Luca Lazzari

UNLABELLED: How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent therapy is unknown. We studied EGFR blockade in colorectal cancer to assess whether tissue and liquid biopsies can be integrated with radiologic imaging to monitor the impact of individual oncogenic alterations on lesion-specific responses. Biopsy of a patient's progressing liver metastasis following prolonged response to cetuximab revealed a MEK1(K57T) mutation as a novel mechanism of acquired resistance. This lesion regressed upon treatment with panitumumab and the MEK inhibitor trametinib. In circulating tumor DNA (ctDNA), mutant MEK1 levels declined with treatment, but a previously unrecognized KRAS(Q61H) mutation was also identified that increased despite therapy. This same KRAS mutation was later found in a separate nonresponding metastasis. In summary, parallel analyses of tumor biopsies and serial ctDNA monitoring show that lesion-specific radiographic responses to subsequent targeted therapies can be driven by distinct resistance mechanisms arising within separate tumor lesions in the same patient. SIGNIFICANCE: Molecular heterogeneity ensuing from acquired resistance drives lesion-specific responses to subsequent targeted therapies. Analysis of a single-lesion biopsy is inadequate to guide selection of subsequent targeted therapies. ctDNA profiles allow the detection of concomitant resistance mechanisms residing in separate metastases and assessment of the effect of therapies designed to overcome resistance.

Background: ALK, ROS1, and NTRK fusions occur in 0.2% to 2.4% of colorectal cancers. Pioneer cases of metastatic colorectal cancer (mCRC) patients bearing rearrangements who benefited from anti-ALK, ROS, and TrkA-B-C therapies have been reported previously. Here we aimed at characterizing the clinical and molecular landscape of ALK, ROS1, and NTRK rearranged mCRC. Methods: Clinical features and molecular characteristics of 27 mCRC patients bearing ALK, ROS1, and NTRK rearranged tumors were compared with those of a cohort of 319 patients not bearing rearrangements by means of Fisher's exact, χ2 test, or Mann-Whitney test as appropriate. Overall survival curves were estimated with the Kaplan-Meier method and compared using the log-rank test. A Cox proportional hazard model was adopted in the multivariable analysis. Deep molecular and immunophenotypic characterizations of rearranged cases, including those described in The Cancer Genome Atlas database, were performed. All statistical tests were two-sided. Results: Closely recalling the "BRAF history," ALK, ROS1, and NTRK rearrangements more frequently occurred in elderly patients (P = .02) with right-sided tumors (P < .001) and node-spreading (P = .03), RAS wild-type (P < .001), and MSI-high (P < .001) cancers. All patients bearing ALK, ROS1, and NTRK fusions had shorter overall survival (15.6 months, 95% confidence interval [CI] = 0.0 to 20.4 months) than negative patients (33.7 months, 95% CI = 28.3 to 42.1 months), both in the univariate (hazard ratio [HR] = 2.17, 95% CI = 1.03 to 4.57, P < .001) and multivariable models (HR = 2.33, 95% CI = 1.10 to 4.95, P = .02). All four evaluable patients with rearrangements showed primary resistance to anti-epidermal growth factor receptor agents. Frequent association with potentially targetable RNF43 mutations was observed in MSI-high rearranged tumors. Conclusions: ALK, ROS1, and NTRK rearrangements define a new rare subtype of mCRC with extremely poor prognosis. Primary tumor site, MSI-high, and RAS and BRAF wild-type status may help to identify patients bearing these alterations. While sensitivity to available treatments is limited, targeted strategies inhibiting ALK, ROS, and TrkA-B-C provided encouraging results.

UNLABELLED: 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. SIGNIFICANCE: 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.

PURPOSE: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models. EXPERIMENTAL DESIGN: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance. RESULTS: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab. CONCLUSIONS: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor-antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies.