Johannes Voortman

Identification of MicroRNA-21 as a Biomarker for Chemoresistance and Clinical Outcome Following Adjuvant Therapy in Resectable Pancreatic Cancer

Background Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The high risk of recurrence following surgical resection provides the rationale for adjuvant therapy. However, only a subset of patients benefit from adjuvant therapy. Identification of molecular markers to predict treatment outcome is therefore warranted. The aim of the present study was to evaluate whether expression of novel candidate biomarkers, including microRNAs, can predict clinical outcome in PDAC patients treated with adjuvant therapy. Methodology/Principal Findings Formalin-fixed paraffin embedded specimens from a cohort of 82 resected Korean PDAC cases were analyzed for protein expression by immunohistochemistry and for microRNA expression using quantitative Real-Time PCR. Cox proportional hazards model analysis in the subgroup of patients treated with adjuvant therapy (N = 52) showed that lower than median miR-21 expression was associated with a significantly lower hazard ratio (HR) for death (HR = 0.316; 95%CI = 0.166–0.600; P = 0.0004) and recurrence (HR = 0.521; 95%CI = 0.280–0.967; P = 0.04). MiR-21 expression status emerged as the single most predictive biomarker for treatment outcome among all 27 biological and 9 clinicopathological factors evaluated. No significant association was detected in patients not treated with adjuvant therapy. In an independent validation cohort of 45 frozen PDAC tissues from Italian cases, all treated with adjuvant therapy, lower than median miR-21 expression was confirmed to be correlated with longer overall as well as disease-free survival. Furthermore, transfection with anti-miR-21 enhanced the chemosensitivity of PDAC cells. Conclusions Significance Low miR-21 expression was associated with benefit from adjuvant treatment in two independent cohorts of PDAC cases, and anti-miR-21 increased anticancer drug activity in vitro. These data provide evidence that miR-21 may allow stratification for adjuvant therapy, and represents a new potential target for therapy in PDAC.

MicroRNA Expression and Clinical Outcomes in Patients Treated with Adjuvant Chemotherapy after Complete Resection of Non–Small Cell Lung Carcinoma

This study determined whether expression levels of a panel of biologically relevant microRNAs can be used as prognostic or predictive biomarkers in patients who participated in the International Adjuvant Lung Cancer Trial (IALT), the largest randomized study conducted to date of adjuvant chemotherapy in patients with radically resected non-small cell lung carcinoma (NSCLC). Expression of miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a was determined by quantitative real-time PCR in formalin-fixed paraffin-embedded tumor specimens from 639 IALT patients. The prognostic and predictive values of microRNA expression for survival were studied using a Cox model, which included every factor used in the stratified randomization, clinicopathologic prognostic factors, and other factors statistically related to microRNA expression. Investigation of the expression pattern of microRNAs in situ was performed. We also analyzed the association of TP53 mutation status and miR-34a/b/c expression, epidermal growth factor receptor and KRAS mutation status, and miR-21 and Let-7a expression. Finally, the association of p16 and miR-29b expression was assessed. Overall, no significant association was found between any of the tested microRNAs and survival, with the exception of miR-21 for which a deleterious prognostic effect of lowered expression was suggested. Otherwise, no single or combinatorial microRNA expression profile predicted response to adjuvant cisplatin-based chemotherapy. Together, our results indicate that the microRNA expression patterns examined were neither predictive nor prognostic in a large patient cohort with radically resected NSCLC, randomized to receive adjuvant cisplatin-based chemotherapy versus follow-up only.

Array comparative genomic hybridization-based characterization of genetic alterations in pulmonary neuroendocrine tumors

The goal of this study was to characterize and classify pulmonary neuroendocrine tumors based on array comparative genomic hybridization (aCGH). Using aCGH, we performed karyotype analysis of 33 small cell lung cancer (SCLC) tumors, 13 SCLC cell lines, 19 bronchial carcinoids, and 9 gastrointestinal carcinoids. In contrast to the relatively conserved karyotypes of carcinoid tumors, the karyotypes of SCLC tumors and cell lines were highly aberrant. High copy number (CN) gains were detected in SCLC tumors and cell lines in cytogenetic bands encoding JAK2, FGFR1, and MYC family members. In some of those samples, the CN of these genes exceeded 100, suggesting that they could represent driver alterations and potential drug targets in subgroups of SCLC patients. In SCLC tumors, as well as bronchial carcinoids and carcinoids of gastrointestinal origin, recurrent CN alterations were observed in 203 genes, including the RB1 gene and 59 microRNAs of which 51 locate in the DLK1-DIO3 domain. These findings suggest the existence of partially shared CN alterations in these tumor types. In contrast, CN alterations of the TP53 gene and the MYC family members were predominantly observed in SCLC. Furthermore, we demonstrated that the aCGH profile of SCLC cell lines highly resembles that of clinical SCLC specimens. Finally, by analyzing potential drug targets, we provide a genomics-based rationale for targeting the AKT-mTOR and apoptosis pathways in SCLC.

