TP53 Pathway Research Articles

1017 - TRANSLATIONAL LESSONS IN OPTIMIZING CLINICAL USE OF BH3 MIMETICS TO TREAT HEMATOLOGICAL MALIGNANCIES

Oncogenic mutations impose stresses on cells which normally would trigger apoptosis. Consequently, many haematological malignancies harbour aberrations that circumvent this, most prominently dysfunction of the TP53 pathway and dysregulation of the BCL2 protein family. The prosurvival proteins BCL2 and MCL1 are commonly highly expressed in diseases like CLL, lymphomas, AML and myeloma. BH3-mimetics are a novel class of anti-cancer drugs which mimic the natural antagonists of the prosurvival proteins and operate downstream of TP53. As such they can directly trigger apoptosis in cells that are dependent on BCL2 or MCL1. Key lessons to date are that: killing of cancer cells by BH3 mimetics is rapid and often dependent on cascading effects requiring the action of endogenous BH3 only proteins; (ii) clinical activity with the specific BCL2 inhibitor, venetoclax, across multiple haematological malignancies was accurately predicted using in vitro assays of primary cells; (iii) BH3 mimetics kill malignant cells independently of TP53 aberrations, but TP53 dysfunction remains prognostic; and (iv) this class of drug is well suited to combination with a range of other targeted therapies in a cell-context dependent manner with clinical trial evidence indicating that deeper and more durable responses can be achieved using rational combinations of venetoclax with monoclonal antibodies, tyrosine kinase inhibitors and hypomethylating agents in CLL, lymphoma and AML respectively. These and other combinations currently studied preclinically will be reviewed to try and provide a sense of how BH3 mimetics may be best used in the future.

Common genetic variants in the TP53 pathway and their impact on cancer.

The TP53 gene is well known to be the most frequently mutated gene in human cancer. In addition to mutations, there are > 20 different coding region single-nucleotide polymorphisms (SNPs) in the TP53 gene, as well as SNPs in MDM2, the negative regulator of p53. Several of these SNPs are known to alter p53 pathway function. This makes p53 rather unique among cancer-critical genes, e.g. the coding regions of other cancer-critical genes like Ha-Ras, RB, and PI3KCA do not have non-synonymous coding region SNPs that alter their function in cancer. The next frontier in p53 biology will consist of probing which of these coding region SNPs are moderately or strongly pathogenic and whether they influence cancer risk and the efficacy of cancer therapy. The challenge after that will consist of determining whether we can tailor chemotherapy to correct the defects for each of these variants. Here we review the SNPs in TP53 and MDM2 that show the most significant impact on cancer and other diseases. We also propose avenues for how this information can be used to better inform personalized medicine approaches to cancer and other diseases.

Genetic profiling as a clinical tool in advanced parathyroid carcinoma.

Parathyroid carcinoma (PC) is a rare endocrine malignancy with no approved systemic therapies for unresectable locally invasive or distant metastatic disease. Understanding the molecular changes in advanced PC can provide better understanding of this disease and potentially help directing targeted therapy. To evaluate tumor-specific genetic changes using next-generation sequencing (NGS) panels. All patients with advanced PC were tested for hot-spot panels using NGS panels including a 50-gene panel, a 409-gene panel if the standard 50-gene panel (Ion Torrent, Life Technology) was negative or a FoundationOne panel. The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. 11 patients with advanced PC were selected to undergo molecular testing. Genetic profiles of advanced PC. Among the 11 patients, 4 patients had the 50-gene panel only, 6 had 409-gene panel after a negative 50-gene panel and 1 had FoundationOne. One patient who had 50-gene panel only also had his metastatic site (esophagus) of his tumor tested with FoundationOne. The most common mutations identified were in the PI3K (PIK3CA, TSC1 and ATM) (4/11 patients) and TP53 (3/11) pathways. Genes not previously reported to be mutated in PC included: SDHA, TERT promoter and DICER1. Actionable mutations were found in 54% (6/11) of the patients. Mutational profiling using NGS panels in advanced PC has yielded important potentially targetable genetic alterations. Larger studies are needed to identify commonly mutated genes in advanced PC patients. Development of novel therapies targeting these cellular pathways should be considered.

Abstract 4612: HOXA13 drives hepatocytes proliferation and liver tumorigenesis in mice

