Single Genetic Alteration Research Articles

Human SNF5 arming of double-deleted vaccinia virus shows oncolytic and cytostatic activity against central nervous system atypical teratoid/rhabdoid tumor cells.

Central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT) is a rare, aggressive tumor that most often affects very young children. The common decisive molecular defect in AT/RT has been shown to be a single genetic alteration, i.e., the loss of hSNF5 gene that encodes for a subunit of the SWI/SNF complex that modulates chromatin remodeling activities. As a result, AT/RT cells display unregulated cell proliferation due to the dysfunction of an important epigenetic control. We have previously demonstrated the preclinical efficacy of the oncolytic double-deleted vaccinia virus (VVDD) against AT/RT. Here we report the establishment of a modified VVDD engineered to express wild type hSNF5 gene. We show that this reconstructed vaccinia virus retains comparable infectivity and in vitro cytotoxicity of the parent strain. However, in addition, hSNF5-arming of VVDD results in a decreased cell cycle S phase population and down-regulation of cyclin D1. These findings suggest that hSNF5-arming of VVDD may increase the efficacy in the treatment of AT/RT and validates, as a proof-of-concept, an experimental approach to enhance the effective use of novel modified oncolytic viruses in the treatment of tumors with loss of a tumor suppressor gene function.

Using genomic data for selecting the treatment of lymphoma patients.

Genomic profiling platforms provide unprecedented genetic information of lymphoma biology, yet information has yet to be readily integrated into clinical medicine. This review summarizes the important concepts of utilizing genomics to aide disease management. A wide range of clinical grade genetic sequencing platforms are available, therefore the selection of sequencing platform should ideally be based on biological and clinical questions, as well as the strength and weaknesses of individual platform. Different evidence-based guidelines exist to aide clinical judgment; however, few have well curated, easy to search platforms. Using one guideline proposed by several regulatory groups, our review summarizes genetic alterations with diagnostic, prognostic and therapeutic potential in the major subtypes of lymphoma. A comprehensive database of genetic alterations that contribute to clinical care in lymphoma is needed. Ideally, a database which accounts for single and pathway-based genetic alterations may be developed to guide development and interventions for management of lymphoma.

Molecular classification of aggressive B-cell lymphoma.

Molecular classification of aggressive B-cell lymphoma.

Kras-driven heterotopic tumor development from hepatobiliary organoids.

Cancers arising from the biliary tract are refractory to conventional therapies, requiring the development of novel therapeutics. However, only a limited number of genetically engineered mouse models have been created, partly due to time-consuming work required. Besides, liver-specific gene manipulation mostly resulted in concurrent development of hepatocellular carcinoma, another type of liver cancer, and gall bladder-restricted gene targeting is still not feasible. Consequently, establishment of cancer type-specific disease modeling remains a technical challenge. To address this issue, we took an alternative cell-based approach to quickly induce tumorigenesis ex vivo. Specifically, murine primary organoids from liver and gall bladder were transduced with lentiviral vectors to reconstitute genetic alterations common in biliary tract cancers, followed by inoculation in immunodeficient mice. Whereas any single genetic alteration did not induce tumors, mutant Kras and repression of major tumor suppressors cooperated for tumor development within two months. Induced lesions varied among normal, dysplastic and papillary lesions to adenocarcinoma, recapitulating multi-step tumorigenesis even in a heterotopic situation. We further demonstrated that two putative oncogenes in intrahepatic cholangiocellular carcinoma, mutant Pik3ca and FGFR2-AHCYL1 fusion, were rather modest drivers for liver-derived organoids, probably requiring additional mutations or hepatic niche to robustly induce full-blown tumors. Thus, we showed that cancer cells could be readily generated from primary cells in the biliary tract, at least in cases where genetic factors play dominant roles. Collectively, the present study will likely contribute to gaining mechanistic insights into biliary carcinogenesis and providing valuable resources for drug discovery.

