Mechanism Of Resistance In Patients Research Articles

CML-363 Recent Perspective of Next-Generation Sequencing (NGS) in Mechanisms of Resistance in Patients With Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have successfully been used to treat CML, some patients develop therapy resistance. Most resistance is due to mutations in the tyrosine kinase domain of BCR::ABL. However, there are still many cases with unknown causes of resistance to TKIs. Determine BCR::ABL-independent biomarkers of resistance to TKIs in patients with CML using NGS. This study included 24 patients (12 men and 12 women) with a median age of 43 years (IQR 26-72 years) with CML resistant to TKI therapy. The control group was 5 patients (3 men and 2 women) with a median age of 54 years (IQR 33-72 years) with CML and optimal response (MMR) to TKI. A myeloid panel of 116 genes with an average reading depth of 200x or 1000x on MiSeq (Illumina) was used. The clinical significance of mutations was evaluated using COSMIC, ClinVar, and VarSome. All studied patients were found to have genetic abnormalities. However, more mutations were detected in the group of patients with resistance than in the control group (average of 5 and 1 mutations, respectively). Multiple mutations were detected in 63% of patients with resistance, although 92% of these mutations had unclear clinical significance. The most common abnormalities were found in tumor suppressor NF1 (10/24), transcription regulators TET2 (10/24) and ATRX (7/24), cohesin complex member STAG2 (6/24), and epigenetic regulator ASXL1 (6/24). Mutations were also found in the intron region of BCR (2/24). Two patients had pathogenic mutations in ASXL1 (p. G646Wfs∗12), which could cause TKI resistance. Despite mutations in NF1 and ATRX being more common in treatment-resistant patients, in our study, these mutations were also noted in optimal responders. The detection of multiple mutations in patients with CML may explain resistance to TKI therapy; however, to better understand the pathogenesis of CML, it is important to know not only the presence of certain mutations but also their level of expression and to study the effects of various regulatory elements, such as transcription factors and miRNAs.

Efficacy and potential resistance mechanisms of afatinib in advanced non-small cell lung cancer patients with EGFR G719X/L861Q/S768I.

Afatinib is the only currently approved EGFR-tyrosine kinase inhibitors for advanced non-small cell lung cancer (NSCLC) patients with EGFR G719X/L861Q/S768I. However, there are limited real-world data concerning the benefits and resistance mechanisms of afatinib in patients with these nonclassical mutations. To fill this gap, the present study was conducted. All NSCLC patients treated with afatinib were screened, and patients with EGFR G719X/L861Q/S768I were enrolled into the analysis. Either tumor tissue or blood specimens were detected by the commercial next-generation sequencing (NGS) panels or amplification-refractory mutation system (ARMS)-polymerase chain reaction (PCR) to figure out the mutation genotype. A total of 106 advanced NSCLC patients with EGFR G719X/L861Q/S768I received afatinib treatment. The benefits of afatinib exhibited heterogeneity in different mutation genotypes. Notably, at baseline, NGS testing was performed in 59 patients, and TP53 was the most frequently coexisting mutation. Patients with TP53 mutations obtained fewer survival benefits than those with TP53 wild-type. A total of 68 patients ultimately experienced progression, and 27 patients received NGS testing to clarify the potential resistance mechanisms. EGFR-T790M, CDK4 amplification, FGFR1 amplification, PIK3CA, MET amplification, RET fusions, HER2, and BRAF mutations were identified in three (11.1%), three (11.1%), three (11.1%), three (11.1%), three (11.1%), one (3.7%), one (3.7%), and one (3.7%) of the cases, respectively. Five patients underwent ARMS-PCR testing for detecting EGFR-T790M mutation, and only one patient was T790M-positive. The present study elucidated the differential benefits of afatinib within different mutation genotypes and first revealed the spectrum of potential resistance mechanisms in patients with EGFR G719X/L861Q/S768I. The results of this study may provide practical clinical information that can guide optimal treatment in this setting.

1206P HER2 copy number variation (CNV), HER2 expression and primary resistance mechanisms in patients (pts) with HER2-positive metastatic gastric or gastroesophageal junction cancer (mGC/GEJC) receiving first-line chemotherapy (CT) + trastuzumab (T) +/- pertuzumab (P) in the JACOB trial

In JACOB trial, P added to T-CT did not significantly improve overall survival (OS) in pts with HER2+ mGC/GEJC despite a 3.3 mos increase vs placebo. We previously showed a negative prognostic role of AMNESIA panel, including KRAS/PIK3CA/MET mutations and KRAS/EGFR/MET amplifications (Pietrantonio et al, CCR 2018), whereas higher HER2 CNV may be associated with better outcomes on T. These biomarkers may allow to better identify pts with higher benefit from HER2 inhibition. Among 780 pts enrolled in JACOB and 580 with available baseline tumor DNA, 327 samples were sequenced by Oncomine Focus DNA assay. In a post-hoc analysis, HER2 CNV, HER2 expression by IHC and AMNESIA were correlated with overall response rate (ORR), progression-free survival (PFS) and OS by uni-/multivariable models. Median HER2 CNV was 4.7 (IQR 2.2-16.9). By pooling treatment arms, HER2-high vs low status using the median as the cut-off value was associated with longer PFS (mPFS: 10.5 vs 6.4 mos; hazard ratio (HR)=0.48, 95%CI: 0.38-0.62; p<.001) and OS (mOS: 20.3 vs 13.0 mos; HR=0.54, 0.42-0.72; p<.001). Combining HER2 CNV and IHC improved the discriminative ability, with better outcomes restricted to HER2-high/HER2 3+ subgroup. AMNESIA positivity was found in 51 (16%), with unadjusted HR=1.35 (0.98-1.86) for PFS; 1.43 (1.00-2.03) for OS. HER2-high subgroup was enriched for AMNESIA-neg and HER2 3+ status. In multivariable models with main variables including treatment arm, only HER2 CNV status remained statistically significant for PFS (p<.001) and OS (p=.004). Higher ORR was significantly associated with IHC 3+ [61% vs 34% in 2+; odds ratio(OR)=3.11 (1.89-5.17)] and HER2-high [59% vs 43% in HER2-low; OR=1.84 (1.16-2.94), with highest OR in the top CNV quartile. The selected biomarkers were not associated with activity and efficacy of P added to T-CT. HER2-high CNV assessed by NGS is associated with significantly better ORR, PFS, OS in a pts subgroup from the JACOB trial, independent from treatment arm and especially if combined with HER2 3+.

