Search

Although BCL2 mutations are reported as later occurring events leading to venetoclax resistance, many other mechanisms of progression have been reported but remain poorly understood. Here we analyze longitudinal tumor samples from eleven patients with disease progression on venetoclax to characterize the clonal evolution of resistance. All patients tested showed increased in vitro resistance to venetoclax at their post-treatment timepoint. We found the previously described acquired BCL2-G101V mutation in only 4/11 patients with 2 patients showing very low variant allele fraction (VAF; 0.03-4.68%). Whole exome sequencing (WES) revealed acquired loss(8p) in 4/11 patients of which 2 patients also have gain (1q21.2-21.3) in the same cells, affecting the MCL-1 gene. In vitro experiments showed that CLL cells from the four patients with loss(8p) were more resistant to venetoclax than those without it, while the cells from two patients also carrying gain (1q21.2-21.3) showed increased sensitivity to MCL-1 inhibition. Progression samples with gain (1q21.2-21.3) were more susceptible to combination MCL-1 inhibitor with venetoclax. Differential gene expression analysis comparing bulk RNAseq data from pre-treatment and progression time points of all patients showed upregulation of proliferation, BCR and NFKB gene sets including MAPK genes. Cells from progression timepoints demonstrated upregulation of surface immunoglobulin M (sIgM) and higher pERK levels compared to the pre-timepoint, suggesting an upregulation of BCR signaling that activates the MAPK pathway. Overall, our data suggest several mechanisms of acquired resistance to venetoclax in CLL that could pave the way for rationally designed combination treatments for venetoclax resistant CLL patients.

Application of Bliss independence to test for synergy effect of TKI combination treatment in vivo

Supplementary Table S2 shows type I and II MET kinase inhibitors and key trials studying their activity lung cancer populations enriched for MET-dependence.

Supplementary Figure S2 shows expression of D1228V mutation in PC9 cells as confirmed by RT-PCR and cDNA sequencing.

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non–small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro. The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

Supplementary Figures S1-3; Supplementary Tables S1-3

Supplementary methods, including croplet digital PCR for MET D1228V; molecular docking study; Molecular Dynamics Simulations.

Supplementary Figure Legends.

Amplified and/or mutated MET can act as both a primary oncogenic driver and as a promoter of tyrosine kinase inhibitor (TKI) resistance in non–small cell lung cancer (NSCLC). However, the landscape of MET-specific targeting agents remains underdeveloped, and understanding of mechanisms of resistance to MET TKIs is limited. Here, we present a case of a patient with lung adenocarcinoma harboring both a mutation in EGFR and an amplification of MET, who after progression on erlotinib responded dramatically to combined MET and EGFR inhibition with savolitinib and osimertinib. When resistance developed to this combination, a new MET kinase domain mutation, D1228V, was detected. Our in vitro findings demonstrate that METD1228V induces resistance to type I MET TKIs through impaired drug binding, while sensitivity to type II MET TKIs is maintained. Based on these findings, the patient was treated with erlotinib combined with cabozantinib, a type II MET inhibitor, and exhibited a response.Significance: With several structurally distinct MET inhibitors undergoing development for treatment of NSCLC, it is critical to identify mechanism-based therapies for drug resistance. We demonstrate that an acquired METD1228V mutation mediates resistance to type I, but not type II, MET inhibitors, having therapeutic implications for the clinical use of sequential MET inhibitors. Cancer Discov; 6(12); 1334–41. ©2016 AACR.See related commentary by Trusolino, p. 1306.This article is highlighted in the In This Issue feature, p. 1293

Tamoxifen is widely used in the adjuvant treatment of estrogen receptor–positive (ER+) breast cancer and is an important drug for pre-menopausal women and post-menopausal patients who cannot tolerate aromatase inhibitors. Despite the clear clinical benefit in improving relapse-free and overall survival in these patients, an adverse effect of tamoxifen is a 2- to 7-fold increased risk of uterine cancer (UC) after 2-5 years of treatment. To date, the mechanism of tamoxifen-driven tumorigenesis is not well understood, and preventive approaches are lacking. Here, to molecularly characterize tamoxifen-associated uterine cancers (TA-UCs) and gain insights into their unique evolution, we performed whole-exome sequencing of 21 TA-UCs (discovery cohort) and droplet digital PCR (ddPCR) of an additional 40 TA-UCs (validation cohort) obtained from the ‘Tamoxifen Associated Malignancies: Aspects of Risk’ (TAMARISK) study. In addition, we used in vivo mouse models to: (i) further investigate tamoxifen-activated molecular pathways that may be involved in TA-UC tumorigenesis; and (ii) offer mechanistic insights. Overall, we discovered that TA-UCs were genomically similar to non–TA-UCs from The Cancer Genome Atlas (TCGA) project, with one profound exception: TA-UCs are characterized by a lower-than-expected frequency of mutations in two highly prevalent UC driver genes in the PI3K pathway: PIK3CA (14% [3/21] vs 48% [265/554] in non–TA-UC; P=0.003, Fisher’s exact test; Q=0.02, Benjamini-Hochberg FDR) and PIK3R1 (0%, [0/21] vs 31% [174/554]; P=0.001; Q=0.01). We used ddPCR in the independent TA-UC validation cohort and confirmed the low frequency of mutations in PIK3CA (7.5% [3/40] vs 21% [144/685] in control UCs from the Dana-Farber contribution to the AACR GENIE project; P=0.04). We next performed mouse in vivo studies and demonstrated that tamoxifen activated the PI3K pathway and increased cell proliferation in normal mouse uterine tissue through paracrine and autocrine effects, both of which were abrogated by the PI3K inhibitor alpelisib. Taken together, we describe a distinct and novel pathway of carcinogenesis in which tamoxifen acts as a driver event in the uterus and promotes tumor development in a mutation-independent manner. Indeed, tamoxifen may increase the risk of UC by activating the PI3K pathway, which can substitute for the early acquisition of oncogenic PIK3CA or PIK3R1 mutations observed in non–TA-UC tumors. Furthermore, the ability of a PI3K inhibitor to reduce cell proliferation in our mouse model raises the possibility that downregulating the PI3K pathway may prevent or significantly reduce TA-UC development, offering a potential future therapeutic and prevention strategy for specific high-risk patients undergoing tamoxifen therapy. Citation Format: Kirsten Kubler, Agostina Nardone, Shankara Anand, Daniel Gorvich, Marjolein Droog, Francisco Hermida-Prado, Tara Akshi, Avery S Feit, Gabriella Cohen, Gwen Dackus, Matthew Pun, Yanan Kuang, Justin Cha, Mendy Miller, William J Gibson, Cloud P Paweletz, Eliezer M Van Allen, Flora E van Leeuwen, Petra Nederlof, Harry Hollema, Quang-Dé Nguyen, Marian JE Mourits, Ignaty Leshchiner, Chip Stewart, Ursula A Matulonis, Wilbert Zwart, Yosef E Maruvka, Gad Getz, Rinath Jeselsohn. Tamoxifen instigates uterine cancer development by activating PI3K signaling and supersedes PIK3CA driver mutations [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS2-09.

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non–small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro. The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

Purpose: Tumor genotyping using cell-free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false-positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype.Experimental Design: ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with nonoverlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in patients with EGFR-mutant lung cancer on a prospective trial of erlotinib.Results: We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic curve analysis of four assays demonstrated an area under the curve in the range of 0.80 to 0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging before objective progression.Conclusions: Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks before radiographic progression. Clin Cancer Res; 20(6); 1698–705. ©2014 AACR.

Supplemental Figure 1: Comparison of targeted sequencing of cfDNA using standard hybridization selection versus Resolution methods. (A) UCSC genome browser depiction of read coverage in the TP53 vicinity. The coverage track representing hybridization selection of cfDNA was created by mapping the reads from sample SRR1197557 of Newman et al 2014. The other coverage track represents unique reads from Sample 511 in this paper. The off-target noise outside of the TP53 gene is higher in SRR1197557 compared with Sample 511. (B) Genome-wide calculation of off-target versus on-target coverage for the two samples. The percentage of read coverage overlapping the declared target regions (widened by an extra 100 nucleotides on each side) was calculated using bedtools. Supplemental Figure 2: Sensitivity of mutant allele detection as a function of dilution. A total of four gene fusions (orange open squares), thirteen point mutations (black circles) and one indels (orange open squares), were assayed at varying mutant allele concentrations (Supplemental Table 1). All 18 lesions were detected in the 2.5%, 1.0%, and 0.4% pools, while fourteen out of the 18 were detected at 0.1%. We expect gene fusions to be more difficult to detect because adequate sequence coverage demands a larger assay footprint than point mutations and probe orientation is unidirectional rather than bidirectional. Consistent with this expectation, the ROS1 fusion in cell line HCC78 and the ALK fusion in cell line H3122 were underrepresented. Supplemental Figure 3: Schematic of predicted genomic junctions for 5 translocations identified in plasma specimens.

Sunitinib (A) and Regorafenib (B) plasma concentration (ng/mL) across several timepoints during cycle 1.

PDF file - 71K

Purpose: Plasma cell-free DNA (cfDNA) genotyping is increasingly used in cancer care, but assay accuracy has been debated. Because most cfDNA is derived from peripheral blood cells (PBC), we hypothesized that nonmalignant mutations harbored by hematopoietic cells (clonal hematopoiesis, CH) could be a cause of false-positive plasma genotyping.Experimental Design: We identified patients with advanced non–small cell lung cancer (NSCLC) with KRAS, JAK2, or TP53 mutations identified in cfDNA. With consent, PBC DNA was tested using droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to test for CH-derived mutations.Results: We first studied plasma ddPCR results from 58 patients with EGFR-mutant NSCLC. Two had KRAS G12X detected in cfDNA, and both were present in PBC, including one where the KRAS mutation was detected serially for 20 months. We then studied 143 plasma NGS results from 122 patients with NSCLC and identified 5 JAK2 V617F mutations derived from PBC. In addition, 108 TP53 mutations were detected in cfDNA; for 33 of the TP53 mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH.Conclusions: We find that most JAK2 mutations, some TP53 mutations, and rare KRAS mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy. Clin Cancer Res; 24(18); 4437–43. ©2018 AACR.See related commentary by Bauml and Levy, p. 4352

Supplemental Table 3: Summary of ddPCR and NGS findings. The table shows the tissue genotype, ddPCR results, ddPCR allele frequency (mut / mut +wt allele frequency), NGS mutation calls and allele frequencies, and sequencing reads.

