Gastrointestinal Cancer Clinical Research Unit

Madrid, Spain

Gastrointestinal Cancer Clinical Research Unit

Madrid, Spain
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Angulo B.,University of San Pablo - CEU | Conde E.,University of San Pablo - CEU | Suarez-Gauthier A.,University of San Pablo - CEU | Plaza C.,University of San Pablo - CEU | And 9 more authors.
PLoS ONE | Year: 2012

The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry), with direct sequencing and to investigate the limit of detection (LOD) of both PCR-based methods. We identified EGFR mutations in 21 (16%) of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%), which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%). Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+) yielded similar results. Immunohistochemistry (IHC) staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity. © 2012 Angulo et al.

Garcia-Garcia E.,University of San Pablo - CEU | Gomez-Martin C.,Gastrointestinal Cancer Clinical Research Unit | Angulo B.,University of San Pablo - CEU | Conde E.,University of San Pablo - CEU | And 6 more authors.
Histopathology | Year: 2011

Amplification of the human epidermal growth factor receptor 2 (HER2) gene has been reported in gastric carcinoma (GC). Accordingly, trastuzumab plus chemotherapy has recently become the new standard treatment for HER2-positive advanced GCs. The aim was to compare the alleged gold standard for hybridization [fluorescence in-situ hybridization (FISH)] with a novel, fully automated brightfield dual-colour silver-enhanced in-situ hybridization (SISH) method. Methods and results: The studies series was comprised of 166 GC samples. Additionally, tumours with discordant results obtained by FISH and SISH were analysed by real-time quantitative polymerase chain reaction (PCR) with the LightMix kit HER-2/neu. Of the samples, 17.5% and 21% were amplified by FISH and SISH, respectively. Heterogeneity was identified in up to 52% of cases. In 96.4% of cases, FISH showed the same results as SISH. All six discordant cases were positive by SISH and negative by FISH. On review of the FISH slides, all contradictory cases were polysomic and were confirmed to be negative for amplification by real-time PCR. Interestingly, all ratios in this latter group were between 2.06 and 2.50, so setting the cut-off for amplification at ≥3 resulted in perfect concordance. Conclusions: Dual-colour SISH represents a novel method for the determination of HER2 status in GC. © 2011 Blackwell Publishing Limited.

Balic A.,Stem Cells and Cancer Group | Sorensen M.D.,Stem Cells and Cancer Group | Sorensen M.D.,University of Aarhus | Trabulo S.M.,Stem Cells and Cancer Group | And 10 more authors.
Molecular Cancer Therapeutics | Year: 2014

Pancreaticductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells in vitro and corresponding patient-derived pancreatic cancer tissue xenografts in vivo. Intriguingly, we found that in vitro treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished in vivo tumorigenicity and invasiveness in a large panel of pancreatic cancers. In vivo treatment in combinationwith gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer.©2014 AACR.

Miranda-Lorenzo I.,Stem Cells and Cancer Group | Dorado J.,Stem Cells and Cancer Group | Lonardo E.,Stem Cells and Cancer Group | Alcala S.,Stem Cells and Cancer Group | And 13 more authors.
Nature Methods | Year: 2014

Cancer stem cells (CSCs) are thought to drive tumor growth, metastasis and chemoresistance. Although surface markers such as CD133 and CD44 have been successfully used to isolate CSCs, their expression is not exclusively linked to the CSC phenotype and is prone to environmental alteration. We identified cells with an autofluorescent subcellular compartment that exclusively showed CSC features across different human tumor types. Primary tumor-derived autofluorescent cells did not overlap with side-population (SP) cells, were enriched in sphere culture and during chemotherapy, strongly expressed pluripotency-associated genes, were highly metastatic and showed long-term in vivo tumorigenicity, even at the single-cell level. Autofluorescence was due to riboflavin accumulation in membrane-bounded cytoplasmic structures bearing ATP-dependent ABCG2 transporters. In summary, we identified and characterized an intrinsic autofluorescent phenotype in CSCs of diverse epithelial cancers and used this marker to isolate and characterize these cells. © 2014 Nature America, Inc.

