Common Genomic Aberrations Research Articles

Complete miRNA-15/16 loss in mice promotes hematopoietic progenitor expansion and a myeloid-biased hyperproliferative state

Dysregulated apoptosis and proliferation are fundamental properties of cancer, and microRNAs (miRNA) are critical regulators of these processes. Loss of miR-15a/16-1 at chromosome 13q14 is the most common genomic aberration in chronic lymphocytic leukemia (CLL). Correspondingly, the deletion of either murine miR-15a/16-1 or miR-15b/16-2 locus in mice is linked to B cell lymphoproliferative malignancies. However, unexpectedly, when both miR-15/16 clusters are eliminated, most double knockout (DKO) mice develop acute myeloid leukemia (AML). Moreover, in patients with CLL, significantly reduced expression of miR-15a, miR-15b, and miR-16 associates with progression of myelodysplastic syndrome to AML, as well as blast crisis in chronic myeloid leukemia. Thus, the miR-15/16 clusters have a biological relevance for myeloid neoplasms. Here, we demonstrate that the myeloproliferative phenotype in DKO mice correlates with an increase of hematopoietic stem and progenitor cells (HSPC) early in life. Using single-cell transcriptomic analyses, we presented the molecular underpinning of increased myeloid output in the HSPC of DKO mice with gene signatures suggestive of dysregulated hematopoiesis, metabolic activities, and cell cycle stages. Functionally, we found that multipotent progenitors (MPP) of DKO mice have increased self-renewing capacities and give rise to significantly more progeny in the granulocytic compartment. Moreover, a unique transcriptomic signature of DKO MPP correlates with poor outcome in patients with AML. Together, these data point to a unique regulatory role for miR-15/16 during the early stages of hematopoiesis and to a potentially useful biomarker for the pathogenesis of myeloid neoplasms.

Genomic and Phenotypic Biomarkers for Precision Medicine Guidance in Advanced Prostate Cancer.

Prostate cancer (PCa) is the second most diagnosed malignant neoplasm and is one of the leading causes of cancer-related death in men worldwide. Despite significant advances in screening and treatment of PCa, given the heterogeneity of this disease, optimal personalized therapeutic strategies remain limited. However, emerging predictive and prognostic biomarkers based on individual patient profiles in combination with computer-assisted diagnostics have the potential to guide precision medicine, where patients may benefit from therapeutic approaches optimally suited to their disease. Also, the integration of genotypic and phenotypic diagnostic methods is supporting better informed treatment decisions. Focusing on advanced PCa, this review discusses polygenic risk scores for screening of PCa and common genomic aberrations in androgen receptor (AR), PTEN-PI3K-AKT, and DNA damage response (DDR) pathways, considering clinical implications for diagnosis, prognosis, and treatment prediction. Furthermore, we evaluate liquid biopsy, protein biomarkers such as serum testosterone levels, SLFN11 expression, total alkaline phosphatase (tALP), neutrophil-to-lymphocyte ratio (NLR), tissue biopsy, and advanced imaging tools, summarizing current phenotypic biomarkers and envisaging more effective utilization of diagnostic and prognostic biomarkers in advanced PCa. We conclude that prognostic and treatment predictive biomarker discovery can improve the management of patients, especially in metastatic stages of advanced PCa. This will result in decreased mortality and enhanced quality of life and help design a personalized treatment regimen.

Serrate RNA Effector Molecule (SRRT) Is Associated with Prostate Cancer Progression and Is a Predictor of Poor Prognosis in Lethal Prostate Cancer.

Arsenite-resistance protein 2, also known as serrate RNA effector molecule (ARS2/SRRT), is known to be involved in cellular proliferation and tumorigenicity. However, its role in prostate cancer (PCa) has not yet been established. We investigated the potential role of SRRT in 496 prostate samples including benign, incidental, advanced, and castrate-resistant patients treated by androgen deprivation therapy (ADT). We also explored the association of SRRT with common genetic aberrations in lethal PCa using immunohistochemistry (IHC) and performed a detailed analysis of SRRT expression using The Cancer Genome Atlas (TCGA PRAD) by utilizing RNA-seq, clinical information (pathological T category and pathological Gleason score). Our findings indicated that high SRRT expression was significantly associated with poor overall survival (OS) and cause-specific survival (CSS). SRRT expression was also significantly associated with common genomic aberrations in lethal PCa such as PTEN loss, ERG gain, mutant TP53, or ATM. Furthermore, TCGA PRAD data revealed that high SRRT mRNA expression was significantly associated with higher Gleason scores, PSA levels, and T pathological categories. Gene set enrichment analysis (GSEA) of RNAseq data from the TCGA PRAD cohort indicated that SRRT may play a potential role in regulating the expression of genes involved in prostate cancer aggressiveness. Conclusion: The current data identify the SRRT's potential role as a prognostic for lethal PCa, and further research is required to investigate its potential as a therapeutic target.