Abstract Introduction: Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third most common cause of cancer related mortality worldwide. For patients suffering from advanced stage disease, the few therapeutic options available are not curative and improve patient survival by only a few months. Therefore, new molecular targets that can be explored as therapeutic options are highly needed. Class I Homeobox (HOX) genes are fundamental components of embryonic patterning and morphogenesis, with expression persisting into adulthood. They are also implicated in neoplastic transformations. However, the role of HOX genes is poorly understood and the functional relationship between the malignant phenotype and abnormal expression of HOX genes is still unclear. In this study we sought to define the role of the HOXA13 gene in hepatocarcinogenesis using in vivo models. Methods: To unravel the molecular mechanism of HOXA13 driven tumorigenesis in liver and its direct oncogenicity in vivo, a murine model of HOXA13 overexpression in liver was generated using hydrodynamic injection coupled with a transposase system. This model led to the stable and specific HOXA13 expression in C57BL6\J mouse hepatocytes up to 5 months post injection. Mouse phenotype was followed over time, from 2 weeks up to 1 year post injection. 16 mice (8 for CTRL vector and 8 for HOXA13) were injected and sacrificed for every time point. RNA sequencing was performed to monitor the transcriptomic changes over time. Results: 1 year post injection 50% (4/8) of the injected mice with HOXA13 developed liver tumors of various histological grades and types, from very well differentiated HCCs to very highly undifferentiated and cholangiocarcinoma like nodules. HOXA13 overexpression in the liver led to highly proliferative hepatocytes after only 2 weeks and the proliferative phenotype was maintained until 5 months post injection, when pre neoplastic lesions began to form. HOXA13 overexpression correlated not only with proliferation but also with the DNA damage marker yH2AX, suggesting a possible mechanism of tumorigenesis driven by genome instability. Gene set enrichment analysis of RNA-seq performed on whole liver extracts of 2 week old mice and tumors showed that the main pathways involved in HOXA13 expression are cell cycle, in particular G2/M transition and mitotic assembly checkpoint, angiogenesis, TP53 pathway, IL6JAKSTAT3 signaling, Notch signaling and epithelial to mesenchymal transition. Conclusion: Our study highlights the key role of HOXA13 as a potential novel oncogene in HCC development and suggests possible mechanisms through which it drives liver tumorigenesis. We expect that the generated data in vivo, coupled with mass spectrometry and ChIP sequencing experiments performed in vitro, will further help us identify downstream effectors of HOXA13 thus providing new potential therapeutic targets for HCC. Citation Format: Gaia Bianco, Hesam Montazeri, Luca Quagliata, Tracy O'Connor, Ursula Ehmer, Rupert Oellinger, Mathias Matter, Christofori Gerhard M., Charlotte K.Y. Ng, Salvatore Piscuoglio, Mathias Heikenwaelder, Luigi M. Terracciano. HOXA13 drives hepatocytes proliferation and liver tumorigenesis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4612.

Abstract 3838: Targeting epigenetic regulator BMI-1 in alveolar rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is an extremely aggressive soft tissue sarcoma which affects mainly children. There are two subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). ARMS is characterized by PAX-FOXO1 fusion proteins, whereas subsets of ERMS harbor alterations within RAS and TP53 pathways. Currently, the outcomes for ARMS (especially when metastatic) remain dismal, thus underscoring the urgent need to identify novel targets for this cancer. The genomic landscape of many pediatric cancers, including ARMS, is relatively sparse. This led us to ask whether key epigenetic factors are driving tumor aggressiveness and could constitute novel approaches for treating ARMS. Notably, the epigenetic complexes PRC1 and PRC2 are overexpressed in a variety of sarcomas and are associated with worse overall survival. We took a hypothesis-based approach and focused on PRC1. We discovered that B lymphoma Mo-MLV insertion region 1 (BMI-1), a protein member of PRC1, is overexpressed in ARMS cells. BMI-1 is a known oncogene in other cancers, but its potential oncogenic role in ARMS and other pediatric malignancies has not yet been interrogated; thus we aim to study it within this context. Methods: To analyze the function of BMI-1 in ARMS, we depleted the protein in ARMS cell line models by both shRNA/siRNA knockdown and measured expression, cell proliferation and apoptosis. We utilized two small molecule inhibitors, PTC-209 and PTC-028, to obtain IC50s in these cell lines, then determined effects on cell proliferation and apoptosis. Results: We examined RNA-Seq tumor datasets and determined that BMI1 is robustly expressed in ARMS tumors. Additionally, we confirmed that BMI-1 is also overexpressed in ARMS cell lines at the levels of RNA and protein. Next, we depleted BMI-1 using multiple shRNAs and siRNAS and found that this led to striking (~70%) decreases in cell growth. We also observed increased levels of apoptosis within knockdown cells. Given these results, we asked whether small molecule inhibitors of BMI-1 mediated similar phenotypes, and so we used the inhibitors PTC-209 and PTC-028. PTC-209 is a first-generation BMI-1 inhibitor, while PTC-028 is a second-generation orally available inhibitor with higher potency. Both compounds inhibited BMI-1 function and greatly reduced cell proliferation in ARMS cell lines within the nanomolar range; however, as expected, PTC-028 showed a more pronounced effect compared to PTC-209. Conclusions: BMI1 supports proliferation and survival in cell line models of ARMS. Both chemical and pharmacologic inhibition of BMI1 led to striking decreases in cell proliferation. Currently, we are further investigating the molecular impact of BMI1 inhibition, with plans to investigate its effectiveness within an in vivo ARMS model. Targeting BMI-1 pharmacologically could provide a novel therapeutic option for patients with ARMS and may apply more broadly to other sarcomas. Citation Format: Cara E. Shields, Selma M. Cuya, Sarah K. Chappell, Komal Rathi, Shiv Patel, Sindhu Potlapalli, Robert W. Schnepp. Targeting epigenetic regulator BMI-1 in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3838.

