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Introduction: In colorectal cancer (CRC) surveillance, adenoma characteristics such as size, number of adenomas, dysplasia, and villous components, are used as indicators for risk of developing metachronous cancer and guide the surveillance interval. The current risk groups (e.g., advanced adenomas) cannot optimally distinguish high risk from low risk, which may result in suboptimal surveillance strategies. Specific DNA copy number aberrations are associated with risk of colorectal adenoma-to-carcinoma progression, but these aberrations are present in only a subset of advanced adenomas. Therefore, we hypothesize that specific DNA copy number aberrations may better predict the risk of CRC than advanced adenoma. Aim: To evaluate whether a molecularly-defined high-risk adenoma is a better risk factor for CRC than the presently used advanced adenoma. Materials and methods: DNA copy number profiles were determined, by means of low-coverage whole genome sequencing, on a series of 529 adenomas, selected from a Norwegian adenoma cohort. We retrieved detailed information on adenoma characteristics and whether adenoma patients were subsequently diagnosed with CRC or not. By univariate and multivariate regression analysis we estimated the odds ratio for association between the development of CRC and baseline presence of advanced adenoma versus presence of a molecularly-defined high-risk adenoma, respectively. Results: Molecular high-risk features were observed in 85/267 (32%) of advanced adenomas and in 27/262 (10%) of non-advanced adenomas. The odds ratio for developing metachronous CRC was 3.58 (p=2.84E-8) and 1.90 (p=0.012), when an advanced adenoma or molecular high-risk adenoma was detected at the baseline colonoscopy, respectively. In the multivariate regression analysis only advanced adenoma was as a significant risk factor for CRC. Conclusion: Molecularly-defined high-risk adenomas are associated with an increased risk of CRC. However, the advanced adenoma is a stronger predictor for risk of CRC. Citation Format: Henriette C. Jodal, Eddymurphy U. Akwiwu, Leticia G. Leon, Margriet Lemmens, Pien Delis-van Diemen, Dagmar Klotz, Meike de Wit, Remond Fijneman, Monique van Leerdam, Evelien Dekker, Manon C. W. Spaander, Gerrit A. Meijer, Michael Bretthauer, Magnus Løberg, Veerle M. H. Coupé, Mette Kalager, Beatriz Carvalho. Molecular adenoma features to predict metachronous colorectal cancer risk: A nested-case control study [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A004.

T-cell prolymphocytic leukemia (T-PLL) is a rare type of neoplasm with an adverse prognosis due to poor responsiveness to conventional chemotherapies. Accurate and rapid diagnosis of T-PLL is problematic due to the heterogeneous nature of the disease. Therefore, further molecular insights of T-PLL are needed. Most of the T-PLL cases (80%) harbor chromosome 14 inversions and translocations or activating mutations in the JAK-STAT pathway. Small non-coding RNAs (sncRNA) are defined as single stranded RNAs that are smaller than 200nt in length and include e.g. microRNAs (miR), small nucleolar RNA (snoRNA) and PIWI-interacting RNAs (piRNA). To increase the molecular understanding of T-PLL, we performed sncRNA expression profiling with the Illumina platform on a well-characterized panel of 21 T-PLL samples. Notably, detailed molecular, cytogenetic, morphologic and clinical data are available of all T-PLL patients included in this study. Of the selected samples, we performed gene expression profiling by microarrays, cytogenetics, VDJ-recombination and multi-color flow cytometric analysis. In short, we sorted CD4+ and CD8+ naïve (CD45RA+, CD27+), effector (CD45RA+, CD27-) and memory (CD45RO+, CD27+) T-cells, and TCRγδ+ T-cells from Ficoll separated PBMCs of buffy coat blood. Total RNA of leukemic cells isolated from the blood was extracted and cDNA libraries for sequencing sncRNAs were generated. For the annotation of the sequence reads we used the DASHR database of sncRNAs and miRbase database of mature miRs. SnoRNA (16%) and ribosomal (r) RNAs (25%) were the most abundant, whereas miRs were only 12% of the total sncRNA fraction. Next, we performed a correlation study using the expression profiles of the different types of sncRNAs and found a clear separation of T-PLL samples from the normal T-cell fractions when miRNA, piRNA and snoRNA expression profiles were used in the analysis. In addition, based on these data we could already identify potential subgroups of T-PLL, indicating that T-PLL is not a homogeneous disease. Finally, we noted aberrant expression of different types of sncRNAs compared to normal T-cell fractions, which may be indicative for essential roles of sncRNA species in the pathogenesis of T-PLL. Currently, we are in the process of characterizing sncRNA species that are aberrantly regulated specifically in T-PLL. Finally, we are selecting candidate sncRNAs for further functional characterization and for testing their diagnostic and/or prognostic value. Citation Format: Leticia G. Leon, Steven C. Koetzier, Fabiënne van Opstal, Joyce Schilperoord-Vermeulen, Anton W. Langerak, Stefan J. Erkeland. A comprehensive genome-wide analysis of small noncoding RNAs in T-cell prolymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2466.