Abstract 3119: A novel tissue resident memory T cell (TRM) signature predicts prognosis and tumor microenvironment of patients with melanoma

Abstract Background: Melanoma is a prime example of how basic and translational research can improve cancer prognosis The tumor immune microenvironment (TIME) of melanoma is a dynamic, heterogeneous mixture of multiple immune cells and malignant and nonmalignant cells. Tissue resident memory T cells (TRM) may be prognostic for improved melanoma survival and proliferate during treatment with immune checkpoint inhibitors. The role of TRM in melanoma and rare melanoma subtypes is unknown. Method: Our goal was to examine the relationship between TRM abundance and melanoma prognosis. We generated TRM signatures using multiple independent single-cell RNA sequencing data from human melanoma cells to infer TRM abundance. Lastly, we built a LASSO Cox regression model that predicts patient survival. The TCGA dataset was used as a test group. Model accuracy was validated using Kaplan-Meier survival analysis, receiver operating characteristic curves, principal component analysis, and t-distributed stochastic neighbor embedding. Findings: We found a significantly longer overall survival in patients with high TRM infiltration. Furthermore, we found that TRM can interact with the tumor microenvironment and genomic features of melanoma, making TRM abundance positively correlate with the prognosis of melanoma patients. Melanoma abundance was also significantly associated with several common genomic aberrations, including the TP53 mutation. In addition, we developed a 20-gene risk score that significantly classifies patients into low- and high-risk categories. We found that in several independent datasets, patients with high risk scores had significantly worse overall survival than patients with low risk scores. Conclusions: Altogether, our findings suggest that TRM abundance may positively correlate with melanoma survival. We developed a high-precision LASSO Cox regression model based on the new TRM abundance characteristics. Our study shows that this model effectively predicts the prognosis of melanoma patients and may provide new therapeutic targets and measure the effectiveness of melanoma-directed therapies. Keywords: Tissue resident memory T cell, tumor immune microenvironment, melanoma, prognosis Citation Format: Chongming Jiang, Jianrong Li, Elena Helman, Xiling Shen, Chao Cheng. A novel tissue resident memory T cell (TRM) signature predicts prognosis and tumor microenvironment of patients with melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3119.

Next-generation sequencing and molecular therapy

Cancers contain a plethora of mutations, few of which are critical to maintaining a state of malignancy. With our ever-expanding understanding of the genomic complexity of cancer, potentially actionable biomarkers whose inhibition could cripple cancer growth are increasingly being elucidated. Modern cancer drug development has largely switched from cytotoxic agents to targeted therapies and immunotherapy, with noteworthy success in several cancer types including non-small-cell lung cancer (NSCLC), breast cancer and melanoma. Next-generation sequencing offers high-throughput, widescale genomic interrogation in a far more efficient and affordable manner than previous sequencing methods. This facilitates detection of potentially actionable mutations and fusions for individual patients and contributes to the identification of novel predictive and prognostic biomarkers in a population. Challenges in the technical aspects of biopsy and sequencing, interpretation, and development of targeted therapies against common genomic aberrations will need to be addressed for personalised medicine to become a reality for more patients with cancer.

