Multiple Molecular Alterations Research Articles

Abstract 794: Molecular analysis of archival inflammatory myofibroblastic tumor tissue samples from EORTC 90101 “CREATE” and correlation with response to crizotinib

Abstract About 50% of inflammatory myofibroblastic tumors (IMFTs) harbor rearrangements of anaplastic lymphoma kinase (ALK), the most common driver in this rare tumor. EORTC 90101 “CREATE” is so far the only prospective, disease-specific phase II clinical trial performed in IMFT. It showed high antitumor activity of crizotinib, an ALK/ROS1/MET-inhibitor. This activity was mainly seen in ALK-rearranged IMFT, but the trial results suggested that other molecular events may also contribute to crizotinib sensitivity. Here we present results of an in-depth molecular analysis of archival IMFTs collected from patients within the CREATE trial with the aim to identify other molecular alterations, which could be responsible for response to crizotinib. DNA from archival IMFTs (primary tumor or metastatic lesion) was used for low-coverage whole genome sequencing to identify genomic regions of gains or losses. Whole exome sequencing (WES) was used to assess the mutational landscape, and mutations affecting cancer consensus genes (CCGs) were analyzed in more detail. Correlative and statistical analysis were used to assess the association between molecular findings and clinical outcomes of IMFT patients. In 24 IMFTs analyzed, the recurrent whole-arm copy number losses were: 22q (58% of cases), 16p (33%), 16q (29%), 13q (29%), 19q (29%), 19p (25%), 6p (25%), 6q (21%) and 18q (21%). Loss of chromosome 19 was found to be associated with shorter progression-free survival in patients receiving crizotinib (p = 0.008). Moreover, 2p21 (including EPAS1, SIX2, EML4 from the CCG set) was frequently amplified (54% of cases), while recurrent losses were observed at 22q12.3 (71%, ISX), 7q36.3 (54%, MNX1), 10q26.3 (54%, MGMT, DUX4), 1p36.32 (50%, RPL22, SKI, TNFRSF14, CAMTA1, PRDM16), 8p23.3 (50%, ARHGEF10), 12q24.33 (46%, POLE), and 11q13.4 (33%, CCND1). No significant correlations were found between focal copy number alterations and crizotinib response. A complex genetic rearrangement involving chromosome 2, identified in 2 ALK-rearranged cases, was likely consistent with chromothripsis, a form of genomic instability related to cancer development. WES was performed in 22 cases with an average of 392 alterations per sample. When considering non-synonymous mutations in CCG, a total of 178 mutations were identified, affecting 143 genes with an average of 7 per case. Mutations in 30 CCGs, identified in ≥ 2 cases, were mainly related to DNA damage/repair mechanism, WNT, RB1-TP53, and RTK-RAS-PI3K signaling. In a case with no ALK rearrangement and ALK immunopositivity, a single base substitution in ALK (p.N571K) was identified. In conclusion, we identified multiple molecular alterations in archival IMFTs and provided further insight in the molecular profile of this ultra-rare malignancy, which may potentially lead to the identification of novel therapeutic targets for IMFT patients. Citation Format: Che-Jui Lee, Patrick Schöffski, Elodie Modave, Bram Boeckx, Diether Lambrechts, Maria Debiec-Rychter, Raf Sciot, Jozef Sufliarsky, Hans Gelderblom, Jean-Yves Blay, Agnieszka Wozniak. Molecular analysis of archival inflammatory myofibroblastic tumor tissue samples from EORTC 90101 “CREATE” and correlation with response to crizotinib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 794.

Efficacy and Safety of Ketamine in Refractory/Super-refractory Nonconvulsive Status Epilepticus: Single-Center Experience.

Refractory/super-refractory nonconvulsive status epilepticus (r/srNCSE) is an acute life-threatening neurocritical entity with significant morbidity. Failure to control SE in its earlier stages leads to multiple molecular alterations in the brain such as downregulation of GABA-A and upregulation of NMDA receptors. Recently ketamine, an NMDA receptor antagonist, has gained increased attention as a therapeutic choice in controlling refractory/super-refractory SE. We aimed to analyze the efficacy and safety profile of ketamine in our center. We retrospectively identified all the patients with nonconvulsive SE who received ketamine during their follow-up in our neurological intensive care unit between 2009 and 2019. Information about demographic, clinical, and laboratory findings; concurrent antiseizure and anesthetic medications; time of initiation, dose and duration of ketamine infusion; any adverse effects and finally prognosis were collected. The effect of day of ketamine initiation and duration of infusion on ketamine efficacy were analyzed statistically. Seven patients (4 males, 3 females; age: 44-86 years) were included in the study. Encephalitis was the most common etiology. Concurrent antiseizure medications varied between 2 and 5. Six patients received midazolam before/during ketamine infusion. Ketamine was initiated 2 to 7 days after the onset of EEG monitoring and lasted 3 to 24 days with a maximum infusion dose ranging between 1 to 5 mg/kg/h. It was definitely effective in 4 patients, and possibly effective in an additional patient. Earlier initiation was correlated with higher efficacy (P = .047). There was a trend toward higher efficacy with longer duration of infusion (P = .285). Overall prognosis was poor with 29% mortality rate. Temporary hepatic failure occurred in 1 patient. Ketamine appears to be a promising drug in r/srNCSE. Earlier and prolonged infusion, as well as combination with benzodiazepines may increase its efficacy. Adverse events are rarely observed.