Abstract 3956: MYC inhibition overcomes IMiD resistance in heterogeneous multiple myeloma populations

Abstract Introduction: Multiple myeloma (MM) is a plasma cell malignancy that remains incurable, with patients enduring multiple relapses and development of drug resistance. Immunomodulatory drugs (IMiDs) are critical anti-MM agents. IMiDs act by inducing CRBN-dependent proteasomal degradation of the transcription factors IKZF1 and IKZF3, which leads to IRF4 and MYC downregulation (collectively termed the “Ikaros axis”). Although CRBN aberrations occur, whether they are a functional mechanism of resistance in patients remains unclear. Based on the importance of CRBN in IMiD response, we hypothesized that IMiD treatment fails to downregulate the Ikaros axis in IMiD-resistant MM cells. Methods: To measure IMiD-induced Ikaros axis downregulation, we designed an intracellular flow cytometry assay that measured relative IKZF1, IKZF3, IRF4 and MYC protein levels in MM cells following ex vivo pomalidomide (Pom) treatment. We established this assay using IMiD-sensitive parental and dose-escalated Pom resistant MM1S and H929 cell lines before utilizing it in patient samples (isolated mononuclear cells). To assess the Ikaros axis in the context of MM intratumoral heterogeneity, we used mass cytometry to simultaneously characterize MM subpopulations in patient samples. Lastly, we determined ex vivo drug sensitivity in patient samples via flow cytometry. Results: Our hypothesis was supported in MM cell lines, as sensitive parental lines showed a significant decrease in all Ikaros axis protein levels following Pom treatment, while resistant lines showed no IMiD-induced decrease in any Ikaros axis protein. However, when assessed in CD38+CD138+ cells from patient samples, Pom treatment caused a significant decrease in IKZF1, IKZF3 and IRF4 regardless of IMiD sensitivity. Mass cytometry in patient samples revealed that individual Ikaros axis proteins were differentially expressed between subpopulations. When correlating this with ex vivo Pom sensitivity of MM subpopulations, we observed that low IKZF1 and IKZF3 corresponded to Pom resistance. Interestingly, most of these resistant populations still expressed MYC. We therefore assessed whether IMiD resistant MM was MYC dependent by treating Pom resistant MM cells with MYCi975. In 88% (7/8) of patient samples tested, IMiD resistant MM cells were sensitive to MYC inhibition. Conclusions: Our findings did not support our initial hypothesis, as IMiD-induced IKZF1 and IKZF3 degradation remains intact in IMiD resistant MM cells from patient samples. However, our data support a mechanism where the Ikaros axis no longer drives MYC expression in IMiD-resistant MM. Therefore, we propose that the critical mediator of IMiD resistance is conversion to a cell state where MYC expression is Ikaros axis independent. This suggests targeting MYC directly or via the as yet uncharacterized mechanism controlling MYC may be an effective strategy to eradicate IMiD resistant MM. Citation Format: Lorraine N. Davis, Zachary J. Walker, Denis Ohlstrom, Brett M. Stevens, Peter A. Forsberg, Tomer M. Mark, Craig T. Jordan, Daniel W. Sherbenou. MYC inhibition overcomes IMiD resistance in heterogeneous multiple myeloma populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3956.

Abstract 5161: Exome-scale longitudinal tracking of emerging therapeutic resistance in GIST via analysis of circulating tumor DNA

Abstract Gastrointestinal stromal tumors (GIST) are lethal tumors characterized by constitutively activating mutations to KIT or PDGFRA. Transient disease control in the first-line setting is achieved via inhibition of tyrosine kinase signaling using the KIT inhibitor imatinib. As patients progress through subsequent lines of therapy a molecularly heterogeneous disease evolves, characterized by distinct subtypes and shifting repertoires of exon-specific KIT variants which directly impact treatment outcomes. Here, we use tumor-informed exome-scale liquid biopsy to identify and track the evolution of multiple resistance mechanisms in patients receiving tyrosine kinase inhibitors (TKIs) to address the unmet need of comprehensive understanding of GIST evolution in response to therapy. Matched tumor, normal and serial plasma samples were obtained from 15 heavily pretreated metastatic GIST patients. Following baseline sample collection, all patients received systemic TKI therapy, and were monitored until disease progression. Exome-scale detection of somatic variants in cfDNA from longitudinal matched plasma samples was achieved using the NeXT Liquid BiopsyTM platform. The ImmunoID NeXT PlatformⓇ, an augmented exome/transcriptome platform and analysis pipeline which generates comprehensive tumor and immune data was used to profile paired tumor and normal samples. Longitudinal whole exome sequencing of plasma identified dynamic shifts in existing clones harboring exon-specific KIT mutations, and evolution of new KIT mutations arising prior to identification of tumor progression using standard imaging techniques. We detected a correlation between the number of damaging mutations detected in baseline ctDNA and tumor exon 11 KIT mutation status, suggesting that plasma mutation profiles may be KIT-variant dependent. ctDNA from patients with shorter overall survival (OS) was enriched for variants in the PI3K-AKT and MAPK pathway, potentially contributing to immune evasion observed in those patients. Additional associations were observed between gene copy-number changes and OS (P = .0097). Previous studies have demonstrated that immune infiltration and activity may be KIT variant specific, here we broaden those findings, identifying a significant correlation between TCRɑ clonality and variants detected only in plasma (P = .04), as well as a significant association between TCRβ diversity and OS (HR = 2.55, log rank P = .04). Comprehensive profiling of paired tumor tissue (WES and RNA-Seq) and WES of serially collected ctDNA sensitively and repeatedly identified evolving KIT mutations and other molecular alterations prior to radiologically confirmed disease progression. These findings suggest plasma-based monitoring of late-stage GIST malignancies may be useful for non-invasive disease tracking, providing treatment guidance prior to traditional approaches. Citation Format: Charles W. Abbott, Niamh Coleman, Jing Wang, Josette Northcott, Jason Pugh, Dan Norton, Fábio C. Navarro, Lee D. McDaniel, Eric Levy, Rachel Marty Pyke, John Lyle, Jason Harris, Gabor Bartha, Filip Janku, John West, Richard O. Chen, Sean Boyle. Exome-scale longitudinal tracking of emerging therapeutic resistance in GIST via analysis of circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5161.