Malignant pleural mesothelioma(MPM) is an aggressive cancer, which incidence has constantly increased over the past two decades and is expected to peak in 2020. The overall prognosis is poor, and predictive biomarkers of drug activity are missing. Given the heterogeneous and complex nature of MPM, it is likely that genomic aberrations changing the expression of several genes, might affect therapeutic response. Therefore, the aim of the present study was to identify genes whose copy number alterations might predict the MPM prognosis. Recurrent copy number alterations of genes were analyzed by high-resolution whole-genome sequencing in DNA obtained from a “discovery cohort” of 26 resected MPM patients treated with pemetrexed-based chemotherapy (8 with progressive disease, vs. 10 with stable disease and 8 with partial response). Prognostic markers identified by Copy Number Variation analysis with Nexus, Control-FREEC and ReadDepth software were validated by PCR gene copy number and gene expression analyses both in the “discovery” and in two “validation” cohorts of pemetrexed-treated and untreated patients (N=45 and 40). The role of emerging genes was evaluated through siRNA and pharmacological studies using proliferation, migration and apoptosis assays in MPM cells. As reported previously we observed copy number loss of CDKN2A (15.4%) and BAP1 (7.7%). Interestingly, copy number gain of NOTCH2 was observed in 50% of samples of the patients who underwent progression, whereas losses of PTP4A3 were associated with clinical benefit (SD+PR). The prognostic relevance of NOTCH2 was confirmed by PCR analysis. NOTCH-2 silencing reduced MPM cell migration and enhanced apoptosis induction by pemetrexed, while a PTP4A3 inhibitor overcame pemetrexed resistance in MPM cells characterized by high NOTCH2 and PTP4A3 expression. These results support the role of NOTCH2 as a novel prognostic/predictive biomarker for MPM, prompting prospective randomized trials for its validation. Moreover, preclinical data suggest that NOTCH2 and PTP4A3 are oncogenes suitable for effective therapeutic targeting in pemetrexed-resistant MPM cells. Citation Format: Leticia G. Leon, Maria Gemelli, Paolo A. Zucali, Christos Zoumadakis, Elisa Giovannetti. Copy number alterations in NOTCH2 and PTP4A3 are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma [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 2460. doi:10.1158/1538-7445.AM2017-2460

Oncogenic KRAS signaling is the main driving force behind pancreatic ductal adenocarcinoma (PDAC); however, targeting this pathway has proven to be difficult. Conversely, the PI3K/Akt pathway represents an exciting new target, because it has been associated with poor prognosis and chemoresistance, and several inhibitors are under development. In particular, perifosine prevents Akt translocation to the cell membrane, while MK-2206 is an Akt allosteric inhibitor and BEZ-235 is a dual PI3K/mTOR inhibitor. Therefore, we investigated the prognostic role of phospho (p)-Akt in PDAC tissues, as well as the molecular mechanisms underlying the interaction of Akt inhibitors with gemcitabine, using PDAC cells, primary cultures and spheroids. Immunohistochemistry of tissue microarrays with specimens from radically-resected patients (n=100) revealed a correlation between high p-Akt1 expression and worse outcome. Patients with low p-Akt expression (as detected by digital scoring) had a median overall survival (OS) of 16.2 months (95% CI, 14.8-20.1), while patients with high expression had a median OS of 12.0 months (95% CI, 9.0-14.9, P=0.03). Parallel immunocytochemistry studies revealed high expression levels in LPC028 primary cells, while LPC006 were characterized by low p-Akt1. Akt inhibitors reduced cancer cell growth in monolayers and spheroids, and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028 (e.g., combination index CI of 0.2, in the gemcitabine-perifosine combination for 72h, at fixed IC50 ratio), while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a 5-fold reduction in the expression of the main gemcitabine target ribonucleotide reductase. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. However, the combination of Akt inhibitors with gemcitabine increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. The Akt signaling is involved in the expression/localization of the key glucose transporter Glut1, and increased glucose metabolism was associated to resistance to axitinib (Hudson et al, Cell Death Dis 2014). Remarkably, the resistant LPC006 cells were characterized by overexpression of Glut1, which was not reduced after exposure to Akt inhibitors. However, the novel Glut1 inhibitor PGL13 enhanced perifosine and perifosine/gemcitabine-induced cell death. In conclusion, our findings support the analysis of phospho-Akt1 expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies. Citation Format: Daniela Massihnia, Amir Avan, Niccola Funel, Carlotta Granchi, Filippo Minutolo, Leticia Leon, Godefridus Peters, Elisa Giovannetti. Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer [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 4731. doi:10.1158/1538-7445.AM2017-4731

