Impact of BRAF Mutation Class on Disease Characteristics and Clinical Outcomes in BRAF-mutant Lung Cancer.

PURPOSE: BRAF mutations are divided into functional classes distinguished by signaling mechanism and kinase activity: V600-mutant kinase-activating monomers (class I), kinase-activating dimers (class II), and kinase-inactivating heterodimers (class III). The relationship between functional class and disease characteristics in BRAF-mutant non-small cell lung cancer (NSCLC) has not been fully explored. EXPERIMENTAL DESIGN: We performed a retrospective analysis of BRAF-mutant NSCLCs treated at 2 institutions from 2005 to 2017 to determine clinicopathologic characteristics, progression-free survival (PFS) on chemotherapy, and overall survival (OS). RESULTS: We identified 236 patients with BRAF-mutant NSCLC (n = 107 class I, n = 75 class II, and n = 54 class III). Patients with class II or III mutations were more likely to have brain metastases (P ≤ 0.01) and RAS coalterations (P ≤ 0.001) than class I. Compared with class I, PFS on chemotherapy was shorter for class II (P = 0.069) and class III (P = 0.034). OS was shorter for class II and III (class I, 40.1 months; class II, 13.9 months; and class III, 15.6 months; I vs. II, P < 0.001; I vs. III, P = 0.023); however, this difference was driven by fewer extrathoracic metastases and higher use of targeted therapies in class I patients. When patients treated with targeted therapy and those with thoracic-only metastases were excluded, there was no difference in OS across the 3 classes. CONCLUSIONS: BRAF-mutant NSCLC is a heterogeneous disease that encompasses 3 distinct functional classes. Classes II and III have more aggressive clinical features leading to less favorable outcomes. The distinct biological characteristics of class II and III tumors suggest that class-specific therapies may be necessary to effectively target these molecular subsets.

Heterogeneity and Coexistence of T790M and T790 Wild-Type Resistant Subclones Drive Mixed Response to Third-Generation Epidermal Growth Factor Receptor Inhibitors in Lung Cancer.

PURPOSE: Third-generation epidermal growth factor receptor (EGFR) inhibitors like nazartinib are active against EGFR mutation-positive lung cancers with T790M-mediated acquired resistance to initial anti-EGFR treatment, but some patients have mixed responses. METHODS: Multiple serial tumor and liquid biopsies were obtained from two patients before, during, and after treatment with nazartinib. Next-generation sequencing and droplet digital polymerase chain reaction were performed to assess heterogeneity and clonal dynamics. RESULTS: We observed the simultaneous emergence of T790M-dependent and -independent clones in both patients. Serial plasma droplet digital polymerase chain reaction illustrated shifts in relative clonal abundance in response to various systemic therapies, confirming a molecular basis for the clinical mixed radiographic responses observed. CONCLUSION: Heterogeneous responses to treatment targeting a solitary resistance mechanism can be explained by coexistent tumor subclones harboring distinct genetic signatures. Serial liquid biopsies offer an opportunity to monitor clonal dynamics and the emergence of resistance and may represent a useful tool to guide therapeutic strategies.

Convergent Therapeutic Strategies to Overcome the Heterogeneity of Acquired Resistance in BRAFV600E Colorectal Cancer.

Clonal heterogeneity associated with acquired resistance presents a critical therapeutic challenge. Whole-exome sequencing of paired tumor biopsies and targeted sequencing of cell-free DNA (cfDNA) from patients with BRAFV600E colorectal cancer receiving BRAF inhibitor combinations identified 14 distinct alterations in MAPK pathway components driving acquired resistance, with as many as eight alterations in a single patient. We developed a pooled clone system to study clonal outgrowth during acquired resistance, in vitro and in vivoIn vitro, the dynamics of individual resistant clones could be monitored in real time in cfDNA isolated from culture media during therapy. Outgrowth of multiple resistant clones was observed during therapy with BRAF, EGFR, and MEK inhibitor combinations. However, ERK inhibition, particularly in combination with BRAF and EGFR inhibition, markedly abrogated clonal outgrowth in vitro and in vivo Thus, convergent, up-front therapy may suppress outgrowth of heterogeneous clones harboring clinically observed resistance alterations, which may improve clinical outcome.Significance: We observed heterogeneous, recurrent alterations in the MAPK pathway as key drivers of acquired resistance in BRAFV600E colorectal cancer, with multiple concurrent resistance alterations detectable in individual patients. Using a novel pooled clone system, we identify convergent up-front therapeutic strategies capable of intercepting multiple resistance mechanisms as potential approaches to suppress emergence of acquired resistance. Cancer Discov; 8(4); 417-27. ©2018 AACR.See related commentary by Janku, p. 389See related article by Corcoran et al., p. 428This article is highlighted in the In This Issue feature, p. 371.