Prediction of outcome of non-small cell lung cancer patients treated with chemotherapy and bortezomib by time-course MALDI-TOF-MS serum peptide profiling

BackgroundOnly a minority of patients with advanced non-small cell lung cancer (NSCLC) benefit from chemotherapy. Serum peptide profiling of NSCLC patients was performed to investigate patterns associated with treatment outcome.Using magnetic bead-assisted serum peptide capture coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), serum peptide mass profiles of 27 NSCLC patients treated with cisplatin-gemcitabine chemotherapy and bortezomib were obtained. Support vector machine-based algorithms to predict clinical outcome were established based on differential pre-treatment peptide profiles and dynamic changes in peptide abundance during treatment.ResultsA 6-peptide ion signature distinguished with 82% accuracy, sensitivity and specificity patients with a relatively short vs. long progression-free survival (PFS) upon treatment. Prediction of long PFS was associated with longer overall survival. Inclusion of 7 peptide ions showing differential changes in abundance during treatment led to a 13-peptide ion signature with 86% accuracy at 100% sensitivity and 73% specificity. A 5-peptide ion signature could separate patients with a partial response vs. non-responders with 89% accuracy at 100% sensitivity and 83% specificity. Differential peptide profiles were also found when comparing the NSCLC serum profiles to those from cancer-free control subjects.ConclusionThis study shows that serum peptidome profiling using MALDI-TOF-MS coupled to pattern diagnostics may aid in prediction of treatment outcome of advanced NSCLC patients treated with chemotherapy.

Molecular Mechanisms Underlying the Role of MicroRNAs in the Chemoresistance of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is an extremely severe disease where the mortality and incidence rates are almost identical. This is mainly due to late diagnosis and limited response to current treatments. The tumor macroenvironment/microenvironment have been frequently reported as the major contributors to chemoresistance in PDAC, preventing the drugs from reaching their intended site of action (i.e., the malignant duct cells). However, the recent discovery of microRNAs (miRNAs) has provided new directions for research on mechanisms underlying response to chemotherapy. Due to their tissue-/disease-specific expression and high stability in tissues and biofluids, miRNAs represent new promising diagnostic and prognostic/predictive biomarkers and therapeutic targets. Furthermore, several studies have documented that selected miRNAs, such as miR-21 and miR-34a, may influence response to chemotherapy in several tumor types, including PDAC. In this review, we summarize the current knowledge on the role of miRNAs in PDAC and recent advances in understanding their role in chemoresistance through multiple molecular mechanisms.

Loss of 18q22.3 Involving the Carboxypeptidase of Glutamate-like Gene Is Associated with Poor Prognosis in Resected Pancreatic Cancer

Purposes: Pancreatic cancer is the fourth leading cause of cancer-related death, and studies on the clinical relevance of its genomic imbalances are warranted. Experimental Design: Recurrent copy number alterations of cytobands and genes were analyzed by array comparative genomic hybridization (aCGH) in 44 resected pancreatic cancer specimens. Prognostic markers identified by aCGH were validated by PCR gene copy number assay in an independent validation cohort of 61 resected pancreatic cancers. The functions of gene identified were evaluated by proliferation, cell cycle, and migration assays in pancreatic cancer cells. Results: We showed recurrent copy number gains and losses in the first cohort. Loss of 18q22.3 was significantly associated with short-term overall survival in the first cohort (P = 0.019). This cytoband includes the carboxypeptidase of glutamate-like (CPGL) gene. CPGL gene deletion was associated with shorter overall survival in the validation cohort (P = 0.003). CPGL deletion and mutations of TP53 or Kras seem to be independent events. A Cox model analysis of the two cohorts combined showed that loss of 18q22.3/deletion of the CPGL gene was an independent poor prognostic factor for overall survival (HR = 2.72, P = 0.0007). Reconstitution of CPGL or its splicing variant CPGL-B into CPGL-negative pancreatic cancer cells attenuated cell growth, migration, and induced G1 accumulation. Conclusion: Loss of 18q22.3/deletion of the CPGL gene is a poor prognostic marker in resected pancreatic cancer, and functional studies suggest the CPGL gene as growth suppressor gene in pancreatic cancer. Clin Cancer Res; 18(2); 524–33. ©2011 AACR.