Cioffi M.,Stem Cells and Cancer Group | Trabulo S.M.,Stem Cells and Cancer Group | Sanchez-Ripoll Y.,Stem Cells and Cancer Group | Miranda-Lorenzo I.,Stem Cells and Cancer Group | And 10 more authors.
Gut | Year: 2015

Objective Cancer stem cells (CSCs) represent the root of many solid cancers including pancreatic ductal adenocarcinoma, are highly chemoresistant and represent the cellular source for disease relapse. However the mechanisms involved in these processes still need to be fully elucidated. Understanding the mechanisms implicated in chemoresistance and metastasis of pancreatic cancer is critical to improving patient outcomes. Design Micro-RNA (miRNA) expression analyses were performed to identify functionally defining epigenetic signatures in pancreatic CSC-enriched sphere-derived cells and gemcitabine-resistant pancreatic CSCs. Results We found the miR-17-92 cluster to be downregulated in chemoresistant CSCs versus non-CSCs and demonstrate its crucial relevance for CSC biology. In particular, overexpression of miR-17-92 reduced CSC self-renewal capacity, in vivo tumourigenicity and chemoresistance by targeting multiple NODAL/ACTIVIN/TGF-β1 signalling cascade members as well as directly inhibiting the downstream targets p21, p57 and TBX3. Overexpression of miR-17-92 translated into increased CSC proliferation and their eventual exhaustion via downregulation of p21 and p57. Finally, the translational impact of our findings could be confirmed in preclinical models for pancreatic cancer. Conclusions Our findings therefore identify the miR-17-92 cluster as a functionally determining family of miRNAs in CSCs, and highlight the putative potential of developing modulators of this cluster to overcome drug resistance in pancreatic CSCs.

Garrido-Laguna I.,University of Utah | Hidalgo M.,Gastrointestinal Cancer Clinical Research Unit
Nature Reviews Clinical Oncology | Year: 2015

Pancreatic cancer is expected to be the second deadliest malignancy in the USA by 2020. The survival rates for patients with other gastrointestinal malignancies have increased consistently during the past 30 years; unfortunately, however, the outcomes of patients with pancreatic cancer have not changed significantly. Although surgery remains the only curative treatment for pancreatic cancer, therapeutic strategies based on initial resection have not substantially improved the survival of patients with resectable disease over the past 25 years; presently, more than 80% of patients suffer disease relapse after resection. Preclinical evidence that pancreatic cancer is a systemic disease suggests a possible benefit for early administration of systemic therapy in these patients. In locally advanced disease, the role of chemoradiotherapy is increasingly being questioned, particularly considering the results of the LAP-07 trial. Novel biomarkers are clearly needed to identify subsets of patients likely to benefit from chemoradiotherapy. In the metastatic setting, FOLFIRINOX (folinic acid, 5-fluorouracil, irinotecan, and oxaliplatin), and nab-paclitaxel plus gemcitabine have yielded only modest improvements in survival. Thus, new treatments are urgently needed for patients with pancreatic cancer. Herein, we review the state-of-the-art of pancreatic cancer treatment, and the upcoming novel therapeutics that hold promise in this disease are also discussed. © 2015 Macmillan Publishers Limited. All rights reserved.

Cioffi M.,Stem Cells and Cancer Group | Trabulo S.,Stem Cells and Cancer Group | Trabulo S.,Barts Cancer Institute | Hidalgo M.,Gastrointestinal Cancer Clinical Research Unit | And 8 more authors.
Clinical Cancer Research | Year: 2015