Predisposition to Alzheimer's and Age-Related Brain Pathologies by PM2.5 Exposure: Perspective on the Roles of Oxidative Stress and TRPM2 Channel.

Accumulating epidemiological evidence supports that chronic exposure to ambient fine particular matters of <2.5 μm (PM2.5) predisposes both children and adults to Alzheimer’s disease (AD) and age-related brain damage leading to dementia. There is also experimental evidence to show that PM2.5 exposure results in early onset of AD-related pathologies in transgenic AD mice and development of AD-related and age-related brain pathologies in healthy rodents. Studies have also documented that PM2.5 exposure causes AD-linked molecular and cellular alterations, such as mitochondrial dysfunction, synaptic deficits, impaired neurite growth, neuronal cell death, glial cell activation, neuroinflammation, and neurovascular dysfunction, in addition to elevated levels of amyloid β (Aβ) and tau phosphorylation. Oxidative stress and the oxidative stress-sensitive TRPM2 channel play important roles in mediating multiple molecular and cellular alterations that underpin AD-related cognitive dysfunction. Documented evidence suggests critical engagement of oxidative stress and TRPM2 channel activation in various PM2.5-induced cellular effects. Here we discuss recent studies that favor causative relationships of PM2.5 exposure to increased AD prevalence and AD- and age-related pathologies, and raise the perspective on the roles of oxidative stress and the TRPM2 channel in mediating PM2.5-induced predisposition to AD and age-related brain damage.

Mucinous Cancer of the Ovary: Overview and Current Status.

Mucinous ovarian cancer (MOC) is a rare subtype of epithelial ovarian carcinoma (EOC). Whereas all EOC subtypes are addressed in the same way, MOC is a distinct entity. Appreciating the pathological features and genomic profile of MOC may result in the improvement in management and, hence, the prognosis. Distinguishing primary MOC from metastatic mucinous carcinoma can be challenging but is essential. Early-stage MOC carries an excellent prognosis, with advanced disease having a poor outcome. Surgical management plays an essential role in the early stage and in metastatic disease. Chemotherapy is usually administered for stage II MOC and beyond. The standard gynecology protocol is frequently used, but gastrointestinal regimens have also been administered. As MOC is associated with multiple molecular alterations, targeted therapy could be the answer to treat this disease.

The Power of Human Cancer Genetics as Revealed by Low-Grade Gliomas.

The human brain contains a vast number of cells and shows extraordinary cellular diversity to facilitate the many cognitive and automatic commands governing our bodily functions. This complexity arises partly from large-scale structural variations in the genome, evolutionary processes to increase brain size, function, and cognition. Not surprisingly given recent technical advances, low-grade gliomas (LGGs), which arise from the glia (the most abundant cell type in the brain), have undergone a recent revolution in their classification and therapy, especially in the pediatric setting. Next-generation sequencing has uncovered previously unappreciated diverse LGG entities, unraveling genetic subgroups and multiple molecular alterations and altered pathways, including many amenable to therapeutic targeting. In this article we review these novel entities, in which oncogenic processes show striking age-related neuroanatomical specificity (highlighting their close interplay with development); the opportunities they provide for targeted therapies, some of which are already practiced at the bedside; and the challenges of implementing molecular pathology in the clinic.

A prognostic 10-lncRNA expression signature for predicting the risk of tumour recurrence in breast cancer patients.

