TAK1-regulated expression of BIRC3 predicts resistance to preoperative chemoradiotherapy in oesophageal adenocarcinoma patients

Background:About 20% of resectable oesophageal carcinoma is resistant to preoperative chemoradiotherapy. Here we hypothesised that the expression of the antiapoptotic gene Baculoviral inhibitor of apoptosis repeat containing (BIRC)3 induced by the transforming growth factor β activated kinase 1 (TAK1) might be responsible for the resistance to the proapoptotic effect of chemoradiotherapy in oesophageal carcinoma.Methods:TAK1 kinase activity was inhibited in FLO-1 and KYAE-1 oesophageal adenocarcinoma cells using (5Z)-7-oxozeaenol. The BIRC3 mRNA expression was measured by qRT–PCR in 65 pretreatment frozen biopsies from patients receiving preoperatively docetaxel, cisplatin, 5-fluorouracil, and concurrent radiotherapy. Receiver operator characteristic (ROC) analyses were performed to determine the performance of BIRC3 expression levels in distinguishing patients with sensitive or resistant carcinoma.Results:In vitro, (5Z)-7-oxozeaenol significantly reduced BIRC3 expression in FLO-1 and KYAE-1 cells. Exposure to chemotherapeutic agents or radiotherapy plus (5Z)-7-oxozeaenol resulted in a strong synergistic antiapoptotic effect. In patients, median expression of BIRC3 was significantly (P<0.0001) higher in adenocarcinoma than in the more sensitive squamous cell carcinoma subtype. The BIRC3 expression significantly discriminated patients with sensitive or resistant adenocarcinoma (AUC-ROC=0.7773 and 0.8074 by size-based pathological response or Mandards tumour regression grade classifications, respectively).Conclusions:The BIRC3 expression might be a valid biomarker for predicting patients with oesophageal adenocarcinoma that could most likely benefit from preoperative chemoradiotherapy.

A circulating TH2 cytokines profile predicts survival in patients with resectable pancreatic adenocarcinoma

ABSTRACT Surgery is the only potentially curative option for patients with pancreatic ductal adenocarcinoma (PDAC), but metastatic relapse remains common. We hypothesized that the expression levels of inflammatory cytokines could predict recurrence of PDAC, thus allowing to select patients who most likely could benefit from surgical resection. We prospectively collected plasma at diagnosis from 287 patients with pancreatic resectable neoplasms. The expression levels of 23 cytokines were measured in 90 patients with PDAC by using a multiplex analyte profiling assay. Levels higher than cutoff identified of the TH2 cytokines interleukin (IL)4, IL5, IL6 of macrophage inflammatory protein (MIP)1α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein (MCP)1, and of IL17α, IFNγ-induced protein (IP)10, and IL1b were significantly associated with a shorter median OS. In particular, levels of IL4 and IP10 higher than cutoff identified, and level of TH1 cytokines TNFα and INFγ, and of IL9 and IL1Rα lower than cutoff identified were significantly associated with a shorter DFS. In the multivariate analysis, high IP10 was confirmed as negatively associated with OS (HR = 3.097, p = 0.014) and IL4 and TNFα remain negatively (HR = 2.75, p = 0.002) and positively (HR = 0.224, p = 0.049) associated with DFS, respectively. Simultaneous expression of low IL4 and high TNFα identified patients with best prognosis (HR = 0.313, p < 0.0001). In conclusion, we demonstrated that, among a series of cytokines, IL4 is the most significant independent prognostic factor for DFS in resectable PDAC patients, and it could be useful to select patients with high risk of early recurrence who may avoid an unnecessary resection.

Allelotype of ampulla of Vater cancer: highly frequent involvement of chromosome 11

Purpose To determine the genetic differences/similarities in ampulla of Vater cancers (AVC) with respect to other pancreatic tumor types.Methods We analyzed eight cases of primary AVC by genome-wide allelotyping on DNA obtained from frozen tissue. A total of 372 microsatellite loci were used for each case, for a total of 2,976 microsatellites analyzed.Results Of the 2,159 informative markers, 400 were allelic losses and 1,759 markers were retained for an average fractional allelic loss of 0.19. Seven cases showed LOH on at least two markers on chromosomal arm 11p, while six cases showed allelic losses on 11q. The high frequency of LOH on chromosome 11 was also confirmed by analysis of an additional 17 paraffin-embedded AVC. Frequent LOH (50% or greater) was also found on chromosome arms 5q, 6q, 9p, 13, 16p, 17p, and 18p.Conclusions It can be inferred that the targets of inactivation on chromosomes 5q, 9p, and 17p appear to be APC, p16, and p53, respectively, while the critical target(s) of inactivation at the other frequently lost loci remain to be characterized. The resulting allelotype reveals that distinctive chromosomal alterations are present in these neoplasms, indicating that it is a tumor entity distinct from pancreatic adenocarcinoma.