Supplemental Table 1: Summary of probe sequences

Purpose:Polyclonal emergence of KIT secondary mutations is a main mechanism of imatinib progression in gastrointestinal stromal tumor (GIST). Approved KIT inhibitors sunitinib and regorafenib have complementary activity against KIT resistance mutations. Preclinical evidence suggests that rapid alternation of sunitinib and regorafenib broadens the spectrum of imatinib-resistant subclones targeted.Patients and Methods:Phase Ib study investigating continuous treatment with cycles of sunitinib (3 days) followed by regorafenib (4 days) in patients with tyrosine kinase inhibitor (TKI)-refractory GIST. A 3+3 dosing schema was utilized to determine the recommended phase II dose (RP2D). Plasma samples were analyzed for pharmacokinetics and circulating tumor DNA (ctDNA) studies using targeted error correction sequencing (TEC-seq) and droplet digital PCR (ddPCR).Results:Of the 14 patients enrolled, 2 experienced dose-limiting toxicities at dose level 2 (asymptomatic grade 3 hypophosphatemia). Sunitinib 37.5 mg/day and regorafenib 120 mg/day was the RP2D. Treatment was well-tolerated and no unexpected toxicities resulted from the combination. Stable disease was the best response in 4 patients, and median progression-free survival was 1.9 months. Combined assessment of ctDNA with TEC-seq and ddPCR detected plasma mutations in 11 of 12 patients (92%). ctDNA studies showed that KIT secondary mutations remain the main mechanism of resistance in TKI-refractory GIST, revealing effective suppression of KIT-mutant subpopulations in patients benefiting from the combination.Conclusions:Sunitinib and regorafenib combination is feasible and tolerable. Rapid alternation of TKIs with complementary activity might be effective when combining drugs with favorable pharmacokinetics, potentially allowing active doses while minimizing adverse events. Serial monitoring with ctDNA may guide treatment in patients with GIST.

Primer/probe mix for KIT-resistant mutations.

XLSX file - 25K, cfDNA results for figures 2, 4, 5, calculated both as concentration per mL plasma and as percent mutant.

PDF file - 365K, Supplemental Figure 1: Assay optimization. For each TaqMan probe, the optimal annealing temperature was determined by testing each assay across a temperature gradient of 55.0 - 65{degree sign}C. Typical FAM plots for EGFR L858R (A) and EGFR exon 19 deletion (B) are shown. Supplemental Figure 2: Assay characteristics. As the sample input increases, the copies/μL output increases in a linear fashion across a wide dynamic range for both the L858R assay (A) and the exon 19 deletion assay (B). Testing for 10 and 50 copies of mutant EGFR in a background of 1000 and 50,000 genome equivalents (GE), the L858R assay demonstrates more consistent sensitivity (C) than the exon 19 deletion assay (D). Supplemental Figure 3: Detection of BRAF V600E in cfDNA from patients with advanced melanoma. Supplemental Figure 4: Correlation of LINE-1 quantitative PCR (QPCR) levels with DNA concentration as measured with PicoGreen across 69 plasma specimens (R2 = 0.94, p

Droplet digital PCR (ddPCR) of the peripheral blood cell DNA from 4 cases with JAK2 V617F found on plasma next-generation sequencing (NGS). In each, the JAK2 mutation was detected (top), consistent with clonal hematopoiesis, while not detected in a control sample.

Supplemental Table 2: Compilation of cell line admixture experimental data. The table shows the number of detected mutant reads, the total number of wild-type reads that correspond to the genomic coordinates of the mutant allele, and the frequency of mutant allele detection.

PDF file - 91K

Supplementary methods

Purpose: Tumor genotyping is a powerful tool for guiding non–small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA).Experimental Design: An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping.Results: NGS could identify mutations present in DNA dilutions at ≥0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification.Conclusions: Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. Clin Cancer Res; 22(4); 915–22. ©2015 AACR.See related commentary by Tsui and Berger, p. 790

Supplementary Figure Legend from Intratumoral Heterogeneity in EGFR-Mutant NSCLC Results in Divergent Resistance Mechanisms in Response to EGFR Tyrosine Kinase Inhibition

Description of additional methods and procedures used in the study.