Abstract 4646: Loss of nuclear HoxA10 is associated with proliferation of testicular germ cell tumors

Abstract Background: HOXA10 is a key transcriptional factor that regulates testis development as reported from previous transgenic mouse models and human inherited diseases. However, whether it also plays important roles in promoting the development of testicular cancer is not well understood. Objective: To study the expression of HOXA10 and its regulated signaling pathways in testicular cancers. Design, Setting, and Participants: A tissue microarray was constructed with benign and cancerous testis. TCam2, NT-2, and NCCIT cell models were applied in this study. Intervention: Immunohistochemistry and immunofluorescence were performed to measure the expression and cellular localization of HOXA10 in testicular cancer tissues and cell models. Cell proliferation and cell cycling rates were determined by BrdU incorporation and flow cytometry assays. HOXA10 transcriptomes were profiled with Ampliseq RNA-seq in testicular cancer cells. Immunoblotting assays were used to detect HOXA10-regulated signaling. Results: HOXA10 is a nuclear protein in benign spermatocytes. Reduced nuclear expression and increased cytoplasmic expression of HOXA10 are associated with testicular cancers. These changes are consistent in both seminoma and non-seminoma. Enhanced HOXA10 expression in testicular cancer cell models inhibits cell proliferation and delays cell cycle progression through G2/M phases. These functions of HOXA10 mainly affect the TP53, cKit, STAT3, AKT and ERK signaling pathways. Conclusions: Loss of nuclear functions of HOXA10 enhances proliferation of testicular cancer cells, suggesting that downregulation of HOXA10 transcription activity may promote the development of testicular cancers. Citation Format: RuiQi Chen, Haolong Li, Yinan Li, Ladan Fazli, Martin Gleave, Lucia Nappi, Xuesen Dong. Loss of nuclear HoxA10 is associated with proliferation of testicular germ cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4646.

Integrated Analysis of TP53 Gene and Pathway Alterations in The Cancer Genome Atlas

The TP53 tumor suppressor gene is frequently mutated in human cancers. An analysis of five data platforms in 10,225 patient samples from 32 cancers reported by The Cancer Genome Atlas (TCGA) enables comprehensive assessment of p53 pathway involvement in these cancers. More than 91% of TP53-mutant cancers exhibit second allele loss by mutation, chromosomal deletion, or copy-neutral loss of heterozygosity. TP53 mutations are associated with enhanced chromosomal instability, including increased amplification of oncogenes and deep deletion of tumor suppressor genes. Tumors with TP53 mutations differ from their non-mutated counterparts in RNA, miRNA, and protein expression patterns, with mutant TP53 tumors displaying enhanced expression of cell cycle progression genes and proteins. A mutant TP53 RNA expression signature shows significant correlation with reduced survival in 11 cancer types. Thus, TP53 mutation has profound effects on tumor cell genomic structure, expression, and clinical outlook.

Utility of Perilymph microRNA Sampling for Identification of Active Gene Expression Pathways in Otosclerosis.

Profiling of microRNA (miRNA) within perilymph samples collected at the time of stapedectomy can be used to identify active gene expression pathways in otosclerosis as compared with controls. miRNAs are small non-coding RNAs that effect gene expression by post-transcription regulation and silencing. Perilymph sampling allows for a novel way to collect material actively involved in the disease process. Perilymph was collected at time of stapedectomy, underwent a microarray analysis, and significantly expressed miRNAs were correlated to known bone morphology pathways using a cochlear transcriptome library. To determine miRNA related specifically to otosclerosis, cochlear implant controls were used for statistical analysis. A total of 321 significantly expressed miRNAs were identified within the four otosclerosis perilymph samples. miRNAs associated with 23 genes involved in bone morphology pathways were significantly expressed. A significant difference in the otosclerotic samples as compared with control was noted in miRNA expression regulating HMGA2, ITGB3, SMO, CCND1, TP53, TP63, and RBL2 gene pathways. No significant difference was noted in miRNAs expression associated with ACE, RELN, COL1A1, and COL1A2 genes which were previously correlated with otosclerosis. Perilymph miRNA profiling obtained at the time of stapedectomy consistently identifies differentially expressed genes compared with controls. Perilymph miRNA sampling with cochlear transcriptome library cross-referencing can be successfully used to identify active gene expression pathways in otosclerosis.

Abstract 3654: Recurrent deletions of 3q13.31 in human osteosarcoma commonly affect TUSC7 and LINC00901

Abstract Background: Osteosarcoma, the most common primary tumor of bone, generally harbors complex structural rearrangements. In this study, we identified frequent mono- and bi-allelic deletions of the two long non-coding RNAs TUSC7 and LINC00901, both located on the long arm of chromosome 3 (3q13.31). Their precise biological function remain largely unknown, although it was suggested that TUSC7 may participate in micro-RNA trapping and thereby could be involved in tumor suppressor gene regulation. Moreover, the presence of a TP53 responsive element upstream of their coding region suggests an involvement in the convoluted TP53 signaling pathway. Methods: Whole genome sequencing data (n=104) and Affymetrix Cytoscan arrays (n=52) were used to derive copy-number profiles of each tumor and interrogated for LINC00901 and TUSC7 deletions. These findings were validated in an independent set of 102 formalin-fixed and paraffin-embedded tissue samples using RNA in situ hybridization techniques. Results: Mono- and bi-allelic deletions were detected in 68 (43.5%) sequenced and array-profiled osteosarcomas. Of these, 34 tumors (50%) acquired deletions in both genes whilst additional focal deletions of TUSC7 and LINC00901 were acquired in 11 (16%) and 19 (28%), respectively. The four remaining cases (6%) revealed copy number losses within the intergenic region between TUSC7 and LINC00901. In addition, reduced amounts or complete losses of hybridisation signals were detected in similar proportions in the independent set of FFPE samples. Conclusions: We identified recurrent 3q13.31 deletions in 68/156 (43,5%) human osteosarcomas which seems remarkable in a tumor well known for its high amount of genomic complexity and intertumoral heterogeneity. TUSC7 and LINC00901 might act as downstream effectors of the TP53 pathway and could be functionally equivalent to TP53 inactivation in case of copy number loss. To further elucidate the consequences of 3q13.31 aberrations we aim to correlate our findings with transcriptome data which is currently ongoing. Citation Format: Baptiste Ameline, Michal Kovac, Karim H. Saba, Maxim Barenboim, Michaela Nathrath, Karolin H. Nord, Daniel Baumhoer. Recurrent deletions of 3q13.31 in human osteosarcoma commonly affect TUSC7 and LINC00901 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3654.

