Immunotherapy using checkpoint-blocking antibodies against PD-1 has produced impressive results in a wide range of cancers. However, the response remains heterogeneous among patients. We used ...noninvasive immuno-positron emission tomography (PET), using 89Zr-labeled PEGylated single-domain antibody fragments (nanobodies or VHHs), to explore the dynamics and distribution of intratumoral CD8⁺ T cells and CD11b⁺ myeloid cells in response to anti–PD-1 treatment in the MC38 colorectal mouse adenocarcinoma model. Responding and nonresponding tumors showed consistent differences in the distribution of CD8⁺ and CD11b⁺ cells. Anti–PD-1 treatment mobilized CD8⁺ T cells from the tumor periphery to a more central location. Only those tumors fully infiltrated by CD8⁺ T cells went on to complete resolution. All tumors contained CD11b⁺ myeloid cells from the outset of treatment, with later recruitment of additional CD11b⁺ cells. As tumors grew, the distribution of intratumoral CD11b⁺ cells became more heterogeneous. Shrinkage of tumors in responders correlated with an increase in the CD11b⁺ population in the center of the tumors. The changes in distribution of CD8⁺ and CD11b⁺ cells, as assessed by PET, served as biomarkers to gauge the efficacy of anti–PD-1 treatment. Single-cell RNA sequencing of RNA from intratumoral CD45⁺ cells showed that CD11b⁺ cells in responders and nonresponders were markedly different. The responders exhibited a dominant population of macrophages with an M1-like signature, while the CD45⁺ population in the nonresponders displayed an M2-like transcriptional signature. Thus, by using immuno-PET and single-cell RNA sequencing, we show that anti–PD-1 treatment not only affects interactions of CD8⁺ T cells with the tumor but also impacts the intratumoral myeloid compartment.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Osimertinib is approved for advanced EGFR-mutated NSCLC, and identification of on-target mechanisms of resistance (i.e., EGFR C797S) to this third-generation EGFR inhibitor are evolving. Whether ...durable control of subsequently osimertinib-resistant NSCLC with the EGFR-sensitizing mutation (SM)/C797S is possible with first-generation EGFR inhibitors (such as gefitinib or erlotinib) remains underreported, as does the resultant acquired resistance profile.
We used N-ethyl-N-nitrosourea mutagenesis to determine the profile of EGFR SM/C797S preclinical models exposed to reversible EGFR inhibitors. In addition, we retrospectively probed a case of EGFR SM lung adenocarcinoma treated with first-line osimertinib, followed by second-line erlotinib in the setting of EGFR SM/C797S.
Use of N-ethyl-N-nitrosourea mutagenesis against the background of EGFR L858R/C797S in conjunction with administration of gefitinib revealed preferential outgrowth of cells with EGFR L858R/T790M/C797S. A patient with EGFR delE746_T751insV NSCLC was treated with osimertinib with sustained response for 10 months before acquiring EGFR C797S. The patient was subsequently treated with erlotinib, with response for a period of 4 months, but disease progression ensued. Liquid biopsy disclosed EGFR delE746_T751insV with T790M and C797S present in cis.
EGFR SM NSCLC can acquire resistance to osimertinib through development of the EGFR C797S mutation. In this clinical scenario, the tumor may respond transiently to reversible first-generation EGFR inhibitors (gefitinib or erlotinib), but evolving mechanisms of on-target resistance—in clinical specimens and preclinical systems—indicate that EGFR C797S along with EGFR T790M can evolve. This report adds to the growing understanding of tumor evolution or adaptability to sequential EGFR inhibition and augments support for exploring combination therapies to delay or prevent on-target resistance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Salivary HPV DNA levels align with local disease burden in HPV oropharyngeal cancer.•The rise and fall of salivary HPV DNA often predicts treatment response or failure.•Plasma HPV cell-free DNA more ...often correlates with distant disease burden.
Quantifying tumor DNA in tissue and circulating in blood permits high-quality molecular monitoring to detect and track cancer progression. Evaluating tumor DNA in both blood and saliva in human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) could provide a non-invasive and clinically actionable method for real-time disease detection.
