PTEN Alterations Research Articles

ALDH-Dependent Glycolytic Activation Mediates Stemness and Paclitaxel Resistance in Patient-Derived Spheroid Models of Uterine Endometrial Cancer.

SummaryUterine endometrial cancer is associated with poor survival outcomes in patients with advanced-stage disease. Here, we developed a three-dimensional cell cultivation method of endometrioid cancer stem-like cells with high aldehyde dehydrogenase (ALDH) activity from clinical specimens. ALDH inhibition synergized with paclitaxel to block cancer proliferation. In the clinical setting, high ALDH1A1 expression was associated with poor survival. A high level of ALDH correlated with an increase of glucose uptake, activation of the glycolytic pathway, and elevation of glucose transporter 1 (GLUT1). Blockade of GLUT1 inhibited characteristics of cancer stem cells. Similarly to ALDH inhibition, GLUT1 inhibition synergized with paclitaxel to block endometrial cancer proliferation. Our data indicated that ALDH-dependent GLUT1 activation and the resulting glycolytic activation are of clinical importance for both prognostic evaluation and therapeutic decision-making in endometrial cancer patients. In addition, the synergistic effects of taxane compounds and ALDH or GLUT1 inhibitors may serve as a new clinical treatment option for endometrial cancer.

PTEN Alterations as a Potential Mechanism for Tumor Cell Escape from PD-1/PD-L1 Inhibition

The recent approval of immune checkpoint inhibitors drastically changed the standard treatments in many advanced cancer patients, but molecular changes within the tumor can prevent the activity of immunotherapy drugs. Thus, the introduction of the inhibitors of the immune checkpoint programmed death-1/programmed death ligand-1 (PD-1/PD-L1), should prompt deeper studies on resistance mechanisms, which can be caused by oncogenic mutations detected in cancer cells. PTEN, a tumor suppressor gene, dephosphorylates the lipid signaling intermediate PIP3 with inhibition of AKT activity, one of the main effectors of the PI3K signaling axis. As a consequence of genetic or epigenetic aberrations, PTEN expression is often altered, with increased activation of PI3K axis. Interestingly, some data confirmed that loss of PTEN expression modified the pattern of cytokine secretion creating an immune-suppressive microenvironment with increase of immune cell populations that can promote tumor progression. Moreover, PTEN loss may be ascribed to reduction of tumor infiltrating lymphocytes (TILs), which can explain the absence of activity of immune checkpoint inhibitors. This review describes the role of PTEN loss as a mechanism responsible for resistance to anti PD-1/PD-L1 treatment. Moreover, combinatorial strategies between PD-1/PD-L1 inhibitors and PI3K/AKT targeting drugs are proposed as a new strategy to overcome resistance to immune checkpoint inhibition.

Abstract 2723: Defective homologous recombination DNA repair as therapeutic target in advanced chordoma

Abstract Chordomas are rare tumors of the axial skeleton and skull base with few therapeutic options and no clinically validated molecular drug targets. The value of comprehensive genomic analyses for guiding medical therapy of patients with advanced-stage chordoma is unknown. We performed whole-exome and genome sequencing of tumor and matched germline control samples from 11 patients with locally advanced or metastatic chordoma within the MASTER program, a prospective clinical sequencing program of the German Cancer Consortium. All patients were pretreated and had progressive disease prior to molecular analysis. Genomic profiling showed that advanced chordomas are frequently characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair. First, DNA copy number profiles showed high numbers of structural variants greater than 10 million base pairs in size in the majority of cases. Second, all patients harbored somatic aberrations of at least 2 genes known to be involved in HR, and 10/11 cases harbored somatic alterations in 3 or more HR pathway genes. For example, 8 patients showed heterozygous BRCA2 deletions, which were associated with heterozygous deletions of ERCC6 in 6 patients and RAD54L in 7 patients, as well as PTEN alterations (heterozygous deletion, heterozygous mutation and deletion of the wildtype allele or loss of heterozygosity). Other recurrently altered HR genes included ATR, CHEK2, FANCC, FANCD2, FANCG, RAD18, RAD51B, and XRCC3. Third, pathogenic germline alterations were detected in 3 patients. A heterozygous BRCA2 frameshift mutation (p.T3085fs*26; ACMG Class 5), a heterozygous NBN frameshift mutation (p.K219Nfs*16; ACMG Class 5), and a heterozygous CHEK2 missense mutation (p.R145W; ACMG Class 4) were accompanied by somatic deletion of the respective wildtype alleles. Fourth, a mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and coincided with genomic instability. The high prevalence of an HR deficiency “footprint” in chordoma patients prompted us to explore the clinical efficacy of the poly(ADP-ribose) polymerase(PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells. Olaparib treatment of a patient whose tumor showed a prominent exposure to an HR deficiency-associated mutational signature, a high degree of genomic instability, and 13 heterozygous HR gene alterations halted tumor growth for 10 months. Whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain, providing novel insight into the mechanisms of PARP inhibitor resistance. In summary, our study has uncovered a key biological feature of advanced chordoma that represents an immediately actionable therapeutic target and provides a rationale for genomics-guided clinical trials of PARP inhibition in this intractable tumor entity. Citation Format: Stefan Gröschel, Daniel Hübschmann, Francesco Raimondi, Peter Horak, Gregor Warsow, Martina Fröhlich, Barbara Klink, Laura Gieldon, Barbara Hutter, Kortine Kleinhenz, David Bonekamp, Oliver Marschal, Priya Chudasama, Jagoda Mika, Marie Groth, Sebastian Uhrig, Stephen Krämer, Christoph Heining, Christoph Heilig, Daniela Richter, Eva Reisinger, Katrin Pfütze, Roland Eils, Stephan Wolf, Christof von Kalle, Christian Brandts, Claudia Scholl, Wilko Weichert, Stephan Richter, Sebastian Bauer, Roland Penzel, Evelin Schröck, Albrecht Stenzinger, Richard Schlenk, Benedikt Brors, Robert Russell, Hanno Glimm, Matthias Schlesner, Stefan Fröhling. Defective homologous recombination DNA repair as therapeutic target in advanced chordoma [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 2723.