Abstract 4142: Conformational dynamics of the chimeric kinase DNAJB1-PRKACA, the driver for fibrolamellar hepatocellular carcinoma

Abstract In fibrolamellar hepatocellular carcinoma (FLC) there is a single common genetic alteration in all tumors: A deletion in one copy of chromosome 19 resulting in the fusion of the first exon of DNAJB1, encoding the J-domain of heat shock protein 40, with exons 2-10 of the catalytic subunit of protein kinase A, PRKACA [1]. This produces an enzymatically active chimeric protein. Expression of the chimera in mouse liver either by recreating the deletion by CRISPR/Cas9, or expression, in trans, by a transposon is sufficient to produce FLC [2]. We used molecular dynamics simulations, NMR, and SAXS to analyze the conformational dynamics of the native and chimeric kinase. The most significant differences were in the amino domain with the chimera in an ensemble of conformations. Some structures had the J-domain tucked under the large lobe of the kinase, similar to that reported in the crystal structure, and in others the J-domain was dislodged from the core of the kinase, swinging free in solution. These simulated dislodged states were captured experimentally both by NMR and small angle X-ray scattering. Modeling of the different conformations onto the RIIβ holoenzyme revealed no obvious steric interactions suggesting that the J-domain does not preclude formation of the holoenzyme. The flexible conformations appear to be independent of the nucleotide/substrate binding mode as they were observed in ATP, ADP, Apo, and ATP-PKI bound states. [1] Honeyman, J. N. et al. Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma. Science 343, 1010-1014 (2014). [2] Kastenhuber, E. R. et al. DNAJB1-PRKACA fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma. Proc. Natl. Acad. Sci. 201716483 (2017). doi:10.1073/pnas.1716483114 Citation Format: Michael D. Tomasini, Yingiie Wang, Adak Karamafrooz, James Hall, Susan S. Taylor, Thijs Beuming, Gianluigi Veglia, Sanford M. Simon. Conformational dynamics of the chimeric kinase DNAJB1-PRKACA, the driver for fibrolamellar hepatocellular carcinoma [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 4142.

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

Genetic alterations related to BRAF-FGFR genes and dysregulated MAPK/ERK/mTOR signaling in adult pilocytic astrocytoma.

Pilocytic astrocytomas occur rarely in adults and show aggressive tumor behavior. However, their underlying molecular-genetic events are largely uncharacterized. Hence, 59 adult pilocytic astrocytoma (APA) cases of classical histology were studied (MIB-1 LI: 1%-5%). Analysis of BRAF alterations using qRT-PCR, confirmed KIAA1549-BRAF fusion in 11 (19%) and BRAF-gain in 2 (3.4%) cases. BRAF-V600E mutation was noted in 1 (1.7%) case by sequencing. FGFR1-mutation and FGFR-TKD duplication were seen in 7/59 (11.9%) and 3/59 (5%) cases, respectively. Overall 36% of APAs harbored BRAF and/or FGFR genetic alterations. Notably, FGFR related genetic alterations were enriched in tumors of supratentorial region (8/25, 32%) as compared with other locations (P = 0.01). The difference in age of cases with FGFR1-mutation (Mean age ± SD: 37.2 ± 15 years) vs. KIAA1549-BRAF fusion (Mean age ± SD: 25.1 ± 4.1 years) was statistically significant (P = 0.03). Combined BRAF and FGFR alterations were identified in 3 (5%) cases. Notably, the cases with more than one genetic alteration were in higher age group (Mean age ± SD: 50 ± 12 years) as compared with cases with single genetic alteration (Mean age ± SD: 29 ± 10; P = 0.003). Immunopositivity of p-MAPK/p-MEK1 was found in all the cases examined. The pS6-immunoreactivity, a marker of mTOR activation was observed in 34/39 (87%) cases. Interestingly, cases with BRAF and/or FGFR related alteration showed significantly lower pS6-immunostatining (3/12; 25%) as compared with those with wild-type BRAF and/or FGFR (16/27; 59%) (P = 0.04). Further, analysis of seven IDH wild-type adult diffuse astrocytomas (DA) showed FGFR related genetic alterations in 43% cases. These and previous results suggest that APAs are genetically similar to IDH wild-type adult DAs. APAs harbor infrequent BRAF alterations but more frequent FGFR alterations as compared with pediatric cases. KIAA1549-BRAF fusion inversely correlates with increasing age whereas FGFR1-mutation associates with older age. Activation of MAPK/ERK/mTOR signaling appears to be an important oncogenic event in APAs and may be underlying event of aggressive tumor behavior. The findings provided a rationale for potential therapeutic advantage of targeting MAPK/ERK/mTOR pathway in APAs.