Background: Approximately 40% of malignant pleural mesothelioma (MPM) patients respond to the combination treatment with the antifolate pemetrexed (PMX) and platinum compounds. Several studies highlighted the predictive role of thymidylate synthase (TS), but additional resistance mechanisms are poorly understood. This study aimed to evaluate the role of the reduced folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) as potential biological markers of intrinsic PMX-resistance in MPM. Materials and methods: PCFT promoter methylation was evaluated by combined bisulfite restriction analysis as well as MethyLight PCR in 14 cell lines and 10 MPM tissues, while PCFT and RFC mRNA levels were studied by Real-Time-PCR in MPM specimens from a test and a validation cohort of 73 and 51 PMX-treated patients, respectively. PCFT protein expression was evaluated by immunohistochemistry in a tissue microarray with representative MPM core biopsies from 35 patients. Data were analyzed by t-test, Fisher/log-rank test and Cox proportional hazards models. Results: PCFT expression inversely correlated with promoter methylation and treatment with 5-Aza-2′-deoxycytidine (5-Aza-dC) induced its marked restoration in MPM cells. Patients with low PCFT mRNA levels had significantly shorter overall survival (OS), in both the test (median OS, 11.3 vs. 17.6 months, P = 0.01) and in the validation cohort (OS, 12.6 vs. 30.3 months, P = 0.02). Multivariate analysis, including age, sex, EORTC prognostic score and histology, confirmed PCFT prognostic relevance (HR, 2.1 and 2.4 in the test and validation cohorts, respectively). Low PCFT protein expression was also associated with significantly shorter OS (11.8 vs. 30.3 months, P = 0.02). Remarkably, the subgroup of patients with both low PCFT and high TS mRNA levels (30% of the patients in the test cohort) had the worse prognosis (median OS, 5.5 months, P Conclusions: These results define PCFT as a novel prognostic factor for MPM patients treated with PMX. Prospective trials for the validation of the prognostic/predictive role of PCFT in MPM patients treated with PMX-based regimens are warranted. Furthermore preclinical data suggest that exposure to 5-Aza-dC might restore PCFT expression and result in efficient eradication of antifolate-resistant cells. Citation Format: Elisa Giovannetti, Paolo A. Zucali, Yehuda G. Assaraf, Niccola Funel, Maria Gemelli, Michal Stark, Leticia G. Leon, Zhanjun Hou, Matteo Perrino, Larry H. Matherly, Godefridus J. Peters. Role of proton-coupled folate transporter expression in resistance of mesothelioma patients treated with pemetrexed. [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 4335. doi:10.1158/1538-7445.AM2015-4335

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal among solid tumors. Only a subset of patients benefit from chemotherapy, and identification of novel prognostic factors is warranted. Recently, miRNAs emerged as a pivotal class of regulators for multiple genes involved in cancer, including PDAC (Giovannetti et al, Cancer Res 2010), and miRNA expression pattern classified tumors better than mRNA data (Lu et al, Nature 2005), suggesting their use for diagnostic purposes. However, high-throughput arrays detecting more than 1000 miRNAs are opening new opportunities to evaluate whether their profiles can also predict clinical outcome. Therefore, the present study evaluated whether comprehensive miRNA expression profiling by microarray technology can predict overall survival (OS) in resected PDAC patients. High-resolution miRNA profiles were obtained with the Toray's 3D-GeneTM miRNA chip, detecting 1200 types of human miRNA. RNA was isolated from formaldehyde fixed paraffin embedded primary tumors of 26 stage-pT3N1Mx homogeneously treated patients (gemcitabine 1000 mg/m2/day on days 1/8/15, every 28 days), selected according to their outcome (OS 30 months). Microarray slides of the 19 samples that passed RNA quality check were scanned using 3D-GeneTM Scanner 3000 with appropriate photomultiplier settings for red channel. Each microarray was scanned three times, and then merged into one dataset analyzed. Highly stringent statistics included t-test, Spearman