Detection and Correlation of Single and Concomitant TP53, PTEN, and CDKN2A Alterations in Gliomas.

Gliomas are the most frequent primary tumors of central nervous system and represent a heterogeneous group of tumors that originates from the glial cells. TP53, PTEN, and CDKN2A are important tumor suppressor genes that encode proteins involved in sustaining cellular homeostasis by different signaling pathways. Though genetic alterations in these genes play a significant role in tumorigenesis, few studies are available regarding the incidence and relation of concomitant TP53, PTEN, and CDKN2A alterations in gliomas. The purpose of this study was to evaluate the occurrence of mutation and deletion in these genes, through single-strand conformational polymorphism, array-comparative genomic hybridization, and fluorescence in situ hybridization techniques, in 69 gliomas samples. Molecular results demonstrated a significant higher prevalence of TP53, PTEN, and CDKN2A alterations in astrocytoma than other tumor subtypes, and heterozygous deletion was the most frequent event. In addition, a significant association was observed between TP53 and CDKN2A alterations (p = 0.0424), which tend to coexist in low grade astrocytomas (5/46 cases (10.9%)), suggesting that they are early events in development of these tumors, and PTEN and CDKN2A deletions (p = 0.0022), which occurred concomitantly in 9/50 (18%) patients, with CDKN2A changes preceding PTEN deletions, present preferably in high-grade gliomas.

Paired genomic analysis of squamous cell carcinoma transformed from EGFR-mutated lung adenocarcinoma

ObjectivesAdenocarcinoma (ADC) to squamous cell carcinoma (SCC) transformation (AST) is reported in epidermal growth factor receptor (EGFR)-mutated non–small cell lung cancer after tyrosine kinase inhibitor (TKI) failure. However, little is known about the underlying genomic changes during the AST process. Materials and methodsWe retrospectively reviewed our tissue database collected after first- or second- generation EGFR TKI resistance (n = 263) and identified 3 cases of AST. The additional case was acquired from the osimertinib resistance sample. Deep target sequencing (381 genes) using paired samples from 4 patients with AST after EGFR TKI treatment was performed. The histology of each sample was confirmed by TTF-1 and p63 immunohistochemistry. The patients received first- or second-generation EGFR TKI as an initial treatment. ResultsOverall incidence of AST was 1.1% (3/263). Transformed SCC acquired genomic alterations related to the PI3K/AKT/mTOR pathway, in addition to the initial EGFR mutation. In a representative case, two separate sub-clones, with a PTEN nonsense mutation and EGFR p.T790 M mutation, were observed without histologic transformation at the time of gefitinib resistance. After subsequent treatment with osimertinib, SCC transformation was observed with the disappearance of the EGFR p.T790 M mutation and acquired copy number loss in PTEN. Adopting the sub-clonal fraction model elucidates the sub-clonal evolution process of the PTEN mutant sub-clone toward AST under the background of EGFR mutation. The rest of the transformed samples also had acquired genomic alterations in PTEN, LKB1, PIK3CA, or RICTOR, which are related to the PI3K/AKT/mTOR pathway. ConclusionsPaired genomic analysis from our sample provides early clinical evidence of the ADC to SCC lineage transition that might be provoked by an alteration in the PI3K/AKT/mTOR pathway during EGFR TKI treatment. This finding could potentially broaden the known spectrum of EGFR TKI resistance mechanisms.

Genomic predictors of benefit of docetaxel (D) and next-generation hormonal therapy (NHT) in metastatic castration resistant prostate cancer (mCRPC).

5018 Background: Predictive genomic biomarkers in mCRPC remain elusive. Prior studies suggest that tumor suppressor (TS) loss is prognostic, and may result in less benefit from NHT, but no impact on D efficacy. We assessed genomic predictors of differential benefit of androgen receptor-targeted therapy and chemotherapy for mCRPC. Methods: Patients with mCRPC and targeted exome sequencing of biopsies obtained after metastatic diagnosis were identified (n=109). Patients with pure small cell histology (n=6) were excluded. Time from NHT or D start to clinical/radiographic progression (time to treatment failure, TTTF) was estimated by Kaplan-Meier method, with censoring at next therapy or last follow-up for non-progressors. Results: 80.1% of patients had bone and/or lymph node-only metastases at mCRPC diagnosis. In total, 87/103 (84.5%) and 61/103 (59.2%) received NHT and D for mCRPC, respectively. Median overall survival was 4.5 years from first mCRPC. The frequency and predictive association of selected recurrently-altered genes are detailed in the table. PTEN alterations (alts) were associated with worse TTTF on NHT, but not D, and a similar trend was observed with BRCA2. Biallelic RB1 loss was strongly predictive, conferring significantly shorter TTTF on both NHT and D. A score based on presence of tumor PTEN alt (1) and/or biallelic RB1 alt (1) was predictive of TTTF on NHT (median TTTF of score 0 vs 1 vs 2: 14.7 vs 12 vs 3.8 months; log rank p=0.003). Conclusions: The presence of single or compound PTEN and RB1 alts predict poorer outcomes with NHT for mCRPC. Chemotherapy may be a preferred therapeutic strategy for this patient population. [Table: see text]