The Promise and the Hype of ‘Personalised Medicine’

Personalised medicine is widely considered as the way of the future for medicine. However, progress in cancer, with a few outstanding exceptions, has fallen below expectations because of the challenges of tumour heterogeneity and clonal evolution. In both benign and malignant disease, diseases caused by single genetic alterations are more amenable to precision medicine approaches. However, most common diseases are caused by a complex interplay of multiple genetic and environmental factors making personalised medicine far more challenging. The current optimism for personalised medicine is distorting clinical consultations, resource allocation and research funding prioritisation. A research active clinician must act both as an agent of change and development, and as a communicator of realism. Thus personalised medicine that includes a sober appreciation of what genomics can achieve, together with continued focus on the individual as a person not just as a genome, will contribute to further improvements in health and healthcare.

Abstract 96: Targeted sequencing from endoscopic ultrasound-guided fine needle aspirates of pancreatic ductal adenocarcinoma

Abstract Next-generation sequencing (NGS) that enables the analyses of massively parallel sequences of DNA can advance the understanding of the underlying molecular pathophysiologies of cancer. Such recent genomic analyses have revealed a complex mutational landscape in surgical specimens of PDAC. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is used in the diagnosis of pancreatic cancer. Several genomic analyses using EUS-FNA in PDAC have been undertaken in a small number of patients. This study evaluated the feasibility of targeted NGS of specimens obtained using EUS-FNA to clarify genetic markers associated with the prognosis of pancreatic cancer. One hundred sixty six patients who underwent EUS-FNA with pathologically confirmed PDAC were included. Genomic DNA was extracted and quality control metrics of DNA analytes were measured. The specimens that passed a quality control test (N = 101, 61%) underwent NGS using a customized cancer panel (CancerSCAN) enriched in the exons of 83 genes. Clinical prognostic factors associated with survival in PDAC were age, performance status, CA 19-9 level, curative resection, chemotherapy, tumor mass size, tumor location, stage and metastasis site in univariate analysis (P = 0.020, <0.001, 0.015, 0.022, <0.001, <0.001, 0.031, 0.024 and 0.028, respectively). Multivariate Cox proportional-hazards analysis revealed chemotherapy (P < 0.001, hazard ratio (HR) = 5.7, 95% CI, 2.8 to 11.3) and tumor mass size (P = 0.001, HR = 0.4, 95% CI, 0.2 to 0.7) as independent prognostic factors. Each patient had different numbers of genetic alterations ranging from 0 to 17, and 99% of the patients had at least a single genetic alteration. KRAS mutation was found in 86 patients (86%); KRAS variant allele frequency ranged from 2 to 79%. The frequencies of major gene mutations responsible for PDACs were KRAS 86%, CDKN2A 22%, TP53 72%, SMAD4 25% and BRCA 14%. Genetic alterations associated with survival were ARID1A, AURKB1, CDK4, ERBB4, HNF1A, JAK3, MPL, PIK3R, PTCH1, STK11 and TOP1 in univariate analysis. In multivariate analysis, CDK4, HNF1A, MPL, PIK3R, PTCH1, STK11 and TOP1 mutations were independently associated with overall survival (P = 0.011, P = 0.001, P = 0.001, P = 0.005, P = 0.041, P = 0.050 and P = 0.003, respectively). KRAS gene (variant allele frequency 50%) and the EphB4 mutation were significantly associated with PDAC metastasis. ATRX and TERT genes were significantly associated with the pattern of metastasis in PDACs. ABL1 and BRAF mutations were significantly associated with stable disease as well as partial response to chemotherapy (P = 0.047 and P = 0.015, respectively). Targeted sequencing using EUS-FNA specimens in PDAC is feasible and shows similar genomic profiles in surgical specimens. Novel genes associated with survival, metastasis and chemotherapy response in PDAC were identified. Citation Format: Joo Kyung Park, Kwang Hyuk Lee, Jong Kyun Lee, Kyu Taek Lee, Woong-Yang Park, Dae-Soon Son. Targeted sequencing from endoscopic ultrasound-guided fine needle aspirates of pancreatic ductal adenocarcinoma. [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 96.