Breast cancer is one of the most frequently diagnosed malignancies and a leading cause of cancer death among females. Multiple molecular alterations are observed in breast cancer. LncRNA transcripts were proved to play important roles in the biology of tumorigenesis. In this study, we aimed to identify lncRNA expression signature that can predict breast cancer patient survival. We developed a 10‐lncRNA signature‐based risk score which was used to separate patients into high‐risk and low‐risk groups. Patients in the low‐risk group had significantly better survival than those in the high‐risk group. Receiver operating characteristic analysis indicated that this signature exhibited excellent diagnostic efficiency for 1‐, 3‐ and 5‐year disease‐relapse events. Moreover, multivariate Cox regression analysis demonstrated that this 10‐lncRNA signature was an independent risk factor when adjusting for several clinical signatures such as age, tumour size and lymph node status. The prognostic value of risk scores was validated in the validation set. In addition, a nomogram was established and the calibration plots analysis indicated the good performance and clinical utility of the nomogram. In conclusion, our results demonstrated that this 10‐lncRNA signature effectively grouped patients at low and high risk of disease recurrence.

Mechanisms of Persistent Neurobiological Changes Following Adolescent Alcohol Exposure: NADIA Consortium Findings.

The Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium has focused on the impact of adolescent binge drinking on brain development, particularly on effects that persist into adulthood. Adolescent binge drinking is common, and while many factors contribute to human brain development and alcohol use during adolescence, animal models are critical for understanding the specific consequences of alcohol exposure during this developmental period and the underlying mechanisms. Using adolescent intermittent ethanol (AIE) exposure models, NADIA investigators identified long‐lasting AIE‐induced changes in adult behavior that are consistent with observations in humans, such as increased alcohol drinking, increased anxiety (particularly social anxiety), increased impulsivity, reduced behavioral flexibility, impaired memory, disrupted sleep, and altered responses to alcohol. These behavioral changes are associated with multiple molecular, cellular, and physiological alterations in the brain that persist long after AIE exposure. At the molecular level, AIE results in long‐lasting changes in neuroimmune/trophic factor balance and epigenetic–microRNA (miRNA) signaling across glia and neurons. At the cellular level, AIE history is associated in adulthood with reduced expression of cholinergic, serotonergic, and dopaminergic neuron markers, attenuated cortical thickness, decreased neurogenesis, and altered dendritic spine and glial morphology. This constellation of molecular and cellular adaptations to AIE likely contributes to observed alterations in neurophysiology, measured by synaptic physiology, EEG patterns, and functional connectivity. Many of these AIE‐induced brain changes replicate findings seen in postmortem brains of humans with alcohol use disorder (AUD). NADIA researchers are now elucidating mechanisms of these adaptations. Emerging data demonstrate that exercise, antiinflammatory drugs, anticholinesterases, histone deacetylase inhibitors, and other pharmacological compounds are able to prevent (administered during AIE) and/or reverse (given after AIE) AIE‐induced pathology in adulthood. These studies support hypotheses that adolescent binge drinking increases risk of adult hazardous drinking and influences brain development, and may provide insight into novel therapeutic targets for AIE‐induced neuropathology and AUDs.

Characterization of glycine-N-acyltransferase like 1 (GLYATL1) in prostate cancer.

Recent microarray and sequencing studies of prostate cancer showed multiple molecular alterations during cancer progression. It is critical to evaluate these molecular changes to identify new biomarkers and targets. We performed analysis of glycine-N-acyltransferase like 1 (GLYATL1) expression in various stages of prostate cancer in this study and evaluated the regulation of GLYATL1 by androgen. We performed in silico analysis of cancer gene expression profiling and transcriptome sequencing to evaluate GLYATL1 expression in prostate cancer. Furthermore, we performed immunohistochemistry using specific GLYATL1 antibody using high-density prostate cancer tissue microarray containing primary and metastatic prostate cancer. We also tested the regulation of GLYATL1 expression by androgen and ETS transcription factor ETV1. In addition, we performed RNA-sequencing of GLYATL1 modulated prostate cancer cells to evaluate the gene expression and changes in molecular pathways. Our in silico analysis of cancer gene expression profiling and transcriptome sequencing we revealed an overexpression of GLYATL1 in primary prostate cancer. Confirming these findings by immunohistochemistry, we show that GLYATL1 is overexpressed in primary prostate cancer compared with metastatic prostate cancer and benign prostatic tissue. Low-grade cancers had higher GLYATL1 expression compared to high-grade prostate tumors. Our studies showed that GLYATL1 is upregulated upon androgen treatment in LNCaP prostate cancer cells which harbors ETV1 gene rearrangement. Furthermore, ETV1 knockdown in LNCaP cells showed downregulation of GLYATL1 suggesting potential regulation of GLYATL1 by ETS transcription factor ETV1. Transcriptome sequencing using the GLYATL1 knockdown prostate cancer cell lines LNCaP showed regulation of multiple metabolic pathways. In summary, our study characterizes the expression of GLYATL1 in prostate cancer and explores the regulation of its regulation in prostate cancer showing role for androgen and ETS transcription factor ETV1. Future studies are needed to decipher the biological significance of these findings.