Abstract 4843: TP53-stabilization with APR-246 enhances antitumor effects of immune checkpoint blockade in preclinical models

Abstract An emerging body of literature suggests a role of TP53 pathway in antitumor immunity including antigen presentation and T cell proliferation. Stabilization of TP53 could therefore alter the immune tumor microenvironment, enabling the immune system to target tumor cells more effectively. APR-246 covalently modifies the core domain of cellular TP53 through the alkylation of thiol groups, leading to reactivation of endogenous TP53 activity. To further study the effect of APR-246 on antitumor immunity we used a B16 pre-clinical melanoma mouse model. In vitro treatment of B16 cells with APR-246 caused intracellular accumulation of TP53, leading to increased apoptosis. However, treatment of B16-melanoma bearing mice with APR-246 monotherapy did not result in a statistically significant change in tumor growth or survival. Analyses of the tumor immune microenvironment showed increased immune potentiating M1 polarized tumor-associated macrophages, and Granzyme B activity in CD8+ T cells, suggesting enhanced anti-tumor immunity. Concomitantly, there was increased PD-L1 expression in the macrophages, PD-1 expression on CD8+ T cells, and Foxp3+ Tregs in tumors from APR-246 treated animals. Therefore, we decided to combine anti-PD-1 antibody (RMP-1) to APR-246 treatment in tumor-bearing mice. The combination led to a significant delay in tumor growth (P < 0.001) and improved survival of B16-bearing mice compared to anti-PD1 or APR-246 monotherapies (P < 0.01). Improved responses were also seen in MC38 colorectal cancer-bearing mice (P < 0.01). The anti-tumor effects of APR-246 and anti-PD1 were T cell dependent and abrogated in hosts lacking T cells. Analyses of the tumor immune microenvironment showed that the combination decreases proliferation but increases cytolytic activity of CD8+ T cells compared to anti-PD1 alone. We next studied the effect of combining APR-246 with anti-PD1+ anti-CTLA-4 (9B9). Combination of APR-246 with dual immune checkpoint blockade using anti-PD1 and anti-CTLA-4 showed further tumor growth inhibition in B16-melanoma compared to monotherapies (P < 0.001). Our studies support a role for restoring TP53 in the tumor microenvironment and provide evidence that reactivation of TP53 by APR-246 can enhance anti-tumor immune responses and inhibit tumor growth in preclinical models. Citation Format: Arnab Ghosh, Judith Michel, Lauren Dong, Nathan Suek, Hong Zhong, Sadna Budhu, Olivier de Henau, Jedd Wolchok, Taha Merghoub. TP53-stabilization with APR-246 enhances antitumor effects of immune checkpoint blockade in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4843.

Abstract 979: Programmed death-ligand 1 expression and tumor-infiltrating immune cells in cancers associated with Li-Fraumeni syndrome

Abstract Purpose: Li-Fraumeni syndrome (LFS), due to TP53 germline mutations, is characterized by high incidence of multiple cancers. Dysregulation of the TP53 pathway is associated with changes in chemokine production and reduced tumor immune infiltration in some cancer types. Recent works also suggested that TP53 negatively regulates PDL1 expression through the non-coding RNA miR-34. To better understand immune evasion of LFS spectrum cancers and to explore the feasibility of PD1/PDL1 targeted therapies as an alternative to genotoxic treatments in LFS patients, we investigated a range of LFS-related tumor types for PDL1 expression and tumor-infiltrating immune cells. Methods: We retrospectively collected a total of 72 tumor samples (osteosarcoma=32, rhabdomyosarcoma=11, adrenocortical carcinoma=11 and breast carcinoma=18) from 60 pediatric and adult patients identified through the LFS French database and direct contact with oncologic centers. Immunochemistry analyses were performed on whole slide sections. PDL1 staining was performed using the clone E1L3N (Cell Signaling Technology) according to the standard automated protocols. Immune infiltrate was characterized by the following antibody panel: CD8, FOXP3 and CD68. For CD8+ and FoxP3+ T cells, cell density per surface unit was determined by cell counting; for CD68+ macrophages, a semi-quantitative scoring (from 0 to 4) was performed. Results: Median age of patients was 13 years (range: 6-23) for osteosarcoma, 1.7 years (range: 0.8-5) for rhabdomyosarcoma, 2.3 years (range: 0.4-6) for adrenocortical carcinoma and 27.5 years (range: 22-54) for breast carcinoma. Seven patients presented ≥2 metachronous tumors. Biopsy was performed at diagnosis for 68 patients, at relapse for 3 patients and at early progression for one patient. None of the samples presented any detectable PDL1 expression neither on tumor cells nor on tumor microenvironment. Low/very low amount of CD8+ cytotoxic T-cells was displayed, regardless of the histology. FoxP3+ regulatory T-cell infiltration varied among tumor types. Thirty-five/72 tumors (49%) did not contain regulatory T cells. Breast carcinomas presented significant amounts of regulatory T cells (16/18, 89%), but at low density for 10 out of 16 samples (62%). Sixty-two/72 tumors (86%) presented CD68+ macrophage infiltrates, with high density in 37/62 (60%) of them (score ≥ 2+). Conclusion: The absence of PDL1 expression on tumor cells and microenvironment as well as the low amount of regulatory T cells do not support the hypothesis of PDL1 induction through p53 inactivation in LFS. The role of innate immunity needs to be explored to identify alternative immunological targets. Citation Format: Emilie Saucier, Claudia Pasqualini, Louise Galmiche, Frédérique Larousserie, Marie Karanian, Véronique Minard-Colin, Birgit Geoerger, Dominique Valteau-Couanet, Laurence Brugières, Jean-Yves Scoazec. Programmed death-ligand 1 expression and tumor-infiltrating immune cells in cancers associated with Li-Fraumeni syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 979.