Introduction: While tumor imaging has a fundamental role in drug development as a determinant of clinical response it may not always be a rapid measure of treatment effect. Changes in plasma ctDNA levels, on the other hand, has the potential to be a more readily available measure of response. Here we report on early plasma response measurements of KRAS G12C ctDNA by digital droplet PCR (ddPCR) in a Phase 2 trial of Adagrasib, a potent, orally available, small molecule covalent inhibitor of KRAS G12C, in patients with advanced non-small cell lung cancer with KRAS G12C mutation. Methods/Results: To study the timing of plasma response, we performed serial ddPCR on 60 KRAS G12C-mutant NSCLC patients that participated in cohort A of the KRYSTAL-1 phase 2 clinical trial. Cohort A evaluated adagrasib at 600 mg orally BID in patients with NSCLC previously treated with platinum-based chemotherapy. We analyzed the change in ctDNA levels at 2 specific intervals: between cycles 1 and 2 (i.e. initial change) and at start of cycles 4. Of 60 pts, 7 pts were excluded because of missing C1D1 draws, 13 pts were excluded for non-detectable KRAS G12C level at C1D1 and 1 patient was excluded because of a missing C2D1 draw leaving us with 39 evaluable patients for plasma response assessment. We found that most of the response in ctDNA could be seen during the initial 3-4 week treatment period, well before the first scan at 6-8 weeks. 38 pts (97.4%) showed any decrease in plasma levels from baseline levels. 35 patients (89.7%) show a decrease >90% and 33 pts (84.6%) achieved complete clearance at C2D1. Patients with complete ctDNA clearance at C2D1 showed improved ORR (60.6% vs 33.3%), while ctDNA clearance at C4 was associated with an improved survival (12.6 months vs 5.4 months) and PFS (HR 0.3). Conclusion: Our results support using early plasma response of KRAS G12C assessed at 2-3 weeks to inform the interpretation of initial imaging performed at 6-8 weeks Citation Format: Cloud Paweletz, Grace A. Heavey, Yanan Kuang, Thian Kheoh, Kenna Anderes, James Christensen, Pasi A. Jänne. Early changes in circulating cell free KRAS G12C as a possible blood-based response biomarker for adagrasib in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A002.

Introduction: There is a need for more nimble tools to evaluate drug activity at early stages of development, and to inform patient treatment decisions. Phase I (dose finding) and Phase II (efficacy) trials are increasingly merging, and newer “Phase IB” trials need to more rapidly assess drug activity with fewer patients. Moreover, emerging immunotherapies can induce a therapeutic effect with atypical response kinetics on imaging that differ from those classically observed with cytotoxic therapies. Noninvasive assessment of response using a blood biomarker such as cell free DNA (cfDNA) could complement tumor imaging as an early marker of response. Here we report on serial measurements of HPV16 E7 cfDNA by digital droplet PCR (ddPCR) from plasma of patients in a Phase I trial of CUE-101 in HPV16+ recurrent/metastatic head and neck squamous cell carcinoma (HNSCC). Methods/Results: 49 patients with HPV16+ HNSCC have been enrolled in the monotherapy arm of this Phase I dose escalation and expansion trial of CUE-101 monotherapy (NCT03978689). CUE-101 is a fusion protein comprised of a human leukocyte antigen (HLA) complex, HLA-A*0201; an immunodominant peptide epitope derived from the HPV16 E7 protein; 4 molecules of a reduced affinity human interleukin-2 (IL-2); and an effector attenuated- human IgG1 Fc domain. The monotherapy dose escalation arm spanned 7 dose levels, ranging from 0.06 to 8 mg/kg administered by IV infusion once every 3 weeks. Plasma samples were collected at screening and longitudinally prior to dosing in subsequent treatment cycles. HPV16 cfDNA was assayed by a previously validated ddPCR assay. Matched screening and cycle 3 plasma samples were available for 33 patients where HPV16 cfDNA was present at detectable levels. A positive plasma response, defined as a decrease of HPV cfDNA from first to second draw, was observed in 12/33 (36.5%) patients, 6 of whom (50.0%) had ≥ 50% decrease in HPV cfDNA. The most robust reductions of HPV16 cfDNA were observed at the 2 mg/kg and 4 mg/kg dose levels, with peak decreases of circulating HPV16 E7 cfDNA of 53.7% and 100%, respectively. Nevertheless, durable stable disease and clinical benefit following CUE-101 treatment was observed in patients with or without HPV cfDNA reduction at C3D1. Ongoing maturation of overall survival data in patients receiving CUE-101 monotherapy will enable exploration of correlations with changes in HPV16 cfDNA at early timepoints following initiation of treatment. Conclusion: Our data support the continued assessment of HPV16 cfDNA as an exploratory biomarker in patients receiving immunotherapy to better understand correlations with radiographic response assessments and overall clinical benefit. Citation Format: Benjamin Hanna, Grace Heavey, Apollina Goel, Steven Quayle, Yanan Kuang, Cloud Paweletz. Circulating free HPV16 DNA as a potential biomarker for immunotherapy-based trials in HPV16+ head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3365.