Microglia-Based Gene Expression Signature Highly Associated with Prognosis in Low-Grade Glioma

Simple SummaryGliomas make up ~80% of malignant brain tumors in adults and are responsible for the majority of deaths from primary brain tumors. Consequently, a better understanding of the malignant features of the TME in glioma is pertinent. The aim of our study was to evaluate the expression of immune-related genes (IRGs) in glioma and their association with patient prognosis. We utilized several approaches to interrogate the glioma immune microenvironment. We found that immune genes are generally negatively associated with survival and that overall survival was significantly lower in those with a high level of microglia infiltration. The microglia abundance was significantly associated with common genomic aberrations. Lastly, we generated a 23-gene expression signature that is highly associated with patient prognosis, independent of clinical variables. These findings are relevant to investigators in the glioma field, those working in biomarker development, but also to individuals working on glioma therapeutics.Gliomas make up ~80% of malignant brain tumors in adults and are responsible for the majority of deaths from primary brain tumors. The glioma tumor microenvironment (TME) is a dynamic, heterogeneous mixture of extracellular matrix and malignant and non-malignant cells. Several ongoing clinical trials are evaluating the efficacy of therapies that target non-malignant cells, particularly immune cells. Consequently, a better understanding of the TME in glioma is pertinent. We utilized several gene expression datasets to evaluate the relationship between immune-related genes (IRGs) and patient prognosis. We generated microglia signatures using single-cell RNAseq data from human and mouse glioma cells to infer microglia abundance. Lastly, we built a LASSO Cox regression model that predicts patient survival. We found that 428 IRGs were negatively associated with survival in glioma patients. Overall survival was significantly lower in those with a high level of microglia infiltration. In addition, we also found that microglia abundance was significantly associated with several common genomic aberrations, including IDH2 and TP53 mutations. Furthermore, we found that patients with high risk scores had significantly worse overall survival than those with low risk scores in several independent datasets. Altogether, we characterized immune features predictive of overall survival in glioma and found that microglia abundance is negatively associated with survival. We developed a 23-gene risk score that can significantly stratify patients into low- and high-risk categories.

Abstract IA26: Understanding CLL and Richter’s syndrome biology through mouse models of human genetics

Abstract Large-scale human next generation sequencing studies have progressively defined the complex genetic landscape of chronic lymphocytic leukemia (CLL) and Richter’s syndrome (RS), yet the difficulties in rapid manipulation of primary CLL cells and the limited availability of cell lines have challenged studies aimed at interrogating the function of the so-called disease “drivers”, defined in human studies through statistical inference. Mouse models represent powerful tools to study mechanisms of normal and malignant B cell biology. Conditional knock-in/knock-out systems have allowed B-cell restricted modeling of CLL genetic lesions, with MDR mice being the first example of a genetically-faithful CLL model, recapitulating the most common genomic aberration of human disease, del(13q). Since then, a series of models generated by our group and others have allowed to uncover key functional changes underlying disease onset and transformation, including in the context of combined mutation in the splicing factor Sf3b1 and the DNA damage response gene Atm, sole mutation in the B cell developmental factor Ikzf3, the nuclear export receptor Xpo1, the ribosomal protein Rps15, and the DNA methyltransferase Dnmt3a (the latter not a CLL lesion per se, but recapitulating disordered methylation patterns typical of CLL). The CRISPR-Cas9 gene editing technology has further enabled B-cell restricted introduction of multiple combinatorial loss-of-function lesions, allowing the generation of faithful models not only of CLL but, importantly, of transformation into RS. In this presentation, I will describe the application of mouse models to studies of CLL and RS pathogenesis and treatment, with a focus on key aspects uncovered through the characterization of genetically- inspired mouse lines of recurrent disease drivers. I will provide a perspective on how these novel models combined with new technology allowed the dissection of mechanisms of disease evolution and response to therapy with greater depth than previously possible, representing valuable platforms for functional genomic analyses. Citation Format: Elisa ten Hacken. Understanding CLL and Richter’s syndrome biology through mouse models of human genetics [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr IA26.

MAP3K8 and ALK alterations in pediatric patients with atypical Spitz tumors and Spitz melanoma.