Prognostic subclass of intrahepatic cholangiocarcinoma by integrative molecular-clinical analysis and potential targeted approach.

Although molecular characterization of iCCA has been studied recently, integrative analysis of molecular and clinical characterization has not been fully established. If molecular features of iCCA can be predicted based on clinical findings, we can approach to distinguish targeted treatment. We analyzed RNA sequencing data annotated with clinicopathologic data to clarify molecular-specific clinical features and to evaluate potential therapies for molecular subtypes. We performed next-generation RNA sequencing of 30 surgically resected iCCA from Korean patients and the clinicopathologic features were analyzed. The RNA sequences from 32 iCCA resected from US patients were used for validation. Patients were grouped into two subclasses on the basis of unsupervised clustering, which showed a difference in 5-year survival rates (48.5% vs 14.2%, p= 0.007) and similar survival outcome in the US samples. In subclass B (poor prognosis), both data sets were similar in higher carcinoembryonic antigen and cancer antigen 19-9 levels, underlying cholangitis, and bile duct-type pathology; in subclass A (better prognosis), there was more frequent viral hepatitis and cholangiolar-type pathology. On pathway analysis, subclass A had enriched liver-related signatures. Subclass B had enriched inflammation-related and TP53 pathways, with more frequent KRAS mutations. CCA cell lines with similar gene expression patterns of subclass A were sensitive to gemcitabine. Two molecular subtypes of iCCA with distinct clinicopathological differences were identified. Knowledge of clinical and pathologic characteristics can predict molecular subtypes, and knowledge of different subtype signaling pathways may lead to more rational, targeted approaches to treatment.

S889 GRANULOCYTES AND MONO‐NUCLEAR CELLS FROM PATIENTS WITH MYELOPROLIFERATIVE DISORDERS HARBOUR PERSISTENT EVIDENCE OF DNA DAMAGE AND TP53 PATHWAY ACTIVATION THAT CORRELATES WITH JAK‐STAT ACTIVATION

Background:The BCR‐ABL‐negative myeloproliferative neoplasms (MPN) are characterised by constitutive activation of the JAK‐STAT pathway in haematopoietic progenitor cells (HPC), typically resulting from somatic mutations in one of three genes; JAK2, MPL or CALR. It is known from murine models that activation of the JAK‐STAT pathways results in hyperproliferation of HPCs in the bone marrow niche, and it has recently been shown that this leads to exhaustion and premature aging in a percentage of HPCs. The impact of the activation of TP53 in the context of cells harbouring MPN‐driver mutations remains unclear however, and whether these ‘TP53‐high HPCs’ contribute to the mature cell populations in MPNs or are out‐competed by ‘TP53‐low’, HPC remains unknown.Aims:To determine the effect of JAK‐STAT activation on DNA damage and TP53 pathways in mature myeloid cells of patients with myeloproliferative disorders.Methods:Peripheral blood samples from patients with essential thrombocythaemia (ET, n = 10), polycythaemia vera (PV, N = 11) and myelofibrosis (MF, n = 7) as well as healthy controls (NC, n = 11) were fractioned by gradient separation and bead selection into granulocyte (PMN), mononuclear (MNC) and T‐cell (TC) fraction. Transcriptomic profiling was performed by expression array and validated by RNA‐seq in a subset of patients. Cell fractionation and purity was confirmed by computational decomposition and supervised differential expression, co‐expression and gene set enrichment analysis was then performed to identify disease specific pathway mis‐regulation.Results:Both PMN and MNC fractions displayed strong enrichment for hallmark TP53 pathway activation and DNA damage pathways. As expected, no TP53 pathway activation was seen in the T‐cell fraction, confirming that this is restricted to the myeloid compartment. Interestingly, TP53 pathway activation directly correlated with JAK‐STAT pathway activation, suggesting the p53 activation results directly from the effect of the MPN‐driver mutations.Given that TP53 is capable of activating a number of biological processes including cell cycle arrest, apoptosis and autophagy we looked at expression levels of a number of transcriptional targets of TP53. There was significantly increased expression of the cyclin‐dependent kinase inhibitor p21 (CDKN1A) a well‐known transcriptional target of p53, in the MNC fraction in all disease groups, and in the PMN fractions of PV and MF. Additionally, we noted upregulation of anti‐apoptotic BCL‐XL (BCL2L1) in a manner that mimicked CDKN1A upregulation.Summary/Conclusion:These data show that mature myeloid lineages in patients with MPNs harbour activation of DNA damage and TP53 pathways with transcriptional activation of down‐stream effectors in PMN and MNC compartments that correlate with JAK‐STAT activation. Typically, engagement of TP53 pathways serves to limit proliferation of mutation harbouring cells, however the upregulation of anti‐apoptotic genes may offer a potential mechanism for the persistent of these cells. These data suggest that MPN clones may develop mechanisms that permit escape from TP53 mediated death, and offers potential insight into novel therapeutic targets.