Abstract 2773: Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to transformation

Abstract Herein, we define a model for how cigarette smoke and chronic inflammation induce human lung cancer via evolution of a co-ordinated pattern of progressive cancer-associated epigenetic abnormalities which prime cells for addiction to a single key genetic alteration, KRAS mutation. Non-clonogenic, non-tumorigenic, epigenetically stable human bronchial epithelial cells (HBEC) were exposed to cigarette smoke condensate (CSC) for 15 months. Earlier studies have established a requirement for the simultaneous disruption of three oncogenes to fully transform these cells. Genome-wide DNA methylation, expression, chromatin changes, as well as binding to chromatin of key epigenetic regulators and cell phenotypic features were examined over time in exposed versus non-exposed cells. CSC exposure acutely causes, within 10 days, a change we have previously associated with DNA damage, tightening of DNA methyltransferase 1 (DNMT1) and EZH2 to chromatin. While the EZH2 binding decreases with prolonged exposure, DNMT1 remains tightly bound to chromatin. Chronic exposure causes progressive, but stochastically variable, global DNA methylation changes which begin by six months and progress over the time course of the study to include hypermethylation of gene promoters which are frequent in human lung cancer. ChIP-seq analyses reveal, preceding the above methylation changes, that promoters of such methylated genes have an initial recruitment of EZH2, which begins at 10 days and then decreases with time. In contrast, recruitment of EZH2 increases with time and remains dominant for these same genes in the non-exposed controls. Following 10 months of exposure, CSC treated cells begin to clone in soft agar, often a feature of transformation, yet do not form tumors in immunodeficient mice. At this time point, gene expression studies show the top signaling pathway change is strong activation of MAP-kinase and KRAS pathways. Yet genome-wide, exome sequencing reveals no known lung cancer driver gene mutations. Remarkably, overexpression of mutant KRAS alone now markedly enlarges the soft agar colonies, and the cells are now fully transformed and form tumors in mice. Our study reveals, in a chronic cigarette exposure model relevant to the time course for evolution of KRAS mutant human lung adenocarcinoma, a key initial role for smoking induced epigenetic changes which facilitate addiction to the oncogene. Citation Format: Michelle P. Vaz, Stephen Y. Hwang, Ashwini Patil, Jillian Phallen, Lauren Murphy, Cynthia A. Zahnow, Victor E. Velculescu, Hariharan Easwaran, Stephen B. Baylin. Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to transformation. [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 2773.