Companion Diagnostics and Predictive Biomarkers for MET-Targeted Therapy in NSCLC

Simple SummaryMET is a receptor tyrosine kinase encoded by the MET proto-oncogene that has a significant role in cancer cell progression. Several drugs targeting MET are under development for the treatment of different cancers, including non-small-cell lung cancer (NSCLC). However, until now, relatively few of these drugs have shown sufficient clinical activity and obtained regulatory approval. One of the reasons for this could be the lack of effective biomarkers to select the right patients for treatment. In a number of clinical trials, different biomarkers have been studied, but so far, MET exon 14 skipping mutation is the only one that has shown sufficient predictive properties. Another interesting biomarker is MET amplification detected by fluorescence in situ hybridization (FISH), which has shown promising results in the treatment of patients with NSCLC. Future clinical research will show whether MET amplification by FISH is an effective predictive biomarker for MET-targeted therapy.Dysregulation of the MET tyrosine kinase receptor is a known oncogenic driver, and multiple genetic alterations can lead to a clinically relevant oncogenesis. Currently, a number of drugs targeting MET are under development as potential therapeutics for different cancer indications, including non-small cell lung cancer (NSCLC). However, relatively few of these drugs have shown sufficient clinical activity and obtained regulatory approval. One of the reasons for this could be the lack of effective predictive biomarkers to select the right patient populations for treatment. So far, capmatinib is the only MET-targeted drug approved with a companion diagnostic (CDx) assay, which is indicated for the treatment of metastatic NSCLC in patients having a mutation resulting in MET exon 14 skipping. An alternative predictive biomarker for MET therapy is MET amplification, which has been identified as a resistance mechanism in patients with EGFR-mutated NSCLC. Results obtained from different clinical trials seem to indicate that the MET/CEP7 ratio detected by FISH possesses the best predictive properties, likely because this method excludes MET amplification caused by polysomy. In this article, the concept of CDx assays will be discussed, with a focus on the currently FDA-approved MET targeted therapies for the treatment of NSCLC.

The FLT3 F691L Gatekeeper Mutation Promotes Clinical Resistance to Gilteritinib + Venetoclax (GILT + VEN) in AML

Background: FMS-like tyrosine kinase (FLT3) is one the most frequently mutated genes in AML and is associated with poor prognosis. FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations occur in up to 30% and 5-10% of AML, respectively. Several small molecule FLT3 inhibitors (FLT3i) have been developed but their use as single agents is limited due to the development of drug resistance. Our lab developed a two-step model of early and late resistance to FLT3i that recapitulates resistance in AML patients (Traer et al. Cancer Res. 2016; Joshi et al. Cancer Cell 2021). Early resistance, also known as AML persistence, is the stage when residual AML cells are dependent upon the marrow microenvironment for survival and patients are clinically responding. Late resistance to GILT was characterized by expansion of intrinsic mutations, with NRAS mutations being the most frequent mutation, in addition to a few gatekeeper FLT3 mutations. Current therapies are looking at combinations to overcome GILT resistance, including chemotherapy, hypomethylating agents (HMAs), and venetoclax (VEN) +/- HMAs. GILT+VEN, in particular, has shown good initial activity in relapsed/refractory FLT3 AML patients (Daver et al. ASH 2020), however the mechanism of resistance to this combination is unknown.Results: Early resistance cell cultures to GILT+VEN were created by exposing MOLM14 cells to GILT 25nM + VEN 25nM alone or supplemented with microenvironmental ligands FGF2 or FLT3 ligand (FL; N=3/group). Media, drugs, and ligands were replenished twice weekly. After 25 weeks, only the cultures exposed to ligand resumed growth (N=1 for FGF2 and N=3 for FL). Ligands were then removed from these early resistant cultures to induce late resistance. There was an initial drop in cell viability but cells resumed growth after only 3.5 weeks (Fig. 1). In contrast, the time to develop early and late resistance to GILT monotherapy was 8 and 15 weeks, respectively. Immunoblot analysis of GILT + VEN early and late resistant cultures demonstrated restoration of FLT3 signaling, as well as phosphorylation of downstream AKT/MAPK pathways. These results also contrasted to late GILT monotherapy resistant cultures, which had downstream AKT/MAPK activation via outgrowth of NRAS mutations. Since FLT3 appeared to be functionally active, we sequenced FLT3 and found that all early and late GILT + VEN resistance cultures had gatekeeper FLT3 F691L mutations. F691L accounted for only in a minority of resistance cultures to GILT monotherapy. To test if FLT3 signaling was important for resistance, we exposed parental cells to higher concentrations of gilteritinib, which have been shown to partly overcome F691L, as well as the FLT3i FF-10101, which binds FLT3 at a different site and is not affected by the F691L mutation. Both of these approaches restored sensitivity to FLT3i in vitro. As expected, the F691L mutation provided broad resistance to most FLT3i (Fig. 2).To validate this mechanism of resistance in patients, we identified a relapsed FLT3-ITD patient who was treated with GILT monotherapy for 5 months, followed by GILT + HMA for 4 cycles, and then GILT + VEN for resistant proliferative disease. After an initial response to GILT + VEN, the leukemia cells began to increase again in the peripheral blood. A repeat genetic test was ordered and the patient was found to have developed a FLT3 F691L mutation at a high variant allele frequency (Fig. 3).Conclusion: We have developed a robust cell line model of early and late resistance to FLT3i that mimics the timing and expansion of resistance mutations in the clinic. Our model of early and late resistance to GILT combinations can prospectively predict mechanisms of resistance. Although uncommon as a mechanism of resistance to GILT monotherapy, our model and early patient data predicts that F691L mutations are more important for GILT + VEN resistance. [Display omitted] DisclosuresTyner: Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Agios: Research Funding; Schrodinger: Research Funding. Traer: ImmunoGen: Membership on an entity's Board of Directors or advisory committees; Schrodinger: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier/Agios: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees.