We previously validated an ultrasensitive droplet-digital (dd)PCR assay targeting the dominant high-risk HPV subtypes causally linked to OPC. Here we enrolled an observational cohort to evaluate the predictive and prognostic potential of paired plasma-salivary tumor DNA among 21 patients with advanced HPV+OPC.
In patients with recurrent, persistent locoregional (LR) disease, median baseline normalized salivary HPV DNA was 10.9 copies/ng total DNA, nearly 20x higher compared with those with distant disease only (p = 0.01). A cutoff of 5 copies/ng yielded 87% sensitivity and 67% specificity for accurately predicting LR disease. Total tumor burden among those with LR disease strongly correlated with salivary HPV DNA levels (R = 0.83, p = 0.02). The rise and fall of salivary HPV DNA predicted treatment failure and response, respectively, in all patients with LR disease, and predated imaging findings. Among paired salivary-plasma (cell-free) cfDNA samples, only higher plasma HPV cfDNA levels were associated with poor outcomes (p < 0.01), suggesting that each bodily fluid provides unique information about HPV disease status.
Salivary HPV DNA provides valuable information about tumor burden and predicts treatment response in advanced HPV+OPC. Paired blood-saliva samples could be used to monitor HPV DNA with broad applications to inform diagnosis, prognosis, and surveillance in HPV-associated diseases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed ...cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.
MET-targeted therapies are clinically effective in
-amplified and
exon 14 deletion mutant (
ex14) 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
The combination of capmatinib/merestinib was evaluated
and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate
and
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.
We report the results from a Foundation for the National Institutes of Health Biomarkers Consortium project to address the absence of well-validated quality control materials (QCMs) for circulating ...tumor DNA (ctDNA) testing. This absence is considered a cause of variance and inconsistencies in translating ctDNA results into clinical actions.
In this phase I study, QCMs with 14 clinically relevant mutations representing single nucleotide variants, insertions or deletions (indels), translocations, and copy number variants were sourced from three commercial manufacturers with variant allele frequencies (VAFs) of 5%, 2.5%, 1%, 0.1%, and 0%. Four laboratories tested samples in quadruplicate using two allele-specific droplet digital polymerase chain reaction and three (amplicon and hybrid capture) next-generation sequencing (NGS) panels.
The two droplet digital polymerase chain reaction assays reported VAF values very close to the manufacturers' claimed concentrations for all QCMs. NGS assays reported most single nucleotide variants and indels, but not translocations, close to the expected VAF values. Notably, two NGS assays reported lower VAF than expected for all translocations in all QCM mixtures, possibly related to technical challenges detecting these variants. The ability to call
copy number amplifications varied across assays. All three QCMs provided valuable insight into assay precision. Each assay across all variant types demonstrated dropouts at 0.1%, suggesting that the QCM can serve for testing of an assay's limit of detection with confidence claims for specific variants.
These results support the utility of the QCM in testing ctDNA assay analytical performance. However, unique designs and manufacturing methods for the QCM, and variations in a laboratory's testing configuration, may require testing of multiple QCMs to find the best reagents for accurate result interpretation.
Plasma circulating tumor DNA (ctDNA) analysis is routine for genotyping of advanced non-small-cell lung cancer (NSCLC); however, early response assessment using plasma ctDNA has yet to be well ...characterized.
Patients with advanced
-mutant NSCLC across three phase I NCI osimertinib combination trials were analyzed in this study, and an institutional cohort of patients with
,
, and
-mutant advanced NSCLC receiving systemic treatment was used for validation. Plasma was collected before treatment initiation and serially before each cycle of therapy, and key driver mutations in ctDNA were characterized by droplet digital polymerase chain reaction. Timing of plasma versus imaging response was compared in a separate cohort of patients with
-mutant NSCLC treated with osimertinib. Across cohorts, we also studied ctDNA variability before treatment start.
In the NCI cohort, 14/16 (87.5%) patients exhibited ≥ 90% decrease in mutation abundance by the first on-treatment timepoint (20-28 days from treatment start) with minimal subsequent change. Similarly, 47/56 (83.9%) patients with any decrease in the institutional cohort demonstrated ≥ 90% decrease in mutation abundance by the first follow-up draw (7-30 days from treatment start). All 16 patients in the imaging cohort with radiographic partial response showed best plasma response within one cycle, preceding best radiographic response by a median of 24 weeks (range: 3-147 weeks). Variability in ctDNA levels before treatment start was observed.