Patterns of progression on osimertinib in EGFR T790M positive NSCLC: A Swiss cohort study

IntroductionOsimertinib is an EGFR tyrosine kinase inhibitor (TKI) with antitumor activity in non-small cell lung cancer (NSCLC) with EGFR T790 M mutations. The incidence of oligo-progression (PD) on osimertinib is unknown. MethodsWe retrospectively analyzed 50 pre-treated EGFR T790M-positive NSCLC patients treated with osimertinib at seven Swiss centers. Oligo-PD was defined as PD in ≤ 5 lesions. Mutational profiling of pre- and post-osimertinib tumor samples was performed. ResultsMedian age was 62 years (37–89), 64% were females, 86% had a PS ≤ 1, 54%/13% were never/current smokers. Median follow-up was 15.3 (IQR: 8.6–21.6) months. Overall response rate was 80%, median progression-free survival 12.1 months (95% CI 8.3–18.3), median overall survival 28 months (95% CI 20.2-not reached [NR]) and median treatment duration 18.8 months (95%CI 16-8-NR). PD occurred in 36 patients (72%). 73% had oligo-PD. Median osimertinib treatment duration in patients with oligo-PD was 19.6 vs. 7 months if systemic PD (p = 0.007). The number of progressive lesions in patients with oligo-PD was 1 (27%), 2 (35%) and 3–5 (39%). Sites of PD included lungs (56%), bones (44%), and brain (17%). Sixteen patients with oligo-PD continued treatment with osimertinib for a median of 6.7 months beyond PD. Thirteen received local ablative treatment (LAT). In pre- and post-PD tumor tissue multiple molecular alterations were detected. ConclusionIn patients with acquired resistance to osimertinib, we observed a high rate (73%) of oligo-PD. Outcomes of patients receiving LAT were favorable, supporting the concept of osimertinib treatment beyond progression in combination with LAT of progressing lesions.

A Role for De Novo Purine Metabolic Enzyme PAICS in Bladder Cancer Progression

Genomic and transcriptome sequencing of bladder cancer (BLCA) has identified multiple molecular alterations during cancer progression. Many of these identified genetic and epigenetic changes play a role in the progression of this disease. Studies have identified molecular subtypes in muscle-invasive bladder cancer (MIBC) with different sensitivities to frontline therapy suggesting the heterogeneity in these tumors and the importance of molecular characterization of MIBC to provide effective treatment. Specifically, it has become increasingly evident, as demonstrated by The Cancer Genome Atlas project, that metabolic enzymes are commonly dysregulated in BLCA. Elevated expression of multiple metabolic enzymes is due to the increased demand from rapidly proliferating BLCA cells requiring extensive nucleotide synthesis. Cancer cells utilize the de novo purine and pyrimidine biosynthetic pathway as a source of their nucleotide needs. In this study, we show that phosphoribosyl aminoimidazole succinocarboxamide synthetase (PAICS), an enzyme involved in de novo purine biosynthetic pathway, is significantly overexpressed in BLCA. Immunohistochemical staining of paraffin-embedded tissue sections showed that PAICS is overexpressed in MIBC. Furthermore, we found that tumor suppressor miR-128 negatively regulated PAICS expression by binding to its 3′-untranslated region. We also found that PAICS induces EMT by positively regulating SNAI1 and by a reduction in E-cadherin expression. Additionally, our in vitro functional studies and in vivo chicken chorioallantoic membrane assay show that PAICS plays a critical role in BLCA cell proliferation, invasion, and tumor growth. Collectively, our data suggest that targeting PAICS may provide a therapeutic option in BLCA.

Prometastatic Molecular Profiles in Breast Tumors From Socially Isolated Women.

BackgroundSocial isolation is associated with accelerated breast cancer progression and increased disease recurrence and mortality, but the underlying biological mechanisms remain poorly understood. In preclinical models, beta-adrenergic signaling from fight-or-flight stress responses can stimulate prometastatic processes in the tumor microenvironment including upregulation of M2 macrophages, epithelial–mesenchymal transition (EMT), and lymphovascular invasion. This study examines whether the same pathways are upregulated in breast tumors from socially isolated cancer patients.MethodsEMT and M1/M2 macrophage gene expression programs were analyzed by genome-wide transcriptional profiling, and lymphatic and vascular density were assessed by immunohistochemistry in primary tumors from 56 early-stage breast cancer patients who were part of the UCLA RISE study. Social isolation was quantified by the Social Provisions Scale, and disease characteristics were assessed by medical record review. General linear models were used to quantify differential gene expression across risk factor groups. Linear regression models were used to examine associations between social isolation and lymphovascular invasion.ResultsTumors from socially isolated patients showed upregulated expression of genes involved in EMT (average score difference = +0.080 log2 mRNA abundance ± 0.034 standard error) and M2 macrophage polarization (+0.033 ± 0.014) as well as increased density of lymphatic vessels (β= –.29) but no difference in blood vessel density. TELiS promoter–based bioinformatics analyses indicated activation of CREB family transcription factors that mediate the gene-regulatory effects of β-adrenergic signaling (log2 fold-difference in promoter binding site prevalence: mean ± standard error = +0.49 ± 0.19). ConclusionsPrimary breast tumors from socially isolated patients show multiple prometastatic molecular alterations, providing a plausible biological pathway through which poor social support may accelerate breast cancer progression and defining new targets for intervention.

PO-507 Deregulated mechanisms downstream of PLCG1 promoting cutaneous T cell lymphomaprogression

IntroductionCutaneous T Cell Lymphoma (CTCL) is a heterogeneous group of non-Hodgkin lymphomas with unfavourable prognosis at advanced stages. The malignant network that control the development of CTCL is now starting to be characterised. NGS-derived studies have identified multiple molecular alterations in specific genes and signalling pathways including PLCG1 and JAK-STAT. It has been shown that up to 50% of CTCL cases develop deregulated PLCG1-downstream signalling as shown by activating PLCG1 mutations and PRKCQ amplifications. Also, frequent mutations in JAK1 and JAK3 and in members of the STAT family have been identified, thus revealing a potentially important role of these signalling pathways in CTCL. We aimed at exploring the interactions between deregulated PLCG1 and JAK-STAT activity in CTCL using pre-clinical models and in samples from CTCL patients at different stages of the disease.Material and methodsHEK-BlueTM IL-6 and CTCL cell lines were used to study PLCG1 downstream signalling towards NFAT and STAT3 activation. Immunohistochemistry (IHC) of NFAT, pSTAT1, pSTAT3, pSTAT5, p50, p52, p65 and RelB wasperformed in 71 patient samples. The status of PRKCQ was analysed in 45 samples by fluorescent in situhybridization (FISH).Results and discussionsDeregulated PLCG1 activity elicited NFAT and STAT3 activation ex vivo. Downstream of PLCG1, pharmacological and genetic inhibition of PRKCQ negatively affected NFAT and STAT3 activation whereas a constitutively active PRKCQ mutant (A148E) was sufficient to trigger their transcriptional activity. Our data shows that downstream of PLCG1, PRKCQ can activate transcription mediated by both NFAT and STAT proteins. We also analysed nuclear accumulation of NFAT, STAT and NF-κB family proteins by IHC in patient samples. Interestingly positive P-STAT3 staining correlated with advanced diseases. Moreover, most CTCL-tumour stage cases studied were positive for PRKCQ amplification and/or 10q23 polysomy.ConclusionOur results support PLCG1 playing a mayor role at controlling CTCL progression towards advanced stages. Downstream of PLCG1 or due to genetic amplification, PRKCQ can mediate the activation of NFAT and STAT3. Thus, mutations in PLCG1, PRKCQ amplifications and/or nuclear NFAT and STAT3 accumulation can serve as diagnostic markers for CTCL. Moreover, these can also provide rational to develop specific therapies targeting PRKCQ alone or in combination with calcineurin or JAK-STAT inhibitors.

Relevance of a molecular tumour board (MTB) for patients’ enrolment in clinical trials: experience of the Institut Curie

BackgroundHigh throughput molecular screening techniques allow the identification of multiple molecular alterations, some of which are actionable and can be targeted by molecularly targeted agents (MTA). We aimed at evaluating the relevance of using this approach in the frame of Institut Curie Molecular Tumor Board (MTB) to guide patients with cancer to clinical trials with MTAs.Patients and methodsWe included all patients presented at Institut Curie MTB from 4 October 2014 to 31 October 2017. The following information was extracted from the chart: decision to perform tumour profiling, types of molecular analyses, samples used, molecular alterations identified and those which are actionable, and inclusion in a clinical trial with matched MTA.Results736 patients were presented at the MTB. Molecular analyses were performed in 442 patients (60%). Techniques used included next-generation sequencing, comparative genomic hybridisation array and/or other techniques including immunohistochemistry in 78%, 51% and 58% of patients, respectively. Analyses were performed on a fresh frozen biopsy in 91 patients (21%), on archival tissue (fixed or frozen) in 326 patients (74%) and on both archival and fresh frozen biopsy in 25 patients (6%). At least one molecular alteration was identified in 280 analysed patients (63%). An actionable molecular alteration was identified in 207 analysed patients (47%). Forty-five analysed patients (10%) were enrolled in a clinical trial with matched MTA and 29 additional patients were oriented and included in a clinical trial based on a molecular alteration identified prior to the MTB analysis. Median time between date of specimen reception and molecular results was 28 days (range: 5–168).ConclusionsThe implementation of an MTB at Institut Curie enabled the inclusion of 10% of patients into a clinical trial with matched therapy.

