Fernando López-Ríos

A direct pancreatic cancer xenograft model as a platform for cancer stem cell therapeutic development

There is an enormous gap between the antiproliferative and in vivo antitumor efficacy of gemcitabine in cell line-based models and its clinical efficacy. This may be due to insensitiveness of the precursor, cancer stem cell (CSC) compartment to cytotoxic agents. The hedgehog pathway is associated with CSC signaling and control. We used a direct xenograft model of pancreatic cancer and a two-stage approach was used to test the hypotheses that targeting CSC could increase the efficacy of gemcitabine. Tumors from a gemcitabine-sensitive xenograft were treated with gemcitabine first, and randomized, after tumor regression to continuing treatment with gemcitabine, a hedgehog inhibitor alone or in combination with gemcitabine. We tested markers described as associated with CSC such as CD24, CD44, ALDH, nestin, and the hedgehog pathway. After induction with gemcitabine, treated tumor showed an enrichment in CSC markers such as ALDH and CD24. Subsequently, a release from gemcitabine prompted a repopulation of proliferating cells and a decrease in such markers to equilibrate from pretreatment levels. Combined treatment with gemcitabine and cyclopamine induced tumor regression and decrease in CSC markers and hedgehog signaling. Cytoplasmic CD24 and ALDH were inversely and strongly associated with growth and were expressed in a minority of cells that we propose constitute the CSC compartment. Hedgehog inhibitors as part of a dual compartment therapeutic approach were able to further reduce tumor growth and decreased both static and dynamic markers of CSC. Direct tumor xenografts are a valid platform to test multicompartment therapeutic approaches in pancreatic cancer. [Mol Cancer Ther 2009;8(2):310–4]

Pulmonary vascular remodeling in pulmonary hypertension due to chronic heart failure.

Pulmonary hypertension (PHT) associated with chronic heart failure (CHF) is a risk factor of right ventricular failure after heart transplantation (HT). Our aim was to study pulmonary vascular changes in patients with CHF and to assess any correlation with haemodynamic data.

Global Gene Expression Profiling of Pleural Mesotheliomas: Overexpression of Aurora Kinases and P16/CDKN2A Deletion as Prognostic Factors and Critical Evaluation of Microarray-Based Prognostic Prediction

Most gene expression profiling studies of mesothelioma have been based on relatively small sample numbers, limiting their statistical power. We did Affymetrix U133A microarray analysis on 99 pleural mesotheliomas, in which multivariate analysis showed advanced-stage, sarcomatous histology and P16/CDKN2A homozygous deletion to be significant independent adverse prognostic factors. Comparison of the expression profiles of epithelioid versus sarcomatous mesotheliomas identified many genes significantly overexpressed among the former, including previously unrecognized ones, such as uroplakins and kallikrein 11, both confirmed by immunohistochemistry. Examination of the gene expression correlates of survival showed that more aggressive mesotheliomas expressed higher levels of Aurora kinases A and B and functionally related genes involved in mitosis and cell cycle control. Independent confirmation of the negative effect of Aurora kinase B was obtained by immunohistochemistry in a separate patient cohort. A role for Aurora kinases in the aggressive behavior of mesotheliomas is of potential clinical interest because of the recent development of small-molecule inhibitors. We then used our data to develop microarray-based predictors of 1 year survival; these achieved a maximal accuracy of 68% in cross-validation. However, this was inferior to prognostic prediction based on standard clinicopathologic variables and P16/CDNK2A status (accuracy, 73%), and adding the microarray model to the latter did not improve overall accuracy. Finally, we evaluated three recently published microarray-based outcome prediction models, but their accuracies ranged from 63% to 67%, consistently lower than reported. Gene expression profiling of mesotheliomas is an important discovery tool, but its power in clinical prognostication has been overestimated.

Evidence against a role for SV40 infection in human mesotheliomas and high risk of false-positive PCR results owing to presence of SV40 sequences in common laboratory plasmids