Abstract P2-01-04: A method for comprehensive genomic analysis of cell free DNA

Abstract Circulating tumor DNA (ctDNA) is released from tumor tissue into the blood, carries tumor specific genetic alterations, and can be analyzed through noninvasive "liquid biopsy" approaches to identify genetic alterations in cancer patients. Liquid biopsies offer a considerable advantage as they may eliminate the need for invasive tissue biopsies and allow for the detection of alterations in multiple metastatic lesions throughout the course of therapy. However, the fraction of ctDNA obtained from a blood sample is often very low (<1.0%) and can be difficult to detect. Additionally, most methods to evaluate circulating tumor DNA (ctDNA) interrogate single hot spot mutations or few genetic alterations. The next generation of ctDNA assays must interrogate multiple gene regions from a single sample with high precision and accuracy and need to evaluate all forms of actionable genomic alterations including point mutations, amplifications, and translocations. To address these issues, we have developed a ctDNA approach called PlasmaSelect to detect somatic sequence mutations, amplifications and translocations at low allele frequencies in the circulation of cancer patients. Utilizing digital genomic approaches, PlasmaSelect achieves high sensitivity and specificity while interrogating >250,000 nucleotides spanning 63 well-established cancer genes. In addition to sequence mutations in the entire coding region of 18 genes and the exons of 40 genes that are frequently mutated in cancer, PlasmaSelect also performs a comprehensive genomic analysis of amplifications in 57 genes and translocations in 10 genes significant in cancer tumorigenesis. To evaluate the PlasmaSelect approach, we performed dilution series using tumor-derived DNA, containing well-characterized somatic mutations, in the presence of wild-type DNA. PlasmaSelect was able to detect genetic alterations with high specificity and a lower level of detection of 0.10% for sequence mutations and translocations, as well as a focal amplification of ERBB2 with a lower level of detection of 0.20%. We evaluated the clinical utility of PlasmaSelect for detection of genetic alterations in the plasma and matched tissue biopsy specimens from late stage cancer patients. These analyses demonstrated high concordance between the somatic sequence mutations, amplifications, and translocations identified in the tumor sample and those identified directly in the plasma, including alterations in both driver genes as well as those related to acquired resistance to targeted therapies. PlasmaSelect provides a non-invasive platform to enable liquid biopsy detection of clinically relevant genetic alterations across a large number of genomic loci. Citation Format: Parpart-Li S, Angiuoli SV, Chesnick B, Galens K, Jones S, Kadan M, Kann L, Lytle K, Murphy D, Nesselbush M, Phallen J, Riley D, Shukla M, Zhang T, Husain H, Velculescu V, Diaz, Jr LA, Sausen M. A method for comprehensive genomic analysis of cell free DNA. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-01-04.

Genetic alterations in uncommon low-grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology.

Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and 'adult-type' diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n=22), diffuse oligodendroglial tumors (d-OTs; n=20), diffuse astrocytomas (DAs; n=17), angiocentric gliomas (n=15), and gangliogliomas (n=17). Most LGNTs (84%) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82% of DNETs and 40% of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87%), and MYB fusion genes were the most common genetic alteration in DAs (41%). A BRAF:p.V600E mutation was present in 35% of gangliogliomas and 18% of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78% of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.

The Two Revolutions in the Treatment of Advanced Non-small Cell Lung Cancer

In parallel with the evolution of therapeutic paradigms for advanced cancers, treatment of advanced non-small cell lung cancer (NSCLC) has been through the revolution of personalised medicine benefiting for a minority of patients whose disease depends on a single oncogenic genetic alteration targetable by specific tyrosine kinase inhibitors. As well as for some other tumour types, targeting immune checkpoints inhibitors has shown encouraging activity for some patients, suggesting the dawn of a second therapeutic revolution for a significant proportion of lung cancer patients.

Impact of Single or Combined Genomic Alterations of TP53, MYC, and BCL2 on Survival of Patients With Diffuse Large B-Cell Lymphomas: A Retrospective Cohort Study.