The clinical efficacy of epidermal growth factor receptor (EGFR)–targeted therapy in EGFR-mutant non–small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor–resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.

A subset of drug-resistant EGFR -mutant MET -amplified lung cancer switches oncogenic dependence to MET and is treatable with MET inhibitor monotherapy.

The development of targeted therapies has revolutionized the treatment of chronic lymphocytic leukemia (CLL). To date, these therapies are generally given continuously, indefinitely, leading to the development of resistance, which is often on target. Venetoclax is the first-in-class BCL-2 inhibitor which was initially approved for continuous therapy in relapsed high-risk CLL. In that context the BCL-2 G101V mutation (mut) was identified in post-progression samples and shown to reduce venetoclax binding to BCL-2, limiting its efficacy. The mut can be identified at low variant allele frequency (VAF) prior to clinical progression. We were therefore interested to identify the frequency of this mut in our cohort of relapsed refractory CLL patients (pts) on continuous venetoclax, and to assess the sensitivity of measurements in blood vs bone marrow. To this end we utilized a ddPCR assay which has LNA probes that specifically bind to either the BCL2 G101wt or G101V sequences, to screen for G101V muts in DNA extracted from patient samples. We also started to investigate additional co-occurring BCL2 muts in G101V positive samples by Sanger sequencing. Our patient cohort included 28 pts, of whom 20 had serial samples collected during venetoclax therapy. The median age of the pts was 66, and they had a median of 3 prior therapies before venetoclax, including chemoimmunotherapy in 67.9% and a BTK inhibitor in 60.7%. Deletion of 17p was present in 43%, with five additional pts having isolated TP53 mut (total with known TP53 aberrancy, 61%). 75% (21/28) of those evaluated had an unmutated IGHV. The median duration of venetoclax treatment was 43.5 months (mos). The timing of the first sample tested was a median of 23.3 mos after venetoclax initiation. We detected the G101V allele in peripheral blood mononuclear cells (PBMCs) in 9 out of 28 pts, at a median allele frequency (AF) of 1.38% (range 0.04%-22.31%), at a median of 44.6 mos on venetoclax. Out of the three pts who had G101V detected at multiple timepoints, two had AF increased with time (7.8 fold increase over 6 mos and 7.7 fold increase over 5 mos, respectively), one had similar AF with time (4.68% at 18.9 mos, 3.43% at 23.8 mos on treatment). Six of these pts also had bone marrow evaluated and all were also positive (at a median AF of 0.21%; range 0.2%-18.66%); one additional patient without a PBMC sample at that timepoint was positive in bone marrow. In order to screen for any co-occurring acquired resistance muts in BCL2 G101V positive samples, we performed Sanger sequencing across the BCL2 open-reading frame. We have identified a duplication mut (R107-R110dup) in one of the samples. In conclusion, this study shows that approximately one-third of pts on continuous venetoclax for 2+ years develop evidence of low-level BCL-2 G101V mut. Further work is underway to identify additional co-existing muts in BCL2 or other genes, and to characterize the additional genetic events at the time of clear clinical progression. Citation Format: Yanan Kuang, Stacey M. Fernandes, Rayan Fardoun, Kevin Vasquez, Abhishek Mogili, Cloud P. Paweletz, Jennifer R. Brown. BCL-2 G101V mutations develop in one-third of patients on continuous venetoclax [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 3960.

BET inhibitors are promising therapeutic agents for the treatment of triple-negative breast cancer (TNBC), but the rapid emergence of resistance necessitates investigation of combination therapies and their effects on tumor evolution. Here, we show that palbociclib, a CDK4/6 inhibitor, and paclitaxel, a microtubule inhibitor, synergize with the BET inhibitor JQ1 in TNBC lines. High-complexity DNA barcoding and mathematical modeling indicate a high rate of de novo acquired resistance to these drugs relative to pre-existing resistance. We demonstrate that the combination of JQ1 and palbociclib induces cell division errors, which can increase the chance of developing aneuploidy. Characterizing acquired resistance to combination treatment at a single cell level shows heterogeneous mechanisms including activation of G1-S and senescence pathways. Our results establish a rationale for further investigation of combined BET and CDK4/6 inhibition in TNBC and suggest novel mechanisms of action for these drugs and new vulnerabilities in cells after emergence of resistance., Effective combination therapies to improve the efficacy of BET inhibitors are currently under investigation. Here, the authors examine palbociclib and paclitaxel as two promising candidates for combination therapies with BET inhibition in breast cancer and investigate the dynamics of resistance to these combinations through DNA barcoding and mathematical modelling.

Background Genomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC). Patients and methods Plasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype. Results A total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R2 = 0.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression. Conclusions This blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.