Molecular profile of early-stage and advanced-stage of renal cell cancer in China.

e16071 Background: The mutation spectrum has been extensively studied in Renal Cell Cancer (RCC), a heterogeneous disease. While major investigations have focused on metastatic RCC (mRCC). This study intended to explore the molecular characteristics between early-stage (stage I/Ⅱ) and advanced-stage (Ⅲ/Ⅳ) of RCC patients (pts). Methods: 36 tumor specimens were obtained from individual pts diagnosed RCC, including 22 tissues paired with blood samples. Somatic mutations were identified via targeted next generation sequencing with Acornmed panel including 808 genes associated with tumor development. Sequencing data were analyzed to call tumor specific single nucleotide variants (SNV), small insertions and deletions (InDels), copy number alterations (CNA) and chromosomal rearrangements. Clinical data of a cohort from TCGA who had RCC was collected and analyzed. Results: Among the 36 pts enrolled, 61% were early-stage group and 39% were advanced-stage group. The most frequently mutated genes in RCC were VHL (50%), PBRM1 (14%), BAP1 (11%), TP53 (11%) and PIK3CA (6%). Truncating mutations were the most frequent alterations accounting for 66% (33/50) of genetic alterations. Mutations in VHL, BAP1, PBRM1 and TP53 were shared by both early and advanced RCC. While mutations in PIK3CA, CDKN2C, EGFR, FAMCA, GRIN2A, KDM6A, SOX17, TSC1 and APC were only detected in early stage group. KNSTRN, KRAS, MYD88, NF2, ATM, PTEN and STAT5B mutations were only detected in advanced group. Among 22 plasma samples, genomic alterations were detected in 40.9% (9/22) of pts postoperatively, such as TP53, PIK3CA, PTEN, and APC. Functional annotation clustering revealed that 4/9 in these genes were tumor suppressors, which negatively regulated apoptosis. Pts with genomic alterations in TP53 (p = 0.0034), PIK3CA (p = 0.0015) or PTEN (p = 0.00017) had worse OS significantly (TCGA). Conclusions: VHL, PBRM1, BAP1 were the most important driver genes mainly detected in all stages of RCC. The heterogeneity between early and advanced-stage may be related with clinical status. Gene alterations in plasma ctDNA postoperatively should have the potential to stratify patients with different prognostic outcome.

Comprehensive Genetic Characterization of Human Thyroid Cancer Cell Lines: A Validated Panel for Preclinical Studies.

Thyroid cancer cell lines are valuable models but have been neglected in pancancer genomic studies. Moreover, their misidentification has been a significant problem. We aim to provide a validated dataset for thyroid cancer researchers. We performed next-generation sequencing (NGS) and analyzed the transcriptome of 60 authenticated thyroid cell lines and compared our findings with the known genomic defects in human thyroid cancers. Unsupervised transcriptomic analysis showed that 94% of thyroid cell lines clustered distinctly from other lineages. Thyroid cancer cell line mutations recapitulate those found in primary tumors (e.g., BRAF, RAS, or gene fusions). Mutations in the TERT promoter (83%) and TP53 (71%) were highly prevalent. There were frequent alterations in PTEN, PIK3CA, and of members of the SWI/SNF chromatin remodeling complex, mismatch repair, cell-cycle checkpoint, and histone methyl- and acetyltransferase functional groups. Copy number alterations (CNA) were more prevalent in cell lines derived from advanced versus differentiated cancers, as reported in primary tumors, although the precise CNAs were only partially recapitulated. Transcriptomic analysis showed that all cell lines were profoundly dedifferentiated, regardless of their derivation, making them good models for advanced disease. However, they maintained the BRAFV600E versus RAS-dependent consequences on MAPK transcriptional output, which correlated with differential sensitivity to MEK inhibitors. Paired primary tumor-cell line samples showed high concordance of mutations. Complete loss of p53 function in TP53 heterozygous tumors was the most prominent event selected during in vitro immortalization. This cell line resource will help inform future preclinical studies exploring tumor-specific dependencies.

Bridging genomics and phenomics of gastric carcinoma.

Genetic alterations are the starting point leading to numerous changes in clinical and pathologic features (phenotypes) of individual cancers; however, their inter-relationships in gastric cancers (GC) are unclear. We performed massive parallel sequencing of 381 cancer-related genes and compared the results with clinical and pathologic findings in 330 GC. High tumor mutation burden (TMB) accounted for 11% of GC (n = 37) and all 19 MSI-H GCs were high TMB. High TMB was significantly more frequent in intestinal-type by Lauren, tumor with higher host cellular immune response, earlier AJCC stage and favorable prognosis. The most significantly mutated genes were TP53 (54%), ARID1A (23%), CDH1 (22%), PIK3CA (12%), RNF43 (10%) and KRAS (9%). For receptor tyrosine kinases, amplifications detected by immunohistochemistry were higher than sequencing (HER2, 9.1% vs. 5.8%; EGFR, 11.2% vs. 6.1%; FGFR2, 4.6% vs. 3.9%, c-MET, 3.4% vs. 0.9%). PTEN protein loss (22%) correlated well with underlying PTEN alterations while ATM loss (27%) was not significantly correlated with genetic alterations of ATM. p53 protein expression predicted alterations of TP53 with high sensitivity (97.8%) and low (15.9%) specificity. The poorly cohesive histology/CDH1-mutant GC subgroup showed the worst survival (p < 0.001). PD-L1 expression was significantly associated with MSI-H, MLH1 loss, ATM loss, MET positivity, higher host immune response, and genetic alterations of ARID1A, BRD3, PIK3CA, KRAS, MAP3K13, CDH2, PTEN and ESR1. The merged clinical, pathology and genomics of GC provide a better understanding of GC and new insights into the treatment of GC.