ranked correlation to generate a distance matrix, and iterative approaches. Unsupervised hierarchical analysis revealed that the PDAC specimens clustered according to their long/short OS classification, while the feature selection algorithm RELIEF identified the top 10 discriminating miRNAs between the two groups. MiR211 emerged as the best discriminating miRNA, with significantly higher expression in long vs. short OS patients. The expression of this miRNA was subsequently assessed by quantitative RT-PCR in an independent cohort of laser microdissected tumors from 60 resected stage-pT3N1Mx PDAC patients treated with the same gemcitabine regimen. Patients with low miR211 expression, according to median value (12.8 a.u. calculated as ΔCt vs. RNU6), had a significantly shorter median OS (14.8, 95%CI=13.1-16.5, vs. 25.7 months, 95%CI=16.2-35.1, log-rank P=0.004). Multivariate analysis demonstrated that low miR211 expression was an independent factor of poor prognosis for OS (hazard ratio 2.3, P=0.03) after adjusting for all the factors influencing clinical outcome. In conclusion, through comprehensive microarray analysis and PCR validation we identified miR211 as prognostic factor in resected PDAC. This miRNA was already associated with melanoma invasiveness, but our results prompt further prospective studies as well as research on miR211 biological role in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 186. doi:1538-7445.AM2012-186

Pemetrexed improves the effect of platinum-compounds against MPM, but biomarkers of response and novel effective combinations are warranted. The present study evaluated the 1) correlation between expression and polymorphisms of pemetrexed's key target TS and outcome of MPM patients treated upfront with carboplatin/pemetrexed, and 2) pharmacological interaction of new targeted compounds (gefitinib, erlotinib, sorafenib, vandetanib, and enzastaurin) with carboplatin-pemetrexed in MPM cell lines. Analysis of TS TSER-2R/3R, mRNA and protein expression was performed by PCR and immunohistochemistry (using H-score) in tumors from 99 patients. The role of TS and other molecular determinants in the interaction of carboplatin, pemetrexed with new targeted compounds was investigated in the H2052, H2452, H28 and MSTO-211H cells. Drug interaction was studied using MTT and SRB assays and evaluated with combination index method. EGFR, Akt and Erk phosphorylation, as well as VEGF secretion, were analyzed with ELISA, whereas RT-PCR and western blot were performed to assess modulation of the expression of TS, E2F-1 and genes involved in DNA repair (ERCC1 and XPD). A significant correlation between low TS protein expression and response (odd ratio, 4.2; p=0.023), longer PFS (8vs6 months; hazard ratio [HR]:0.60: p=0.023), or OS (18vs9 months; HR:0.59; p=0.029) was found when patients were categorized according to median H-score. Similarly, patients with TS mRNA below the median had longer PFS and OS (p Vandetanib emerged as the targeted compound with the most potent cell growth inhibitory effects, and interacted synergistically with carboplatin and pemetrexed in all cell lines, increasing apoptotic indices. Pemetrexed enhanced EGFR phosphorylation, but reduced Akt phosphorylation and ERCC1 and XPD expression. Conversely, vandetanib significantly downregulated EGFR, Erk and Akt phosphorylation, as well as E2F-1 and TS expression (eg, H28 cells had 4.8-fold TS mRNA decrease). In conclusion, low TS protein and mRNA levels were associated to response, longer PFS and OS. Furthermore, vandetanib improved pemetrexed-carboplatin activity against MPM cells, through modulation of critical molecular mechanisms, such as EGFR-Akt phosphorylation and TS expression. These data support further prospective trials for the validation of the prognostic/predictive role of TS in patients treated with pemetrexed-based regimens, as well as future studies on the integration of vandetanib in the treatment of MPM. 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 5041. doi:10.1158/1538-7445.AM2011-5041