MYC and BCL2 translocations as well as TP53 deletion/mutation are known risk factors in diffuse large B-cell lymphoma (DLBCL) but their interplay is not well understood.In this retrospective cohort study, we evaluated the combined prognostic impact of TP53 deletion and mutation status, MYC and BCL2 genomic breaks in tumor samples of 101 DLBCL patients. The cohort included 53 cases with MYC rearrangements (MYC+).TP53 deletions/mutations (TP53+) were found in 32 of 101 lymphomas and were equally distributed between MYC+ and MYC- cases (35.8% vs. 27.1%). TP53+ lymphomas had lower responses to treatment than TP53- (complete remission 34.4% vs. 60.9%; P = 0.01). TP53 alteration was the dominant independent prognostic factor in multivariate analysis (P = 0.01). Overall survival (OS) varied considerably between subgroups with different genomic alterations: Patients with sole MYC translocation, and interestingly, with triple MYC+/BCL2+/TP53+ aberration had favorable outcomes (median OS not reached) similar to patients without genomic alterations (median OS 65 months). In contrast, patients with MYC+/BCL2+/TP53- double-hit lymphomas (DHL) (28 months), MYC+/BCL2-/TP53+ lymphomas (10 months) or sole TP53 mutation/deletion (12 months) had a poor median OS. Our findings demonstrate differences in OS of DLBCL patients depending on absence or presence of single or combined genetic alterations of MYC, BCL2, and TP53. Cooccurrence of TP53 and BCL2 aberrations ameliorated the poor prognostic impact of single TP53+ or BCL2+ in MYC positive patients.This pilot study generates evidence for the complex interplay between the alterations of genetic pathways in DLBCL, which goes beyond the concept of DHL. The variable survival of DLBCL patients dependent on single or combined alterations in the TP53, MYC, and BCL2 genes indicates the need for comprehensive genomic diagnosis.

Is it Possible to Predict Benefit from 5FU Adjuvant Therapy in Stage III Colon Cancer Patients?

For more than three decades patients diagnosed with stage III colorectal cancers have undergone adjuvant chemotherapy based on fluoropyrimidines (5-FU) and more recently in combination with oxaliplatin (Dienstmann et al., 2015). Overall that treatment has reduced the risk of tumor recurrence and improved survival for patients with resected colon cancer, but at present no validated biomarkers are available to predict the benefit of adjuvant chemotherapy in that group of patients. Up to now most efforts in CRC have been made to predict response to anti-EGFR therapy which is recommended now only to wild type KRAS and NRAS patients, but will most likely be followed by BRAF and PIK3CA in the near future (De Stefano and Carlomagno, 2014). For conventional 5-FU chemotherapy no companion biomarkers are available to predict therapy response, either for adjuvant or for curative protocols. Molecular heterogeneity of colon cancers is the main factor affecting the differential response to adjuvant chemotherapy. There are a lot of scientific articles reporting on biomolecules which can predict the response of that therapeutic approach. Among them MSI status (microsatellite instability) has already been shown to influence survival and response to adjuvant infusional 5-FU in colon cancer patients (Sargent et al., 2010) since patients with MSI tumors have better outcomes as compared with patients with microsatellite stable (MSS) tumors. However, the improved prognosis is abrogated in the face of adjuvant 5-FU treatment (Sargent et al., 2010). In addition, thymidylate synthetase, EGFR polymorphisms and many others have been shown to influence progression free survival and therefore response to adjuvant treatment to 5-FU, but frequently with controversial results. In spite of the extensive scientific production no consensus on the use of a particular biomarker for therapy benefit prediction has been obtained. One of the reasons is that in several studies the impact of adjuvant 5-FU chemotherapy referred to specific biomarkers was not addressed to specific subgroups (stage II versus stage III patients). The discrimination between stages II and III is relevant because these two stages differ both at the clinical–biological level and at the molecular level. Moreover, prognostic markers for stage III have been reported to have no significance for stage II cancers (Javle, 2010). In this issue of EBioMedicine, Kandioler et al. analyzed TP53 mutations which produce a partial functionality of the protein (decreasing its transcriptional activity to less than 75%) in stage III colon cancer patients (Kandioler et al., in press). Relevant of this study is that the authors confined their analyses only to stage III patient and that they sub-classify that stage with respect to the number of the involved lymph nodes. They found a significant survival benefit of 5FU adjuvant chemotherapy only in N1 wild type patients in comparison to TP53 mutated ones. That benefit was lost when considering N2 patients highlighting that subgrouping is also relevant for N classes. There is the possibility as in breast cancers that N1 tumors behave as N0 ones. With respect to TP53 it plays at least 2 separate roles in the responses to therapeutic agents: it is an important component of cellular checkpoints, and it can mediate apoptosis. The response to individual drugs is determined by which of these 2 functions is paramount and for 5-FU the apoptotic effect is predominant (Bunz et al., 1999). The effect of mutation reducing TP53 activity seems to cause resistance to the apoptotic effects of FU, hence a diminished therapy benefit (Bunz et al., 1999). Nonetheless, the mutational status at TP53 could predict the benefit to adjuvant chemotherapy only in stage III patients with less than 3 positive lymph nodes and not in patients with more than 3 lymph nodes involved, for whom the authors did not expect any benefit from adjuvant chemotherapy. Surely, the response to all drugs, including 5-FU, is complex and unlikely to be completely explained by any single genetic alteration. Sub-classification in lymph node classes has proved to be a meaningful variable to understand response to 5-FU adjuvant chemotherapy. Possibly, tumors' heterogeneity with further additional “spatial” alterations in different metastatic sites could account for this result. The present study (Kandioler et al., in press) highlights that “early” advanced stage III tumors are different from N2 tumors and that N1 patients with TP53 mutations are less likely to respond to 5-FU adjuvant therapy in comparison to wild type. If confirmed it could have a considerable impact on clinical practice. Although those results are relevant additional studies are needed to sub-classify stage III colon cancer patients and define the group of patients who will benefit from adjuvant treatment in routine clinical practice.