Identification of genetic alterations associated with primary resistance to EGFR-TKIs in advanced non-small-cell lung cancer patients with EGFR sensitive mutations

BackgroundIdentification of activated epidermal growth factor receptor (EGFR) mutations and application of EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have greatly changed the therapeutic strategies of non-small-cell lung cancer (NSCLC). However, the long-term efficacy of EGFR-TKI therapy is limited due to the development of drug resistance. The aim of this study was to investigate the correlation between the aberrant alterations of 8 driver genes and the primary resistance to EGFR-TKIs in advanced NSCLC patients with activated EGFR mutations.MethodsWe retrospectively reviewed the clinical data from 416 patients with stage III/IV or recurrent NSCLC who received an initial EGFR-TKI treatment, from April 2004 and March 2011, at the Sun Yat-sen University Cancer Center. Several genetic alterations associated with the efficacy of EGFR-TKIs, including the alterations in BIM, ALK, KRAS, PIK3CA, PTEN, MET, IGF1R, and ROS1, were detected by the routine clinical technologies. The progression-free survival (PFS) and overall survival (OS) were compared between different groups using Kaplan–Meier survival analysis with the log-rank test. A Cox regression model was used to estimate multivariable-adjusted hazard ratios (HRs) and their 95% confidence intervals (95% CIs) associated with the PFS and OS.ResultsAmong the investigated patients, 169 NSCLC patients harbored EGFR-sensitive mutations. EGFR-mutant patients having PTEN deletion had a shorter PFS and OS than those with intact PTEN (P = 0.003 for PFS, and P = 0.034 for OS). In the combined molecular analysis of EGFR signaling pathway and resistance genes, we found that EGFR-mutant patients coexisted with aberrant alterations in EGFR signaling pathway and those having resistant genes had a statistically poorer PFS than those without such alterations (P < 0.001). A Cox proportional regression model determined that PTEN deletion (HR = 4.29,95% CI = 1.72–10.70) and low PTEN expression (HR = 1.96, 95% CI = 1.22–3.13), MET FISH + (HR = 2.83,95% CI = 1.37–5.86) were independent predictors for PFS in patients with EGFR-TKI treatment after adjustment for multiple factor.ConclusionsWe determined that the coexistence of genetic alterations in cancer genes may explain primary resistance to EGFR-TKIs.

Age-related variation in gene alteration frequency in metastatic prostate cancer.

178 Background: Despite advances in therapy, metastatic prostate cancer (mPCA) remains lethal. Younger men with mPCA have a significantly greater risk of mPCA-related death compared to men diagnosed later in life. This suggests that early-onset mPCA is a uniquely aggressive subtype of disease, however, the genomic drivers have not been explored. Here, we analyze data from 2,196 mPCA specimens profiled as part of routine clinical care. Methods: Comprehensive genomic profiling was performed by hybridization capture of exonic regions from either 236, 315 or 324 cancer-related genes and select introns from 19 genes commonly rearranged in cancer. Libraries were sequenced to high, uniform coverage and assessed for all classes of genomic alterations. Tumor mutational burden (TMB) was determined from 1.1 Mb of sequence and microsatellite instability (MSI) from 114 loci. Results: 336 (15.3%) men were &lt; 60 years old (yo), and 1,860 (84.7%) were ≥60 yo at time of biopsy. Lymph node, liver, and bone were the three most common metastatic sites in both groups, accounting for 75% of all specimens. Patients &lt; 60 yo harbored significantly fewer AR and PTEN point mutations when compared to patients ≥60 yo (OR = 0.48, p &lt; 0.001; OR = 0.51, p = 0.01). Copy number alterations in PTEN and CDKN2A/B were significantly more common among young patients (OR = 1.46, p = 0.003; OR = 1.91, p = 0.04). AR copy number alterations were significantly less common among young patients (OR = 0.57, p &lt; 0.001). Although uncommon in mPCA, markers of genomic instability including MSH2 mutations and MSI were both significantly less common among patients &lt; 60 yo (0.00% vs. 1.29%, p = 0.04; 0.89% vs. 3.06%, p = 0.03). There were no significant differences in the frequency of TP53, APC, BRCA2, ATM, and CDK12 point mutations, MYC copy number alterations, or TMPRSS2-ERG fusions. The average TMB was not significantly different between young and old patients, with 3.26 and 5.79 mutations per Mb, respectively (p = 0.19). Conclusions: Younger men with mPCA appear to represent a genomically distinct disease subtype. To better define this population, we are prospectively investigating the genomic, genetic, and environmental risk factors through an ongoing PCF funded clinical trial.

Gliosarcoma Is Driven by Alterations in PI3K/Akt, RAS/MAPK Pathways and Characterized by Collagen Gene Expression Signature.