PURPOSE: High-throughput arrays are opening new opportunities for our understanding of the molecular aberrations underlying the outcome of colorectal cancer (CRC) in response to specific therapies. The aim of the present study was to evaluate whether genome-wide DNA copy number profiles of CRC specimens can predict the clinical outcome to irinotecan and 5-fluorouracil (5-FU) or pemetrexed treatments. EXPERIMENTAL DESIGN: High-resolution DNA copy number profiles were obtained by 44K oligonucleotide-based array-comparative-genomic-hybridization (aCGH). DNA was isolated from formaldehyde-fixed paraffin-embedded primary tumors of 34 patients with advanced CRC, treated within a randomized phase II trial of pemetrexed+irinotecan (ALIRI) vs. leucovorin-modulated-5-FU + irinotecan (FOLFIRI). RESULTS: Unsupervised hierarchical cluster analysis of the aCGH data revealed two clusters significantly correlated with response status (P=0.03). Most frequently observed chromosomal aberrations (>50%) in the 9 responders were losses of 18q (78%), losses of 18p (72%), 8p22-p23.1 (56%), and 17p11.2 (67%), and gains of 20p13-q13.3 (73%), the whole chromosome 13 (78%), 7p11.2-22.3 (56%), and 8q24.21 (56%). After multiple correction gains in 8q24.21and in 20p13-q13 as well as losses of 18p and 17p11 were significantly associated with response. The analysis of patients categorized for different treatments showed that tumors of patients with better response after FOLFIRI had specific profiles of chromosomal aberrations, including significantly more losses at chromosome 18, fewer losses at chromosome 17, and more gains at chromosomes 20 and 8. Similar results were observed in the ALIRI arm. However, in contrast to the results observed in the patients treated in the ALIRI arm, response to FOLFIRI was correlated with a higher number of losses at chromosome 1, including the 1p36 region (P CONCLUSIONS: Genome-wide DNA profiling of CRC revealed several genomic loci, of which the copy number status may serve as predictive marker for clinical outcome after the commonly used FOLFIRI regimen, and large prospective studies are ongoing. Some of these aberrations, but also other specific aberrations, affected the activity of the pemetrexed-irinotecan combination, and should be further studied to explain the biological basis of response to these drugs and optimize their use. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 2146.

MiR-21 was reported to be overexpressed in PDAC and contributed to tumor invasion and resistance to gemcitabine. Further studies are needed to test whether miR-21 could be used to stratify patients for treatment and provide mechanistic insights for new therapeutic targets. The aim of this study was to evaluate whether miR-21 expression was associated with the overall survival (OS) of PDAC patients treated with gemcitabine, and investigate its role in PDAC invasive behaviour and chemoresistance. MiR-21 expression was evaluated in cells (including 7 PDAC cell lines, 7 primary PDAC cultures, fibroblasts and a normal pancreatic ductal cell line) and tissues (neoplastic specimens from 77 PDAC patients (32 in metastatic/inoperable and 45 in adjuvant setting) and normal ductal samples) isolated by laser microdissection. The role of miR-21 on the pharmacological effects of gemcitabine was studied in cells transfected with a specific miR-21 precursor (pre-mir). Modulation of apoptosis, Akt activation and VEGF secretion was analyzed by Annexin and ELISA assays, respectively. Quantitative PCR evaluated the correlation of miR-21 with invasion-related genes and VEGF. Inhibitors of PI3K and mTOR (LY294002 and rapamycin) were used to test whether modulation of these signalling pathways affected molecular mechanisms activated by miR-21 and gemcitabine. Patients with high miR-21 expression had a significantly shorter OS both in adjuvant and inoperable/metastatic setting, while a trend toward correlation was found between low expression and clinical benefit. Multivariate analysis in all patients with adequate follow-up (N=58), confirmed the negative prognostic significance of high miR-21 expression (HR:2.6, 95%CI:1.7-3.4; P:0.001). MiR-21 expression was correlated with the expression in their respective tissues in the primary cultures and with gemcitabine resistance in all the PDAC cells. Treatment with gemcitabine resulted in a significant increase of miR-21 expression, ranging from 2 to 19-fold, in 13 PDAC cell cultures. Pre-mir transfection significantly decreased apoptosis induction by gemcitabine and up-regulated metalloproteinase-2/−9 and VEGF mRNA expression in 2 selected PDAC cultures. In contrast, addition of LY294002 and rapamycin resulted in reduction of phospho-Akt and prevented pre-mir induced resistance to gemcitabine. In conclusion, miR-21 expression was correlated with OS in PDAC gemcitabine-treated patients. In addition, miR-21 expression is associated with reduced apoptosis, increased Akt phosphorylation and expression of genes involved in invasive behaviour resulting in gemcitabine chemoresistance, which was prevented by Akt inactivation. Our findings indicate that inactivation of PI3K/mTOR pathway may increase gemcitabine sensitivity in PDAC and warrants clinical studies. 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 4819.