Molecular Determinants of Decitabine Response in Chronic Myelomonocytic Leukemia

The nucleoside analog (na) 5-aza-2’-deoxycytidine (dac) has good, but heterogeneous, efficacy in the therapy of chronic myelomonocytic leukemia (cmml). Given that no standard therapy has been identified so far for cmml, there is urgent need to identify molecular markers that could support the choice of dac therapy and identify patients more prone to respond. Recently, we demonstrated that in mds patients the expression of uck1, involved in the activation of azacitidine, may influence the clinical response to this treatment (valencia et al, leukemia 2013). In the present study, we assessed the role of dac metabolizing enzymes as well as genetic alterations common to cmml patients in response to dac in a uniformly and prospectively treated cohort of cmml patients.Methods:Patients Forty cmml patients were treated with dac 20mg/m2/day for 5 days every 28 d. DNA and rna were extracted from bm mononuclear cells of 19 pts defined as responders to dac (r), and 21 as non-responders (nr). Hematological response was evaluated according to iwg 2006 criteria.Gene mutation. the fifteen most frequently mutated genes in cmml were sequenced at a mean depth of coverage of 520x (range 169–714x).Functional studies. the role of two main genes involved in dac metabolism: dck (dac activation) and dctd (dac deactivation) was evaluated by silencing both genes with sirnas. The experiments were performed in the mds-skm1 cell line and in primary cells of 6 cmml cases exposed in vitro to dac 10um for 48h.Gene expression. the expression level of genes hent1, hent2, dctd, hcnt3, cn-ii, dck and cda, involved in dac metabolism was evaluated by qrt-pcr in 38/40 cmml patients and by rnaseq in 14/40 patients. CDNA libraries were sequenced using the hiseq 2000. The counts of endogenous genes were normalized by ercc spike-in library controls, and differential expression analysis was performed using edger (v3.4.2) glm model. The differentially expressed genes were identified at the fdr cutoff of 0.05 and absolute fold change greater than 2.Results: in skm-1 cells and in cmml primary mononuclear cells, dctd silencing increased dac- induced apoptosis (annexin v-positive cells 20.2%±0.8% vs control 13.8%±0.5%; p=0.01). dck silencing led to a decrease in dac-induced apoptosis (annexin v-positive cells 8.8%±0.1% vs control 13.8%±0.5%; p=0.05). We could not detect by rnaseq a significant difference in the expression levels of na metabolizing enzymes between responders and non responders to dac. qrt-pcr confirmed these observations.The mutational frequencies (figure) in this cohort of cmml cases were: srsf2 50%, tet2 44.7%, asxl1 39.4%, nras 18.4%, runx1 and dnmt3a 10.5%, u2af1 and tp53 7.8%, kras, jak2 and kit 5.2%, ezh2, idh1, idh2 and sf3b1 2.6%. No single genetic alteration was significantly associated with shorter overall survival or resistance to dac.Conclusions: although we could clearly demonstrate that in vitro expression of dac metabolizing enzymes influenced response to dac, clinical resistance does not appear to be directly correlated with the expression of genes involved in dac metabolism nor to specific gene mutations and is likely determined by other clinical/molecular variables that remain to be identified. [Display omitted] DisclosuresFinelli:Janssen: Speaker, Speaker Other; Novartis: Speaker, Speaker Other; Celgene: Research Funding, Speaker Other.