Gliosarcoma is a very rare brain tumor reported to be a variant of glioblastoma (GBM), IDH-wildtype. While differences in molecular and histological features between gliosarcoma and GBM were reported, detailed information on the genetic background of this tumor is lacking. We intend to fill in this knowledge gap by the complex analysis of somatic mutations, indels, copy number variations, translocations and gene expression patterns in gliosarcomas. Using next generation sequencing, we determined somatic mutations, copy number variations (CNVs) and translocations in 10 gliosarcomas. Six tumors have been further subjected to RNA sequencing analysis and gene expression patterns have been compared to those of GBMs. We demonstrate that gliosarcoma bears somatic alterations in gene coding for PI3K/Akt (PTEN, PI3K) and RAS/MAPK (NF1, BRAF) signaling pathways that are crucial for tumor growth. Interestingly, the frequency of PTEN alterations in gliosarcomas was much higher than in GBMs. Aberrations of PTEN were the most frequent and occurred in 70% of samples. We identified genes differentially expressed in gliosarcoma compared to GBM (including collagen signature) and confirmed a difference in the protein level by immunohistochemistry. We found several novel translocations (including translocations in the RABGEF1 gene) creating potentially unfavorable combinations. Collected results on genetic alterations and transcriptomic profiles offer new insights into gliosarcoma pathobiology, highlight differences in gliosarcoma and GBM genetic backgrounds and point out to distinct molecular cues for targeted treatment.

Interaction between IGF2-PI3K axis and cancer-associated-fibroblasts promotes anal squamous carcinogenesis.

Anal squamous cell carcinoma (ASCC) is a rare tumour, but its incidence is increasing. Standard chemoradiotherapy fails to cure 20% of patients and no targeted therapy is currently approved for recurrent ASCC. The PI3K/Akt/mTOR pathway is frequently altered in this poorly characterised carcinoma. IGF2 was identified here as a key factor in ASCC oncogenesis, as IGF2 was shown to play a crucial role in the PI3K pathway with frequent (~60%) and mutually exclusive genomic alterations in IGF2, IGF1R, PTEN and PIK3CA genes. We also demonstrated that IGF2 expression is mainly due to cancer-associated fibroblasts and that IGF2 secreted by cancer-associated fibroblasts from ASCC samples promotes proliferation of a human ASCC cell line via IGF2 paracrine signalling. Altogether, these results highlight the key role of the IGF2/PI3K axis, and the major role of cancer-associated fibroblasts in tumour growth via IGF2 secretion, suggesting a major role of IGF2/IGF1R inhibitors in ASCC therapies.

Abstract P5-12-02: PTEN alterations and tumor mutational burden (TMB) as potential predictors of resistance or response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC)

Abstract Purpose: To date no biomarker has been identified that predicts response to ICI in mTNBC. This study aimed to explore if tumor genomic alterations correlate with efficacy of PD-1/PD-L1 inhibition in patients (pts) with mTNBC. Methods: Demographic, treatment response, and long-term outcome data were collected on patients with mTNBC treated at Dana-Farber Cancer Institute (DFCI) under several clinical trials incorporating PD-1/PD-L1 inhibitors, given as monotherapy or combined with chemotherapy (CT). Pts included in this analysis had available results of targeted exon sequencing performed using Oncopanel, our institutional gene sequencing panel, on archival tumor tissue. TMB was calculated by determining the number of non-synonymous somatic mutations that occur per megabase of exonic sequence data across all genes on the panel. High TMB was defined as 310 mutations/megabase. TMB and gene alterations were correlated with objective response rate (ORR) per RECIST 1.1, progression-free (PFS) and overall survival (OS). Results: A total of 50 pts with mTNBC were included in this analysis. At baseline, the median age was 55.9 years (31.8–75.9), 60% had ECOG 0 and 40% had ECOG 1, 72% had visceral metastasis, and 46% had received 31 prior lines of systemic therapy in the metastatic setting. While 26% of pts received monotherapy [pembrolizumab (n=7, NCT02447003); atezolizumab (n=6; NCT01375842)], 74% received combination with CT [pembrolizumab plus eribulin (n=31; NCT02513472); atezolizumab plus nab-paclitaxel (n=6; NCT01633970)]. PTEN alterations were present in 30% of pts (mutations = 7; one copy number loss = 7; two copy number loss = 1). Median follow-up was 14 months (1–40). Pts with tumors harboring PTEN alterations had lower ORR (7% vs 57%; P&amp;lt;0.001), shorter median PFS (2.3 vs 6.3 months; P=0.027), and shorter median OS (8.1 vs 20.1 months; P=0.012) compared to pts without PTEN alterations. The median TMB was 6.6 mut/Mb (1.2–50.8), and 23% of pts had a high TMB. While high TMB was not associated with higher ORR (P=0.56), it was associated with better median PFS (16.5 vs 2.4 months; P=0.017), and better median OS (not reached vs 13.5 months; P=0.026). Both PTEN status and TMB remained significantly associated with PFS in the multivariable model. Only PTEN status remained significantly associated with OS in the multivariable analysis with the same covariables. Ongoing analysis to better understand if these predictors are specific for predicting benefit to immunotherapy and/or a marker of chemotherapy resistance will be presented at the symposium. Conclusion: PTEN genomic alterations and TMB may impact benefit from PD-1/PD-L1 inhibitors largely administered with chemotherapy in mTNBC. These observations warrant prospective validation and may inform the importance of stratifying pts according to these characteristics in future randomized studies with ICI. Table 1.Multivariable analysis for PFS Hazard ratioConfidence Intervalp-valueCombination therapy0.420.16 – 1.130.009Visceral metastasis1.310.63 – 2.770.46Previous lines of therapy1.020.09 – 0.700.85ECOG 12.11.06 – 1.280.034PTEN altered3.741.65 – 8.440.002Hypermutated tumors0.850.75 – 0.970.011 Citation Format: Barroso-Sousa R, Tyekucheva S, Pernas-Simon S, Exman P, Jain E, Garrido-Castro AC, Hughes M, Bychkovsky B, Di Lascio S, Umeton R, Files J, Lindeman NI, MacConaill LE, Hodi FS, Krop IE, Dillon D, Winer EP, Wagle N, Lin NU, Mittendorf EA, Tolaney SM. PTEN alterations and tumor mutational burden (TMB) as potential predictors of resistance or response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-02.

