PIK3CA H1047R Research Articles

The H1047R PIK3CA oncogene induces a senescence-like state, pleiotropy and acute HSP90 dependency in HER2+ mammary epithelial cells.

In pre-clinical models, co-existence of Human Epidermal Growth Factor Receptor-2 (HER2)-amplification and PI3K catalytic subunit (PIK3CA) mutations results in aggressive, anti-HER2 therapy-resistant breast tumors. This is not always reflected in clinical setting. We speculated that the complex interaction between the HER2 and PIK3CA oncogenes is responsible for such inconsistency. We performed series of biochemical, molecular and cellular assays on genetically engineered isogenic mammary epithelial cell lines and breast cancer cells expressing both oncogenes. In vitro observations were validated in xenografts models. We showed that H1047R, one of the most common PIK3CA mutations, is responsible for endowing a senescence-like state in mammary epithelial cells overexpressing HER2. Instead of imposing a permanent growth arrest characteristic of oncogene-induced senescence, the proteome secreted by the mutant cells promotes stem cell enrichment, angiogenesis, epithelial-to-mesenchymal transition, altered immune surveillance and acute vulnerability toward HSP90 inhibition. We inferred that the pleiotropism, as observed here, conferred by the mutated oncogene, depending on the host microenvironment, contributes to conflicting pre-clinical and clinical characteristics of HER2+, mutated PIK3CA-bearing tumor cells. We also came up with a plausible model for evolution of breast tumors from mammary epithelial cells harboring these two molecular lesions.

PI3K activated tumors evade tumor immunity by promoting an inhibitory myeloid microenvironment

Abstract Oncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood. Here we use an in vivo screen of known cancer-associated somatic mutations in immunocompetent mouse tumor models treated with checkpoint blockade to identify oncogenes that confer immune evasion. We found that a catalytically active mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A>G (H1047R) confers selective growth advantage after immunotherapy. Pharmacologic PI3K inhibition resensitizes mutant tumors to immunotherapy with anti-PD-1. The tumor microenvironment (TME) in PIK3CA H1047R-expressing tumors has fewer infiltrating CD8+ T cells after immunotherapy but is enriched for immune inhibitory myeloid cells. Inhibition of myeloid infiltration in PIK3CA H1047R tumors results in increased sensitivity to PD-1 checkpoint blockade. Thus PI3K has a role in tumor immune evasion mediated by establishment of an inhibitory myeloid microenvironment in addition to its well-described, cell-intrinsic oncogenic role. Activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy. More generally, our data suggest a rationale to combine PI3K inhibition with immunotherapy of PI3KCA mutant tumors.

Low-Frequency Mutational Heterogeneity of Invasive Ductal Carcinoma Subtypes: Information to Direct Precision Oncology.

Information regarding the role of low-frequency hotspot cancer-driver mutations (CDMs) in breast carcinogenesis and therapeutic response is limited. Using the sensitive and quantitative Allele-specific Competitor Blocker PCR (ACB-PCR) approach, mutant fractions (MFs) of six CDMs (PIK3CA H1047R and E545K, KRAS G12D and G12V, HRAS G12D, and BRAF V600E) were quantified in invasive ductal carcinomas (IDCs; including ~20 samples per subtype). Measurable levels (i.e., ≥ 1 × 10−5, the lowest ACB-PCR standard employed) of the PIK3CA H1047R, PIK3CA E545K, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E mutations were observed in 34/81 (42%), 29/81 (36%), 51/81 (63%), 9/81 (11%), 70/81 (86%), and 48/81 (59%) of IDCs, respectively. Correlation analysis using available clinicopathological information revealed that PIK3CA H1047R and BRAF V600E MFs correlate positively with maximum tumor dimension. Analysis of IDC subtypes revealed minor mutant subpopulations of critical genes in the MAP kinase pathway (KRAS, HRAS, and BRAF) were prevalent across IDC subtypes. Few triple-negative breast cancers (TNBCs) had appreciable levels of PIK3CA mutation, suggesting that individuals with TNBC may be less responsive to inhibitors of the PI3K/AKT/mTOR pathway. These results suggest that low-frequency hotspot CDMs contribute significantly to the intertumoral and intratumoral genetic heterogeneity of IDCs, which has the potential to impact precision oncology approaches.

Establishment and molecular characterization of a human ovarian clear cell carcinoma cell line (FDOV1)

BackgroundOvarian clear cell carcinoma is a distinct histologic subtype with grave survival. The underlying molecular mechanism is not fully elucidated. However, we don’t have many cell lines, which are useful experimental tools for research. We describe the establishment and characterization of a new ovarian clear cell carcinoma cell line from a Chinese patient.ResultsFDOV1 has been subcultured for more than 80 generations. Monolayer cultured cells are polygonal in shape, showing a transparent cytoplasm full of vacuoles. The number of chromosomes ranges from 45 to 90. FDOV1 cells produces CA-125, but not CA-199. The cells could be transplanted and produced tumors mimicking the donor tumor morphologically and immunohistochemically. Whole exome sequence showed both FDOV1 and tissue block harbored PIK3CA H1047R mutation and ARID1A frameshift mutations (p.L2106 fs, p.N201 fs). More interestingly, we observed SPOP mutation (p.D82H) and ZNF217 (chromosome 20q13) amplification in FDOV1, which are quite novel.ConclusionsOnly a few patient-derived ovarian clear cell carcinoma cell lines have been reported in the literature. FDOV1 is the very first one, to the best of our knowledge, from a Mainland Chinese patient. It showed infinite multiplication until now and tumorigenicity in vivo. FDOV1 has co-existing PIK3CA and ARID1A mutations. It also harbored SPOP mutation and ZNF217 amplification, which would probably be a good model for exploring the molecular mechanism of ovarian clear cell carcinoma.