e21569 Background: Pediatric atypical Spitz tumors and Spitz melanoma (AST/SM) are characterized by structural variants (rearrangements/truncations) of various kinase genes more commonly involving MAP3K8 and ALK. The clinical features associated with these genomic abnormalities have not been well characterized. Methods: We reviewed the clinical characteristics and outcomes of children and adolescents with AST/SM who were enrolled on our institutional comprehensive registry for melanocytic lesions MACMEL (Cancer 127:3825, 2021) and abstracted clinical and outcome data for patients with MAP3K8, ALK, and other less common genomic aberrations. Results: Table shows the characteristics and outcomes of patients with AST/SM who had MAP3K8, ALK, and other genomic aberrations. There were 49 patients of whom 21 had MAP3K8 rearrangements/ truncations (n = 11;22%) or ALK rearrangements (n = 10;20%). The remaining 28 patients had fusions involving ARAF (1), ROS1 (2), NTRK1 (3), ERBB2 (1) or rare mutations (ATM = 1) or copy number changes (n = 14) or no identifiable genomic abnormality (n = 6). Female sex was more common in all three groups (32 female and 17 male). The median age for the entire cohort was 7.5 years. Most lesions arose in the extremities (n = 32; 65%). About half had stage III disease. 40 patients underwent sentinel node biopsy (SLNB), 25 had at least one positive node (7/9 MAP3K8, 5/8 ALK, 13/23 other), Forty-four patients had FISH analysis for CDKN2A and homozygous loss was seen in 9/11 MAP3K8-rearranged, 2/6 ALK-rearranged, and 11/27 other tumors. No TERT promoter mutation was detected in any of the 45 tumors that were analyzed. Two patients experienced local recurrence, one with a MAP3K8-STX fusion and one with a FLOT2-ARAF fusion and activating RAC1 mutation. All patients are alive and currently disease free (median follow up of 2.33 years). There were no statistically significant associations between gene mutations and tumor site, gender, or stage. Conclusions: Approximately 40% of patients with AST/SM harbored ALK or MAP3K8 alterations in our pediatric melanoma registry. We found no significant association between the individual genes and any specific clinical features in this limited cohort.[Table: see text]

Tumor cell intrinsic and extrinsic features predictprognosis in estrogen receptor positive breast cancer.

Although estrogen-receptor-positive (ER+) breast cancer is generally associated with favorable prognosis, clinical outcome varies substantially among patients. Genomic assays have been developed and applied to predict patient prognosis for personalized treatment. We hypothesize that the recurrence risk of ER+ breast cancer patients is determined by both genomic mutations intrinsic to tumor cells and extrinsic immunological features in the tumor microenvironment. Based on the Cancer Genome Atlas (TCGA) breast cancer data, we identified the 72 most common genomic aberrations (including gene mutations and indels) in ER+ breast cancer and defined sample-specific scores that systematically characterized the deregulated pathways intrinsic to tumor cells. To further consider tumor cell extrinsic features, we calculated immune infiltration scores for six major immune cell types. Many individual intrinsic features are predictive of patient prognosis in ER+ breast cancer, and some of them achieved comparable accuracy with the Oncotype DX assay. In addition, statistical learning models that integrated these features predicts the recurrence risk of patients with significantly better performance than the Oncotype DX assay (our optimized random forest model AUC = 0.841, Oncotype DX model AUC = 0.792, p = 0.04). As a proof-of-concept, our study indicates the great potential of genomic and immunological features in prognostic prediction for improving breast cancer precision medicine. The framework introduced in this work can be readily applied to other cancers.

Common Genomic Aberrations in Mouse and Human Breast Cancers with Concurrent P53 Deficiency and Activated PTEN-PI3K-AKT Pathway.

Simultaneous P53 loss and activation of the PTEN-restricted PI3K-AKT pathway frequently occur in aggressive breast cancers. P53 loss causes genome instability, while PTEN loss and/or activating mutations of PIK3CA and AKT promote cancer cell proliferation that also increases incidences of genomic aberrations. However, the genomic alterations associated with P53 loss and activated PTEN-PI3K-AKT signaling in breast cancer have not been defined. Spatiotemporally controlled breast cancer models with inactivation of both P53 and Pten in adult mice have not been established for studying genomic alterations. Herein, we deleted both floxed Pten and Tp53 genes in the mammary gland epithelial cells in adult mice using a RCAS virus-mediated Cre-expressing system. These mice developed small tumors in 21 weeks, and poorly differentiated larger tumors in 26 weeks. In these tumors, we identified 360 genes mutated by nonsynonymous point mutations and small insertions and deletions (NSPMs/InDels), 435 genes altered by copy number amplifications (CNAs), and 450 genes inactivated by copy number deletions (CNDs). Importantly, 22.2%, 75.9% and 27.3% of these genes were also altered in human breast tumors with P53 and PTEN losses or P53 loss and activated PI3K-AKT signaling by NSPMs/InDels, CNAs and CNDs, respectively. Therefore, inactivation of P53 and Pten in adult mice causes rapid-growing breast tumors, and these tumors recapitulate a significant number of genetic aberrations in human breast tumors with inactivated P53 and activated PTEN-PI3K-AKT signaling. Further characterization of these commonly altered genes in breast cancer should help to identify novel cancer-driving genes and molecular targets for developing therapeutics.