Abstract P2-08-19: Exploratory biomarker analyses of FAIRLANE, a double-blind placebo (PBO)-controlled randomized phase II trial of neoadjuvant ipatasertib (IPAT) + paclitaxel (PAC) for early triple-negative breast cancer (TNBC)

Abstract Background: The oral AKT inhibitor IPAT is being evaluated in cancers with a high prevalence of PI3K/AKT pathway activation. In the PBO-controlled randomized phase II FAIRLANE trial (NCT02301988), adding IPAT to PAC as neoadjuvant therapy for TNBC led to a numerical increase in pathologic complete response (pCR) in unselected patients (17.1% vs 13.3%), with a greater treatment effect in patients with PIK3CA/AKT1/PTEN-altered tumors (17.9% vs 11.8%). The addition of IPAT also led to an increase in complete response (CR) by MRI (27.6% vs 13.3%) that was enhanced in patients with PIK3CA/AKT1/PTEN-altered tumors (39.3% vs 8.8%) [Oliveira, AACR 2018]. We report an exploratory analysis performed to provide better understanding of potential biomarkers for response. Methods: Pretreatment tumor samples were evaluated for genomic alterations using the FoundationOne® (Foundation Medicine) assay (n=144) and gene expression by RNA-Seq (n=92). Samples were classified into TNBC subtypes based on the method developed by Lehmann and Pietenpol [Lehmann, J Clin Invest 2011]. Tumor-infiltrating lymphocytes (TILs) were quantified using the Salgado method [Salgado, Ann Oncol 2015] (n=135). Results: Of 62 patients (43%) with PIK3CA/AKT1/PTEN-altered tumors, 21 had an activating mutation in PIK3CA or AKT1 and 47 had an alteration in PTEN (6 [3 in each arm] had both PIK3CA mutation and PTEN alteration). Although only 3 patients with PIK3CA/AKT1-mutant tumors achieved a pCR, there was an increased rate of MRI CR with the addition of IPAT to PAC [Table]. In patients with PTEN alterations, both pCR rate and MRI CR rate were increased with IPAT. In patients treated with PBO + PAC, all 4 pCR patients evaluable by RNA-Seq were of the immunomodulatory (IM) subtype. However, in the IPAT + PAC arm, pCRs were also seen in patients with basal-like 1 (BL-1), mesenchymal (M), and mesenchymal stem-like (MSL) subtypes. Consistent with this observation, in the PBO + PAC arm, samples from patients achieving a pCR had significantly higher levels of stromal TILs than those from patients who did not have a pCR, while no difference was observed in the IPAT + PAC arm. Response, n (%)PIK3CA/AKT mutation (n=21)PTEN alteration (n=47) IPAT + PAC (n=11)PBO + PAC (n=10)IPAT + PAC (n=21)PBO + PAC (n=26)pCR1 (9%)2 (20%)4 (19%)3 (12%)CR by MRI5 (45%)1 (10%)8 (38%)2 (8%) Conclusions: This retrospective exploratory biomarker analysis of the phase II FAIRLANE trial of neoadjuvant IPAT for TNBC provides insight into the potential heterogeneity of response and resistance to taxane therapy. The results also hint that response to PAC alone is dependent on baseline immune infiltration and that this dependency might be relieved with the addition of AKT inhibition. Citation Format: Wongchenko MJ, Oliveira M, Saura C, Nuciforo P, Calvo I, Andersen J, Passos Coelho JI, Gil Gil M, Bermejo B, Patt DA, Ciruelos E, Singel SM, Maslyar DJ, Xu N, de la Peña L, Baselga J, Gendreau S, Isakoff SJ. Exploratory biomarker analyses of FAIRLANE, a double-blind placebo (PBO)-controlled randomized phase II trial of neoadjuvant ipatasertib (IPAT) + paclitaxel (PAC) for early triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-19.

Abstract P5-11-01: Phamacodynamic and circulating tumor DNA evaluation in a phase I study of GDC-0927, a selective estrogen receptor antagonist/ degrader (SERD)