Abstract 3472: Dual PI3K/mTOR inhibition in PIK3CA mutant pancreatic cancers

Abstract Background: Pancreatic cancer is one of the most deadly cancer diagnoses with limited long-term survival for patients, even with early-stage disease. 3-5% of these cancers have mutations in the phosphoinositide 3-kinase (PI3K) signaling pathway. There is growing interest in targeting cancers with this mutation using specific pathway inhibitors. We have previously proven that dual PI3K/mTOR inhibition is sufficient to decrease tumor growth in mice with a PI3K p110* transgene. Here we aim to determine if this treatment can induce a response in pancreatic cancers with the human PIK3CA H1047R hotspot mutation. Methods: Transgenic mice were developed using a cre lox-p system (Pdx1-cre) to develop PIK3CA H1047R (PCPK mice) pancreatic adenocarcinomas. Tumors were harvested and grown in spheroid culture. These were treated with increasing concentrations of BEZ235, a dual PI3K/mTOR inhibitor. Treatment groups were compared using spheroid diameter changes and metabolic activity via NADH and FAD+ quantitation. Additionally, immunofluorescence staining was performed and quantified on treated spheroid cultures. PET/CT diagnostics were performed and analyzed pre- and post- treatment with BEZ-235 on a cohort of these experimental mice. Treated tissues were resected for immunohistochemistry and Western blot analyses. Results: Organotypic PCPK spheroid cultures were treated with BEZ235 in the culture media for 48 hours. Pre- and post-brightfield imaging demonstrated a modest reduction in the percent change in spheroid diameter compared to control (control: 41.8%, BEZ235 100nM: 9%, BEZ235 200nM: -7.7%, BEZ235 400nM: -14.3%; p<0.001). This stability of the spheroid size correlated with minimal differences between the control and treated spheroids using the optical metabolic imaging, including nonsignificant changes in NADH lifetime, FAD+ lifetime and the optical redox ratio (p=NS). PCPK mice (n=10) aged until they developed cancers and then were treated with BEZ235 for 14 days. Pre- and post-positron emission tomography/computed tomography (PET/CT) were performed. Nonsignificant changes in tumor volume and PET avidity were seen between the treatment groups. Both in vitro and in vivo treatments with BEZ235 proved to have no significant change in tumor avidity in this model. Immunoblotting demonstrated persistent activation of the PI3K pathway despite BEZ235 treatment in these spheres and mice as measured by persistent phosphorylation of RPS6 and 4EBP1. Conclusions: Despite significant responses being previously detected in mice with pancreatic tumors initiated by the PI3K p110* transgene, BEZ235 did not result in a similar treatment effect in PCPK mice possessing the human PIK3CA H1047R hotspot mutation. Further studies will examine the potential resistance mechanisms of these tumors to this therapy and potential ways in which to overcome this resistance. Citation Format: Susan Payne, Mitchell Depke, Alex Yueh, Joseph T. Sharick, Peter F. Favreau, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Melissa Skala, Dustin A. Deming. Dual PI3K/mTOR inhibition in PIK3CA mutant pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3472.

Abstract 1428: Application of ONCO/REVEAL Lung & Colon Cancer Panel in CRC samples and effect of DNA damage in FFPE samples

Abstract Introduction: The ONCO/REVEAL Lung&Colon Caner Panel (LC103, Pillar Biosciences Inc.) interrogates regions in 22 genes that are frequently mutated in NSCLC and CRC. To evaluate the performance of ONCO/REVEAL Lung&Colon Cancer Panel, we assessed a cohort of 207 colorectal cancer FFPE samples collected by a top-tier hospital in Shanghai between 2015 and 2016. Among them, 27 FFPE samples showed abnormally high numbers of low frequency variants (<2%) and were further investigated to assess the effect of DNA damage in somatic variant detection. Materials and Methods: DNA library preparation and sequencing: 10-20 ng of FFPE DNA (Quantitated by Qubit, Life Tech.) was used to prepare libraries using the LC103 panel. All libraries were subsequently sequenced on Illumina MiSeq sequencer. FFPE DNA repair: DNA extracted from 27 FFPE samples with a high number of low VAF (<2%) variants was treated with NEBNext FFPE DNA Repair Mix prior to library preparation. FFPE and DNA repaired FFPE were compared to analyze the effect of DNA damage in variant detection. Matched fresh frozen tumor tissue samples were also tested. Data analysis: PiVATTM (Pillar Biosciences Inc.) was used for data analysis. PCR errors and sequencing errors are reduced to be well below 1% VAF through the PiVAT error correction algorithm. Results: 100% successful rate of library preparation and sequencing: All of 207 FFPE samples yielded high quality sequencing data that detected mutant alleles at frequencies as low as 1%. Variant detection: A total of 414 somatic variants, including SNV and small indels, were identified above 2% VAF in 193 out of 207 samples. TP53 (38%), KRAS (24%), PIK3CA (12%), FBXW7 (9%) and PTEN (4%) were the most frequently mutated genes. CNVs were identified in EGFR, MET, ERBB2, KRAS and FGFR1 genes. The effect of DNA damage: The majority of detected variants between 1% and 2% VAF, are not known hotspot mutations. C:G>T:A mutations account for 73.8% of variants between 1% and 2% VAF. DNA repair by NEBNext FFPE DNA Repair Mix or other UDG enzymes reduced 1-2% VAF calls significantly, indicating that these variants are false positive calls. However, DNA repair enzymes used in our study could not eliminate false positive calls completely. Other mechanisms are suspected to contribute to the remaining low frequency calls. Variant between 2% and 5% VAF: In total, 33 somatic variants were detected at 2-5% VAF. There are many clinically actionable mutations or common driver mutations, including KRAS G12C (2.10%), KRAS G12D (2.21%), KRAS Q61L (2.74%), BRAF G469E (2.51%), PIK3CA E545K (2.08%, 2.39%, 4.78%), PIK3CA E545G (4.61%), PIK3CA Q546K (4.51%), PIK3CA H1047R (2.80%). Conclusions: The ONCO/REVEAL Lung&Colon Caner Panel is a robust and sensitive NGS Assay for the detection of somatic variants. DNA damage confounds variant identification in FFPE samples between 1 and 5% frequency. Citation Format: Jianfeng Xiao, Jiajie Tang, Xiangzhi Liu, Xiaoyan Tian. Application of ONCO/REVEAL Lung & Colon Cancer Panel in CRC samples and effect of DNA damage in FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1428.