Abstract 2995: MicroRNA expression and outcome of adjuvant chemotherapy in patients with completely resected non-small cell lung cancer: International Adjuvant Lung Cancer Trial Biologic Program (IALT-Bio)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The aim of our study was to assess whether expression levels of a panel of biologically relevant microRNAs can be used as prognostic and/or predictive biomarkers in patients who participated in the International Adjuvant Lung Cancer Trial (IALT), the largest randomized study conducted so far of adjuvant chemotherapy in patients with radically resected NSCLC stage I-III. Expression of miR-21, miR-29b, miR-34a/b/c, miR-155 and Let-7a was determined by quantitative real-time PCR in paraffin embedded formalin fixed tumor specimens from 639 IALT patients, using small nuclear RNA U66 as the endogenous normalization control. From 79 patients sufficient tumor adjacent normal tissue was available as well. The prognostic and predictive value of microRNA expression and chemotherapy for survival were studied using a Cox model, which included every factor used in the stratified randomization plus clinical and histological prognostic factors as well as other factors statistically related to microRNA expression. Association of p53 mutation status and miR-34a/b/c expression was analyzed, as well as association of EGFR and K-Ras mutation status with miR-21 and Let-7a expression, respectively. Finally association of p16 and miR-29b expression was assessed. In situ hybridization was performed for validation and to determine histological expression patterns. Overall, we found that there was no significant association between the expression profile of any of the tested microRNAs and survival. However, for miR-21, miR-34b and miR-34c, there is a suggested deleterious prognostic effect of lower values on survival. No single microRNA expression profile or a combination of microRNA expression profiles, as determined by cluster analysis, predicted response to adjuvant cisplatin-based chemotherapy. Concluding, expression levels of the tested microRNAs were neither predictive nor prognostic in this patient cohort. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2995.

Abstract 2989: High-dose, intermittent sunitinib as an alternative treatment strategy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Increased exposure to sunitinib treatment is associated with improved outcome indicative of the importance of dose intensity in the efficacy/toxicity balance. The currently approved schedule (50 mg daily, 4 weeks on, 2 weeks off) precludes further dose intensification. We hypothesized that high doses of sunitinib would improve tumor response with limited toxicity, when applied intermittently. Methods: In vitro proliferation was studied with MTT assays, one week after 3-9 hrs exposure of a panel of cancer cell lines (786-0, HT29, MDA-MB231, HCC827, A431) to high (5 and 20uM) concentrations of tyrosine kinase inhibitors (TKIs), namely sunitinib, sorafenib, erlotinib, pazopanib and axitinib. Apo-ONETM Homogenous Caspase 3/7 Assay (Promega) was applied to detect activation of apoptosis. The chick embryo chorioallantoic membrane (CAM) in vivo model was employed to further test the efficacy of the new scheduling, where sunitinib was topically administered on HT29 tumors growing on the CAM either daily at the commonly used dose of 40 mg/kg or once weekly at 200 mg/kg. Microvessel density (MVD) was determined by CD31 immunohistochemical staining. Plasma sunitinib concentrations were determined by LC-MS-MS from refractory patients with solid tumors participating in a phase I clinical trial (classical 3+3 design, EudraCT No: 2012-005756-41) in which high-dose, intermittent sunitinib treatment is being studied. Results: Sunitinib was identified as one of the most potent inhibitors of cell growth from the panel of TKIs; 6 hrs exposure to 20 uM of sunitinib resulted in tumor cell death. This effect was tumor cell line-independent and mediated via activation of caspase 3/7 pathway. High dose treatment of CAM tumors resulted in significant decrease in tumor growth compared to the control (mean 0.17 mg vs. 0.23 mg respectively, p=0.04), irrespective of angiogenesis inhibition as denoted by lack of inhibitory effect on MVD. It also led to higher intratumoral sunitinib concentrations compared to the daily schedule (14.3 vs. 8 ng/mg tissue, respectively), while blood concentrations were identical between the two schedules, averaging at 170 ng/ml. Patients enrolled in the first cohort of the clinical trial received 200 mg sunitinib orally weekly. This schedule was well tolerated and no dose limiting toxicities occurred. Overall, sunitinib exhibited moderate interpatient variability. Maximum plasma concentrations on Day 1 averaged on 161 ng/ml (range 118-239.5 ng/ml) and were reached approximately 6 hours after administration. Plasma drug concentrations decreased to undetectable from one cycle to the other. We have proceeded to escalate the dose for the next cohort of patients. Conclusion: Optimization of therapy with targeted agents remains an unmet challenge. Thus far, high-dose, intermittent sunitinib is well tolerated. These findings open new avenues that might contribute to improved treatment with sunitinib. Citation Format: Maria Rovithi, Richard R. de Haas, Richard J. Honeywell, Johannes Voortman, Arjan W. Griffioen, Mariette Labots, Anne Marije Luik, Godefridus J. Peters, Henk J. Broxterman, Henk M.w. Verheul. High-dose, intermittent sunitinib as an alternative treatment strategy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2989. doi:10.1158/1538-7445.AM2014-2989

MicroRNA Expression and Outcome in Resected NSCLC—Response

We are grateful to Kageyama and colleagues for their interest in our article and for their comments concerning several methodologic aspects of our study. It is argued that accuracy of obtained results could be increased by the use of multiple tissue sections per patient as well as microdissection