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a cancer of the exocrine pancreas with unmet medical need and is strongly promoted by tumor-Associated macrophages (TAM). The presence of TAMs is associated with poor clinical outcome, and their overall role, therefore, appears to be protumorigenic. The don't eat me signal CD47 on cancer cells communicates to the signal regulatory protein-A on macrophages and prevents their phagocytosis. Thus, inhibition of CD47 may offer a new opportunity to turn TAMs against PDAC cells, including cancer stem cells (CSC), as the exclusively tumorigenic population. Experimental Design: We studied in vitro and in vivo the effects ofCD47inhibition on CSCs using a large set of primary pancreatic cancer (stem) cells as well as xenografts of primary human PDAC tissue. Results: CD47 was highly expressed on CSCs, but not on other nonmalignant cells in the pancreas. Targeting CD47 efficiently enhanced phagocytosis of a representative set of primary human pancreatic cancer (stem) cells and, even more intriguingly, also directly induced their apoptosis in the absence of macrophages during long-Term inhibition of CD47. In patient-derived xenograft models, CD47 targeting alone did not result in relevant slowing of tumor growth, but the addition of gemcitabine or Abraxane resulted in sustained tumor regression and prevention of disease relapse long after discontinuation of treatment. Conclusions: These data are consistent with efficient in vivo targeting of CSCs, and strongly suggest that CD47 inhibition could be a novel adjuvant treatment strategy for PDAC independent of underlying and highly variable driver mutations. © 2015 American Association for Cancer Research.

Garrido-Laguna I.,University of Houston | Hidalgo M.,Gastrointestinal Cancer Clinical Research Unit | Kurzrock R.,University of Houston
Nature Reviews Clinical Oncology | Year: 2011

In the past, clinical phase I trials often suffered from low response rates and inadequate experimental drug doses. Over the past decade, however, phase I trials have evolved from simple dose-finding studies to trials that might provide clinically relevant therapeutic opportunities for patients with advanced-stage cancer for which no standard therapies are available. In the future, the routine use of modern technologies such as large-scale genome sequencing will help to unravel the specific biology of a patient's cancer. Such tools will expand our knowledge about genetic aberrations and might provide opportunities for the development of novel, molecular targeted therapies for patients with refractory cancer. Increasingly, the focus will likely turn from carrying out large randomized trials in unselected patients to conducting smaller biomarker-driven trials in selected patients with known molecular aberrations. We expect that these new strategies will enhance response rates as appropriate patients are targeted, therefore sparing those patients who are unlikely to benefit. © 2011 Macmillan Publishers Limited. All rights reserved.

Lonardo E.,Stem Cells and Cancer Group | Cioffi M.,Stem Cells and Cancer Group | Sancho P.,Stem Cells and Cancer Group | Sanchez-Ripoll Y.,Stem Cells and Cancer Group | And 5 more authors.
PLoS ONE | Year: 2013

Pancreatic ductal adenocarcinomas contain a subset of exclusively tumorigenic cancer stem cells (CSCs), which are capable of repopulating the entire heterogeneous cancer cell populations and are highly resistant to standard chemotherapy. Here we demonstrate that metformin selectively ablated pancreatic CSCs as evidenced by diminished expression of pluripotency-associated genes and CSC-associated surface markers. Subsequently, the ability of metformin-treated CSCs to clonally expand in vitro was irreversibly abrogated by inducing apoptosis. In contrast, non-CSCs preferentially responded by cell cycle arrest, but were not eliminated by metformin treatment. Mechanistically, metformin increased reactive oxygen species production in CSC and reduced their mitochondrial transmembrane potential. The subsequent induction of lethal energy crisis in CSCs was independent of AMPK/mTOR. Finally, in primary cancer tissue xenograft models metformin effectively reduced tumor burden and prevented disease progression; if combined with a stroma-targeting smoothened inhibitor for enhanced tissue penetration, while gemcitabine actually appeared dispensable. © 2013 Lonardo et al.

Aparicio S.,University of British Columbia | Hidalgo M.,Gastrointestinal Cancer Clinical Research Unit | Kung A.L.,Columbia University
Nature Reviews Cancer | Year: 2015

Patient-derived xenograft (PDX) models are now being widely used in cancer research and have the potential to greatly inform our understanding of cancer biology. However, many questions remain, especially regarding the ability of PDX models to affect clinical decision making. With these points in mind, we asked three scientists to give their opinions on the generation and uses of PDX models and the future of this field. © 2015 Macmillan Publishers Limited. All rights reserved.

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