Abstract 706: In vivo tumor-associated mutation screen identifies PI3K activation as a mechanism of resistance to PD-1 blockade

Abstract Known genomic correlates of response to immunotherapy do not perfectly predict clinical outcome, supporting the existence of unknown mechanisms of resistance to tumor immunity. We hypothesize that somatic, cancer-associated mutations account for heterogeneity in spontaneous response to tumors and response to immunotherapy. We have undertaken a systematic in vivo screen to identify mechanisms of resistance to tumor immunity in order to define a comprehensive set of therapeutic targets and provide biomarkers of sensitivity to immunotherapy. Mouse tumor cell lines (MC38 colon carcinoma or B16 melanoma) were engineered to express a library of barcoded open reading frames (ORFs) mutagenized to encode known cancer-associated somatic mutations. Tumor-bearing animals were treated with anti-PD-1. A mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A>G, increased in representation in anti-PD-1 treated tumors but not in immunodeficient animals, suggesting that activity of the mutant allele conferred selective growth advantage in the setting of tumor immunity. This mutation encodes a constitutively active mutant catalytic domain, PIK3CA H1047R. MC38 tumors homogeneously expressing H1047R and implanted into wild-type mice failed to respond to anti-PD-1 therapy, while tumors expressing a control gene regressed. Pharmacologic PI3K inhibition resensitized tumors to treatment with anti-PD-1. PD-1-treated PIK3CA H1047R tumors had fewer infiltrating CD8+ T cells as measured by immunohistochemistry and flow cytometry. Single-cell RNA-seq of tumor-infiltrating immune cells revealed a population of myeloid cells expressing known immune inhibitory proteins that differentially enriched in PIK3CA H1047R-expressing tumors. Our data suggest that PI3K has, in addition to its well-described oncogenic role, a role in tumor immune evasion mediated by establishment of an inhibitory myeloid microenvironment. As such, activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy, and these studies provide a rationale for therapeutic combination trials of PI3K inhibition with checkpoint blockade and other myeloid-targeting immunotherapies. Citation Format: Natalie B. Collins, Brian C. Miller, Kevin Bi, Rose Al Abosy, Kathleen Yates, Yashaswi Shrestha, John Doench, Jesse Boehm, W Nicholas Haining. In vivo tumor-associated mutation screen identifies PI3K activation as a mechanism of resistance to PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 706.

Abstract 2173: Testing neratinib-containing drug combination regimens on HER2 mutated non-amplified, ER+ breast cancer patient-derived xenografts

Abstract Introduction: HER2 activating mutations are a novel, druggable genomic alteration in metastatic breast cancer (MBC). These HER2 mutations are predominantly found in HER2 gene amplification negative, hormone receptor positive breast cancers. We have previously demonstrated that HER2 mutations can be potently inhibited by the second generation, irreversible pan-HER tyrosine kinase inhibitor, neratinib (Bose et al., Cancer Discovery 2013). Further, we performed a phase II clinical trial to treat HER2 mutated MBC and we found that neratinib monotherapy produced a clinical benefit rate of 31% and progression free survival of 16 weeks in a heavily pre-treated patient population (Ma et al., Clinical Cancer Research 2017). The objective of the current pre-clinical study is to develop combination strategies to improve the anti-tumor activity of neratinib in HER2 mutated breast cancer. As ER and CDK4/6 signaling activation are known resistance mechanisms to HER2 targeted agents in HER2 positive breast cancer, we hypothesized that neratinib in combination with agents that target ER (fulvestrant) or CDK4/6 (palbociclib) will induce synergistic anti-tumor effect in HER2 mutated MBC. In addition, we hypothesized that dual HER2 targeting (with trastuzumab) or in combination with chemotherapy agents (vinorelbine), may be more effective than neratinib alone. Since these partnering agents are well established in the treatment of breast cancer and are without overlapping toxicities with neratinib, rapid clinical translation is possible for the treatment of patients with HER2 mutated, ER+ MBC. Description of our Human-in-Mouse Patient Derived Xenografts (PDX): We developed two HER2 mutated breast cancer PDX lines. Both lines come from HER2 mutation positive, HER2 gene amplification negative, ER+ MBC patients. PDX-51 has a HER2 exon 20 insertion mutation (ERBB2 G776insYVMA), a PIK3CA H1047R mutation and is wild-type for TP53. PDX-64 has a HER2 kinase domain missense mutation (ERBB2 L869R) and is wild-type for both PIK3CA and TP53. Both PDX lines are grown in female NSG strain mice without any exogenous estrogen supplementation. Results: We tested combinations of neratinib with fulvestrant, palbociclib, trastuzumab, or vinorelbine for their anti-tumor activity and effects on cell proliferation and survival pathway signaling activities, including ER, CDK4/6 and HER2 pathways, in vivo. We will present data on the efficacy of these drug combinations. Tumor size measurements and reverse phase protein array (RPPA) data on the treated PDX lines will be shown. Citation Format: Shunqiang Li, Tina M. Primeau, Stephanie L. Pratt, Katherine R. Harrill, Francesca Avogadri-Connors, Alshad S. Lalani, Cynthia X. Ma, Ron Bose. Testing neratinib-containing drug combination regimens on HER2 mutated non-amplified, ER+ breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2173.

Abstract 3650: Use of unique molecular identifiers to gain insight about the true positive mutations in cfDNA of breast cancer patients for implementation of personalized treatment