Abstract Background: Modulation of estrogen activity and/or synthesis is the mainstay therapeutic strategy in the treatment of ER positive breast cancer. However, despite the effectiveness of available endocrine therapies, many patients ultimately relapse or develop resistance to these agents via estrogen-dependent and estrogen-independent mechanisms, including mutations in ESR1 affecting the ER ligand binding domain that drive ER-dependent transcription and proliferation in the absence of estrogen. Based on preclinical and clinical data, SERDs are expected be effective in patients harboring ESR1 mutations. Biomarker analysis was performed on plasma and tumor samples from the Phase I study of GDC-0927 in metastatic breast cancer (Dickler et al, SABCS 2017) with the goal of evaluating activity in both ESR1 mutant and wildtype tumors, and to assess ER pathway modulation. Methods: Hotspot mutations in ESR1, PIK3CA, and AKT1 were analyzed in baseline, on-treatment and end of treatment plasma derived circulating tumor DNA (ctDNA) using the BEAMing assay in patients treated at multiple dose levels of GDC-0927. A subset of samples was analyzed with Foundation Medicine's next generation sequencing ctDNA assay (FACT), which covers genomic alterations in 62 commonly altered genes. Paired pre- and on-treatment biopsies were collected to assess ER pathway modulation. ER, PR, and Ki67 protein levels were analyzed by immunohistochemistry. Gene expression analysis was performed using Illumina's RNA Access library preparation kit followed by paired-end (2x50b, 50M reads) sequencing on the HiSeq. Results: Baseline and on-treatment plasma samples were available for 40 patients. ESR1 and PIK3CA mutations were observed in 52% and 33% of patient baseline samples, respectively (BEAMing method). Mutant allele frequencies (MAF) generally declined in the first on-treatment samples collected for both ESR1 (16 out of 21 samples) and PIK3CA (7 out of 12 samples). The majority of the reductions were greater than 95% relative to baseline. Increases in ESR1 MAFs were observed in later time-points and were not associated with any particular ESR1 mutation. There were six instances for which an ESR1 mutation was detected in an on-treatment sample that was not detected in the baseline sample, three at L536P and one each at D538G, L536H, and S463P, and four out of six with MAFs close to the limit of detection. The FACT assay also detected alterations in CDH1, NF1, PTEN, and TP53 in baseline samples. The relationship between MAF changes and clinical benefit to GDC-0927 will be presented. A predefined, experimentally-derived set of ER target genes were evaluated in pre- and on-treatment tumor biopsy pairs from six patients. Four of the six patients showed evidence of suppression in ER pathway activity, one patient treated at the 1000 mg dose level and three at the 1400 mg dose. The degree of pathway suppression was associated with pre-treatment pathway levels and decreases of ER and Ki67 protein levels. Conclusions: We report here evidence of consistent reduction of ESR1 and PIK3CA ctDNA in patients treated with GDC-0927. ER pathway suppression was observed at both the transcript and protein level confirming pharmacodynamic activity of the SERD. Citation Format: Spoerke JM, Daemen A, Chang C-W, Giltnane J, Metcalfe C, Dickler MN, Bardia A, Perez Fidalgo JA, Mayer IA, Boni V, Winer EP, Hamilton EP, Bellet M, Urruticoechea A, Gonzalez Martin A, Cortes J, Martin M, Gates M, Cheeti S, Fredrickson J, Wang X, Friedman LS, Liu L, Li R, Chan IT, Mueller L, Milan S, Lauchle J, Humke EW, Lackner MR. Phamacodynamic and circulating tumor DNA evaluation in a phase I study of GDC-0927, a selective estrogen receptor antagonist/ degrader (SERD) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-11-01.

Association of DNA damage response and repair genes (DDR) mutations and microsatellite instability (MSI), PD-L1 expression, tumor mutational burden (TMB) in gastroesophageal cancers.

60 Background: DDR mutations are associated with higher neoantigen load and tumor infiltrating lymphocytes, and are a potential biomarker for immunotherapy. We investigated the association of DDR mutations in gastric (GC), esophageal (EC), and gastroesophageal junction (GEJ) cancers with MSI, PD-L1, and TMB, known predictors for immune checkpoint inhibitors. Methods: 20 DDR mutations were tested by Next-Generation Sequencing (NGS) with a 592-gene panel on a total of 1935 (709 EC; 831 GC; 355 GEJ) cancers. TMB was assessed by NGS, MSI by NGS or fragment analysis, and PD-L1 by IHC (22c3 for CPS or SP142). Results: GC had the highest DDR mutation rate compared to EC and GEJ (27% vs. 20%, p = 0.0005 and 17%, p = 0.0002, respectively). MSI-High (MSI-H) was significantly more common in the DDR mutated cohort (DDR-M) compared to non-mutated cancers (18% vs. 1%; p &lt; 0.0001). TMB-High (≥ 10 mutations/megabase [mt/MB]) was higher in DDR-M (35% vs. 21%; p &lt; 0.0001); in DDR-M cohort, GC had the highest TMB compared to DDR-M EC and GEJ (mean: 13.8 vs. 9.4 vs. 10 mt/MB, respectively; p &lt; 0.0001). DDR mutations were more frequent in the PD-L1 combined positive score (CPS) ≥ 50 group than CPS 0 (42.9% vs. 24%; p = 0.037) and CPS 1-9 (42.9% vs. 20.6%; p = 0.005). ARID1A, ATRX, BRCA2, and PTEN were the most prevalent DDR mutations in MSI-H (87%, 31%, 25%, 24%, respectively); ARID1A, ATRX, BRCA2, and PTEN in TMB-High (47%, 7.7%, 6.7%, 6.8%) and ARID1A, BRCA2, RAD50, and WRN in PD-L1 high (CPS ≥ 10) (48.5% vs. 5.2% vs. 2.5% vs. 3.4%). Conclusions: MSI-H, TMB-high and high PD-L1 expression were significantly more prevalent in the DDR-M cohort compared to non-DDR-mutated cancers, most pronounced in GC. Alterations in ARID1A, ATRX, BRCA2, and PTEN were correlated with MSI-H and TMB-high while ARID1A, BRCA2, RAD50, and WRN were correlated with increased PD-L1 expression. Our findings may help identify patients for tailored immunotherapy approaches in future clinical trials.