RNA Polymerase II Is a Therapeutic Target to Overcome FLT3 Inhibitor Resistance Mediated By the Bone Marrow Microenvironment

Background: Gilteritinib is a clinically active FLT3 tyrosine kinase inhibitor (TKI) approved for relapsed/refractory FLT3-mutant AML, but nearly all patients treated with gilteritinib and other FLT3 TKIs eventually develop clinical resistance. Activating RAS/MAPK pathway mutations are a predominant non-FLT3 dependent resistance mechanism in patients treated with gilteritinib. AML blasts can also develop FLT3 TKI resistance secondary to paracrine MAPK activation stimulated by FLT3 Ligand, FGF2, or other protective cytokines within the bone marrow microenvironment (BME). To identify potential targets that sensitize AML cells to gilteritinib-induced apoptosis in a model of the BME, we performed a genome-wide CRISPR/Cas9 death screen in MOLM-14 FLT3-ITD+ human AML cells cultured in bone marrow stromal cell conditioned media. We hypothesize that identified genes represent promising combinatorial therapeutic targets that can enhance clinical efficacy of FLT3 TKIs in AML.Methods: To model stroma-mediated TKI resistance, we used the HS5 human bone marrow stromal cell line that secretes multiple cytokines (G-CSF, GM-CSF, FGF2) and supports myeloid progenitor proliferation in co-culture. MOLM-14 CRISPRi cells transduced with CRISPRi-v2 genome-wide sgRNA library were cultured in HS5 conditioned media for 24 hours and then treated with gilteritinib 250 nM. Cells were stained with a fluorogenic caspase 3/7 reagent and then fixed after 24 hours of drug treatment. Caspase-3 positive cells were sorted from the entire drug-treated cell population by FACS and guide RNAs enriched or depleted in this sample as compared to an untreated T0 sample were determined by NGS.Results: We identified several gene-level hits that were enriched in the apoptotic population (FDR <0.2). Among these, we identified multiple transcription factors or regulators of transcriptional activation. The latter included multiple components of RNA pol II machinery (POLR2G, RTF1) and multiple subunits of Mediator (MED12, MED30, MED21, MED11), a complex that regulates RNA pol II activity and has been shown to modulate super-enhancer-associated genes in AML cells. To validate select hits, we transduced MOLM-14 and MV411 CRISPRi cells with a tetracycline-inducible sgRNA expression vector. Using this system, we found that conditional knockdown of MED12, the top scoring Mediator subunit in our screen, significantly sensitized MOLM-14 (FgH1) cells to gilteritinib while modestly augmenting the cytotoxicity of gilteritinib in MV411 (FgH1) cells when compared to a non-targeting sgRNA. Functionally, MED12 associates with MED13, Cyclin C, and CDK8 to form the CDK8 kinase module of Mediator. MED12 knockdown, as expected, led to suppression of STAT1 S7272 and STAT5 S726 phosphorylation, known targets of CDK8. Based on these results, we hypothesized that CDK8 inhibition would augment apoptosis induced by gilteritinib in HS5 conditioned media. Using SEL120, a novel CDK8 inhibitor already in early phase AML clinical trials, we performed an 8 x 8 dose matrix drug synergy analysis of gilteritinib and SEL120 in multiple FLT3-mutant AML cell lines using Bliss independence modeling. We found that SEL120 was synergistic with gilteritinib in inducing apoptosis in MOLM-14 and MV411 cells in HS5 conditioned media (Bliss synergy scores of 2.44 and 11.45 with most synergistic area scores of 10.91 and 26.85, respectively). We also found combinatorial activity against MOLM-14 cells harboring secondary NRAS activating mutations (G12C and Q61K), suggesting the therapeutic combination could potentially overcome cell intrinsic and extrinsic MAPK-activating resistance mechanisms. Lastly, we found that gilteritinib and SEL120 combined to impart greater cytotoxicity than either drug alone in a primary sample (AML #1) from a patient with newly diagnosed AML possessing a FLT3-ITD mutation at high mutant allele ratio.Conclusions: The results and validation of our CRISPRi screen suggest that combined CDK8 and FLT3 inhibition is a novel strategy for augmenting gilteritinib cytotoxicity. Assessment of the activity of the combination in additional primary AML samples and in vivo murine models of AML is planned. Additional candidate targets already described and other Mediator and RNA pol II subunits from our screen are also being further evaluated to precisely define the transcriptional programs that influence FLT3 inhibitor resistance. DisclosuresLogan: Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding; Amgen, Pfizer, AbbVie: Consultancy. Gilbert: Denali Therapeutics: Ended employment in the past 24 months, Other: Spouse/Significant Other's Employment; GSK: Consultancy, Research Funding; AstraZeneca: Research Funding; Chroma Medicine: Consultancy, Other: Co-founder. Smith: Revolutions Medicine: Research Funding; AbbVie: Research Funding; Daiichi Sankyo: Consultancy; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

First-in-Class Anti-immunoglobulin-like Transcript 4 Myeloid-Specific Antibody MK-4830 Abrogates a PD-1 Resistance Mechanism in Patients with Advanced Solid Tumors.