Abstract Background: Blood analytes, as liquid biopsies, are discussed to be surrogate markers for therapy stratification, as serial sampling enabled by the minimal invasive nature of blood draw facilitates monitoring of clonal evolution. Mutational analysis of cell-free DNA (cfDNA) in plasma of breast cancer (BC) patients (pts) may predict the ideal therapy strategy. In this regard, PIK3CA mutations in BC pts' cfDNA were already shown to correlate with treatment response. However, in Next Generation Sequencing (NGS) workflows, used for cfDNA analysis, artefacts are often induced during library construction. Here we used unique molecular identifiers (UMIs) to verify true positive PIK3CA mutations in cfDNA of BC pts. Patients and methods: cfDNA was isolated from 54 plasma samples of 38 BC patients by affinity-based binding to magnetic beads (QIAamp MinElute ccfDNA Kit, QIAGEN, Germany). 30ng - 60ng cfDNA was used for library construction with the QIAseq Targeted DNA Panel for Illumina (QIAGEN, Germany) with integrated UMIs. Sequencing was executed on the NextSeq ® 500 platform (Illumina, US). Data analysis was performed by QIAseq Targeted Sequencing Data Analysis Portal and the Biomedical Genomics Workbench. As a reference, the PIK3CA mutational status of matched tumor tissue DNA (analyzed by Sanger sequencing) was consulted. Results: Library preparation was successful (yield >15ng) in 52/54 cases. Mean coverage was ~20.000x (mean UMI coverage ~2.500x) and >10.000x in 48/52 cases. The minimal allele frequency found for PIK3CA hotspot mutations (P539S, E545K, H1047R) by UMI analysis was 0.72%. In total, 133 mutations of the PIK3CA gene were identified as true positive mutations by UMI analysis in all 52 samples, which is a reduction of 69% (294/427) of all PIK3CA mutations incorrectly called by conventional data analysis. 59% of all different PIK3CA mutations called by UMI analysis appeared in over 5% of all pts. In the cohort of pts with PIK3CA hotspot mutant tumor tissue, 32% (6/19) showed the mentioned mutations also in matched cfDNA, whereas 16% of pts (3/19) without PIK3CA mutant tumor tissue were identified with true positive PIK3CA hotspot mutations in their plasma. Longitudinal analysis across two years during therapy revealed the increase in allele frequency (0%;11%;39%) of the PIK3CA H1047R mutation in one pt, whereas another pt showed a stable allele frequency of the PIK3CA P539S mutation (52%; 51%; 48%). Results will be expanded by consideration of mutations in BC hotspot genes despite PIK3CA and correlation to clinical parameter. Conclusions: Unique molecular identifiers enable the identification of true positive mutations in cfDNA and can thus, be used in clinical practice to determine molecular drivers of individual cancer progression and to employ personalized therapy. Citation Format: Corinna Keup, Karim Benyaa, Peter Hahn, Siegfried Hauch, Pawel Mach, Mitra Tewes, Hans-Christian Kolberg, Sabine Kasimir-Bauer. Use of unique molecular identifiers to gain insight about the true positive mutations in cfDNA of breast cancer patients for implementation of personalized treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3650.

Abstract 2943: In vivo combination of miransertib (ARQ 092) with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab and paclitaxel

Abstract Dysregulation of the PI3K-AKT signaling pathway has been implicated as a key driver in cancer initiation and progression. AKT is a serine/threonine kinase and a critical component mediating the PI3K-AKT signaling axis. Although AKT inhibitors have been extensively studied, clinical outcome has not been impressive. Interestingly, it has been shown that PI3K/AKT pathway has been involved in resistance to conventional chemotherapy, and inhibition of AKT enhances targeted therapy and sensitizes radiation therapy. Miransertib is a potent and selective pan-AKT inhibitor and currently in early clinical studies. In this study, we assessed combined effect of Miransertib with immune checkpoint inhibitor, anti-MEK and anti-HER2 agents, and a chemotherapeutic agent in vivo. Miransertib at doses of range from 20mg/kg to 120mg/kg was tested in combination with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab, or paclitaxel. Anti-tumor efficacy was assessed in syngeneic mouse CT-26 colon and 4T1 breast tumor models and xenografts models with endometrial (AN3CA) and breast (HCC1954, KPL-4, ZR-75-1) tumors, two patient-derived (PDX) models of endometrial cancer (with PIK3CAH1047R and R93W+D350G mutations) and in one vemurafenib-resistant melanoma PDX (with BRAFV600E and PIK3CA H1047R mutations). Combinability and efficacy of Miransertib and anti-PD-1 antibody was assessed in syngeneic mouse CT-26 colon tumor model. Miransertib at 60 mg/kg and anti-PD-1 antibody at 10mg/kg were combinable. Combined dosing of Miransertib with Anti-PD-1 antibody exerted superior anti-tumor activity in comparison to the single agents (TGI: 65% for combination, 50% for Miransertib and 55% for anti-PD-1 antibody after dosing for 9 days and more significant difference after 12 day dosing). In 4T1 breast tumor model, combination of Miransertib and anti-PD-1 antibody showed a very modest anti-tumor activity whereas there is no effect as single agents. In one of the endometrial PDX models, the combination of Miransertib with trametinib enhanced anti-tumor activity of each drug alone, reducing tumor growth by 67% compared to single-agent tumor reductions by 43% for either Miransertib or trametinib. In a vemurafenib-resistant melanoma PDX model, tumor growth was reduced by 73% when Miransertib was combined with trametinib, while trametinib alone only reduced tumor growth by 26% for trametinib and by 16% by Miransertib. Miransertib enhanced efficacy of in combination with trastuzumab, lapatinib, or paclitaxel, by reducing tumor growth as much as 92%, 73% and 85% respectively, while single agent comparators reduced tumors by less than 50%. Miransertib is combinable with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab, and paclitaxel and exhibits enhanced anti-tumor activity. These results provide us rationale for the combination study of Miransertib in a clinical setting. Citation Format: Yi Yu, Terence Hall, Sudharshan Eathiraj, Ron E. Savage, Brian Schwartz. In vivo combination of miransertib (ARQ 092) with anti-PD-1 antibody, trametinib, lapatinib, trastuzumab and paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2943.

A simple and robust real-time qPCR method for the detection of PIK3CA mutations

PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E545K being the most common of these, accounting together for around 60% of all PIK3CA mutations and have promising therapeutic implications. Given the low sensitivity and the high cost of current genotyping methods we sought to develop fast, simple and inexpensive assays for PIK3CA H1047R and E545K mutation screening in clinical material. The methods we describe are based on a real-time PCR including a mutation specific primer combined with a non-productive oligonucleotide which inhibits wild-type amplification and a parallel internal control reaction. We demonstrate consistent detection of PIK3CA H1047R mutant DNA in genomic DNA extracted from frozen breast cancer biopsies, FFPE material or cancer cell lines with a detection sensitivity of approximately 5% mutant allele fraction and validate these results using both Sanger sequencing and deep next generation sequencing methods. The detection sensitivity for PIK3CA E545K mutation was approximately 10%. We propose these methods as simple, fast and inexpensive diagnostic tools to determine PIK3CA mutation status.

