DNA fragments released from cancer cells into the blood can be used to generate molecular profiles of tumors. Non-invasive 'liquid biopsies' can be used to monitor minimal residual disease and detect ...the emergence of drug resistance.
Abstract
Mutant-selective KRASG12C inhibitors, such as MRTX849 (adagrasib) and AMG 510 (sotorasib), have demonstrated efficacy in KRASG12C-mutant cancers, including non–small cell lung cancer ...(NSCLC). However, mechanisms underlying clinical acquired resistance to KRASG12C inhibitors remain undetermined. To begin to define the mechanistic spectrum of acquired resistance, we describe a patient with KRASG12C NSCLC who developed polyclonal acquired resistance to MRTX849 with the emergence of 10 heterogeneous resistance alterations in serial cell-free DNA spanning four genes (KRAS, NRAS, BRAF, MAP2K1), all of which converge to reactivate RAS–MAPK signaling. Notably, a novel KRASY96D mutation affecting the switch-II pocket, to which MRTX849 and other inactive-state inhibitors bind, was identified that interferes with key protein–drug interactions and confers resistance to these inhibitors in engineered and patient-derived KRASG12C cancer models. Interestingly, a novel, functionally distinct tricomplex KRASG12C active-state inhibitor RM-018 retained the ability to bind and inhibit KRASG12C/Y96D and could overcome resistance.
Significance:
In one of the first reports of clinical acquired resistance to KRASG12C inhibitors, our data suggest polyclonal RAS–MAPK reactivation as a central resistance mechanism. We also identify a novel KRAS switch-II pocket mutation that impairs binding and drives resistance to inactive-state inhibitors but is surmountable by a functionally distinct KRASG12C inhibitor.
See related commentary by Pinnelli and Trusolino, p. 1874.
This article is highlighted in the In This Issue feature, p. 1861
(HER2) amplification is an emerging biomarker in colon cancer, conferring sensitivity to combination anti-HER2 therapy. Measurement of HER2 copy number is typically performed using surgical ...specimens, but cell-free circulating tumor DNA (ctDNA) analysis may be a noninvasive alternative. We determined the sensitivity of plasma copy number (pCN) for detecting
amplifications and whether pCN correlated with tissue-detected copy number. We also assessed response to HER2-targeted therapy based on pCN and suggest a pCN threshold predictive of response.
Forty-eight pretreatment and progression plasma samples from 29 HER2-positive patients in the HERACLES A clinical trial were tested using the Guardant360 cfDNA assay. We correlated
pCN with progression-free survival (PFS) and best objective response (BOR) and applied an adjustment method based on tumor DNA shedding using the maximum mutant allele fraction as a surrogate for tumor content to accurately determine the pCN threshold predictive of response.
Forty-seven of 48 samples had detectable ctDNA, and 46 of 47 samples were
-amplified on the basis of cfDNA 2.55-122 copies; 97.9% sensitivity (95% confidence interval, 87.2%-99.8%). An adjusted
pCN of ≥25.82 copies correlated with BOR and PFS (
= 0.0347).
cfDNA is a viable alternative to tissue-based genotyping in the metastatic setting. The cfDNA platform utilized correctly identified 28 of 29 (96.6%) of pretreatment samples as
-amplified and predicted benefit from HER2-targeted therapy. In this study, an observed pCN of 2.4 and an adjusted pCN of 25.82 copies of
are proposed to select patients who will benefit from HER2-targeted therapy.