BACKGROUND PCR-based evidence of infection by simian virus 40 (SV40) has been reported in varying proportions of pleural mesotheliomas and other tumours, but data are conflicting and reproducibility limited. During a study of SV40 in relation to homozygous deletion of CDKN2A in mesotheliomas, we became concerned by inconsistent results and therefore used several independent techniques to investigate SV40 in these tumours. METHODS High-quality DNA and RNA were extracted from 71 frozen mesothelioma samples. DNA PCR was done with four sets of primers for the SV40 T-antigen gene. RNA transcripts were examined by RT-PCR. FINDINGS The first two primer sets for DNA PCR gave positive results in proportions similar to those reported in positive studies (56-62%) but there were unusual reproducibility difficulties. These primers were in a region of the T-antigen gene (nucleotides 4100-4713) that is present in many common laboratory plasmids. In assays with PCR primers not included within that region, only four cases (6%) showed products but these were too faint to suggest clonal infection. Further PCR assays confirmed that the SV40 sequences in the tumour samples had a deletion found only in plasmids, not in native functional SV40. Review of previous studies showed a similar pattern of discrepancies between SV40 T-antigen DNA PCR results obtained with primers within and beyond the region 4100-4713. All 71 mesotheliomas were negative for T-antigen transcripts by RT-PCR, and lacked T-antigen-positive tumour cells by immunohistochemistry. INTERPRETATION Our data based on three independent experimental approaches do not support a significant role for SV40 in human mesotheliomas. The risk of false-positive results due to contamination by common laboratory plasmids containing SV40 sequences has been underestimated. Studies of SV40 based on PCR methods require careful primer design to reduce this risk. RELEVANCE TO PRACTICE This paper presents several lines of evidence against the proposed link between SV40 infection and human mesotheliomas. Studies reporting a high prevalence of SV40 DNA in human tumours have been based on molecular assays prone to false-positive results. Because SV40 appears unlikely to have a major role, if any, in human mesotheliomas, clinicians should continue to consider asbestos exposure as the most likely and most thoroughly established aetiological factor in individuals with this cancer.

EML4-ALK testing in non-small cell carcinomas of the lung: a review with recommendations

In non-small cell lung cancer, epidermal growth factor receptor gene mutations and anaplastic lymphoma kinase (ALK) gene rearrangements have a major impact upon the level of response to treatment with specific tyrosine kinase inhibitors. This review describes the molecular basis of ALK inhibition, summarizes current data on the effectiveness and safety of ALK inhibition therapy, describes the different testing methodologies with their advantages and disadvantages, provides a suggested testing algorithm and puts forward a proposal for an external quality assessment program in ALK testing.

Loss of the Mitochondrial Bioenergetic Capacity Underlies the Glucose Avidity of Carcinomas

The down-regulation of the catalytic subunit of the mitochondrial H+-ATP synthase (beta-F1-ATPase) is a hallmark of most human carcinomas. This characteristic of the cancer cell provides a proteomic signature of cellular bioenergetics that can predict the prognosis of colon, lung, and breast cancer patients. Here we show that the in vivo tumor glucose uptake of lung carcinomas, as assessed by positron emission tomography in 110 patients using 2-deoxy-2-[18F]fluoro-d-glucose as probe, inversely correlates with the bioenergetic signature determined by immunohistochemical analysis in tumor surgical specimens. Further, we show that inhibition of the activity of oxidative phosphorylation by incubation of cancer cells with oligomycin triggers a rapid increase in their rates of aerobic glycolysis. Moreover, we show that the cellular expression level of the beta-F1-ATPase protein of mitochondrial oxidative phosphorylation inversely correlates (P < 0.001) with the rates of aerobic glycolysis in cancer cells. The results highlight the relevance of the alteration of the bioenergetic function of mitochondria for glucose capture and consumption by aerobic glycolysis in carcinomas.

The challenge of NSCLC diagnosis and predictive analysis on small samples. Practical approach of a working group

Until recently, the division of pulmonary carcinomas into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) was adequate for therapy selection. Due to the emergence of new treatment options subtyping of NSCLC and predictive testing have become mandatory. A practical approach to the new requirements involving interaction between pulmonologist, oncologist and molecular pathology to optimize patient care is described. The diagnosis of lung cancer involves (i) the identification and complete classification of malignancy, (ii) immunohistochemistry is used to predict the likely NSCLC subtype (squamous cell vs. adenocarcinoma), as in small diagnostic samples specific subtyping is frequently on morphological grounds alone not feasible (NSCLC-NOS), (iii) molecular testing. To allow the extended diagnostic and predictive examination (i) tissue sampling should be maximized whenever feasible and deemed clinically safe, reducing the need for re-biopsy for additional studies and (ii) tissue handling, processing and sectioning should be optimized. Complex diagnostic algorithms are emerging, which will require close dialogue and understanding between pulmonologists and others who are closely involved in tissue acquisition, pathologists and oncologists who will ultimately, with the patient, make treatment decisions. Personalized medicine not only means the choice of treatment tailored to the individual patient, but also reflects the need to consider how investigative and diagnostic strategies must also be planned according to individual tumour characteristics.