Abstract PD4-13: Estrogen receptor-negative breast adenomyoepitheliomas are driven by co-occurring HRAS hotspot and PI3K pathway gene mutations: A genetic and functional analysis

Abstract Introduction:Adenomyoepithelioma (AME) of the breast is a rare biphasic tumor, characterized by epithelial and myoepithelial differentiation. Although AMEs have an indolent clinical course, a subset may progress to carcinoma and metastasize. We sought to define the mutational landscape of AMEs and investigate the functional impact of recurrent pathogenic mutations identified in these tumors. Methods: Thirty-one AMEs were subjected to whole-exome sequencing (WES, n=8) or massively parallel sequencing targeting all coding regions of 410 key cancer genes and intronic and regulatory regions of selected genes (n=23). Somatic genetic alterations were defined using state-of-the-art bioinformatics algorithms. In an additional set of 12 AMEs, Sanger sequencing analysis of HRAS, PIK3CA and AKT1 was performed. Non-tumorigenic estrogen receptor (ER)-negative mammary epithelial cells (i.e. MCF10A, MCF10A with a PIK3CA H1047R mutation knock-in and MCF12A) were utilized for functional studies using both conventional monolayer and three-dimensional (3D) culture assays. Results: 27 (63%) and 16 (37%) AMEs were ER-positive and ER-negative, respectively. ER-negativity was significantly associated with histologic features predictive of a more aggressive behavior, with a higher number of mutations and copy number alterations, and with a distinct mutational profile as compared to ER-positive AMEs. Of the 27 ER-positive AMEs, 12 cases (44%) harbored PIK3CA hotspot mutations, and 5 PIK3CA wild-type cases displayed E17K AKT1 hotspot mutations. By contrast, of the 16 ER-negative AMEs, 9 (56%), 9 (56%) and 3 (19%) harbored HRAS, PIK3CA (mostly E545K and H1047R hotspots) and PIK3R1 mutations, respectively. Strikingly, all HRAS mutations were restricted to ER-negative AMEs, affected the hotspot codon Q61 (Q61R/K), and all but one co-occurred with PIK3CA or PIK3R1 mutations. In addition, HRAS Q61 hotspot mutations were significantly associated with necrosis (p=0.01) and high mitotic rates (p=0.03). CDKN2A homozygous deletions were also detected only in ER-negative AMEs (19%) and found to be significantly associated with progression to carcinoma (p=0.001). Forced expression of HRAS Q61R in MCF10A and MCF12A cells resulted in i) increased proliferation and transformation, ii) an irregular growth pattern in 3D organotypic cell cultures, iii) partial loss of the epithelial phenotype, and iv) acquisition of myoepithelial differentiation, which was more overt in PIK3CA-mutant MCF10A cells. HRAS Q61Rinduced hyperactivation of the PI3K pathway, but both PI3K and MAPK pathways likely contributed to the RAS-mediated proliferation, which was completely arrested by combined AKT and MEK inhibition. Conclusion: AMEs are phenotypically and genetically heterogeneous. Whilst pathogenic mutations in PI3K pathway-related genes occur across the spectrum of lesions, HRAS Q61 hotspot mutations are restricted to ER-negative AMEs. Our genomic and functional analyses indicate that HRAS Q61 mutations are driver events in the pathogenesis of ER-negative AMEs and, in conjunction with mutant PIK3CA, may lead to the acquisition of myoepithelial differentiation in breast epithelial cells. Citation Format: Geyer FC, Li A, Papanastasiou AD, Smith A, Selenica P, Burke KA, Edelweiss M, Wen H-C, Piscuoglio S, Schultheis AM, Martelotto LG, Pareja F, Kumar R, Brandes A, Lozada J, Macedo GS, Muenst S, Terracciano LM, Jungbluth A, Foschini MP, Wen HY, Brogi E, Palazzo J, Rubin BP, Ng CKY, Norton L, Varga Z, Ellis IO, Rakha E, Chandarlapatty S, Weigelt B, Reis-Filho JS. Estrogen receptor-negative breast adenomyoepitheliomas are driven by co-occurring HRAS hotspot and PI3K pathway gene mutations: A genetic and functional analysis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-13.

Abstract B072: Liquid biopsy analysis of NSCLC patient plasma using ddPCR and NGS

Abstract Introduction: Analysis of cfDNA has potential applications in therapy selection and disease monitoring in the clinical management of NSCLC. Here we use EGFR ddPCR and the Oncomine Lung cfDNA Assay to evaluate the mutation profile and allele burden in a set of plasma samples collected from NSCLC patients. Methods: Plasma samples were collected at Dana-Farber Cancer Institute (DFCI). cfDNA was extracted using the QIAamp DSP circulating NA kit. EGFR ddPCR assays were designed by DFCI. One third of cfDNA extracted from 1 ml plasma was used for each EGFR ddPCR assay (L858R, T790M and ex19del). The L858R and T790M ddPCR assays detect mutations using mutant specific probes. The ex19del assay allows for the detection of a broad range of exon 19 deletions via the loss of wild-type exon 19 signal. Twenty-two plasma samples from patients with EGFR WT tumors were analyzed to assess the background noise (Limit of Blank). The other 32 plasma samples were analyzed in a blinded fashion. ddPCR analysis was performed on the QX200 system and data was analyzed using QuantaSoft software. A subset of 12 plasma samples had sufficient cfDNA for additional analysis using the Oncomine Lung cfDNA Assay on the Ion S5. This NGS panel is designed to detect 150 hotspot mutations in 11 lung cancer-related genes. Torrent Suite Software 5.2 was used for NGS data analysis. The HorizonDx cfDNA Multiplex Reference Standard (AF 5%, 1%, 0.1%, and WT) was also included in the ddPCR and NGS studies. Results: Based on the Limit of Blank data, the positive mutation cut-off for each EGFR ddPCR assay was set at 3 mutant copies with minimum 3000 wild type copies. Studies using the HorizonDx cfDNA standard demonstrated that the analytical sensitivity of the ddPCR assays increases with cfDNA input: the lowest detected allele frequency (AF) is 1%, 0.5%, and 0.25% for 3, 6, and 12ng input, respectively. In plasma samples, EGFR mutations were detected as low as 0.08% AF using ddPCR (4.5 L858R mutant copies/5827 wild type copies). L858R and exon 19 deletions were detected in 13/54 and 21/54 plasma samples, respectively. T790M mutations were detected in 19/54 plasma samples, and coexisted with L858R or ex19del in all cases. The Oncomine™ Lung cfDNA Assay showed 100% concordance with EGFR ddPCR results for the 12 plasma samples analyzed by both methods. Mutations were detected as low as 0.18% AF with 8.5ng plasma cfDNA input in the NGS assay. Using the HorizonDx cfDNA multiplex standard mutations were detected as low as 0.1% AF using 20ng input. In addition, PIK3CA mutations were found to coexist with EGFR ex19 deletions in 3/12 plasma samples using NGS. The AF of PIK3CA mutations and EGFR mutations was comparable in the same samples (TRL23: 24.3% for EGFR del19 and 10.4% T790M vs 20.2% for PIK3CA H1047R; TRL48: 7.8% for EGFR del19 vs 3.4% for PIK3CA E545K; TRL51: 0.85% for EGFR del19 vs 1.02% for PIK3CA E545K). Conclusions: Both ddPCR and NGS were able to detect mutations at 0.1% AF with sufficient cfDNA input. The Oncomine panel confirmed EGFR ddPCR results and detected coexisting PIK3CA mutations. EGFR and PIK3CA mutations were present at a similar AF, suggesting that EGFR and PIK3CA mutations may develop at the same stage of tumorigenesis in these patients. Our study results demonstrate that comprehensive profiling of cfDNA using NGS could be critical for the selection of appropriate therapies for NSCLC patients while ddPCR is an ideal platform for monitoring disease burden during the course of therapy. Citation Format: WeiHua Liu, Peng Fang, Chad Galderisi, Cindy Spittle, Jin Li. Liquid biopsy analysis of NSCLC patient plasma using ddPCR and NGS [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B072.

Variation in organ-specific PIK3CA and KRAS mutant levels in normal human tissues correlates with mutation prevalence in corresponding carcinomas.

Large‐scale sequencing efforts have described the mutational complexity of individual cancers and identified mutations prevalent in different cancers. As a complementary approach, allele‐specific competitive blocker PCR (ACB‐PCR) is being used to quantify levels of hotspot cancer driver mutations (CDMs) with high sensitivity, to elucidate the tissue‐specific properties of CDMs, their occurrence as tumor cell subpopulations, and their occurrence in normal tissues. Here we report measurements of PIK3CA H1047R mutant fraction (MF) in normal colonic mucosa, normal lung, colonic adenomas, colonic adenocarcinomas, and lung adenocarcinomas. We report PIK3CA E545K MF measurements in those tissues, as well as in normal breast, normal thyroid, mammary ductal carcinomas, and papillary thyroid carcinomas. We report KRAS G12D and G12V MF measurements in normal colon. These MF measurements were integrated with previously published ACB‐PCR data on KRAS G12D, KRAS G12V, and PIK3CA H1047R. Analysis of these data revealed a correlation between the degree of interindividual variability in these mutations (as log10 MF standard deviation) in normal tissues and the frequencies with which the mutations are detected in carcinomas of the corresponding organs in the COSMIC database. This novel observation has important implications. It suggests that interindividual variability in mutation levels of normal tissues may be used as a metric to identify mutations with critical early roles in tissue‐specific carcinogenesis. Additionally, it raises the possibility that personalized cancer therapeutics, developed to target specifically activated oncogenic products, might be repurposed as prophylactic therapies to reduce the accumulation of cells carrying CDMs and, thereby, reduce future cancer risk. Environ. Mol. Mutagen. 58:466–476, 2017. © 2017 This article is a U.S. Government work and is in the public domain in the USA. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society

Abstract 4887: Comprehensive molecular analysis of pediatric thalamic tumors

Abstract Childhood thalamic tumors are relatively rare cancers, accounting for 5% of all pediatric brain tumors and categorized as midline gliomas such as diffuse intrinsic pontine gliomas (DIPGs). We and others have shown that mutations in genes encoding for histone 3.3 (H3F3A), histone 3.2 (HIST2H3C) and histone 3.1 (HIST1H3B) along with their obligate partner mutations are the major driver mutations in DIPGs. Where recent studies have identified major histone partner mutations associated with DIPGs, more research is required to provide a clear landscape of genomic aberrations associated with thalamic tumors. We hypothesize that comprehensive whole genome sequence, methylation and proteome analysis of a large cohort of thalamic tumors will map differentially regulated pathways and identify potential novel driver and obligate partner mutations associated with thalamic gliomas. We have established a cohort (CNHS and CBTTC) of 128 thalamic specimens, including 56 pediatric and adolescent primary thalamic tumors with median age at diagnosis of 5.6 years (range 0-20 years); 40 normal controls with matched age and gender; and 32 midline tumors with potential thalamic involvement with median age at diagnosis of 7.6 years (range 0-19 years). Our cohort of primary thalamic tumors contained 31 (55.3%) and 19 (33.9%) tumors reviewed as high and low grade gliomas, respectively. From our midline tumors with potential thalamic involvement, 22 (68.7%) were classified as primary DIPG and 10 (31.2%) were other midline gliomas. Where available, MRI reviews and histopathological analysis were performed. Preliminary results showed that 7 of the extended tumors presented hypercellularity and positive histone 3 K27M staining, confirming that these tumors in fact extended to the thalamus as MRI showed. Additionally, Whole Exome Sequencing (WES) from one DIPG sample extending to the thalamus showed the same mutations found on the primary pons tumor: H3.1 K27M; ACVR1 G328V; PIK3CA H1047R; MAX R51Q and PTEN A126S. The remaining primary and extended thalamic tumors will be analyzed by WES to understand the molecular changes associated with this disease. In addition to our results, we analyzed the genomic landscape from pediatric thalamic tumors previously published (188), showing H3.3/H3.1 K27M (51%); BRAF (10.6%) and TP53 (8%) as the most frequent mutations among thalamic high and low grade astrocytomas. Further studies will allow us to compare comprehensive molecular analysis of thalamic tumors (primary and metastatic) and non-thalamic midline tumors (specimen and data already in hand) and identify similarities and differences in genomic, epigenomic and proteomic expression pattern which may guide a better characterization of thalamic tumor as a separate entity. Citation Format: Heloisa H. Moser, Susanne Yoon, Madhuri Kambhampati, Sridevi Yadavilli, Angela J. Waanders, Adam Resnick, Roger J. Packer, Javad Nazarian. Comprehensive molecular analysis of pediatric thalamic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4887. doi:10.1158/1538-7445.AM2017-4887

Abstract 5692: Cross-platform detection and quantification of actionable mutations in cell-free DNA shows high concordance and correlation between next-generation sequencing and droplet digital PCR