Colorectal cancers (CRCs) evolve by a reiterative process of genetic diversification and clonal evolution. The molecular profile of CRC is routinely assessed in surgical or bioptic samples. ...Genotyping of CRC tissue has inherent limitations; a tissue sample represents a single snapshot in time, and it is subjected to spatial selection bias owing to tumor heterogeneity. Repeated tissue samples are difficult to obtain and cannot be used for dynamic monitoring of disease progression and response to therapy. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment with the epidermal growth factor receptor (EGFR)-specific antibodies cetuximab or panitumumab. We identified alterations in ctDNA of patients with primary or acquired resistance to EGFR blockade in the following genes: KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1. Mutated KRAS clones, which emerge in blood during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells that had acquired resistance to cetuximab reveals that upon antibody withdrawal KRAS clones decay, whereas the population regains drug sensitivity. ctDNA profiles of individuals who benefit from multiple challenges with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results indicate that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of rechallenge therapies based on EGFR blockade.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain ...of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Most solid tumors, including colorectal cancers, shed cell-free DNA (ctDNA) in the blood. ctDNA can be analyzed to generate molecular profiles which capture the heterogeneity of the disease more ...comprehensively then tumor tissue biopsies. This approach commonly called ‘liquid biopsy’ can be applied to monitor response to therapy, to assess minimal residual disease and to uncover the emergence of drug resistance. This review will discuss current and future developments of ctDNA analysis in the clinical management of colorectal cancer patients.
•Liquid biopsies can be used to define – non invasively-the molecular profiles of the disease.•ctDNA analysis can be used to assess minimal residual disease and to monitor response and resistance to therapy.•Liquid, as compared to tissue biopsies, capture more comprehensively the molecular heterogeneity of colorectal cancers.•Radiological measurements and ctDNA assessments can be successfully combined to tailor treatments.•Large prospective studies are needed to incorporate liquid biopsies in the management of colorectal cancer patients.
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FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Blockade of the epidermal growth factor receptor (EGFR) with the monoclonal antibodies cetuximab or panitumumab is effective in a subset of colorectal cancers (CRCs), but the emergence of resistance ...limits the efficacy of these therapeutic agents. At relapse, the majority of patients develop RAS mutations, while a subset acquires EGFR extracellular domain (ECD) mutations. Here we find that patients who experience greater and longer responses to EGFR blockade preferentially develop EGFR ECD mutations, while RAS mutations emerge more frequently in patients with smaller tumour shrinkage and shorter progression-free survival. In circulating cell-free tumour DNA of patients treated with anti-EGFR antibodies, RAS mutations emerge earlier than EGFR ECD variants. Subclonal RAS but not EGFR ECD mutations are present in CRC samples obtained before exposure to EGFR blockade. These data indicate that clonal evolution of drug-resistant cells is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies.
Even if
wild-type and
-negative metastatic colorectal cancer (mCRC) patients frequently respond to anti-EGFR mAbs, acquired resistance almost invariably occurs. Mechanisms of resistance to EGFR ...blockade include the emergence of
,
, and
extracellular domain mutations as well as
alterations. However, these findings derive from retrospective studies that analyzed one single resistance mechanism at a time; moreover, it is still unclear how molecular heterogeneity affects clonal evolution in patients. In this work, we aimed at extensively characterizing and correlating the molecular characteristics of tissue- and blood-based data in a prospective cohort of patients with mCRC who received anti-EGFR antibodies.
Twenty-two
-
wild-type,
-negative mCRC patients progressing on anti-EGFR therapy after initial response underwent rebiopsy. Next-generation sequencing and silver
hybridization (SISH)/IHC analyses were performed both on archival tumors and postprogression samples. Circulating tumor (ctDNA) molecular profiles were obtained in matched tissue-plasma samples.
mutations and
amplification were the most frequently detected resistance mechanisms in both tissue and blood sample analysis. On the other hand,
and
ectodomain mutations were much rarer. Patients with acquired
amplification showed worse PFS on anti-EGFRs. We detected both intralesion heterogeneity, as suggested by co-occurrence of different resistance mechanisms in the same sample, and interlesion heterogeneity. The combined analysis of tissue and blood (ctDNA) results highlights the complexity of clonal evolution triggered by EGFR blockade.
Our results indicate that it may be extremely challenging to target the complex landscape of molecular heterogeneity associated with emergence of resistance to targeted therapies in patients with mCRC.
.
Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an ...LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next-generation TRKA inhibitors.
We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identification of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the first time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements.
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