PS1057 INDUCTION WITH FLUGA SCHEME AND MAINTENANCE WITH DECITABINE IN PATIENTS WITH ACUTE MYELOID LEUKEMIA OF 65 YEARS AND OLDER

Background: Chromosome 17 (ch17) abnormalities, especially loss of the 17p region and P53 gene mutations (p53mut) result in very low rates of cure for patients with acute myeloid leukemia (AML) treated with conventional chemotherapy or allogenic stem cell transplant (ASCT). Aims: We assessed whether such poor outcomes extend to patients with all types of ch17 abnormalities and whether p53mut has further prognostic value. Methods: We retrospectively analyzed 98 patients at our institution with AML/myelodysplastic syndrome (MDS) who had ch17 abnormalities noted on cytogenetics. p53mut was analyzed by next generation sequencing (NGS). Results: 61 patients with AML and 37 patients with MDS were included. Patient characteristics are in Table 1. 95 out of the 98 patients had 17p abnormalities as part of a complex karyotype, whereas 67 out of the 98 patients had a monosomal karyotype. Complete remission (CR) with first line treatment was similar between induction chemotherapy or hypomethylating agents, 22.9% vs 21.6 % (p = 0.33). Median overall survival (OS) for all patients was 10 months. Ch17 abnormalities were classified into those resulting in deletion of the p53 loci such as 17p deletion and monosomy 17 (17pdel/mono17) and all other ch17 abnormalities (ch17oth). Amongst patients with 17pdel/mono17with NGS available, 19 of 26 had p53mut, while for ch17oth, 13 of 17 had p53mut (p = 0.72).Patients having both p53mut and abnormal ch17 exhibited poorer OS compared to patients with no p53mut (10 vs 23 months, p = 0.02). 30 patients (19 AML, 11 MDS) underwent ASCT, with a median post-transplant OS of 11 months. For AML ASCT patients, 18 were in CR (13 with cytogenetic remission) and 1 had refractory disease at transplant, whereas for MDS patients, 3 were in CR (2 with cytogenetic remission) and 8 had stable disease. The post-ASCT survival between AML/MDS cohorts did not differ (p = 0.6), although cytogenetic CR at time of ASCT trended towards improved OS (17 vs 8 months; p = 0.6). At last follow-up, only 5 of 30 patients were alive, 4 in CR.Summary/Conclusion: AML/MDS patients with all types of ch17 abnormalities have a poor prognosis with or without ASCT, representing a distinct subset of patients amongst that that have complex cytogenetics. TP53 abnormalities detected by NGS are not limited to patients with 17pdel/mono17 and can be seen with other ch17 abnormalities. Drugs targeting the TP53 pathway and improving depth of response prior to ASCT are needed for improved outcomes in these patients.

PS1056 ACUTE MYELOID LEUKEMIA OR MYELODYSPLASTIC SYNDROME WITH CHROMOSOME 17 ABNORMALITIES AND LONG‐TERM OUTCOMES WITH OR WITHOUT HEMATOPOIETIC STEM CELL TRANSPLANTATION

Background:Chromosome 17 (ch17) abnormalities, especially loss of the 17p region and P53 gene mutations (p53mut) result in very low rates of cure for patients with acute myeloid leukemia (AML) treated with conventional chemotherapy or allogenic stem cell transplant (ASCT).Aims:We assessed whether such poor outcomes extend to patients with all types of ch17 abnormalities and whether p53mut has further prognostic value.Methods:We retrospectively analyzed 98 patients at our institution with AML/myelodysplastic syndrome (MDS) who had ch17 abnormalities noted on cytogenetics. p53mut was analyzed by next generation sequencing (NGS).Results:61 patients with AML and 37 patients with MDS were included. Patient characteristics are in Table 1. 95 out of the 98 patients had 17p abnormalities as part of a complex karyotype, whereas 67 out of the 98 patients had a monosomal karyotype.Complete remission (CR) with first line treatment was similar between induction chemotherapy or hypomethylating agents, 22.9% vs 21.6 % (p = 0.33).Median overall survival (OS) for all patients was 10 months. Ch17 abnormalities were classified into those resulting in deletion of the p53 loci such as 17p deletion and monosomy 17 (17pdel/mono17) and all other ch17 abnormalities (ch17oth). Amongst patients with 17pdel/mono17with NGS available, 19 of 26 had p53mut, while for ch17oth, 13 of 17 had p53mut (p = 0.72).Patients having both p53mut and abnormal ch17 exhibited poorer OS compared to patients with no p53mut (10 vs 23 months, p = 0.02).30 patients (19 AML, 11 MDS) underwent ASCT, with a median post‐transplant OS of 11 months. For AML ASCT patients, 18 were in CR (13 with cytogenetic remission) and 1 had refractory disease at transplant, whereas for MDS patients, 3 were in CR (2 with cytogenetic remission) and 8 had stable disease. The post‐ASCT survival between AML/MDS cohorts did not differ (p = 0.6), although cytogenetic CR at time of ASCT trended towards improved OS (17 vs 8 months; p = 0.6). At last follow‐up, only 5 of 30 patients were alive, 4 in CR.Summary/Conclusion:AML/MDS patients with all types of ch17 abnormalities have a poor prognosis with or without ASCT, representing a distinct subset of patients amongst that that have complex cytogenetics. TP53 abnormalities detected by NGS are not limited to patients with 17pdel/mono17 and can be seen with other ch17 abnormalities. Drugs targeting the TP53 pathway and improving depth of response prior to ASCT are needed for improved outcomes in these patients.image