Functional impact of titin (TTN) mutations in ocular surface squamous neoplasia

Mutations in the titin (TTN) gene are among the most common genomic aberrations in ocular surface squamous neoplasia (OSSN), the most common cancer of the external eye. Further, TTN mutations are associated with resistance to standard therapy with topical interferon alpha-2b (IFN-α2b). However, it remains unclear how TTN mutations drive OSSN pathogenesis and treatment resistance. TTN encodes the largest protein in the human body and its best understood function is as a myofibril scaffold in striated muscle. However, recent evidence indicates that TTN has additional functions in non-muscle cells and in cancer. Here, we performed a disorder-based bioinformatics analysis which revealed that intrinsically disordered protein regions are abundant in TTN and provide mechanistic insights into its function as a nuclear protein in epithelial cells. Specific mutations found in OSSN are predicted to affect its intrinsically disordered protein regions (IDPRs), promoting chromosomal instability, oncogenesis, and altered response to IFN-α2b treatment.

Predictive Genomic Biomarkers of Hormonal Therapy Versus Chemotherapy Benefit in Metastatic Castration-resistant Prostate Cancer

BackgroundBiomarkers predicting second-generation novel hormonal therapy (NHT) benefit relative to taxanes are critical for optimized treatment decisions for metastatic castration-resistant prostate cancer (mCRPC) patients. These associations have not been reported simultaneously for common mCRPC genomic biomarkers. ObjectiveTo evaluate predictive associations of common genomic aberrations in mCRPC using an established comprehensive genomic profiling (CGP) system. Design, setting, and participantsA retrospective cohort study used data from a deidentified US-based clinicogenomic database comprising patients treated in routine clinical practice between 2011 and 2020, evaluated with Foundation Medicine CGP in tissue biopsies obtained around the time of treatment decision. The main cohort included 180 NHT and 179 taxane lines of therapy (LOTs) from 308 unique patients. The sequential cohort comprised a subset of the main cohort NHT LOTs immediately followed by taxane from 55 unique patients. Outcome measurements and statistical analysisProstate-specific antigen (PSA) response, time to next treatment (TTNT), and overall survival (OS) were assessed. Main cohort analyses were adjusted for known treatment assignment biases via inverse probability of treatment weighting (IPTW) in treatment interaction models. Results and limitationsIn the main cohort, patients with AR amplification (ARamp) or PTEN aberrations (PTENalt) had worse relative PSA response on NHT versus taxanes compared with patients without. Patients with ARamp, PTENalt, or RB1 aberrations (RB1alt) also had worse relative TTNT and OS on NHT but not on taxanes. In multivariable models for TTNT and OS adjusted via IPTW, ARamp, PTENalt, and RB1alt were shown as poor prognostic factors overall and demonstrated significant treatment interactions, indicating reduced hazards of therapy switch and death on taxanes versus NHT. Consistent associations favoring increased benefit from subsequent taxane despite prior NHT treatment line were observed only for ARamp in the sequential cohort, in which very few patients had RB1alt for assessment. ConclusionsARamp status is a candidate biomarker to predict poor effectiveness of NHT relative to taxanes in mCRPC in scenarios where both options are considered. Patient summarySpecific alterations in the DNA of tumors may assist in choosing between novel oral hormonal therapies and standard chemotherapy in advanced prostate cancer patients.