Abstract 3265: Homeobox B9 (HOXB9) sustains anti-VEGF treatment resistance in gastrointestinal tumors

Resistance to anti-angiogenic therapies poses one of the greatest challenges in gastrointestinal tumors research. We recently identified several proinflammatory secreted factors, including interleukin-1 (IL1), CXC ligand (CXCL) 1 and 8, Transforming Growth Factor β (TGFβ)1, and Angiopoietin-like Protein 2 (ANGPTL2), that were overexpressed in murine models of tumors resistant to the anti-VEGF antibody bevacizumab (BEV). These factors induced epithelial-to-mesenchymal transition (EMT) and increased aggressiveness. HOXB9 has been identified as a key transcription factor in common for these factors. Here, we hypothesized that HOXB9 might be responsible for BEV resistance in gastrointestinal tumors. HOXB9 expression and activation were measured in BEV-sensitive COLO357FG and in their BEV-resistant counterpart FGBR pancreatic cancer cell lines, and in LOVO, MDST8, LIM2099, CCK81, GP5D, and SNUC4 colon cancer cell lines by EMSA and DAPA. Serum levels of IL1, CXCL1 and 8, TGFβ1 and ANGPTL2 in nude mice bearing tumors were measured by multiplex xMAP technology. Immunohistochemical analyses were performed in pre- vs. post-progression biopsies from colorectal cancer patients receiving BEV. HOXB9 protein was more expressed and activated in FGBR than did in COLO357FG cells. shRNA to knock down HOXB9 significantly (P In conclusion, we identified HOXB9 as crucial transcription factor to sustain BEV resistance in gastrointestinal tumors. Silencing of HOXB9 is a promising approach to modulate this resistance. Our results candidate HOXB9 as potential biomarker for selecting patients with colorectal and pancreatic cancer for antiangiogenic therapy. Citation Format: Carmine Carbone, Geny Piro, Francesca Simionato, Fotios Loupakis, Chiara Cremolini, Gabriella Fontanini, Federica Di Nicolantonio, Elisa Moratti, Francesca Ligorio, Marco Zanotto, Raffaela Santoro, Maria Mihaela Mina, Aldo Scarpa, Alberto Bardelli, Giampaolo Tortora, Davide Melisi. Homeobox B9 (HOXB9) sustains anti-VEGF treatment resistance in gastrointestinal tumors. [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 3265.

Abstract 579: TAK1-regulated expression of BIRC3 is responsible for chemoradiotherapy (CRT) resistance in esophagogastric junction (EGJ) adenocarcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Preoperative CRT followed by surgery is the most common approach for patients with resectable esophageal and EGJ cancers. Based on the histology, patients with adenocarcinoma consistently demonstrated significantly lower rate of pathological complete response if compared with patients with squamous cell carcinoma, thus the need for accurate biomarkers to select the patients with esophageal and EGJ adenocarcinoma most likely to benefit from preoperative CRT has become even more critical. We recently demonstrated that the MAP3K TGF-βeta-activated kinase-1 (TAK1) is responsible for the resistance to the proapoptotic effect of chemotherapeutic agents by increasing the transcription of the member of the inhibitor of apoptosis proteins family BIRC3 in preclinical models of pancreatic cancer. Here, we hypothesized that the TAK1-regulated expression of BIRC3 might be responsible for the resistance to CRT in EGJ adenocarcinoma. Materials and methods: TAK1 kinase activity was targeted in FLO-1 and KYAE-1 esophageal cells by using (5Z)-7oxozeaenol. To test the effect of reducing BIRC3 expression on the resistance to CRT, FLO-1 and KYAE-1 cells were treated with increasing doses of cisplatin, 5-fluorouracil, paclitaxel, or radiotherapy in combination with (5Z)-7oxozeaenol. Drug interactions were studied for synergism according to Chou and Talalay method. Apoptotic induction was studied by western blot analysis of PARP and caspase 3 cleavages as well as by AnnexinV staining. BIRC3 expression was measured in 33 pretreatment biopsies from patients with EGJ adenocarcinoma and 34 from patients with esophageal squamous cell carcinoma receiving neoadjuvant CRT by Real-Time PCR. Tumor response was evaluated by Tumour regression grade (TRG) and by Size-based Pathological Response (SPR) scores. Correlation between BIRC3 expression and treatment response was analysed by ROC curve analysis. Results: In vitro, (5Z)-7oxozeaenol significantly reduced BIRC3 expression in FLO-1 and KYAE-1 esophageal cells. Exposure to sublethal equitoxic doses of chemotherapeutic agents plus (5Z)-7oxozeaenol pretreatment resulted in a strong synergistic anti-proliferative effect. Baseline expression of BIRC3 was significantly higher in patients with EGJ adenocarcinoma if compared with the more sensitive squamous-cell carcinoma subtype. Moreover, patients with EGJ adenocarcinoma expressing higher pretreatment levels of BIRC3 had a significantly poorer treatment response than did those with lower expression, indicating that BIRC3 expression significantly correlates with response to preoperative CRT (AUC-ROC = 0.777 and 0.807 for SPR and TRG, respectively). Conclusions: TAK1-regulated expression of BIRC3 might be a valid biomarker to predict resistance to CRT in EGJ adenocarcinoma patients. Citation Format: Geny Piro, Simone Giacopuzzi, Maria Bencivenga, Carmine Carbone, Giuseppe Verlato, Melissa Frizziero, Maria Mihaela Mina, Marco Zanotto, Valeria Merz, Giovanni De Manzoni, Giampaolo Tortora, Davide Melisi. TAK1-regulated expression of BIRC3 is responsible for chemoradiotherapy (CRT) resistance in esophagogastric junction (EGJ) adenocarcinoma. [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 579. doi:10.1158/1538-7445.AM2015-579