In this first-in-human study (NCT03564691) in advanced solid tumors, we investigated a novel first-in-class human IgG4 monoclonal antibody targeting the immunoglobulin-like transcript 4 (ILT4) receptor, MK-4830, as monotherapy and in combination with pembrolizumab. Patients with histologically/cytologically confirmed advanced solid tumors, measurable disease by RECIST v1.1, and evaluable baseline tumor sample received escalating doses of intravenous MK-4830 every 3 weeks as monotherapy (parts A and B) and in combination with pembrolizumab (part C). Safety and tolerability were the primary objectives. Pharmacokinetics, objective response rate per RECIST v1.1, and molecular biomarkers were also evaluated. Of 84 patients, 50 received monotherapy and 34 received combination therapy. No dose-limiting toxicities were observed; maximum tolerated dose was not reached. MK-4830 showed dose-related target engagement. Eleven of 34 patients in the dose-escalation phase who received combination therapy achieved objective responses; 5 previously had progressive disease on anti-PD-1/PD-L1 therapies. Exploratory evaluation of the association between response and pretreatment gene expression related to interferon-gamma signaling in tumors suggested higher sensitivity to T-cell inflammation with combination therapy than historically expected with pembrolizumab monotherapy, with greater response at more moderate levels of inflammation. This first-in-class MK-4830 antibody dosed as monotherapy and in combination with pembrolizumab was well tolerated with no unexpected toxicities, and demonstrated dose-related evidence of target engagement and antitumor activity. Inflammation intrinsic to the ILT4 mechanism may be facilitated by alleviating the myeloid-suppressive components of the tumor microenvironment, supporting the target of ILT4 as a potential novel immunotherapy in combination with an anti-PD-1/PD-L1 agent.

LIMITing tumours with an immunogenic lncRNA.

Overcoming immune resistance mechanisms in patients with cancer is critical for successful immunotherapy. A new study now identifies LIMIT as an immunogenic long non-coding RNA (lncRNA) that intrinsically regulates anti-tumour immune responses, thereby highlighting the therapeutic potential of targeting lncRNAs to improve the outcome of cancer immunotherapy.

Heterogeneity in Nectin-4 expression across molecular subtypes of urothelial cancer mediates sensitivity to enfortumab vedotin.

463 Background: Enfortumab vedotin (EV) is an antibody-drug conjugate (ADC) targeting Nectin-4 (encoded by the PVRL4/NECTIN4 gene ) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In the present study, we sought to 1) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and 2) determine if Nectin-4 expression mediates EV sensitivity or resistance. Methods: NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1912 total patients) were used to compare relative NECTIN4 expression across molecular subtypes. The outcome of the gene expression analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in multiple bladder cancer cell lines. NECTIN4 was stably over-expressed or knocked down in basal (TCCSUP and UMUC-3) and luminal (HT-1376, HT-1197 and UMUC-9) bladder cancer cell lines, respectively, and EV dose-response assays were performed, as measured by cell proliferation and clonogenic assays. Results: NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes (p &lt; 0.001). NECTIN4 expression is positively correlated with the luminal markers GATA3, FOXA1, and PPARG across cohorts (Spearman’s rank correlation r = 0.57, p &lt; 0.0001 for GATA3, r = 0.37, p &lt; 0.0001 for FOXA1, and r = 0.56, p &lt; 0.0001 for PPARG). NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 led to EV resistance, and EV-resistant cell lines expressed decreased levels of Nectin-4. Conclusions: Results of this pre-clinical study suggest that sensitivity to EV is mediated by expression of NECTIN4, which is significantly enriched in luminal subtypes of bladder cancer. Downregulation of NECTIN4 leads to resistance to EV. These findings have implications for biomarker development, patient selection and the inclusion of molecular subtyping in ongoing and future EV clinical trials. Further investigation into Nectin-4 loss as a mechanism of resistance in patients treated on EV is warranted.

Genomic analysis of circulating tumor DNA in 3,334 patients with advanced prostate cancer to identify targetable BRCA alterations and AR resistance mechanisms.