Stromal disrupting effects of nab-paclitaxel in pancreatic cancer

Background:Nab-paclitaxel and gemcitabine have demonstrated a survival benefit over gemcitabine alone in advanced pancreatic cancer (PDA). This study aimed to investigate the clinical, biological, and imaging effects of the regimen in patients with operable PDA.Methods:Patients with operable PDA received two cycles of nab-paclitaxel and gemcitabine before surgical resection. FDG-PET and CA19.9 tumour marker levels were used to measure clinical activity. Effects on tumour stroma were determined by endoscopic ultrasound (EUS) elastography. The collagen content and architecture as well as density of cancer-associated fibroblasts (CAFs) were determined in the resected surgical specimen and compared with a group of untreated and treated with conventional chemoradiation therapy controls. A co-clinical study in a mouse model of PDA was conducted to differentiate between the effects of nab-paclitaxel and gemcitabine.Results:A total of 16 patients were enrolled. Treatment resulted in significant antitumour effects with 50% of patients achieving a >75% decrease in circulating CA19.9 tumour marker and a response by FDG-PET. There was also a significant decrement in tumour stiffness as measured by EUS elastography. Seven of 12 patients who completed treatment and were operated had major pathological regressions. Analysis of residual tumours showed a marked disorganised collagen with a very low density of CAF, which was not observed in the untreated or conventionally treated control groups. The preclinical co-clinical study showed that these effects were specific of nab-paclitaxel and not gemcitabine.Conclusion:These data suggest that nab-paclitaxel and gemcitabine decreases CAF content inducing a marked alteration in cancer stroma that results in tumour softening. This regimen should be studied in patients with operable PDA.

Molecular Context of the EGFR Mutations: Evidence for the Activation of mTOR/S6K Signaling

Purpose: Activating somatic mutations in the epidermal growth factor receptor (EGFR) gene are present in a small subset of lung adenocarcinomas. These mutations cluster in specific regions and confer sensitivity to inhibitors of the tyrosine kinase activity of EGFR. To further determine the genetic and molecular characteristics of tumors carrying EGFR gene mutations, we investigated the EGFR gene status in lung adenocarcinomas and evaluated its association with specific characteristics of the patients and tumors, such as mutations at KRAS and p53, EGFR and ErbB2 gene amplification, levels of EGFR and HER2 proteins, and levels of downstream effectors of EGFR, such as phospho–extracellular signal-regulated kinase and phospho-S6 proteins. Experimental Design: The mutational status of EGFR was determined by direct sequencing in 86 primary lung adenocarcinomas and 12 lung cancer cell lines, and was correlated with a number of variables relating to the tumor and patient. A tissue microarray containing 37 lung tumors was constructed to determine, by fluorescence in situ hybridization analysis, the number of copies of EGFR and ErbB2 genes and, by immunohistochemistry, the levels of EGFR, HER2, phospho-ERK, and phospho-S6 proteins. Results:EGFR gene mutations were identified in 13% of the primary tumors. The type and clustering of the mutations were identical to those previously reported. Amplification of the EGFR occurred in 14% of the tumors and could arise in tumors with EGFR mutations. Interestingly, mTOR activation, as measured indirectly by augmented levels of phospho-S6 protein, was more frequent in tumors with gene alterations in either EGFR or KRAS (P = 0.00005; Fishers exact test) than in their wild-type counterparts. Conclusions: Our data agree with the accumulation of EGFR mutations in a subset of patients with lung cancer. Moreover, we report EGFR gene amplification in EGFR-mutant tumors and a positive correlation between EGFR or KRAS alterations and activation of mTOR signaling.

A Commercial Real-Time PCR Kit Provides Greater Sensitivity than Direct Sequencing to Detect KRAS Mutations: A Morphology-Based Approach in Colorectal Carcinoma

KRAS mutation testing has become a standard procedure in the management of patients with carcinomas. The most frequently used method for KRAS testing is direct sequencing of PCR products. The development of commercial real-time quantitative PCR kits offers a useful alternative since they are in theory much more sensitive than direct sequencing and they avoid post- PCR handling. We present our experience as a reference center for the study of KRAS mutations, comparing direct sequencing and the use of a commercial real-time quantitative PCR kit, as well as determining the sensitivity of both procedures in clinical practice. The TheraScreen K-RAS Mutation Kit identified mutations in 75 (44%) of the 170 tumors. Three cases were tested positive using TheraScreen K-RAS Mutation Kit and negative by direct sequencing. We then compared the sensitivity of the kit and that of direct sequencing using 74 mutant tumors. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in 13.5% of the tumors and, in 84%, KRAS mutation was identified at a dilution of 5%. Sequencing was able to detect KRAS mutations when the mutant DNA represented 10% of the total DNA in 20/74 (27%) of the tumors. When the mutant DNA represented 30% of the total DNA, sequencing could detect mutations in 56/74 (76%).