Impact of EML4-ALK Variant on Resistance Mechanisms and Clinical Outcomes in ALK-Positive Lung Cancer.

Purpose Advanced anaplastic lymphoma kinase ( ALK) fusion-positive non-small-cell lung cancers (NSCLCs) are effectively treated with ALK tyrosine kinase inhibitors (TKIs). However, clinical outcomes in these patients vary, and the benefit of TKIs is limited as a result of acquired resistance. Emerging data suggest that the ALK fusion variant may affect clinical outcome, but the molecular basis for this association is unknown. Patients and Methods We identified 129 patients with ALK-positive NSCLC with known ALK variants. ALK resistance mutations and clinical outcomes on ALK TKIs were retrospectively evaluated according to ALK variant. A Foundation Medicine data set of 577 patients with ALK-positive NSCLC was also examined. Results The most frequent ALK variants were EML4-ALK variant 1 in 55 patients (43%) and variant 3 in 51 patients (40%). We analyzed 77 tumor biopsy specimens from patients with variants 1 and 3 who had progressed on an ALK TKI. ALK resistance mutations were significantly more common in variant 3 than in variant 1 (57% v 30%; P = .023). In particular, ALK G1202R was more common in variant 3 than in variant 1 (32% v 0%; P < .001). Analysis of the Foundation Medicine database revealed similar associations of variant 3 with ALK resistance mutation and with G1202R ( P = .010 and .015, respectively). Among patients treated with the third-generation ALK TKI lorlatinib, variant 3 was associated with a significantly longer progression-free survival than variant 1 (hazard ratio, 0.31; 95% CI, 0.12 to 0.79; P = .011). Conclusion Specific ALK variants may be associated with the development of ALK resistance mutations, particularly G1202R, and provide a molecular link between variant and clinical outcome. ALK variant thus represents a potentially important factor in the selection of next-generation ALK inhibitors.

Tumor Heterogeneity and Lesion-Specific Response to Targeted Therapy in Colorectal Cancer.

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.

Molecular Heterogeneity and Receptor Coamplification Drive Resistance to Targeted Therapy in MET-Amplified Esophagogastric Cancer.

UNLABELLED: MET inhibition is effective in some patients with MET-amplified esophagogastric cancer (EGC), but understanding acquired and de novo resistance mechanisms will be critical to improving therapy. We identified KRAS mutation as a novel cause of acquired resistance in a patient after a 2-year response to a MET inhibitor. We also observed that 40% to 50% of patients with MET-amplified EGC harbor coamplification of HER2 and/or EGFR concurrently in the same tumor cells, which can drive de novo resistance. One patient with concurrent MET and HER2 amplification was refractory to HER2 blockade, but responded to combined MET/HER2 inhibition. We also found striking heterogeneity in MET amplification between distinct metastatic lesions and primary tumors in individual patients with EGC. In these patients, MET inhibition led to mixed responses and disease progression through outgrowth of non-MET-amplified clones, which could be monitored in circulating tumor DNA. Thus, receptor coamplification and molecular heterogeneity may be key drivers of clinical resistance in MET-amplified EGC. SIGNIFICANCE: Coamplification of driver oncogenes occurs frequently in EGC and can drive therapeutic resistance, supporting a role for comprehensive molecular analysis prior to targeted therapy. EGCs can also exhibit extensive heterogeneity in gene amplification between distinct tumor lesions within the same patient, suggesting that molecular profiling of a single-lesion biopsy may be insufficient to guide targeted therapy selection.

Mechanisms of acquired crizotinib resistance in ALK-rearranged lung Cancers.

Most anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancers (NSCLCs) are highly responsive to treatment with ALK tyrosine kinase inhibitors (TKIs). However, patients with these cancers invariably relapse, typically within 1 year, because of the development of drug resistance. Herein, we report findings from a series of lung cancer patients (n = 18) with acquired resistance to the ALK TKI crizotinib. In about one-fourth of patients, we identified a diverse array of secondary mutations distributed throughout the ALK TK domain, including new resistance mutations located in the solvent-exposed region of the adenosine triphosphate-binding pocket, as well as amplification of the ALK fusion gene. Next-generation ALK inhibitors, developed to overcome crizotinib resistance, had differing potencies against specific resistance mutations. In addition to secondary ALK mutations and ALK gene amplification, we also identified aberrant activation of other kinases including marked amplification of KIT and increased autophosphorylation of epidermal growth factor receptor in drug-resistant tumors from patients. In a subset of patients, we found evidence of multiple resistance mechanisms developing simultaneously. These results highlight the unique features of TKI resistance in ALK-positive NSCLCs and provide the rationale for pursuing combinatorial therapeutics that are tailored to the precise resistance mechanisms identified in patients who relapse on crizotinib treatment.