Abstract A060: Targeting glioma-macrophage interplay via LOX in PTEN-deficient glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most lethal form of brain cancer in adults. The median survival of GBM patients is only about one year after initial diagnosis. Genomic profiling has stratified GBM into various subgroups, which are driven by specific genetic alternations of core signaling pathways, including RTK/RAS/PI3K/PTEN, P53/ARF/MDM2 and RB/CDKN2A pathways. However, targeted therapies, such as therapy against EGFR, have failed in the clinic, and no effective therapeutic drugs are available to target tumor suppressors. A key reason for therapeutic failure is inter- and intra-tumoral cancer cell genetic instability and heterogeneity, resulting in aberrant activation of multiple signaling pathways within and across tumors. Stromal/immune cells in the tumor microenvironment (TME) are genetically stable, which not only play a pivotal role in GBM progression by affecting multiple cancer hallmarks, but can also be educated by cancer cells. However, whether and how the behavior and function of specific stromal/immune cells in the TME are regulated by cancer cell with specific genetic alterations in GBM remain relatively undefined. Utilizing a large scale of bioinformatic analysis in TCGA GBM patients, we revealed that genetic alteration (deletion/mutation) of PTEN in GBM patients specifically triggers immune response by promoting macrophage recruitment, without affecting macroglia and other immune cells. Using unbiased transcriptome profiling following functional validation, we identified that lysyl oxidase (LOX) is preferentially secreted by PTEN-deficient cancer cells. In vitro transwell migration assay and in vivo Matrigel Plug assay demonstrated that LOX is a potent macrophage chemoattractant. Transcriptome profiling following Gene Set Enrichment Analysis (GSEA) and functional validation demonstrated that activation of SRC and AKT signaling pathways drives LOX upregulation in PTEN-deficient cancer cells. Genetic and pharmacologic inhibition of LOX in PTEN-deficient cancer cells does not affect tumor cell proliferation in vitro, but markedly inhibits macrophage density and tumor growth in vivo. Using the bioinformatics analysis in clinical GBM samples, we demonstrated that LOX is enriched in GBM patients with higher macrophage density, and that these patients show lower survival. Together, our findings highlight the significance of PTEN-LOX axis in macrophage infiltration in GBM, and demonstrate a possibility of improving GBM treatment by targeting this axis-mediated macrophage recruitment. Citation Format: Peiwen Chen, Alan Wang, Ronald DePinho. Targeting glioma-macrophage interplay via LOX in PTEN-deficient glioblastoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A060.

Interplay Among PI3K/AKT, PTEN/FOXO and AR Signaling in Prostate Cancer.

The PI3K signaling pathway is activated in a majority of cancer types. It promotes tumorigenesis by regulating nutrient metabolism, cell proliferation, survival, migration, and angiogenesis. The underlying mechanisms of PI3K/AKT activation are mainly due to deletions or mutations in its key negative regulator gene-PTEN. However, mutations in other pathway genes, such as the tumor suppressor gene SPOP, may contribute indirectly to the activation of this pathway. Interestingly, a mutually exclusive relationship exists between genomic alterations in PTEN and mutations in SPOP in prostate cancer patients, suggesting that altered functions of these two tumor suppressors might share similar or at least partially overlapping mechanisms in tumorigenesis. Activated AKT can phosphorylate directly a number of downstream effectors and thereby inhibit or activate their functions. An important target of PI3K/AKT signaling is FOXO1 protein that can be phosphorylated directly by AKT leading to translocation of FOXO1 from the cytoplasm to the nucleus. This not only impairs FOXO1 activities on transactivation of downstream target genes, but also abolishes its transcriptional activity-independent inhibitory effect on other targetssuch as AR, ERG and RUNX2. Interestingly, heterozygous deletion of Pten, or mutation of Spop alone has minimal effects on tumorigenesis in the mouse prostate, suggesting that PI3K/AKT pathway interacts with other pathways to drive prostate cancer progression. Indeed, the cross talk between PI3K/AKT and other pathways, such as AR, WNT, and ERK signaling pathways is known to play essential roles in disease progression and drug resistance in prostate cancer. Therefore, co-targeting the PI3K/AKT signaling pathway and its cooperating pathways may be critical for improving the anti-cancer efficacy of PI3K/AKT inhibitors in the clinic.

Increased RNA Expression of von Willebrand Factor Gene Is Associated With Infiltrating Lobular Breast Cancer and Normal PAM50 Subtype.

Infiltrating lobular carcinoma (ILC) is the second most common histologicaI subtype of breast cancer, accounting for 10% of all cases. ILC has a characteristic genomic profile. ILC shows a high frequency of cadherin 1 (CDH1) mutations, along with loss of phosphatase and tensin homolog (PTEN), activation of alpha serine/threonine kinase (AKT), and mutations in T-box transcription factor (TBX3) and forkhead box protein A1 (FOXA1). We suspected that another gene, von Willebrand factor (VWF), might also be part of the profile, since coagulation tests reveal significant differences in patients with breast cancer. For newly-diagnosed breast cancer, the association between VWF and histology in the GDC Breast Cancer dataset in The Cancer Genome Atlas (TCGA) was evaluated. The following were used to access and analyze the data: Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/); Xena browser (https://xenabrowser.net); cBioportal (http://cbioportal.org); Oncomine (https://oncomine.org); and Prediction Analysis of Microarray 50 (PAM50). Patients with ILC had higher VWF RNA expression than patients with infiltrating ductal carcinoma and other histology. The difference of expression was present to the same degree in both pre-menopausal and post-menopausal patients. Nine alterations in VWF and PTEN were significantly co-occurrent. Considering all histologies in 843 samples, Tukey's honest significant difference post hoc test showed that VWF RNA expression of the normal subtype was significantly greater than that of the other subtypes (p<0.001). Our finding of significantly higher VWF RNA expression in the PAM50 normal (non-basal-like) breast cancer subtype suggests that VWF protein measurement might complement or corroborate PAM50 results. VWF and PAM50 results both suggesting a low risk of recurrence might make the decision whether to give chemotherapy easier, especially if VWF protein were an independent predictor.