Evaluation of MLPA as a comprehensive molecular cytogenetic tool to detect cytogenetic markers of chronic lymphocytic leukemia in Egyptian patients

BackgroundChronic lymphocytic leukemia (CLL) is the most common form of adult leukemia. This disease is genetically heterogeneous, and approximately 85% of patients with CLL harbor chromosomal aberrations that are considered effective prognostic biomarkers. The most frequent aberrations include deletions in 13q14, followed by trisomy 12, and deletions in 11q22.3 and 17p13 (TP53). Currently, fluorescence in situ hybridization (FISH) is the most widely used molecular cytogenetic technique to detect these aberrations. However, FISH is laborious, time-consuming, expensive, and has a low throughput. In contrast, multiplex ligation-dependent probe amplification (MLPA) is a reliable, cost-effective, and relatively rapid technique that can be used as a first-line screening tool and complement with FISH analysis. This study aimed to evaluate the contributions of MLPA as a routine standalone screening platform for recurrent chromosomal aberrations in CLL in comparison to other procedures. Thirty patients with CLL were screened for the most common genomic aberrations using MLPA with SALSA MLPA probemix P038-B1 CLL and FISH. ResultsIn 24 of the 30 cases (80%), the MLPA and FISH results were concordant. Discordant results were attributed to a low percentage of mosaicism. Moreover, the MLPA probemix contains probes that target other genomic areas known to be linked to CLL in addition to those targeting common recurrent CLL aberrations. ConclusionsThe usage of MLPA as the first screening platform followed by FISH technique for only the negative cases is the most appropriate approach for CLL diagnosis and prognosis.

Cutaneous Metastatic Cancer: Carcinoma Hemorrhagiectoides Presenting as the Shield Sign.

Cutaneous metastases can be either the initial presentation of an unsuspected internal neoplasm in a cancer-free individual, or the harbinger of recurrent malignancy in an oncology patient who had achieved remission of his cancer, or a sign of progressive disease in a cancer patient who is currently receiving antineoplastic treatment. The cutaneous presentation of skin metastases is pleomorphic and can mimic not only benign conditions and tumors of the skin but also infections and inflammation of the skin. Carcinoma erysipelatoides, carcinoma hemorrhagiectoides, and carcinoma telangiectoides are the three subtypes of inflammatory cutaneous metastatic cancer. The former masquerades as a cutaneous streptococcal infection whereas the latter mimics idiopathic telangiectasias. In contrast, the morphology of carcinoma hemorrhagiectoides is distinctive: it appears similar in shape to a medieval knight’s shield and its presence is referred to as a positive shield sign. To the best of our knowledge, carcinoma hemorrhagiectoides has been reported in four oncology patients whose skin metastases presented with the shield sign: two men with salivary duct carcinoma and two women with breast cancer. In conclusion, the shield sign may not only be a pathognomonic clinical feature of carcinoma hemorrhagiectoides but also reflect a common genomic aberration of these metastatic tumors.

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities.

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.

Biomarkers of outcome to weekly paclitaxel in epithelial ovarian cancer

ObjectiveWe sought to evaluate the role of intrinsic chromosomal aberrations in determining favorable outcome to weekly paclitaxel (WP) in patients with epithelial ovarian cancer (EOC). MethodsWe evaluated the common genomic aberrations of two patients with EOC and exceptional WP response in the GENIUS study (NCT03740503). We then searched for potential markers of unusual outcomes to WP in a validation cohort. We performed shallow whole genome sequencing (sWGS) in the tumor tissue of women with EOC considered as short-responders (SR; progression with ≤3 cycles) and long-responders (LR; response at ≥8 cycles) to WP monotherapy. ResultsWe identified two women with exceptional response to WP, lasting over four years, who shared chromosome 8 gain as a common genomic aberration. In order to validate our findings, we reviewed 188 patients with EOC treated with WP and selected 61 women (39 SR, 22 LR) with unusual responses. By sWGS, there was no differential alterations in the copy number changes in chromosome 8, or in genes related to angiogenesis, tubulin superfamily, cell-cycle, apoptosis and paclitaxel metabolism or transportation pathways. Amongst the LR group, we identified six exceptionally long responders (ExLR), with responses lasting over a year. In an exploratory analysis, there was increased amplification of angiogenesis (VEGFB, MMP9), tubulin superfamily (TSC2) and apoptosis related genes (BCL2L1, BAD) in ExLR compared to SR. We identified one patient with a complete response to WP for over 7 years. Molecular profiling identified unique amplifications in interleukin related genes (CXCR1, CXCR2, IL1A, IL1B), not detected in other patients. ConclusionIntrinsic tumor pathways may impact outcome with weekly paclitaxel monotherapy and further investigations are required.