Purpose: Polyclonal emergence of KIT secondary mutations is a main mechanism of imatinib progression in gastrointestinal stromal tumor (GIST). Approved KIT inhibitors sunitinib and regorafenib have complementary activity against KIT resistance mutations. Preclinical evidence suggests that rapid alternation of sunitinib and regorafenib broadens the spectrum of imatinib-resistant subclones targeted. Patients and Methods: Phase Ib study investigating continuous treatment with cycles of sunitinib (3 days) followed by regorafenib (4 days) in patients with tyrosine kinase inhibitor (TKI)-refractory GIST. A 3+3 dosing schema was utilized to determine the recommended phase II dose (RP2D). Plasma samples were analyzed for pharmacokinetics and circulating tumor DNA (ctDNA) studies using targeted error correction sequencing (TEC-seq) and droplet digital PCR (ddPCR). Results: Of the 14 patients enrolled, 2 experienced dose-limiting toxicities at dose level 2 (asymptomatic grade 3 hypophosphatemia). Sunitinib 37.5 mg/day and regorafenib 120 mg/day was the RP2D. Treatment was well-tolerated and no unexpected toxicities resulted from the combination. Stable disease was the best response in 4 patients, and median progression-free survival was 1.9 months. Combined assessment of ctDNA with TEC-seq and ddPCR detected plasma mutations in 11 of 12 patients (92%). ctDNA studies showed that KIT secondary mutations remain the main mechanism of resistance in TKI-refractory GIST, revealing effective suppression of KIT-mutant subpopulations in patients benefiting from the combination. Conclusions: Sunitinib and regorafenib combination is feasible and tolerable. Rapid alternation of TKIs with complementary activity might be effective when combining drugs with favorable pharmacokinetics, potentially allowing active doses while minimizing adverse events. Serial monitoring with ctDNA may guide treatment in patients with GIST.

Purpose: Tumor genotyping is a powerful tool for guiding non–small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). Experimental Design: An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. Results: NGS could identify mutations present in DNA dilutions at ≥0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Conclusions: Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. Clin Cancer Res; 22(4); 915–22. ©2015 AACR. See related commentary by Tsui and Berger, p. 790

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy.

ESR1 mutations were recently found to be an important mechanism of endocrine resistance in ER-positive (ER + ) metastatic breast cancer. To determine the clinicopathological features driving the emergence of the ESR1 mutations we studied plasma cfDNA and detailed clinical data collected from patients with metastatic breast cancer. Droplet Digital PCR was performed for the detection of the most common ESR1 mutations and PIK3CA mutations. Among the patients with ER + /HER2- disease, ESR1 mutations were detected in 30% of the patients. There were no associations between the pathological features of the primary disease or time to distant recurrence and the emergence of ESR1 mutations in metastatic disease. The prevalence of the ESR1 mutations was significantly associated with prior treatment with an aromatase inhibitor in the adjuvant or metastatic setting. The prevalence of the ESR1 mutations was also positively associated with prior fulvestrant treatment. Conversely, the prevalence of ESR1 mutations was lower after treatment with a CDK4/6 inhibitor. There were no significant associations between specific systemic treatments and the prevalence of PIK3CA mutations. These results support the evolution of the ESR1 mutations under the selective pressure of treatment with aromatase inhibitors in the adjuvant and metastatic settings and have important implications in the optimization of adjuvant and metastatic treatment in ER + breast cancer., Genetics: Drug treatment spurs new resistance mutations Treatment with aromatase inhibitors, a class of drugs that suppress the synthesis of estrogen, can drive the evolution of mutations in the estrogen receptor gene ESR1, leading to tumor resistance against hormone therapies. To better understand the emergence of ESR1 mutations, Rinath Jeselsohn from the Dana-Farber Cancer Institute in Boston, Massachusetts, USA, and coworkers tested tumor DNA contained within blood samples from 155 women with metastatic breast cancer. They found ESR1 mutations rarely in women with any molecular subtype of cancer other than estrogen receptor-positive disease. Nothing about the primary tumor predicted who would develop ESR1 mutations; however, treatment with an aromatase inhibitor was associated with mutations arising. The findings highlight the need to develop therapeutic regimens that reduce the selective pressure for ESR1 mutations and/or target these mutations directly.

Background Measuring cell-free (cf)DNA in blood and tissues holds significant potential as a minimally invasive method for disease monitoring in cancer. Cancers arising in the oropharynx and causally linked to human papillomavirus (HPV) represent an ideal model in which to interrogate these methods. Patients and methods We designed an ultrasensitive and quantitative droplet digital (dd)PCR assay to detect the five dominant high-risk HPV subtypes linked to oropharyngeal cancer (OPC). We enrolled a pilot observational cohort of 22 patients with advanced HPV+ OPC to evaluate the clinical utility of our assay and explore its predictive and prognostic potential. Results Total tumor burden (TTB) strongly correlated with HPV cfDNA levels (R = 0.91, P = 2.3×10−6) at this cohort size, and in most cases more distant anatomic disease locations predicted increasing HPV cfDNA levels. All participants demonstrated a corresponding change in their HPV cfDNA levels at a median of 16 days (range 12–38) before restaging scans confirming treatment response or progression. Patients with locoregional disease in the head and neck or pulmonary-only metastases had worse outcomes (P = 0.01). Both TTB and median plasma HPV cfDNA levels negatively correlated with survival (R=−0.65, P = 0.01; R=−0.48, P = 0.05, respectively). Conclusion(s) Plasma HPV cfDNA monitoring recapitulates fluctuations in disease status. While blood-based HPV DNA monitoring does not currently have a role in managing HPV+ OPC, these data speak to their broad clinical potential in an era of precision medicine.