Plasma ctDNA response is an early phenomenon, with the majority of change detectable within the first cycle of therapy. These kinetics may offer an opportunity for early insight into treatment effect before standard imaging timepoints.
Abstract
Purpose: The human papillomavirus (HPV) is linked to the majority of oropharyngeal squamous cell carcinomas (OPSCC). We have previously shown that tumor-derived HPV cell-free (cf)DNA can be ...detected and quantified in circulation with high sensitivity and specificity using droplet digital (dd)PCR1. Further, we have shown that plasma cfDNA levels correlate with disease burden and indicate treatment response in advanced OPSCC. In this study we pair plasma with salivary HPV cfDNA to understand its clinical application in monitoring locoregional spread of disease.
Methods: We present a proof-of-concept prospective observational cohort of recurrent, metastatic (R/M) HPV+ OPSCC patients treated with standard systemic therapies or on active surveillance. We utilized ultrasensitive ddPCR to identify and quantify both plasma and salivary HPV cfDNA (subtypes 16, 18, 31, 33 and 45) at multiple time points. Salivary HPV cfDNA was normalized to total DNA concentration as measured by fluorometric quantification. We then compared matched plasma and normalized salivary HPV cfDNA concentrations at various timepoints with clinical parameters, such as tumor burden and therapeutic response.
Results: Clinicopathologic data from 15 R/M patients revealed a male cohort with a median age of 57 at diagnosis. Five (33%) were on immunotherapy and 6 (40%) on standard chemotherapy during the study period (3 months). Salivary HPV cfDNA was detected in 13/15 (87%) samples (range 0-729 copies/mL) in at least one timepoint during the study (plasma cfDNA was detected in 12/15 or 80% of the cohort, range 0-12,949 copies/mL). All patients with measurable locoregional (LR) disease had detectable salivary cfDNA. While matched plasma and salivary cfDNA levels showed no correlation among individual patients, plasma cfDNA levels correlated with site of disease (LR, pulmonary or extra-pulmonary) (p < 0.001), while median salivary cfDNA levels strongly correlated with LR tumor burden (R = 0.81, p < 0.001). Salivary cfDNA levels declined by 80% of their baseline value within a median of 6.5 days among all patients with LR disease experiencing a response to treatment.
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Conclusion: Our results suggest that high sensitivity salivary HPV cfDNA levels correlate with locoregional disease burden and can be an early indicator of locoregional treatment response, while our prior work has supported the role of plasma HPV cfDNA levels in monitoring metastatic disease. Evaluating paired plasma and salivary HPV cfDNA levels in a curative HPV OPSCC population is underway to further validate their prognostic and predictive potential.
1Hanna GJ, Supplee JG, Kuang Y, Mahmood U, Lau CJ, Haddad RI, et al. Plasma HPV cell-free DNA monitoring in advanced HPV-associated oropharyngeal cancer. Ann Oncol. 2018;29:1980–6.
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Citation Format: Glenn J. Hanna, Christie J. Lau, Umair Mahmood, Robert I. Haddad, Cloud P. Paweletz, Pasi A. Janne. Salivary HPV cell free DNA levels predict locoregional disease burden and response in oropharyngeal cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 452.