25 Background: Comprehensive genomic profiling (CGP) by next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) from plasma provides a minimally invasive method to identify targetable genomic alterations (GAs) and resistance mechanisms in patients with metastatic castration-resistant prostate cancer (mCRPC). The circulating tumor fraction in patients with mCRPC and the clinical validity of GAs detected in plasma remain unknown. We evaluated the landscape of GAs using ctDNA-based CGP and assessed concordance with tissue-based CGP. Methods: Plasma from 3,334 patients with advanced prostate cancer (including 1,674 mCRPC screening samples from the TRITON2/3 trials and 1,660 samples from routine clinical CGP) was analyzed using hybrid-capture-based gene panel NGS assays. Results were compared with CGP of 2,006 metastatic prostate cancer tissue biopsies. Concordance was evaluated in 837 patients with both tissue (archival or contemporaneous) and plasma NGS results. Results: 3,127 patients [94%] had detectable ctDNA. BRCA1/2 were mutated in 295 patients [8.8%]. In concordance analysis, 72/837 [8.6%] patients had BRCA1/2 mutations detected in tissue, 67 [93%] of whom were also identified by ctDNA, and 20 patients were identified using ctDNA but not tissue [23% of all patients identified using ctDNA]. ctDNA detected subclonal BRCA1/2 reversions in 10 of 1,660 [0.6%] routine clinical CGP samples. AR alterations, including amplifications and hotspot mutations, which were detected in 940/2,213 patients [42%]. Rare AR compound mutations, rearrangements, and novel in-frame deletions were identified. Altered pathways included PI3K/AKT/mTOR [14%], WNT/β-catenin [17%], and RAS/RAF/MEK [5%]. Microsatellite instability was detected in 31/2,213 patients [1.4%]. Conclusions: In the largest study of mCRPC plasma samples conducted to date, CGP of ctDNA recapitulated the genomic landscape detected in tissue biopsies, with a high level of agreement in detection of BRCA1/2 alterations. It also identified patients who may have gained somatic BRCA1/2 alterations since archival tissue was collected. ctDNA detected more acquired resistance GAs than tissue, including novel AR-activating variants. The large percentage of patients with rich genomic signal from ctDNA, and the sensitive, specific detection of BRCA1/2 alterations position liquid biopsy as a compelling clinical complement to tissue CGP for patients with mCRPC.

OA05.02 Analysis of Resistance Mmechanisms to Pralsetinib in Patients with RET Fusion-Positive Non-Small Cell Lung Cancer (NSCLC) from the ARROW Study

Chromosomal rearrangements involving the RET receptor tyrosine kinase are validated oncogenic drivers in 1–2% of patients with NSCLC. Pralsetinib is a selective RET tyrosine kinase inhibitor that has demonstrated clinical activity in patients with NSCLC harboring a RET fusion. Here, we report potential mechanisms of resistance in patients with NSCLC whose disease progressed on pralsetinib.

Usefulness of point-of-care multiplex PCR to rapidly identify pathogens responsible for ventilator-associated pneumonia and their resistance to antibiotics: an observational study

BackgroundThe use of multiplex PCR to shorten time to identification of pathogens and their resistance mechanisms for patients with ventilator-associated pneumonia (VAP) is attractive, but poorly studied. The multiplex PCR–based Unyvero pneumonia cartridge assay can directly identify 20 bacteria and one fungus, amongst the most frequently causing VAP, and 19 of their resistance markers in clinical specimens (bronchoalveolar lavage or tracheal aspirate), with a turnaround time of 4–5 h. We performed this study to evaluate the concordance between the multiplex PCR–based Unyvero pneumonia cartridge assay and conventional microbiological techniques to identify pathogens and their resistance mechanisms in patients with VAP.MethodsAll patients suspected of having VAP (January 2016 to January 2019), who underwent fiberoptic bronchoscopy with bronchoalveolar lavage fluid (BALF) and whose BALF microscopy examination revealed intracellular bacteria, were included. BALF conventional cultures (gold standard), antimicrobial susceptibility testing and processing for the Unyvero pneumonia cartridge were done. Culture and Unyvero results were compared.ResultsCompared to cultures of the 93 samples processed for both techniques, Unyvero correctly identified pathogens in 68 (73%) proven VAP episodes, was discordant for 25 (27%), detected no pathogen in 11 and overdetected a not otherwise found pathogen in six. For the eight remaining discordant results, the pathogen responsible for VAP was not included in the Unyvero cartridge panel or it grew at a non-significant level in culture. Amongst the 31 (33%) resistance mechanism discordances observed, 22 were resistance detection failures and 24 concerned Pseudomonas aeruginosa.ConclusionsCompared to conventional microbiological cultures, the Unyvero pneumonia cartridge had poor diagnostic performance: it correctly identified pathogens and their resistance mechanisms in 73% and 67% of VAP cases, respectively. The lack of performance on the resistance mechanism was more pronounced when the pathogen detected was a Pseudomonas aeruginosa.

Abstract A28: SPACEWALK: Plasma NGS for remote evaluation of ALK drug resistance in advanced NSCLC

Abstract Introduction: Precision therapy for cancer drug resistance requires detection of resistance mutations and treatment with appropriate targeted therapies. This paradigm is well established in EGFR-mutant NSCLC, yet our understanding of drug resistance in ALK-positive NSCLC is limited. Next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA) now permits noninvasive interrogation of drug resistance. To facilitate improved understanding of ALK drug resistance and the effectiveness of treatment strategies, we launched this remote participation study. Methods: The SPACEWALK study offers plasma NGS to patients with advanced NSCLC with systemic progression on a next-generation ALK TKI to assess for mechanisms of resistance. Patients consent, enroll, and participate remotely, permitting a larger cohort of ALK positive patients to be studied. Remote phlebotomists can be dispatched to the patient’s home when necessary to ease the burden of study participation and to ensure study compliance. Blood is sent to Resolution Biosciences for ctDNA extraction and hybrid capture NGS, with results returned to the study team and the patient’s physician. Patients are followed for 2 years and blood is collected again after starting a new treatment or at progression. This study is supported by the Addario Lung Cancer Medical Institute (ALCMI). Results: Of 143 patients contacting us via the study website, 61 were offered consent and 53 signed consent. 46 enrolled from 23 different US states and 38 different medical centers. Plasma NGS results were returned a median of 13 days after blood draw (range 8-21). Of the 46 patients, the known ALK fusion was detected in 20 (43%) with a median ALK fusion AF of 3.5% (range 0.3%-37%). 10 patients also underwent plasma NGS with Guardant360, with a median time between the two plasma NGS draws of 1.5 weeks (range 0-22.6). In 4 cases, Resolution Bioscience identified an ALK fusion (AF 0.2-37%) not detected on Guardant360. Among the 20 patients with ALK fusions detected, 12 (60%) were found to carry ALK resistance mutations, 5 with G1202R; 2 patients had 2 or more ALK mutations detected. In addition, 6 participants (30%) had a MET amplification detected with a median of 8 copies (range 3-22) and 1 had a KRAS G12V mutation detected (10% AF). One patient with high MET amplification received combination therapy with alectinib and crizotinib and had dramatic radiographic response. Conclusions: Plasma NGS permits the detection of targetable resistance mechanisms in patients with ALK-positive NSCLC and drug resistance. Sensitivity of different plasma NGS assays for ALK fusions varies. This assay may help guide oncologists across the country to select best treatment options after resistance. Such remote-participation studies may offer a new mechanism for characterizing resistance to emerging targeted therapies in rare cancer populations. Citation Format: Marissa N. Lawrence, Rubii M. Tamen, Alicia Sable-Hunt, Seyed Ali Hosseini, George R. Simon, Jonathan W. Riess, Geoffrey R. Oxnard. SPACEWALK: Plasma NGS for remote evaluation of ALK drug resistance in advanced NSCLC [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A28.

