Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients

Abstract

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.

Figure 1. Identification of genetic alterations associated with resistance to anti-EGFR antibodies in plasma samples

The tables list putative genetic mechanism of primary (a) and acquired (b) resistance to anti-EGFR antibodies which were identified in circulating tumor DNA of 26 patients in total. EGFR mutational analysis was not performed in AOUP-CRC04, AOUP-CRC05, AOUP-CRC01, AOUP-CRC06 and AOUP-CRC02 due to limited sample amount. Green colour highlights actionable targets. CETUX: cetuximab; PANIT: panitumumab; IRINO: irinotecan. N.I. not identified.

Figure 2. KRAS mutant alleles emerge in circulating DNA during anti-EGFR therapy and decline when treatment is suspended

Detection of KRAS mutations (a–e) and MET amplification (f) in circulating DNA of patients who developed acquired resistance to first-line chemotherapy plus anti-EGFR and then received other lines of treatment. Grey bars represent the variation of tumor load, compared to baseline, during systemic treatments specified in arrows below the graphs. Tumor load is calculated as follows: measurable disease at the initiation of treatment (baseline) is assumed as 100%; responses or progression are calculated as % of tumor load as compared to baseline, as per RECIST criteria. Relevant clinical events are indicated in grey boxes below the graphs. Black lines indicate the frequency of KRAS mutation (% of alleles) or MET copy number alteration, detected in circulating DNA at the time points indicated below the graphs. Dotted blue line indicates CEA values. CETUX: cetuximab; PANIT: panitumumab; BEV: bevacizumab; IRINO: irinotecan. In patient AOUP-CRC01 the first CT scan revaluation assessed complete response (no measurable disease). KRAS mutational frequency is plotted on the primary axis while % of tumor load is plotted on the secondary axis. In patient ONCG-CRC71 the first CT scan revaluation not available. CEA levels were used as a surrogate marker of response.

Figure 3. Anti-EGFR antibodies re-challenge in colorectal cancer cells and patients

(a) Two colorectal cancer cell populations (DiFi A and DiFi B) that developed KRAS amplification as a resistance mechanism to cetuximab were allowed to replicate in the absence of the antibody for 160 days. Top panel: KRAS amplification was assessed by qPCR in the indicated cell models (parental/sensitive, resistant derivatives and resistant cells after 160 days of antibody withdrawal). Grey bars indicate KRAS gene copy number (CNA). Statistical differences were calculated by Student’s t-Test. Data are expressed as means ± SD of three independent experiments. *** p value ≤0.001; ** p value ≤0.01. Bottom panel: cetuximab sensitivity assay. The indicated cell populations were treated for 1 week, with increasing concentrations of cetuximab. Cell viability was measured by the adenosine triphosphate (ATP) assay. Data points represent means ± SD of three independent experiments. (b) Clinical synopsis of a patient treated with irinotecan plus cetuximab achieving SD in lung and bone metastases that lasted approximately 6 months. At progression, the patient began treatment with capecitabine plus oxaliplatin (XELOX) with progression of the disease after 3 months of treatment. The patient was subsequently re-treated with irinotecan plus cetuximab achieving a PR. Grey area represents tumor load (% of baseline); dotted blue line indicates CEA values. (c) Clinical synopsis of a patient treated with cetuximab as third-line for pelvic relapse of rectal cancer, achieving partial response lasting 13 months; the patient then refused further therapy for skin toxicity. At disease progression, one year later, she was treated with radiotherapy and 5-fluorouracil with partial response and then progression after 6 months. She therefore received rechallenge with anti-EGFR (panitumumab monotherapy) achieving long-lasting SD (7 months). Grey area represents tumor load (% of baseline); dotted blue line indicates CEA values. CETUX: cetuximab.

Figure 4. KRAS mutant clones dynamically evolve in response to pulsatile anti-EGFR antibodies therapy

(a–c) Dynamics of KRAS mutant clones in plasma samples of three CRC patients receiving the indicated therapies. Grey bars represent variation of tumor load, compared to baseline, during treatments as specified below the graphs. Tumor load is calculated as follows: measurable disease at the initiation of treatment (baseline) is assumed as 100%; responses or progression are calculated as % of tumor load as compared to baseline, as per RECIST criteria. Relevant clinical events are indicated in grey boxes below the individual graphs. Black and red lines indicate the frequency of KRAS mutation (% of alleles), detected in circulating DNA at the indicated time points. Black stars represent analyzed tissue samples. Dotted blue line indicates CEA values. CETUX: cetuximab; PANIT: panitumumab; REGO: regorafenib.