Abstract only
9507
Background: First-line treatment with an EGFR tyrosine kinase inhibitor (TKI) is standard of care for patients (pts) with EGFR-mutated NSCLC. The EGFR TKI osimertinib is active ...against the acquired gefitinib-resistant mutation EGFR T790M, as is gefitinib against the osimertinib-resistant EGFR C797S. Preclinical evidence suggests dual EGFR inhibition with gefitinib + osimertinib may delay emergence of acquired resistance. Methods: This ongoing phase I/II study enrolled pts with stage IV EGFR-mutated (L858R or del19) NSCLC, without prior therapy for metastatic disease. Treatment in dose escalation (n = 6): concurrent osimertinib 40 mg or 80 mg + gefitinib 250 mg daily. In dose expansion (n = 21): osimertinib + gefitinib at the maximum tolerated dose (MTD). Prior to protocol amendment 6 pts received alternating monthly cycles of TKI monotherapy and were excluded from this analysis. The primary endpoints in the dose escalation and expansion phases were, respectively, identification of the MTD and feasibility, defined as receipt of combination therapy for ≥ 6 four-week cycles. Secondary endpoints included overall response rate (ORR), survival outcomes, plasma EGFR mutation clearance (cell free DNA by droplet digital PCR (ddPCR)), and mechanisms of acquired resistance. Results: From May 2017 to July 2019 27 pts were enrolled and evaluable for the primary endpoints. The MTD was osimertinib 80 mg plus gefitinib 250 mg orally daily. In feasibility analysis, 81.5% completed ≥6 cycles combination therapy (1 pt discontinued for progression, 4 for toxicity). The ORR was 85.2% (95% CI 67.5%-94.1%). Best response: 85.2% partial response, 14.8% stable disease. The most common treatment-related adverse effects (TRAEs) (% any grade, % grade 3) were rash (96.3%, 3.7%), diarrhea (85.2%, 11.1%) and dry skin (70.4%, 0%). Plasma ddPCR (n = 25 pts) detected the driver EGFR mutation at baseline in 68% of pts. In these pts, plasma EGFR cleared to undetectable at 2 weeks treatment in 82.4%. At 14.8 months median follow up the median progression free survival was not yet reached. Conclusions: Combination therapy with osimertinib and gefitinib is tolerable for first-line treatment of EGFR-mutated NSCLC and resulted in rapid plasma clearance of the EGFR mutation. The observed ORR is consistent with previously reported first-line response rates to osimertinib. Analysis of survival outcomes and acquired resistance mechanisms are pending data maturity and will facilitate understanding of the role of first-line dual EGFR TKI therapy for this pt population. Clinical trial information: NCT03122717 .
Abstract only
9057
Background: Osi (3
rd
gen EGFR TKI) has robust activity in 1
st
line EGFR mutant NSCLC and TKI resistant T790M
pos
NSCLC but progressive disease (PD) occurs and outcomes with Osi ...alone are poor in T790M
neg
, C797X
pos
and EGFR exon 20 insertion (ins20) disease. This study examined the EGFR monoclonal antibody Neci with Osi in select settings of EGFR TKI resistance. Methods: Using a 3+3 design, Neci was examined in advanced EGFR mutant NSCLC at dose levels (DL) of 600 mg (DL1) & 800 mg (DL2) D1, D8 IV q21 days + Osi 80 mg qd. Four expansion cohorts (ExC; 18 each) included: A) T790M
neg
PD on 1
st
/2
nd
gen TKI as last therapy, B) T790M
neg
PD on 3
rd
gen TKI, C) T790M
pos
PD on 3
rd
gen TKI, D) EGFR ex20ins PD on chemotherapy. Central T790M testing by ddPCR in ExC A-C. Additional studies performed include: NGS panel of > 400 genes, EGFR FISH, plasma for PK and serial EGFR ctDNA by ddPCR as exploratory analyses. Adverse events (AEs) graded (Gr) by CTCAEv5 with ORR and PFS by RECIST 1.1. Results: Dose escalation and ExC B completed accrual. In total 55 pts were evaluable (Table). 1 pt had DLT at DL2, Gr 3 sinus bradycardia. Drug related Gr 3 AEs were seen in 27% (15) of pts, mainly rash (7;13%). ctDNA showed decreased mutant allele frequency with therapy in all pts studied with detected EGFR at baseline, with complete plasma clearance in a pt with detectable C797S. Conclusions: The RP2D (Osi 80 mg qd and Neci 800 mg D1, D8 IV on q21d cycle) is feasible and tolerable. In ExC A,T790M
neg
pts with 1
st
/2
nd
gen EGFR TKI as last treatment, using curtailed sampling, the pre-specified efficacy signal was reached for Osi + Neci comparing favorably to Osi alone in analogous pts from the AURA trial. Clinical activity was also seen in EGFR-dependent resistance (T790M
pos
C797S
pos
) after PD on 3
rd
gen TKI and in EGFR ins 20. Clinical trial information: NCT02496663. Table: see text
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