Efficacy of immune-checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC) patients harboring activating molecular alterations.

172 Background: Two revolutions recently occur in the treatment of advanced NSCLC: the development of targeted therapies and of ICI). Both strategies have been shown to outperform chemotherapy. Patients with molecular alterations are usually considered as poor candidate for immunotherapy. Here, we aimed to analyze the efficacy of immunotherapy in NSCLC patients with oncogenic addiction. Methods: We conducted a retrospective multicentric study, on patients treated with ICI and carrying an activating molecular abnormality: EGFR, ALK, KRAS, ROS1, HER2, BRAF, MET and RET. We have collected anonymized data, which were evaluated for clinical characteristics and outcome (progression free survival duration of ICI treatment and overall survival since initiation of ICI). Results: 209 patients were registered from 7 centers in France and Switzerland. 198 patients had adenocarcinoma (95.2%), 6 large cell carcinoma (2.9%), 161 were former or current smokers (81.3%), 99 were female (47.4%), median age at diagnosis was 59 yrs. (range 30-79). 33 patients had EGFR mutations (15.8%), 132 KRAS mutation (63.2%), 6 ALK rearrangement (2.9 %), 2 ROS1 rearrangement (1 %), 5 HER2 mutation (2.4 %), 12 BRAF mutations (5.7 %), 7 MET alteration (3.3 %), 3 RET rearrangements (1.4 %), and 9 had concomitant multiple molecular alterations (4.3%). After validated treatment 200 patients were treated with PD1 inhibitors (nivolumab, 97.5% and pembrolizumab, 2.5%), 4 with anti CTLA4 (tremelimumab), 5 with anti PDL1 (4 atezolizumab). The median PFS was 2.8 m. [95%CI 2.3;3.5] for the whole population 2.1 for EGFR [1.7;2.8], 3.5 for KRAS [2.5;4.9], 2.7 for BRAF [1.5;NR] and not estimable for other alterations. Median exposure to ICI was 1.89 m. [0;18.5]. The median overall survival for the whole population was 13.0 m. [9.4; 15.6], for EGFR 13.3 m. [4.7; NR], KRAS 11.3 m. [8.2; 16], BRAF 10.7 m. [1.5; NR] and not yet estimable for the other alterations. Conclusions: PFS (2.8 m.) and OS (13 m.) are very similar from the ones observed in pretreated unselected NSCLC patients. ICI is associated with better PFS in patients with KRAS mutation than in EGFR mutated patients. Analysis of outcome in other molecular subgroups is ongoing.

Interaction of the Wnt/β-catenin and RAS-ERK pathways involving co-stabilization of both β-catenin and RAS plays important roles in the colorectal tumorigenesis.

Cancer development is usually driven by multiple genetic and molecular alterations rather than by a single defect. In the human colorectal cancer (CRC), series of mutations of genes are involved in the different stages of tumorigenesis. For example, adenomatous polyposis coli (APC) and KRAS mutations have been known to play roles in the initiation and progression of the tumorigenesis, respectively. However, many studies indicate that mutations of these two genes, which play roles in the Wnt/β-catenin and RAS-extra-cellular signal regulated kinase (ERK) pathways, respectively, cooperatively interact in the tumorigenesis in several different cancer types including CRC. Both Apc and Kras mutations critically increase number and growth rate of tumors although single mutation of these genes does not significantly enhance the small intestinal tumorigenesis of mice. Both APC and KRAS mutations even result in the liver metastasis with inductions of the cancer stem cells (CSCs) markers in a mice xenograft model. In this review, we are going to describe the history for interaction between the Wnt/β-catenin and RAS/ERK pathways especially related with CRC, and provide the mechanical basis for the cross-talk between the two pathways. The highlight of the crosstalk involving the stability regulation of RAS protein via the Wnt/β-catenin signaling which is directly related with the cellular proliferation and transformation will be discussed. Activation status of GSK3β, a key enzyme involving both β-catenin and RAS degradations, is regulated by the status of the Wnt/β-catenin signaling dependent upon extracellular stimuli or intracellular abnormalities of the signaling components. The levels of both β-catenin and RAS proteins are co-regulated by the Wnt/β-catenin signaling, and these proteins are overexpressed with a positive correlation in the tumor tissues of CRC patients. These results indicate that the elevation of both β-catenin and RAS proteins is pathologically significant in CRC. In this review, we also will discuss further involvement of the increments of both β-catenin and RAS especially mutant KRAS in the activation of CSCs and metastasis. Overall, the increments of β-catenin and RAS especially mutant KRAS by APC loss play important roles in the cooperative tumorigenesis of CRC.