CtDNA resistance landscape of lazertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI).

9601 Background: While EGFR mutant ( EGFRm) non-small cell lung cancer (NSCLC) patients usually experience improved clinical benefit with EGFR TKIs, most eventually progress. Understanding mechanisms of resistance (MoR) may allow for more personalized treatment. Lazertinib is an irreversible third generation EGFR TKI for which MoR are unknown. Obtaining sufficient tumor tissue for genotyping at progression is often difficult. Therefore, we utilized plasma ctDNA from patients treated with lazertinib to explore MoR. Methods: Plasma samples from 47 NSCLC patients in the phase 2 trial of lazertinib (NCT03046992) were collected at screening and progressive disease (PD) and underwent ctDNA NGS of 74 genes using Guarant360. All patients were positive for an EGFR Ex19del or L858R ( EGFRm) and T790M by tissue testing at screening. Acquired, nonsynonymous, characterized mutations detected in a PD sample but not in the screening sample from the respective patient were considered putative MoR, excluding aneuploidy. Patients with detectable plasma EGFRm and/or T790M at screening were evaluable. Results: ctDNA was detected in 47 (100%) screening samples and 43/45 (96%) PD samples (two failed sequencing). An EGFRm was detected in 85% of patients at screening (n = 40), 38 of which had PD ctDNA results and were included in analysis. T790M was detected in 30 patients at screening and subsequently not detected at PD in 21 of these patients, 55% of all 38 included patients. Among the ten patients with T790M detected at PD, on-target MoR were detected in 7 (18% of all included patients) including EGFR C797S (n = 3, 8%), EGFR amplification (n = 3, 8%), and EGFR T854A (n = 1, 3%). All C797S were in cis with T790M. No on-target MoR were detected in patients without T790M detected at PD. Off-target MoR were seen in 34% of patients (13/38) including mutations in PIK3CA (13%; 2 E545K, 2 E542K, 1 E81K), ERBB2 (5%; 1 D769H, 1 V777L), KRAS (3%; 1 G12C), and BRAF (3%; 1 G469A). Gene amplifications were detected in CCND1 (n = 1, 3%) , CCNE1 (n = 2, 5%) , ERBB2 (n = 1, 3%) , FGFR1 (n = 1, 3%) , MET (n = 4, 11%) , and PIK3CA (n = 1, 3%), with some patients having multiple MoR. Conclusions: The spectrum of MoR identified in this cohort of patients treated with lazertinib is similar to that reported in other third generation EGFR TKIs, but with some differences in frequencies. The most common resistance mechanisms are T790M loss and PIK3CA alterations which may address the mechanism of action. Our findings suggest putative MoR of lazertinib and show that ctDNA NGS is an effective way to identify MoR in patients progressing on targeted therapy. Clinical trial information: NCT03046992 .

Abstract GS2-05: Microscaled proteogenomic methods for precision oncology

Abstract Cancer proteogenomics combines genomics, transcriptomics and mass spectrometry-based proteomics to gain insights into cancer biology and treatment responsiveness. While proteogenomics analyses have already shown great potential to deepen our understanding of cancer tissue complexity and signaling, how a patient’s tumor changes upon treatment has largely been the province of genomics. This is due to technical difficulties associated with doing proteogenomic analysis on clinic-derived core-needle biopsies. To address this critical need, we have developed a “microscaled” proteogenomics approach for tumor-rich OCT-embedded core needle biopsies. Tissue-sparing specimen processing (“Biopsy Trifecta EXTraction”, BioTExt) and microscaled proteomics (MiProt) methodologies allowed generation of deep-scale proteogenomics datasets, with copy number and transcript information for &amp;gt;20,000 genes and mass spectrometry-based identification and quantification of nearly all expressed proteins in a tumor (&amp;gt;10,000 proteins) and more than &amp;gt;20,000 phosphosites starting with just 25 micrograms of protein per sample. In order to understand the capabilities and limitations our our approach relative to more conventional deepscale proteomics requiring &amp;gt;10X more starting material, we compared preclinical patient derived xenograft (PDX) models at conventional scale with data obtained by core-needle biopsy of the same tissues. Comparable depth and biological insights were obtained from the cores relative to surgically resected tumors. As a proof-of-concept for implementation in clinical trials, we applied microscaled proteogenomic methods to a small-scale clinical study where biopsies were accrued from patients with ERBB2+ locally advanced breast cancer before and 48 to 72 hours after the first dose of neoadjuvant Trastuzumab-based chemotherapy. Multi-omics comparisons were conducted between samples associated with residual disease versus samples associated with complete pathological response. Integrative, microscaled proteogenomic analyses efficiently diagnosed the molecular bases of diverse candidate treatment resistance mechanisms including: 1) absence of ERBB2 amplification (false-ERBB2+); 2) insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification (pseudo-ERBB2+); 3) resistance features in true-ERBB2+ cases including androgen receptor signaling, mucin expression and an inactive immune microenvironment; 4) lack of acute phospho-ERBB2 down-regulation in non-pCR cases. In summary, we have developed a robust proteogenomics pipeline well suited for large-scale cancer clinical studies to identify potential resistance mechanism in patients. We conclude that microscaled cancer proteogenomics could improve diagnostic precision in the clinical setting. Citation Format: Shankha Satpathy, Eric Jaehnig, Krug Karsten, Beom-Jun Kim, Alexander Saltzman, Doug Chan, Kimberly Holloway, Meenakshi Anurag, Chen Huang, Purba Singh, Ari Gao, Noel Namai, Yongchao Dou, Bo Wen, Suhas Vasaikar, David Mutch, Mark Watson, Cynthia Ma, Foluso Ademuyiwa, Mothaffar Rimawi, Jeremy Hoog, Samuel Jacobs, Anna Malovannaya, Terry Hyslop, D.R Mani, Charles Perou, George Miles, Bing Zhang, Michael Gillette, Steven Carr, Matthew Ellis. Microscaled proteogenomic methods for precision oncology [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-05.