The identification of oncogenic driver mutations has led to the rapid rise of genotype-directed treatments. However, genetic analysis of tumors remains cumbersome and a morbid experience for patients. Noninvasive assessment of tumor genotype, so-called "liquid biopsy," such as plasma genotyping represents a potentially transformative tool. Here we describe a genotyping protocol of cell-free plasma DNA (cfDNA) using Droplet Digital™ PCR (ddPCR™). ddPCR emulsifies DNA into ~20,000 droplets in which PCR is performed to endpoint in each droplet for both mutant and wild-type DNA. Droplets are run through a modified flow cytometer where mutant and wild-type DNA emit different colored signals. The count of these signals upon Poisson distribution analysis allows sensitive quantification of allelic prevalence.

Purpose: Plasma cell-free DNA (cfDNA) genotyping is increasingly used in cancer care, but assay accuracy has been debated. Because most cfDNA is derived from peripheral blood cells (PBC), we hypothesized that nonmalignant mutations harbored by hematopoietic cells (clonal hematopoiesis, CH) could be a cause of false-positive plasma genotyping. Experimental Design: We identified patients with advanced non–small cell lung cancer (NSCLC) with KRAS, JAK2, or TP53 mutations identified in cfDNA. With consent, PBC DNA was tested using droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to test for CH-derived mutations. Results: We first studied plasma ddPCR results from 58 patients with EGFR-mutant NSCLC. Two had KRAS G12X detected in cfDNA, and both were present in PBC, including one where the KRAS mutation was detected serially for 20 months. We then studied 143 plasma NGS results from 122 patients with NSCLC and identified 5 JAK2 V617F mutations derived from PBC. In addition, 108 TP53 mutations were detected in cfDNA; for 33 of the TP53 mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH. Conclusions: We find that most JAK2 mutations, some TP53 mutations, and rare KRAS mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy. Clin Cancer Res; 24(18); 4437–43. ©2018 AACR. See related commentary by Bauml and Levy, p. 4352

The identification of oncogenic driver mutations has led to the rapid rise of genotype-directed treatments. However, genetic analysis of tumors remains cumbersome and a morbid experience for patients. Noninvasive assessment of tumor genotype, so-called "liquid biopsy," such as plasma genotyping represents a potentially transformative tool. Here we describe a genotyping protocol of cell-free plasma DNA (cfDNA) using Droplet Digital™ PCR (ddPCR™). ddPCR emulsifies DNA into ~20,000 droplets in which PCR is performed to endpoint in each droplet for both mutant and wild-type DNA. Droplets are run through a modified flow cytometer where mutant and wild-type DNA emit different colored signals. The count of these signals upon Poisson distribution analysis allows sensitive quantification of allelic prevalence.

Non–small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy. Cancer Res; 75(20); 4372–83. ©2015 AACR.

Amplified and/or mutated MET can act as both a primary oncogenic driver and as a promoter of tyrosine kinase inhibitor (TKI) resistance in non–small cell lung cancer (NSCLC). However, the landscape of MET-specific targeting agents remains underdeveloped, and understanding of mechanisms of resistance to MET TKIs is limited. Here, we present a case of a patient with lung adenocarcinoma harboring both a mutation in EGFR and an amplification of MET, who after progression on erlotinib responded dramatically to combined MET and EGFR inhibition with savolitinib and osimertinib. When resistance developed to this combination, a new MET kinase domain mutation, D1228V, was detected. Our in vitro findings demonstrate that METD1228V induces resistance to type I MET TKIs through impaired drug binding, while sensitivity to type II MET TKIs is maintained. Based on these findings, the patient was treated with erlotinib combined with cabozantinib, a type II MET inhibitor, and exhibited a response. Significance: With several structurally distinct MET inhibitors undergoing development for treatment of NSCLC, it is critical to identify mechanism-based therapies for drug resistance. We demonstrate that an acquired METD1228V mutation mediates resistance to type I, but not type II, MET inhibitors, having therapeutic implications for the clinical use of sequential MET inhibitors. Cancer Discov; 6(12); 1334–41. ©2016 AACR. See related commentary by Trusolino, p. 1306. This article is highlighted in the In This Issue feature, p. 1293