Abstract 475: Role of microRNA-34a in regulating oncoprotein STMN1

Abstract MicroRNAs, as negative regulators of gene expression, play a major role in cellular homeostasis. Downregulation of miRs is common in cancer which results in upregulation of multiple oncogenes in cancer. Here we show that stathmin (STMN1), which is over-expressed and oncogenic in wide variety of cancers, is regulated by microRNA-34 in prostate cancer. Earlier studies suggest that stathmin is involved in vital cellular processes such as cell proliferation, motility and metastasis. In the present study, we show elevated expression of stathmin in metastatic prostate cancer. Functionally, knockdown of stathmin in prostate cancer cells resulted in reduced cell proliferation and invasion in vitro, and tumor growth and metastasis in vivo. We also show that tumor suppressor microRNA miR-34a targets stathmin and mir-34 is regulated by transcriptional corepressor CtBP1 (C-Terminal Binding Protein 1). Further, Microarray analyses using stathmin modulated prostate cancer cell line RNA revealed multiple molecular alterations which may play a critical role in STMN1-mediated oncogenic activity. Thus, our study suggests that CtBP1-regulated miR-34a modulates stathmin expression in prostate cancer. This study therefore demonstrates functional role of CtBP1-miR-34a-Stathmin axis in prostate cancer progression. Citation Format: Balabhadrapatruni V.S.K. Chakravarthi, Rohit Mehra, Rui Wang, Darshan Shimoga Chandrashekar, Sai Akshaya Hodigere Balasubramanya, Irfan A. Asangani, Robert J. Lonigro, Arul M. Chinnaiyan, Sooryanarayana Varambally. Role of microRNA-34a in regulating oncoprotein STMN1 [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 475. doi:10.1158/1538-7445.AM2017-475

Next-generation sequencing for patients with non-obstructive azoospermia: implications for significant roles of monogenic/oligogenic mutations.

Azoospermia affects up to 1% of adult men. Non-obstructive azoospermia is a multifactorial disorder whose molecular basis remains largely unknown. To date, mutations in several genes and multiple submicroscopic copy-number variations (CNVs) have been identified in patients with non-obstructive azoospermia. The aim of this study was to clarify the contribution of nucleotide substitutions in known causative genes and submicroscopic CNVs in the genome to the development of non-obstructive azoospermia. To this end, we conducted sequence analysis of 25 known disease-associated genes using next-generation sequencing and genome-wide copy-number analysis using array-based comparative genomic hybridization. We studied 40 Japanese patients with idiopathic non-obstructive azoospermia. Functional significance of molecular alterations was assessed by in silico analyses. As a result, we identified four putative pathogenic mutations, four rare polymorphisms possibly associated with disease risk, and four probable neutral variants in 10 patients. These sequence alterations included a heterozygous splice site mutation in SOHLH1 and a hemizygous missense substitution in TEX11, which have been reported as causes of non-obstructive azoospermia. Copy-number analysis detected five X chromosomal or autosomal CNVs of unknown clinical significance, in addition to one known pathogenic Y chromosomal microduplication. Five patients carried multiple molecular alterations. The results indicate that monogenic and oligogenic mutations, including those in SOHLH1 and TEX11, account for more than 10% of cases of idiopathic non-obstructive azoospermia. Furthermore, this study suggests possible contributions of substitutions in various genes as well as submicroscopic CNVs on the X chromosome and autosomes to non-obstructive azoospermia, which require further validation.

The progressive fragmentation of the KIT/PDGFRA wild-type (WT) gastrointestinal stromal tumors (GIST)

Recent advances in molecular biology have revolutionized the concept of KIT/PDGFRA wild type (WT) gastrointestinal stromal tumors (GIST) than the past. Indeed, from being defined as GIST without KIT or PDGFRA mutations, we are now faced with the opposite scenario, where KIT/PDGFRA WT GIST are “positively” defined according to their specific molecular alterations. In particular, if until recently KIT/PDGFRA GIST without abnormalities of KIT, PDGFRA, SDH, and the RAS signaling pathway were referred as quadruple WT GIST, today also this small subset of GIST is emerging out as a group of heterogeneous distinct entities with multiple different molecular alterations. Therefore, given this still growing and rapidly evolving scenario, the progressive molecular fragmentation may inevitably lead over the time to the disappearance of KIT/PDGFRA WT GIST, destined to be singularly defined by their molecular fingerprint.