Abstract Background: Non-invasive genotyping of cell-free DNA (cfDNA) provides physicians with the ability to identify genomic alterations, evaluate response to treatment, and uncover potential mechanisms of resistance. Here, we analyze the concordance of next-generation sequencing (NGS) and droplet digital PCR (ddPCR) in detecting and quantifying actionable mutations from cfDNA of lung, colorectal, and breast cancer patients. Methods: We studied a cohort of 221 advanced cancer cfDNA samples with completed Guardant360 digital sequencing results. Variant allele frequencies (% AFs) ranged from less than 0.2% to greater than 90% for common driver and resistance mutations. Blinded to plasma NGS results, we performed ddPCR using our validated assays for EGFR L858R, exon19 del, T790M (113 presumed lung cases); BRAF V600E, KRAS G12X (77 colorectal cases); ESR1 D538G, Y537C and PIK3CA H1047R (31 breast cases). Following unblinding, qualitative and quantitative accuracy were compared. Results: Qualitative results (Table 1) show a high concordance between NGS and ddPCR, with 98.3% (576/586) of all assays run being concordant. 9/22 of samples that had an AF below 0.4% were discordant, while only one sample above 0.4% AF was discordant. AFs calculated by NGS and ddPCR were strongly correlated by linear regression (R between 0.951 and 0.999). Notably, the slope of the ddPCR AF versus NGS AF graph for EGFR ex19del was 0.725, which improved to 0.95 with the adoption of an alternate calling method. Conclusions: This interlaboratory comparison of actionable mutations in cfDNA from lung, colorectal, and breast cancers by orthogonal genotyping platforms demonstrates a substantial concordance in detection and quantification between NGS and ddPCR. Our cross-platform study establishes the feasibility of standardization of qualitative and quantitative measurements of cfDNA-detected alterations. Table 1LungEGFR L858REGFR Ex19 delEGFR T790M(n=113)G360+G360-G360+G360-G360+G360-ddPCR+580510530ddPCR-055458456BreastESR1 Y537CESR1 D538GPIK3CA H1047R(n=31)G360+G360-G360+G360-G360+G360-ddPCR+1022020ddPCR-03009029ColorectalBRAF V600EKRAS G12X(n=78)G360+G360-G360+G360-ddPCR+220571ddPCR-055118 Citation Format: Bryan C. Ulrich, Rebecca J. Nagy, Justin I. Odegaard, Richard B. Lanman, Geoffrey R. Oxnard, Cloud P. Paweletz. Cross-platform detection and quantification of actionable mutations in cell-free DNA shows high concordance and correlation between next-generation sequencing and droplet digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5692. doi:10.1158/1538-7445.AM2017-5692

Abstract 2715: Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA

Abstract Detecting and quantifying mutations from circulating tumor DNA (ctDNA) is a new approach for longitudinally monitoring cancer patients throughout the course of treatment. However, general limitations to the clinical utility of ctDNA include sample concentration, overall assay cost, and result turnaround time. We have developed a novel molecular assay that utilize single color digital droplet PCR (ddPCR) to both genotype and quantify the number of tumor derived DNA molecules in a given sample. Our assay routinely detects as few as three mutant DNA molecules per a reaction volume, can be tested efficiently for fewer than ten dollars per reaction, and generates useable mutation information within four hours. Additionally, the assay can be rapidly configured to detect different mutations specifically identified in any cancer patient. To demonstrate the single molecule sensitivity and specificity for clinically relevant hotspot mutations, we have validated the assay using multiple input sources including high quality cell line DNA, formalin-fixed paraffin embedded tissue (FFPET) DNA, and ctDNA. Our ddPCR assay utilizes a novel primer design that is not rely on fluorescent probes. The overall simplicity of assay design enables one to detect nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. We have created customized precision mutation assays for two individual cancer patients. After extracting ctDNA from 500µL of plasma we prepare a controlled mixed wild type and mutant standard curves which contain between 150-3 detectable mutant molecules of interest per reaction. We then assay the patient ctDNA sample in parallel to the controlled standard curve and generate clustering confidence intervals. We have designed precision mutation assays targeting KRAS A146V, AKT1 E17K, and TP53 R175H for Patient A as well as KRAS G12V and PIK3CA H1047R for Patient B. The results of our single-color precision mutation quantification assays support the original mutation findings from the primary tumors. In one case, from 1µL of un-amplified ctDNA equivalent to less than 0.5ng of DNA, we identified 4 mutant molecules per ddPCR reaction volume. We have verified the presence of hotspot mutations from un-amplified patient derived ctDNA and quantified the number of mutant molecules present in each ctDNA sample. This assay has many features that make it amenable to automation. We are currently developing a massively high throughput process to design assays for tens of thousands of mutations in a matter of weeks. Overall this is an extremely low cost, highly sensitive and scalable diagnostic technology. We anticipate that this technology will be a valuable tool for rapidly monitoring ctDNA longitudinally in cancer patients. Citation Format: Christina M. Wood-Bouwens, Christine Handy, Billy Lau, Hanlee Ji. Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2715. doi:10.1158/1538-7445.AM2017-2715

Abstract 4461: Detection of somatic mutations in exosomal RNA from human oral squamous cell carcinoma cells