Abstract SY45-02: Evaluation of targeted therapies in advanced breast cancer

Abstract SY45-02: Evaluation of targeted therapies in advanced breast cancer

29IN - Emerging Druggable Targets in Colorectal Cancer

ABSTRACT The analysis of the Tumor Cancer Genome Atlas (TCGA) network on colorectal cancer (CRC) samples has identified a plethora of genetic alterations that deregulate five main pathways: WNT, TGF-b, p53, PI3K and Receptor Tyrosine Kinase (RTK)-RAS signaling. Three or more pathways can be concomitantly disregulated in each tumor. How to exploit this knowledge? Among the low-hanging fruits, there are molecular alterations affecting several genes encoding for RTKs (including RET, ERBB3, FGFRs, TRKs, PDGFRA, ALK and KIT), which have been identified to occur at low prevalence (e.g. 0.5-2%) by the TCGA. Larger studies are needed to establish their prevalence in the general CRC population as well as in molecularly distinct subtypes. Additional druggable targets may be represented by genes disregulated in PI3K signaling, including IGF2, PIK3CA, PTEN or PIK3R1. However, caution should be taken to define therapeutic strategies based on the molecular status of a single gene. The complexity of the CRC molecular landscape strongly suggest a single genetic alteration may not per se represent a promising drug target, unless it is analyzed within the context of co-existing alterations leading to simultaneous activation of multiple oncogenic signaling pathways. Functional studies on the role of specific RTKs in CRC progression are largely missing and are needed to discriminate whether the above mentioned kinases could represent valuable targets in this disease. Nevertheless, it is likely that targeting one gene (one pathway) may not be sufficient to induce prolonged tumor remission, and that combined inhibition of two key signaling pathways in CRC could be more effective. Finally, recent transcriptomic analyses have highlighted the existence of 3-5 molecularly distinct CRC subtypes based on the expression of genes encoding for epithelial or mesenchymal (stem-like) phenotypes, or the presence of inflammatory or goblet-like signatures. These studies also suggest that the ensuing molecular subtypes may be differentially responsive to distinctive therapies. Therefore, it is possible that future drug targets will emerge from screening chemical or RNA interference libraries against preclinical CRC models recapitulating the molecular features of each subtype. Disclosure: The author has declared no conflicts of interest.

The combinatorial complexity of cancer precision medicine.

Precision medicine approaches have recently been developed that offer therapies targeting mainly single genetic alterations in malignant tumors. However, next generation sequencing studies have shown that tumors normally harbor multiple genetic alterations, which could explain the so far limited successes of personalized medicine, despite considerable benefits in certain cases. Combination therapies may contribute to a solution, but will pose a major challenge for clinical trials evaluating those therapies. As we discuss here, reasons include the low abundance of most of the relevant mutations and particularly the combinatorial complexity of possible combination therapies. Our report provides a systematic and quantitative account of the implications of combinatorial complexity for cancer precision medicine and clinical trial design. We also present an outlook on how systems biological approaches may be harnessed to contribute to a solution of the complexity challenge by predicting optimal combination therapies for individual patients and how clinical trial design may be adapted by combining and extending basket and umbrella design features.