Introduction: Genotype-directed therapies are revolutionizing care for gastrointestinal stromal tumors (GIST). Somatic mutations in KIT exons (ex) 11 and 9 induce constitutive kinase activity and account for ~95% of the primary oncogenic events in KIT-driven GIST; KIT mutations correlate with prognosis and predict clinical activity of tyrosine kinase inhibitors (TKI), such as imatinib. After a median of 24 months on imatinib, 85% of patients (pts) develop resistance and progress multifocally, mostly due to secondary KIT mutations in ex 13 and/or ex 17. We describe here a rapid, clinical grade droplet digital PCR (ddPCR) assay to detect and quantify KIT mutations associated with TKI resistance in cfDNA from plasma of metastatic (met) GIST pts. Unlike allele-specific ddPCR that requires splitting input DNA into many reactions, we designed pan-ex 17 KIT assay to capture the polyclonal mutational landscape of KIT in a single reaction. Methods: Tumor and plasma samples and clinical data were collected from pts with met GIST. ddPCR assays were developed for the detection in cfDNA of all common secondary KIT mutations associated with imatinib resistance (V654A, T670I, A829P and multiple ex 17 mutations). The pan-ex 17 mutation drop-off assay uses a FAM-labeled mutant (mut) probe that spans sequences encoding amino acids 819~824, and a VIC-labeled reference probe that is shared by both wild-type (wt) and mut alleles. Common ex 17 mutations were detected via cluster separation. V654A, T670I, A829P are detected with allele-specific assays that use a FAM-labeled mut probe and a VIC-labeled wt probe. To demonstrate analytical sensitivity/specificity of each assay, ddPCR cycling conditions were optimized to yield the maximum fluorescence signal with minimal noise signal. Results: The KIT ddPCR assays detect a mutation prevalence of 0.01 - 0.05%, with a sensitivity of 5 - 50 KIT mut copies in a background of 10,000 KIT wt copies, depending on the mutation assayed, with absolute specificity. Results were validated in a pilot observational cohort of pts who were treated with imatinib and progressed, for which matching plasma (1 ml) and tumor samples were analyzed to reveal identical mutations. In a separate cohort, ddPCR results were validated by sensitive plasma NGS. Conclusions: We have developed rapid, highly sensitive, and 100% specific quantitative assays to enable plasma-based tumor genotyping for secondary KIT resistance mutations, which has been technically optimized for translation into clinical practice. Analysis of cfDNA by ddPCR can successfully detect KIT secondary mutations in met GIST pts. In an ongoing study, serial monitoring of KIT mutations in cfDNA from pts with advanced GIST should allow for early detection of secondary mutations and optimization of genotype-targeted therapeutic management. Citation Format: Adrian Marino-Enriquez, Suzanne George, Julianna Supplee, Grace Heavey, Pasi A. Janne, Chandrajit Raut, Jonathan Fletcher, George D. Demetri, Cloud P. Paweletz, Yanan Kuang. Development of a rapid clinical grade assay to detect and monitor secondary KIT mutations in circulating free DNA (cfDNA) for personalization of targeted therapy for gastrointestinal stromal tumors (GIST) [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 4578.

Purpose: Human papillomavirus (HPV) is associated with the majority of oropharyngeal squamous cell carcinomas (OPSCC), with a rising incidence of OPSCC predominantly among middle-aged men. Tumor-derived HPV cell-free (cf)DNA can be detected and quantified in circulation with high sensitivity and specificity. Although cfDNA has limited clinical application in monitoring locoregional spread of these tumors, up to 10% of HPV+ OPSCC patients present or recur with distant, metastatic disease. The potential of HPV DNA monitoring in this setting remains largely unexplored. Methods: We present a proof-of-concept prospective observational cohort of recurrent, metastatic (R/M) HPV+ OPSCC patients treated with standard systemic therapies. We utilized droplet digital (dd)PCR to identify and quantify HPV cfDNA (strains 16, 18, 31, 33 and 45) at multiple time points throughout treatment. We then compared HPV cfDNA concentration at various timepoints with clinical parameters such as disease burden and treatment response. Results: Clinicopathologic data from 12 R/M patients revealed a predominantly male cohort (11/12, 92%) with a median age of 55 at diagnosis. Eight (67%) were on immunotherapy treatments and the other four on standard chemotherapy during the study period (2.5 months). Plasma HPV cfDNA was detected in 6/12 (50%) samples (range 0-4460 copies/mL) in at least one timepoint during the study. All patients with undetectable HPV cfDNA had evidence of stable, low burden disease or no measurable lesions (near-complete response) on their current therapy. HPV cfDNA levels strongly correlated with both disease burden (p < 0.01) and distant disease sites (p < 0.01). In three cases, HPV DNA levels declined prior to contrast enhanced imaging findings which later confirmed treatment response (up to 14 days prior to planned imaging). Conclusion: Our results suggest that high sensitivity plasma HPV cfDNA levels can be an early indicator of treatment response. HPV cfDNA monitoring could significantly impact clinical decision-making and improve patient outcomes by informing treatment response or failure. Further studies are underway to validate these findings and determine how early cfDNA can detect metastatic disease. Citation Format: Glenn J. Hanna, Julianna G. Supplee, Yanan Kuang, Kate Geary, Pasi A. Janne, Robert I. Haddad, Cloud P. Paweletz. Plasma HPV cell free DNA as an earlier predictor of treatment response in advanced oropharyngeal cancer [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 2581.