Prognostic and predictive impact of genetic markers in patients with CLL treated with obinutuzumab and venetoclax

AbstractGenetic parameters are established prognostic factors in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy, but are less well studied with novel compounds. We assessed immunoglobulin heavy variable chain (IGHV) mutation status, common genomic aberrations, and gene mutations in 421 untreated patients within the CLL14 trial (NCT02242942), comparing obinutuzumab+chlorambucil (GClb) vs obinutuzumab+venetoclax (VenG). The incidences of genomic aberrations considering the hierarchical model were del(17p) 7%, del(11q) 18%, +12 18%, and del(13q) 35%, whereas IGHV was unmutated in 60% of patients. NOTCH1 mutations were most common (23%), followed by SF3B1 (16%), ATM (13%), and TP53 (10%). Although the overall response rate (ORR) for GClb was lower in patients with del(17p), del(11q), mutated TP53, ATM, and BIRC3, none of these parameters reduced complete remission (CR) rate and ORR with VenG. At a median follow-up of 28 months, del(17p) and mutated TP53 were the only abnormalities with an effect on progression-free survival (PFS) for both treatment groups: GClb (hazard ratio [HR], 4.6 [P < .01]; HR, 2.7 [P < .01], respectively) and VenG (HR, 4.4 [P < .01]; HR, 3.1 [P < .01], respectively). No other factors affected outcome with VenG, whereas for GClb del(11q), BIRC3, NOTCH1, and unmutated IGHV were associated with shorter PFS. Multivariable analysis identified del(17p), del(11q), unmutated IGHV, and mutated TP53, BIRC3, and SF3B1 as independent prognostic factors for PFS with GClb, whereas for VenG, only del(17p) was significant. VenG was superior to GClb across most genetic subgroups. Patients with adverse genetic markers had the strongest benefit from VenG, particularly subjects with unmutated IGHV, which was identified as a predictive factor in a multivariable treatment-interaction analysis.

ES03.02 Targeting CDKN2A in Mesothelioma

Interrogation of the genomic landscape in mesothelioma has revealed considerable inter-patient genomic heterogeneity, however development of molecularly targeted stratified therapy is currently in its infancy. Somatic copy number loss involving the chromosome 9p21-3 locus occurs at high frequency (45% deep deletion in the TCGA cohort), and its expression may be further suppressed by DNA methylation. 9p21-3 harbours CDKN2A. Conditional Knockout of CDKN2A is sufficient to initiate mesotheliomas in vivo consistent with a role in tumorigenesis. This is supported by emerging phylogenetic analysis in which 9p21-3 occurs as an early (clonal or truncal) homozygous event during tumour evolution, in around 20% of cases. Late homozygous and heterogzyous losses also occur, with evidence of parallel evolution. Importantly, CDKN2A is a major negative prognostic factor associated with earlier progression following surgical resection. CDKN2A encodes two genes; 1, the inhibitor of cyclin dependent kinases 4 and 6 known as p16ink4A, and 2. MDM2, an inhibitor of the p53-MDM2 interaction. Early preclinical studies showed that re-introducing p16ink4A transgene in a CDKN2A negative mesothelioma exhibited anti-tumour activity. Small molecule inhibition of CDK4/6 essentially phenocopies p16ink4a (in the presence of wild type RB1). Large scale drug-gene interaction studies have revealed CDKN2A dependent sensitivity to CDK4/6 inhibition suggesting a possible strategy in mesothelioma. We have therefore developed the MiST stratified umbrella trial (NCT NCT03654833) is therefore evaluating the CDK4/6 inhibitor abemaciclib in p16ink4a negative relapsed mesothelioma (arm 2) Loss of p14ARF expression promotes MDM2, an E3 ubiquin ligase targeting p53. Given the low mutation rate for p53 in mesothelioma (around 16% in the TCGA), MDM2 inhibitors may, in the context of p14ARF loss, de-repress p53 to mediate tumour suppression. Preclinical data supports this hypothesis which has translational potential. Co-deletion of the gene MTAP with CDKN2A is common, and coincident with around 80% of CDKN2A losses. MTAP (methylthioadenosine phosphorylase) may represent a potential molecular target in 9p21-3 deleted mesothelioma. This tumour suppressor salvages the essential amino acid methionine, as well as adenine, and plays a critical role in the polyamine salvage pathway. Recently it has been shown that loss of MTAP leads to an increase level in its substrate, methylthioadenosine (MTA). This metabolite directly interacts with and inhibits the epigenetic modifier, PRMT5 (protein arginine methyltransferase 5) that mediates symmetrical arginine di-methylation of histone H4 (H4R3me2s). A direct consequence of this is a reduction in the pool of functional PRMT5 enzyme, revealing a vulnerability to 1. Inhibition of PRMT5 expression or 2. Inhibition of PRMT5 substrate (SAM) synthesis via MAT2A (which converts methionine to SAM). 1st generation small molecule PRMT5 inhibitors are currently undergoing phase 1 clinical evaluation, however MTA dependent allosteric antagonism of such inhibitors presents a challenge to achieving efficacy. Small molecule transcriptional suppression may present a solution. Accordingly, using the connectivity map, we have identified quinacine as a PRMT5 transcriptional suppressor capable of suppressing PRMT5 transcription (which is c-jun dependent), mediating MTAP selective loss of viability with commensurate reduction in H4R3me2s, consistent with an on-target effect. In summary, 9p21-3 loss encompassing CDKN2A and MTAP is one of the most common genomic aberrations in mesothelioma. Novel strategies are emerging with significant translational potential to deliver targeted therapies for mesothelioma. CDKN2A MTAP synthetic lethality

