Anti-EGFR-resistant clones decay exponentially after progression: implications for anti-EGFR re-challenge.

BACKGROUND: Colorectal cancer (CRC) has been shown to acquire RAS and EGFR ectodomain mutations as mechanisms of resistance to epidermal growth factor receptor (EGFR) inhibition (anti-EGFR). After anti-EGFR withdrawal, RAS and EGFR mutant clones lack a growth advantage relative to other clones and decay; however, the kinetics of decay remain unclear. We sought to determine the kinetics of acquired RAS/EGFR mutations after discontinuation of anti-EGFR therapy. PATIENTS AND METHODS: We present the post-progression circulating tumor DNA (ctDNA) profiles of 135 patients with RAS/BRAF wild-type metastatic CRC treated with anti-EGFR who acquired RAS and/or EGFR mutations during therapy. Our validation cohort consisted of an external dataset of 73 patients with a ctDNA profile suggestive of prior anti-EGFR exposure and serial sampling. A separate retrospective cohort of 80 patients was used to evaluate overall response rate and progression free survival during re-challenge therapies. RESULTS: Our analysis showed that RAS and EGFR relative mutant allele frequency decays exponentially (r2=0.93 for RAS; r2=0.94 for EGFR) with a cumulative half-life of 4.4 months. We validated our findings using an external dataset of 73 patients with a ctDNA profile suggestive of prior anti-EGFR exposure and serial sampling, confirming exponential decay with an estimated half-life of 4.3 months. A separate retrospective cohort of 80 patients showed that patients had a higher overall response rate during re-challenge therapies after increasing time intervals, as predicted by our model. CONCLUSION: These results provide scientific support for anti-EGFR re-challenge and guide the optimal timing of re-challenge initiation.

Clinical utility of plasma-based digital next-generation sequencing in patients with advance-stage lung adenocarcinomas with insufficient tumor samples for tissue genotyping.

BACKGROUND: Approximately 30% of tumor biopsies from patients with advanced-stage lung adenocarcinomas yield insufficient tissue for successful molecular subtyping. We have analyzed the clinical utility of next-generation sequencing (NGS) of cell-free circulating tumor DNA (ctDNA) in patients with inadequate tumor samples for tissue genotyping. PATIENTS AND METHODS: We conducted the study in a multi-institutional prospective cohort of clinically unselected patients with advanced-stage lung adenocarcinomas with insufficient tissue for EGFR, ALK or ROS1 genotyping across 12 Spanish institutions (n = 93). ctDNA NGS was carried out by Guardant Health (Guardant360, Redwood City, CA), using a hybrid-capture-based 73-gene panel. Variants were deemed actionable if they were part of the OncoKB precision oncology knowledge database and classified in four levels of actionability based on their clinical or preclinical evidence for drug response. RESULTS: Eighty-three out of 93 patients (89%) had detectable levels of ctDNA. Potentially actionable level 1-4 genomic alterations were detected in 53 cases (57%), of which 13 (14%) had level 1-2A alterations (Food and Drug Administration-approved and standard-care biomarkers according to lung cancer guidelines). Frequencies of each genomic alteration in ctDNA were consistent with those observed in unselected pulmonary adenocarcinomas. The majority of the patients (62%), particularly those with actionable alterations (87%), had more than one pathogenic variant in ctDNA. The median turnaround time to genomic results was 13 days. Twelve patients (13%) received genotype-matched therapies based on ctDNA results, deriving the expected clinical benefit. Patients with co-occurring pathogenic alterations had a significantly shorter median overall survival as compared with patients without co-occurring pathogenic alteration (multivariate hazard ratio = 5.35, P = 0.01). CONCLUSION: Digital NGS of ctDNA in lung cancers with insufficient tumor samples for tissue sequencing detects actionable variants that frequently co-occur with other potentially clinically relevant genomic alterations, allowing timely initiation of genotype-matched therapies.

Impact of genomic alterations on lapatinib treatment outcome and cell-free genomic landscape during HER2 therapy in HER2+ gastric cancer patients.