Abstract 3605: Combined inhibition of IL-1, CXCR1/2, and TGFβ signaling pathways modulates in vivo acquired resistance to anti-VEGF treatment

Introduction: Resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. We recently identified interleukin (IL)-1, CXC receptors (CXCR)1/2 ligands, and Transforming Growth Factor β (TGFβ) among the proinflammatory factors that were expressed at higher levels in murine models resistant to the anti-VEGF antibody bevacizumab. Here, we hypothesized that the combined inhibition of these proinflammatory signaling pathways might reverse the resistance to anti-VEGF treatment. Methods: Bevacizumab-resistant FGBR orthotopic tumor bearing mice received bevacizumab alone or in combination with a recombinant human IL-1 receptor antagonist, a monoclonal antibody against TGFβ Receptor type II, and a recombinant antibody binding CXCR1/2 ligands. Western blot analysis was performed to assess the activity of p65 NFB, Smad2 and on the expression of IL-6 in FGBR cells in culture. Immunohistochemical analysis of FFPE sections from FGBR tumors was performed to assess Smad2 phosphorylation and IL-6 expression. Wound healing assay was performed to assess cell motility in FGBR cells in culture. Immunohistochemical analysis was performed to assess the expression of E-Cadherin and Vimentin in FGBR tumors. Immunohistochemical analysis was performed to estimate the presence of Cd11b+ cells. Results: The combination of these agents with bevacizumab reduced the tumor burden and significantly prolonged mice survival if compared with single agent bevacizumab. Tumors from mice receiving the combination treatment demonstrated significantly lower expression of IL-6 and phosphorylation of p65 and Smad2 when compared with control. FGBR cells treated in vitro with the three agents plus bevacizumab had significantly higher levels of E-cadherin and lower levels of Vimentin, and exhibited significantly lower migration rates than did their bevacizumab- treated controls. Consistently, tumors from mice receiving the combination treatment demonstrated significantly higher expression of E-cadherin, lower levels of Vimentin, and a significantly lower infiltration by CD11b+ cells when compared with bevacizumab -treated controls. Conclusion: This study suggests that inhibition of IL-1, CXCR1/2, and TGFβ signaling pathways is a potential therapeutic approach to modulate the acquired resistance to anti-VEGF treatment by reversing EMT and inhibiting CD11b+ proangiogenic myeloid cells tumor infiltration. Citation Format: Carmine Carbone, Anna Tamburrino, Geny Piro, Marco Zanotto, Maria Mihaela Mina, Silvia Zanini, Federico Boschi, Aldo Scarpa, Giampaolo Tortora, Davide Melisi. Combined inhibition of IL-1, CXCR1/2, and TGFβ signaling pathways modulates in vivo acquired resistance to anti-VEGF treatment. [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 3605. doi:10.1158/1538-7445.AM2015-3605