Gain of 20q11.21 in Human Pluripotent Stem Cells Impairs TGF-β-Dependent Neuroectodermal Commitment

SummaryGain of 20q11.21 is one of the most common recurrent genomic aberrations in human pluripotent stem cells. Although it is known that overexpression of the antiapoptotic gene Bcl-xL confers a survival advantage to the abnormal cells, their differentiation capacity has not been fully investigated. RNA sequencing of mutant and control hESC lines, and a line transgenically overexpressing Bcl-xL, shows that overexpression of Bcl-xL is sufficient to cause most transcriptional changes induced by the gain of 20q11.21. Moreover, the differentially expressed genes in mutant and Bcl-xL overexpressing lines are enriched for genes involved in TGF-β- and SMAD-mediated signaling, and neuron differentiation. Finally, we show that this altered signaling has a dramatic negative effect on neuroectodermal differentiation, while the cells maintain their ability to differentiate to mesendoderm derivatives. These findings stress the importance of thorough genetic testing of the lines before their use in research or the clinic.

Targeted next-generation sequencing (tNGS) of metastatic castrate-sensitive prostate cancer (M1 CSPC): A pilot molecular analysis in the STAMPEDE multi-center clinical trial.

5019 Background: The STAMPEDE trial recruits men with high risk prostate cancer commencing first line systemic therapy. In a pilot study to ascertain the feasibility of tNGS and the prevalence of common genomic aberrations, we tested a commercial clinically-accredited assay on tumor blocks and present data obtained in the largest cohort of treatment-naïve M1 CSPC to date. Methods: Archival FFPE blocks were retrieved from trial participants and a single block submitted for sequencing by a Foundation Medicine. Inc. tNGS assay that includes 395 genes. Results: We successfully obtained tNGS data on 115 (62%) of 186 patients enrolled between Nov-2011 and April-2017 at 15 UK participating centers. The median age was 70 years (IQR 44-85); 97% had de novo M1 disease and 83% Gleason score ≥8. We observed PTEN deficiency (34%) due to copy-number loss (25%) or mutation (9%); TP53 mutation or loss (33%) and aberrations in PI3K signaling (16%), genes involved in DNA repair (14%), Wnt signaling (14%) and cell cycle control (6%). In total, these aberrations were observed in 76% of patients, with 35% harboring two or more. No androgen receptor ( AR) mutations were detected. Conclusions: The prevalence of PTEN deficiency is comparable with that observed in mCRPC consistent with this being a feature of metastatic disease. In contrast, AR mutations are not observed in this treatment-naïve group. The prevalence of DNA repair deficiency is less than observed in mCRPC but more than reported in prostatectomy cohorts. Although it is possible to use FFPE biopsies for tNGS, the test failure-rate poises challenges to evaluating treatments in low prevalence biomarker-defined groups. These data will inform the design and conduct of biomarker-directed trials.