Background: To identify predictive markers for responders in lapatinib-treated patients and to demonstrate molecular changes during lapatinib treatment via cell-free genomics. Patients and methods: We prospectively evaluated the efficacy of combining lapatinib with capecitabine and oxaliplatin as first line neoadjuvant therapy in patients with previously untreated, HER2-overexpressing advanced gastric cancer. A parallel biomarker study was conducted by simultaneously performing immunohistochemistry and next-generation sequencing (NGS) with tumor and blood samples. Results: Complete response was confirmed in 7/32 patients (21.8%), 2 of whom received radical surgery with pathologic-confirmed complete response. Fifteen partial responses (46.8%) were observed, resulting in a 68.6% overall response rate. NGS of the 16 tumor specimens demonstrated that the most common co-occurring copy number alteration was CCNE1 amplification, which was present in 40% of HER2+ tumors. The relationship between CCNE1 amplification and lack of response to HER2-targeted therapy trended toward statistical significance (66.7% of non-responders versus 22.2% of responders harbored CCNE1 amplification; P = 0.08). Patients with high level ERBB2 amplification by NGS were more likely to respond to therapy, compared with patients with low level ERBB2 amplification (P = 0.02). Analysis of cfDNA showed that detectable ERBB2 copy number amplification in plasma was predictive to the response (100%, response rate) and changes in plasma-detected genomic alterations were associated with lapatinib sensitivity and/or resistance. The follow-up cfDNA genomics at disease progression demonstrated that there are emergences of other genomic aberrations such as MYC, EGFR, FGFR2 and MET amplifications. Conclusions: The present study showed that HER2+ GC patients respond differently according to concomitant genomic aberrations beyond ERBB2, high ERBB2 amplification by NGS or cfDNA can be a positive predictor for patient selection, and tumor genomic alterations change significantly during targeted agent therapy.

Acquired Resistance to Poly (ADP-ribose) Polymerase Inhibitor Olaparib in BRCA2-Associated Prostate Cancer Resulting From Biallelic BRCA2 Reversion Mutations Restores Both Germline and Somatic Loss-of-Function Mutations.

PARP1/2 inhibitors are effective against BRCA2-deficient tumors. The PARP inhibitor (PARPi) olaparib received FDA breakthrough designation for treatment of metastatic castration-resistant prostate cancers (CRPC) carrying mutations in BRCA1/2 or ATM genes. Emergent resistance to PARPi has been associated with tumor-specific BRCA2 mutations that revert the normal open reading frame rescuing homologous recombination. We describe a case of metastatic CRPC with germline BRCA2 mutation with acquired resistance to olaparib related to biallelic BRCA2 reversion mutations of both the germline and somatic loss of function alleles detected by circulating tumor DNA testing. We also summarize a retrospective analysis of 1,534 prostate cancer cases with ctDNA analysis showing a 1.6% incidence of germline BRCA2 mutations. Within the germline BRCA2-positive cases exposed to platinum chemotherapy or PARP inhibition, the prevalence of reversion mutations was 40%. This report documents the frequency of reversion mutations in a large cohort of prostate cancer patients carrying of BRCA mutations. It also shows the potential utility of ctDNA analyses for early detection of reversion mutation driving tumor resistance.

Polyclonal RB1 mutations and acquired resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer.

Background: While deregulation of the cyclin D1-CDK4/6-retinoblastoma pathway is common in hormone receptor positive (HR+) breast cancer, Rb is usually intact in HR+ breast cancer, and targeted CDK 4/6 inhibitors that act upstream of Rb, are routinely being utilized in clinical practice. However, factors that can lead to clinical resistance to CDK 4/6 inhibitors are not known. Patients and methods: We identified patients who had pre- and post-genotyping in tissue and peripheral blood samples after receiving CDK 4/6 inhibitors. Genotyping was carried out in tumor tissue or blood collected before start of CDK 4/6 inhibitor and after disease progression on CDK 4/6 inhibitor, covering more than 90% of the coding region in RB1. Results: We identified detectable acquired RB1 mutations in circulating tumor DNA (ctDNA) after exposure to CDK4/6 inhibitor (palbociclib, palbociclib, ribociclib) for 5, 8, and 13 months, respectively, in three patients. The RB1 mutations included substitution in donor splicing site of exon 8 of the RB1 gene in patient #1; substitution in donor splicing site of exon 22 of RB1 gene, exon 19 deletion, exon 3 insertion in patient #2; and RB1 exon 16 H483Y mutation in patient #3. None of these RB1 mutations were present in the pre-CDK 4/6 specimen highlighting these molecular alterations, which lead to functional loss of Rb1, likely emerged under selective pressure from the CDK4/6 inhibitor potentially confering therapeutic resistance. Conclusion: This is the first clinical report to describe the emergence of somatic RB1 mutations after exposure to palbociclib or ribociclib, in patients with metastatic breast cancer. Further research is needed to validate these findings, identify how these mutations temporally emerge under selective pressure of CDK 4/6 inhibitor, and develop rational therapeutic strategies.