Ultra-deep next-generation sequencing of plasma cell-free DNA in patients with advanced lung cancers: results from the Actionable Genome Consortium

BackgroundNoninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration. Patients and methodsPlasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50000× raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases. ResultsIn 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r=0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification. ConclusionsUltra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping.

Genetic instability and recurrent MYC amplification in ALK-translocated NSCLC: a central role of TP53 mutations.

The anaplastic lymphoma kinase (ALK) rearrangement defines a distinct molecular subtype of non‐small cell lung cancer (NSCLC). Despite the excellent initial efficacy of ALK inhibitors in patients with ALK+ lung cancer, resistance occurs almost inevitably. To date, there is no reliable biomarker allowing the identification of patients at higher risk of relapse. Here, we analysed a subset of 53 ALK+ tumours with and without TP53 mutation and ALK+ NSCLC cell lines by NanoString nCounter technology. We found that the co‐occurrence of early TP53 mutations in ALK+ NSCLC can lead to chromosomal instability: 24% of TP53‐mutated patients showed amplifications of known cancer genes such as MYC (14%), CCND1 (10%), TERT (5%), BIRC2 (5%), ORAOV1 (5%), and YAP1 (5%). MYC‐overexpressing ALK+ TP53‐mutated cells had a proliferative advantage compared to wild‐type cells. ChIP‐Seq data revealed MYC‐binding sites within the promoter region of EML4, and MYC overexpression in ALK+ TP53‐mutated cells resulted in an upregulation of EML4–ALK, indicating a potential MYC‐dependent resistance mechanism in patients with increased MYC copy number. Our study reveals that ALK+ NSCLC represents a more heterogeneous subgroup of tumours than initially thought, and that TP53 mutations in that particular cancer type define a subset of tumours that harbour chromosomal instability, leading to the co‐occurrence of pathogenic aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract 4952: Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to ER-directed therapies

Abstract Despite the reduction in cancer recurrence and mortality provided by therapies that target the estrogen receptor (ER), resistant ER+ breast cancer remains the most common cause of breast cancer death. Beyond mutations in ER in 25-30% of patients treated with aromatase inhibitors, which deplete circulating estrogen, our understanding of clinical mechanisms of resistance to agents that target the ER remains incomplete. We used whole exome sequencing (WES) of metastatic biopsies to identify mechanisms of resistance in patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-directed agents, including aromatase inhibitors, tamoxifen, and fulvestrant. We noted mutations in human epidermal growth factor receptor 2 (HER2) in metastatic biopsies from 12 patients out of 168. Of these, eight mutations had previously been described as activating. Examination of the treatment-naïve primary tumors in the five patients with activating mutants where WES from the matched treatment-naïve primary tumor was available revealed no evidence of pre-existing mutations in four of the five patients, suggesting that these four mutations were acquired under the selective pressure of ER-directed therapy. These acquired HER2 mutations were mutually exclusive with ER mutations, suggesting a distinct mechanism of resistance to ER-directed therapies. In vitro analysis through expression in ER+/HER2- cell lines confirmed that these mutations conferred estrogen independence. In addition, and in contrast to ER mutations, these HER2 mutations resulted in resistance to tamoxifen, fulvestrant, and the CDK4/6 inhibitor palbociclib. The mutants result in increased AKT and ERK phosphorylation in ER+ cell lines, while simultaneously repressing ER levels and ER-dependent transcriptional targets. One mutation observed in two patients, S653C, occurring in the transmembrane domain and not previously observed in breast cancer, was shown to likely function through constitutive dimerization, a mechanism of activation not previously described for this mutation. Resistance caused by all four mutations was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib, suggesting a novel effective treatment strategy in these patients. Thus, we have shown that acquired activating HER2 mutations can confer endocrine resistance in ER+/HER2- breast cancer, and that such resistance can be effectively reversed by combination therapies that include an anti-HER2 inhibitor. Citation Format: Utthara Nayar, Ofir Cohen, Christian Kapstad, Adrienne Waks, Seth A. Wander, Corrie Painter, Samuel Freeman, Priyanka Ram, Nicole Persky, Lori Marini, Karla Helvie, Nelly Oliver, Cynthia X. Ma, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to ER-directed therapies [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 4952.