Characteristics of POLE and POLD1 gene variations in Chinese cancer patients.

e14031 Background: POLE and POLD1 gene variations have been suggested as potential markers for immunotherapy due to their significant association with the tumor mutation burden (TMB), an effective indicator for response prediction in immunotherapy. Methods: In this study, we systematically studied the spectrum and characteristics of POLE and POLD1 gene variations from 1,392 Chinese cancer patients, and their correlation with existing immunotherapeutic markers and cancer associated genes. Results: We found that the frequency of POLE variations was not statistically different from that in COSMIC database, while the frequency of POLD1 variations in Chinese lung cancer patients was significantly higher than that in COSMIC database. Variations frequency analysis showed that c.857C > G and c.2091dupC were potential high frequency variations in Chinese cancer patients. Patients carrying POLE damaging variations were significantly younger, and patients carrying POLD1 damaging variations exhibited significantly higher frequency of MSI than POLE/POLD1 WT patients. Further studies found that patients carrying POLE exonuclease domain damaging variations, POLD1 exonuclease and non-exonuclease domain damaging variations exhibited significantly higher TMB than POLE/POLD1 WT patients, while no such difference was found in patients carrying POLE and POLD1 neutral variations. Moreover, patients with POLE exonuclease domain damaging variations showed significantly higher frequency of MMR gene and driver gene variations than POLE/POLD1 WT patients, including genes associated with RTK/RAS/RAF pathway. Patients with damaging POLD1 variations also exhibited higher frequency of MMR gene variations than POLE/POLD1 WT patients. The high frequency variant genes in patients with POLE exonuclease domain damaging variations or POLD1 damaging variations were associated with MMR, RTK/RAS/RAF, TGFβ, WNT, PI3K-Akt and TP53 pathways. Conclusions: This study identified key characteristics and domains of POLE and POLD1 gene variations that related to TMB, MSI, MMR gene variations and key driver gene variations, and provided theoretical and practical basis for patient selection based on POLE or POLD1 gene status in immunotherapy.

Integrative molecular analysis of metastatic Merkel cell carcinoma to identify predictive biomarkers of response to avelumab.

9569 Background: Avelumab, an FDA-approved human anti–PD-L1 IgG1 monoclonal antibody for patients (pts) with metastatic Merkel cell carcinoma, showed an objective response rate (ORR; by RECIST v1.1) of 31.8% in a second-line phase 2 trial (NCT02155647). We assessed the association of tumor mutational burden (TMB; nonsynonymous somatic variants/megabase), PD-L1 expression, Merkel cell polyomavirus (MCPyV) status, and CD8+ tumor-infiltrating T-cell density with ORR and survival. Molecular profiles (RNAseq and WEX) also were analyzed. Methods: Baseline tumors (n = 36) were profiled using RNAseq and WEX sequencing. PD-L1 expression (≥1% cutoff), MCPyV status, and CD8+ T-cell density at the tumor invasive margin were evaluated by IHC. MHC locus expression was measured with OptiType and loss of heterozygosity (LOH) with LOHHLA. Results: Of 36 pts profiled, 12 had a response, 27 were PD-L1+, and 23 were MCPyV+. The TMB upper tertile and quartile values were 1.34 and 3.16, respectively. Consistent with literature, MCPyV− pts had a higher median TMB (2.72) than MCPyV+ pts (0.49). PD-L1+ tumors trended toward a higher TMB. An empirical cohort-specific TMB cutoff of ≥2 was chosen to include sufficient pts per subgroup. Pts with TMB ≥2 vs TMB < 2 had higher ORR (5 of 11 [45.5%] vs 7 of 25 [28.0%]) and 6-mo PFS rates (60% vs 38%). Among pts with TMB ≥2, the highest ORRs were reported in MCPyV− (4 of 7 pts), PD-L1+ (5 of 9 pts), and CD8+ T-cell density higher than median (5 of 6 pts) subgroups. MHC expression trended with ORR and survival. Higher mean MHC expression was found in pts with CD8+ T-cell density higher than median (p < 0.05). Mutations in antigen presentation genes were detected: LOH at the HLA locus in 9 of 30 pts (28%), including 4 with a response; an NK cell activation signature was also associated with response. These data may suggest that ADCC contributes to response. Factorial analysis of gene signature scores identified signatures (eg, IFNγ, TP53 pathway) associated with MCPyV status and response. Conclusions: Responses in this data set were not attributed to any specific biomarker alone. Future analysis is focused on validating these results and identifying rational drug combinations with avelumab. Clinical trial information: NCT02155647.

IL2-STAT5 immune signatures to predict responses to PD-1 inhibition and azacitidine treatment in acute myeloid leukemia (AML): A subset analysis of a phase 2 study.