Abstract Exosomes are small vesicles, ranging from 30 to 100 nm in size, secreted from most types of cells. They contain proteins, lipids, and nucleic acids (e.g. DNA, mRNA, and microRNA) and are thought to play an important role in cell proliferation, invasion, and metastasis in human malignancies. Recently, circulating exosomes have also been of interest as a source for liquid biopsies. In this study, we examined the usefulness of exosomal RNA as a biomarker and attempted to detect the tumor-specific gene mutations from exosomal RNA derived from human oral squamous cell carcinoma (OSCC) cells. We used 3 human OSCC cells (HSC2, KT-T, KT-N). KT-T and KT-N cells were established from the lower gingival tumor and lymph node metastasis, respectively. First, we detected the somatic mutations in genomic DNA extracted from these cells by next-generation sequencing (NGS) with the use of HaloPlex Cancer Research Panel (Agilent Technologies). PIK3CA H1047R mutation was identified in all OSCC cells, and KT-N cells had the additional HRAS Q61R mutation. We confirmed these mutations of PIK3CA and HRAS in OSCC cells by TaqMan® SNP Genotyping Assays (Thermo Fisher Scientific). Subsequently, we evaluated the gene mutations in exosomal RNA from these OSCC cells. Exosomal RNA was isolated from the conditioned medium using exoRNeasy Serum/Plasma Maxi Kit (Qiagen) according to the manufacture’s protocol. In exosomal RNA, we found the PIK3CA H1047R mutation from all OSCC cells and HRAS Q61R mutation from KT-N cells by genotyping assays. Furthermore, we assessed the detection of these mutations in tumor xenograft mouse models. KT-T and KT-N cells (5 x 106) complexed with Matrigel® (BD) in 100-µl aliquots were injected subcutaneously space in the flank of male athymic nude mice. Exosomal RNA was isolated from the serum of tumor-bearing nude mice using exoRNeasy Serum/Plasma Midi Kit (Qiagen). By genotyping assays using serum exosomal RNA, PIK3CA H1047R mutation was observed in both types of xenograft mice and HRAS Q61R mutation was positive in KT-N xenograft mice. Cancer cell-derived exosomal RNA had the same gene mutations in those cells, and could be detected in the serum of tumor-bearing nude mice. These results suggest that exosomal RNA is useful for detecting somatic mutations in OSCC cells. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Detection of somatic mutations in exosomal RNA from human oral squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4461. doi:10.1158/1538-7445.AM2017-4461

A mouse-human phase I co-clinical trial of taselisib in combination with TDM1 in advanced HER2-positive breast cancer (MBC).

1030 Background: PI3K blockade has the potential to revert resistance to anti-HER2 therapies. Methods: Pre-clinical experiments were conducted in parallel to a phase Ib study with the PI3-kinase inhibitor Taselisib + T-DM1 in HER2+ MBC. Results: Pre-clinically, HER2+ cell lines (HCC1954, MDA-MB-361, MDA-MB453) treated with T-DM1 until resistance had decreased HER2 expression. Tx with Taselisib restored HER2 levels in T-DM1 resistant cells. In vivo, the combination of T-DM1 + Taselisib was superior to either agent alone in transgenic mammary carcinomas driven by HER2 with or without a PIK3CA H1047R transgene. The phase Ib study enrolled 26 pts with HER2+ MBC. Median age was 57 (38-95), ER+ (58%), ER- (42%). PIK3CA wild type (35%) mutant (23%) pending results (42%). Pts had a median of 2 lines of prior tx for MBC. Prior T-DM1 was given to 38.5% of pts. Median duration of tx among 18 pts was 4 cycles (2-19). 8 pts remain on tx. No DLTs were observed in the 2 dose levels explored: T-DM1 (3.6mg/Kg) q3 wk with either Taselisib 2mg QD or Taselisib 4mg QD. 20 patients were included in the expansion phase. Most common G3 G4 AEs were thrombocytopenia (19.2%), diarrhea (15.4%), vomiting (7.7%), lipase increase (7.7%) and hyperglycemia (7.7%). Pneumonitis was reported in 4 cases (3 G1, 1 G2); further evaluation revealed elevated beta-glucan in 3 of the 4 cases raising suspicion for pneumocystis pneumonia (PJP). 24/26 pts were evaluable for confirmed response: (4% CR, 29% PR, 50% SD). Confirmed PR/CR according to prior TMD1 (yes 30% no 36%) and PIK3CA mut status (40% mut, 22% wild type; 9/23 PIK3CA results pending). Median PFS was 7.6 mos (95% CI 2.9 to NR). Conclusions: The combination of Taselisib + T-DM1 is superior to either agent alone in mouse models of HER2+ BC. In the phase Ib study, T-DM1 + Taselisib had an acceptable safety profile although PJP prophylaxis should be considered for this regimen. Thrombocytopenia was reversible with no bleeding events. Preliminary evidence of efficacy was observed regardless of PIK3CA status or prior tx with T-DM1. This co-clinical trial allowed for the rapid translation of lab observations to the clinical setting and provided data to facilitate interpretation of the clinical results. Clinical trial information: NCT02390427.

Phase II study of panitumumab in KRAS wild-type metastatic adenocarcinoma of the small bowel or ampulla of vater.

e15799 Background: Given the benefit noted with epidermal growth factor receptor (EGFR) antibodies in KRAS wild-type colorectal cancer, we conducted a phase II trial to evaluate the efficacy of panitumumab in metastatic small bowel adenocarcinoma (SBA) and ampullary adenocarcinoma (AAC). Methods: This is a single-center open-label single arm two-stage phase II study. The primary objective was response rate (RR). Secondary objectives included overall progression-free survival (PFS), overall survival (OS) and toxicity. Eligibility included: KRAS exon 2 wild-type, ECOG PS 0-1, metastatic disease, adequate organ function and prior progression or intolerance to fluoropyrimidine with oxaliplatin. Panitumumab was administered at a dose of 6mg/kg IV every 14 days. Next generation sequencing panels were used for genomic analysis. Results: 9 patients [M/F 7/2, median age: 61 yrs (range: 40-74), ECOG PS 0/1: 2/7] were enrolled from September 2013 to October 2015. 1 pt had AAC (pancreaticobiliary subtype) and 8 pts had SBA (duodenal in 3, jejunal/ileal in 5). The most common toxicity was grade 1 acneiform rash in all patients. The most common grade 2/3 toxicities were anemia (33%), fatigue (22%), hypomagnesemia (22%) and skin infection (22%). None of the patients had a response, 2 patients had SD while the remaining 7 patients had PD. At a median follow-up time of 16.6 months, the median PFS and median OS were 2.4 months and 5.6 months, respectively. Extended RAS mutational testing identified 0/9 patients with mutations in extended KRAS or NRAS. Further genomic analysis of genes relevant to anti-EGFR activity (BRAF, PIK3CA and ERBB2) identified 2/9 patients with BRAF G469A mutation, 1/9 patients with PIK3CA H1047R mutation, and 0/9 patients with ERRB2 mutations. Conclusions: Although well tolerated, panitumumab had minimal clinical activity in patients with metastatic SBA and AAC. Given recent findings suggesting that right-sided colon cancers (hindgut derivation) benefit less from anti-EGFR therapy compared to left-sided colon cancers (midgut derivation), we propose that our findings may relate to the primarily foregut (proximal duodenum) and midgut (distal duodenum to ileum) derivation of the small bowel. Clinical trial information: NCT01202409.