Heterogeneity in the colorectal primary tumor and the synchronous resected liver metastases prior to and after treatment with an anti-EGFR monoclonal antibody.

Molecular heterogeneity between primary tumors (PTs) and synchronous resected liver metastasis in colorectal cancer (CRC) has potential relevance in treatment strategies. Next-generation sequencing (NGS) may be able to increase the chances of identifying multiple molecular driver alterations, calling for therapy. The aim of the present study was to evaluate mutations in PT and synchronous resected liver metastases for patients with Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) exon 2 wild-type metastatic (m)CRC who underwent chemotherapy (CT) featuring an anti-epidermal growth factor receptor (EGFR) monoclonal antibody. Genomic analysis was performed on 54 lesions from 7 patients with mCRC. For each patient, a PT biopsy or a surgical specimen was obtained prior to CT, and the PT and all liver metastases resected following CT were analyzed. DNA libraries were generated using the Ion AmpliSeq Colon and Lung Cancer Panel, assessing the most frequent somatic mutations in 22 genes involved in colon tumorigenesis, and sequencing was performed on an Ion Personal Genome Machine system. A partial response was achieved in all the patients, with a median progression free survival time of 11 months (range, 3-21 months). All the patients were subjected to surgical liver metastasis resection. The median overall survival time was 31 months (range, 4-46 months). Molecular analysis of the genes correlated with the target therapy, suggesting significant intratumor heterogeneity, as revealed by the different mutational landscape of certain PTs and synchronous resected liver metastases following systemic therapy when compared with the PT prior to treatment. In particular, the loss and acquisition of mutations in KRAS, neuroblastoma RAS viral oncogene homolog (NRAS), tumor protein p53 (TP53), the p110α catalytic subunit of phosphoinositide 3-kinase (PIK3CA), F-box/WD repeat-containing protein 7 (FBXW7) and phosphatase and tensin homolog (PTEN) were observed. In addition, one patient developed a mucinous pattern following systemic CT. Taken together, the results of the present study demonstrated that intratumor heterogeneity is likely to affect the response to therapy, and to drive acquired resistance to targeted agents. The preliminary data also suggest a potential role for NGS in the evaluation of biological drug resistance, affecting future sequential treatment strategies.

Characteristics and Outcomes of Patients with Lung Cancer Harboring Multiple Molecular Alterations: Results from the IFCT Study Biomarkers France

IntroductionLittle is known about the prevalence, prognosis, and response to treatment of advanced NSCLC harboring multiple genomic alterations. MethodsThe French Biomarkers France database, which includes 17,664 patients, was used. The prevalence of multiple alterations, their associations, their impact on prognosis (overall survival [OS]), and their response to targeted or conventional treatments (progression-free survival [PFS] and objective response rate) were assessed and compared with those of patients harboring single or no mutation. ResultsWe identified 162 patients (0.9%) with double alterations and three with triple mutations. Multiple molecular alterations preferentially involved KRAS (67.3%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA) (53.3%), and EGFR (42.4%). Patients with multiple alterations were more likely to be male (56.4%), be never-smokers (25.8 versus 34.7%, p < 0.001), and exhibit adenocarcinomas (83.6%). OS did not differ between single and multiple alterations. Patients with EGFR/KRAS and EGFR/PIK3CA mutations experienced worse PFS than did patients with only EGFR mutations (7.1 and 7.1 versus 14.9 months, p = 0.02 and 0.002, respectively). Concomitant mutations in patients harboring anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement bore little impact on OS (17.7 versus 20.3 months, p = 0.57) or PFS (10.3 versus 12.1 months, p = 0.93). Patients harboring KRAS mutations plus another alteration had an OS time (13.4 versus 11.2 months, p = 0.28), PFS time (6.4 versus 7.2 months, p = 0.78), and objective response rate under first-line chemotherapy (41.7% versus 37.2%) similar to those of patients harboring KRAS mutations only. ConclusionsWith almost 1% of patients harboring multiple alterations, the dogma of mutually exclusive mutations should be reconsidered. Although double mutations do not decrease OS, they do alter PFS under first-line treatment for patients with EGFR mutations. Among limited numbers of patients, therapies targeting the dominant oncogene seem to usually remain active.