7041 Background: Combining PD-1/PD-L1 blockade with hypomethylating agents (HMA) shows encouraging preliminary efficacy in AML, but immune features predictive of response are lacking. Methods: We treated 11 relapsed/refractory (R/R) AML patients with azacitidine (AZA) and nivolumab (Nivo) in a phase 2 clinical trial ( Daver N et al Cancer Discovery 2018). Patient characteristics: median (med) age 65 years (47-73), 63% adverse cytogenetics, 27% TP53 mutated. Pretreatment bone marrow aspirates had immune-phenotypic 17-color flow analysis and NanoString RNA quantification of 1469 immune-relevant genes. Results were correlated with clinical, pathological and molecular data. Results: The median courses of AZA+Nivo administered was 3 (range 1-17). The CR/CRi rate was 45% (including 2 CR, 1 CRi, 1 CRN and 1 CRP), with a median time to response of 1.8 months (range 0.8-4.9 months). The median overall survival was 13 months with 27% patients alive at 1 year. We found significant positive correlations between proportions of T-effector cells at baseline, and CD3+, CD8+, and T-regulatory cells at end of cycle (EOC) 1 (r > 0.75, p < 0.01 for all). At EOC2, these correlations were no longer significant. However, there was a significant positive correlation between T-effector cells at baseline and T-regulatory cells (r = 1, p < 0.001) at EOC4. Using NanoString analysis, we found 105 differentially expressed genes (fold change = 1.5, p < 0.05) between responders (5/11) and non-responders (6/11) at pretreatment. IL2-STAT5, TP53 and TNF Hallmark pathways and immune response from GO gene sets were highly enriched (q < 5x10-4) in responders. We then utilized z-score distribution analysis to quantify the degree of activation of known immunologic pathways. We found that signatures highly specific to neutrophils, NK cells, T-cells and eosinophils were significantly (p < 0.05) upregulated in patients with CR compared to non-responders at pretreatment. Conclusions: Our data demonstrates that a signature suggestive of lymphocyte activation in the pretreatment BM may be associated with augmented clinical response to PD-1 based therapies. Similar underlying pathways that have consistently predicted for responses to PD-1 inhibition in solid cancers, primarily IL2-STAT5 genes, may have predictive relevance in AML. Such pretreatment flow and NanoString signatures may help select AML patients most likely to benefit from PD-1 blockade plus HMA, further enhancing the benefit-risk ratio with such therapies.

Identification of genomic alterations by circulating tumor DNA in leiomyosarcoma: A molecular analysis of 73 patients.

11044 Background: Leiomyosarcoma is a malignant mesenchymal tumor of cells of smooth muscle lineage arising commonly in retroperitoneum, uterus, large veins, and the limbs. In contrast to many sarcomas, the genetics of leiomyosarcomas are complex and there is very limited understanding of common driver mutations. Circulating Tumor DNA (ctDNA) offers a rapid and non-invasive method of next-generation sequencing (NGS) that could be utilized for diagnosis, therapy and detection of recurrence. Methods: ctDNA testing was performed using Guardant360, which detects single nucleotide variants, amplifications, fusions, and specific insertion/deletion mutations in 73 genes utilizing NGS. Blood samples from patients with leiomyosarcoma were analyzed, and results from December 2014-December 2018 were reviewed. Results: Of the 90 samples collected and analyzed, alterations were detected in 73 ctDNA samples. After exclusion of variants of uncertain significance (VUS), 63 ctDNA samples harbored cancer-associated genomic alterations. Of 73 patients, 59 were found to have one or more cancer-associated genomic alteration. 76% (45) were female with a median age of 63 (range, 38-87) years. All samples were designated metastatic. The following table shows common alterations and types of mutations. The other alterations included RAF1, ERBB2, MET, PTEN, TERT, APC and NOTCH1. In 24 of the 73 patients (33%) the genomic alterations detected by ctDNA are potentially targetable by an FDA-approved or clinical trial therapy. There were 4 (5%) patients who were found to have incidental germline TP53 mutations. Conclusions: NGS of ctDNA allows identification of genomic alterations in plasma from patients with leiomyosarcoma. Unfortunately, there is limited activity of targeted agents in leiomyosarcomas. These results suggest opportunities to develop therapy against TP53, cell cycle, and kinase signaling pathways. Further validation and prospective evaluation is warranted to investigate the clinical utility of ctDNA for patients with leiomyosarcoma.[Table: see text]

Transcriptional and functional consequences of TP53 splice mutations in colorectal cancer

TP53 mutations are common in colorectal cancer (CRC). Most TP53 sequencing studies have been restricted to coding regions, but recent studies have revealed that splice mutations can generate transcript variants with distinct tumorigenic and prognostic properties. Here, we performed unrestricted sequencing of all coding sequences and splice regions of TP53 in a single-hospital series of 401 primary CRCs. TP53 splice mutations were detected in 4% of the cases (N = 16), considerably more frequent than reported in major databases, and they were mutually exclusive to exon mutations. RNA sequencing revealed high-level expression of aberrant transcript variants in the majority of splice mutated tumors (75%). Most variants were predicted to produce truncated TP53 proteins, including one sample expressing the potentially oncogenic and druggable p53ψ isoform. Despite heterogeneous transcript structures, downstream transcriptional profiling revealed that TP53 splice mutations had similar effects on TP53 target gene expression and pathway activity as exonic mutations. Intriguingly, TP53 splice mutations were associated with worse 5-year relapse-free survival in stage II disease, compared to both TP53 wild-type and exon mutations (P = 0.007). These data highlight the importance of including splice regions when examining the biological and clinical consequences of TP53 mutations in CRC.