Jessica J. Lin

Targeting ALK: Precision Medicine Takes on Drug Resistance

Anaplastic lymphoma kinase (ALK) is a validated molecular target in several ALK-rearranged malignancies, including non-small cell lung cancer. However, the clinical benefit of targeting ALK using tyrosine kinase inhibitors (TKI) is almost universally limited by the emergence of drug resistance. Diverse mechanisms of resistance to ALK TKIs have now been discovered, and these basic mechanisms are informing the development of novel therapeutic strategies to overcome resistance in the clinic. In this review, we summarize the current successes and challenges of targeting ALK. SIGNIFICANCE Effective long-term treatment of ALK-rearranged cancers requires a mechanistic understanding of resistance to ALK TKIs so that rational therapies can be selected to combat resistance. This review underscores the importance of serial biopsies in capturing the dynamic therapeutic vulnerabilities within a patients tumor and offers a perspective into the complexity of on-target and off-target ALK TKI resistance mechanisms. Therapeutic strategies that can successfully overcome, and potentially prevent, these resistance mechanisms will have the greatest impact on patient outcome. Cancer Discov; 7(2); 137-55. ©2017 AACR.

Clinical Activity of Alectinib in Advanced RET-Rearranged Non-Small Cell Lung Cancer

Introduction Chromosomal rearrangements involving rearranged during transfection gene (RET) occur in 1% to 2% of NSCLCs and may confer sensitivity to rearranged during transfection (RET) inhibitors. Alectinib is an anaplastic lymphoma kinase tyrosine kinase inhibitor (TKI) that also has anti‐RET activity in vitro. The clinical activity of alectinib in patients with RET‐rearranged NSCLC has not yet been reported. Methods We have described four patients with advanced RET‐rearranged NSCLC who were treated with alectinib (600 mg twice daily [n = 3] or 900 mg twice daily [n = 1]) as part of single‐patient compassionate use protocols or off‐label use of the commercially available drug. Results Four patients with metastatic RET‐rearranged NSCLC were identified. Three of the four had received prior RET TKIs, including cabozantinib and experimental RET inhibitors. In total, we observed two (50%) objective radiographic responses after treatment with alectinib (one confirmed and one unconfirmed), with durations of therapy of 6 months and more than 5 months (treatment ongoing), respectively. Notably, one of these two patients had his dose of alectinib escalated to 900 mg twice daily and had clinical improvement in central nervous system metastases. In addition, one patient (25%) experienced a best response of stable disease lasting approximately 6 weeks (the drug discontinued for toxicity). A fourth patient who was RET TKI–naive had primary progression while receiving alectinib. Conclusions Alectinib demonstrated preliminary antitumor activity in patients with advanced RET‐rearranged NSCLC, most of whom had received prior RET inhibitors. Larger prospective studies with longer follow‐up are needed to assess the efficacy of alectinib in RET‐rearranged NSCLC and other RET‐driven malignancies. In parallel, development of more selective, potent RET TKIs is warranted.

Five-Year Survival in EGFR-Mutant Metastatic Lung Adenocarcinoma Treated with EGFR-TKIs

Introduction: Activating mutations in the epidermal growth factor receptor gene (EGFR) predict for prolonged progression‐free survival in patients with advanced non–small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (EGFR‐TKIs) versus chemotherapy. Long‐term survival outcomes, however, remain undefined. The objective of this study was to determine the 5‐year survival in these patients and identify clinical factors associated with overall survival (OS). Methods: Patients with EGFR‐mutant metastatic lung adenocarcinoma who had been treated with erlotinib or gefitinib at Dana‐Farber Cancer Institute between 2002 and 2009 were included. OS was analyzed. Results: Among 137 patients, median progression‐free survival and OS were 12.1 months (95% CI: 10.2–13.5) and 30.9 months (95% CI: 28.2–35.7), respectively. Twenty patients (14.6%) were 5‐year survivors. In multivariate analysis, exon 19 deletions (hazard ratio [HR] = 0.63, 95% CI: 0.44–0.91, p = 0.01), absence of extrathoracic (HR = 0.62, 95% CI: 0.41–0.93, p = 0.02) or brain metastasis (HR = 0.48, 95% CI: 0.30–0.77, p = 0.002), and not a current smoker (HR = 0.23, 95% CI: 0.09–0.59, p = 0.002) were associated with prolonged OS. Age; sex; stage at diagnosis; liver, bone, or adrenal metastasis; specific TKI; and line of TKI therapy were not associated with OS. Conclusions: Our data suggest that the rate of 5‐year survival among patients with EGFR‐mutant metastatic lung adenocarcinoma treated with erlotinib or gefitinib is 14.6%. Exon 19 deletions and absence of extrathoracic or brain metastasis are associated with prolonged survival. On the basis of our findings, clinicians can gain an enhanced estimation of long‐term outcomes in this population.

Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells.

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.

ROS1 Fusions Rarely Overlap with Other Oncogenic Drivers in Non–Small Cell Lung Cancer

Introduction: Chromosomal rearrangements involving the gene ROS1 define a distinct molecular subset of NSCLCs with sensitivity to ROS1 inhibitors. Recent reports have suggested a significant overlap between ROS1 fusions and other oncogenic driver alterations, including mutations in EGFR and KRAS. Methods: We identified patients at our institution with ROS1‐rearranged NSCLC who had undergone testing for genetic alterations in additional oncogenes, including EGFR, KRAS, and anaplastic lymphoma receptor tyrosine kinase gene (ALK). Clinicopathologic features and genetic testing results were reviewed. We also examined a separate database of ROS1‐rearranged NSCLCs identified through the commercial FoundationOne assay (Foundation Medicine, Cambridge, MA). Results: Among 62 patients with ROS1‐rearranged NSCLC evaluated at our institution, none harbored concurrent ALK fusions (0%) or EGFR activating mutations (0%). KRAS mutations were detected in two cases (3.2%), one of which harbored a concurrent noncanonical KRAS I24N mutation of unknown biological significance. In a separate ROS1 fluorescence in situ hybridization–positive case, targeted sequencing failed to confirm a ROS1 fusion but instead identified a KRAS G13D mutation. No concurrent mutations in B‐Raf proto‐oncogene, serine/threonine kinase gene (BRAF), erb‐b2 receptor tyrosine kinase 2 gene (ERBB2), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha gene (PIK3CA), AKT/serine threonine kinase 1 gene (AKT1), or mitogen‐activated protein kinase kinase 1 gene (MAP2K1) were detected. Analysis of an independent data set of 166 ROS1‐rearranged NSCLCs identified by FoundationOne demonstrated rare cases with co‐occurring driver mutations in EGFR (one of 166) and KRAS (three of 166) and no cases with co‐occurring ROS1 and ALK rearrangements. Conclusions: ROS1 rearrangements rarely overlap with alterations in EGFR, KRAS, ALK, or other targetable oncogenes in NSCLC.

Patterns of Metastatic Spread and Mechanisms of Resistance to Crizotinib in ROS1-Positive Non–Small-Cell Lung Cancer

PURPOSE The ROS1 tyrosine kinase is activated through ROS1 gene rearrangements in 1-2% of non-small cell lung cancer (NSCLC), conferring sensitivity to treatment with the ALK/ROS1/MET inhibitor crizotinib. Currently, insights into patterns of metastatic spread and mechanisms of crizotinib resistance among ROS1-positive patients are limited. PATIENTS AND METHODS We reviewed clinical and radiographic imaging data of patients with ROS1- and ALK-positive NSCLC in order to compare patterns of metastatic spread at initial metastatic diagnosis. To determine molecular mechanisms of crizotinib resistance, we also analyzed repeat biopsies from a cohort of ROS1-positive patients progressing on crizotinib. RESULTS We identified 39 and 196 patients with advanced ROS1- and ALK-positive NSCLC, respectively. ROS1-positive patients had significantly lower rates of extrathoracic metastases (ROS1 59.0%, ALK 83.2%, P=0.002), including lower rates of brain metastases (ROS1 19.4%, ALK 39.1%; P = 0.033), at initial metastatic diagnosis. Despite similar overall survival between ALK- and ROS1-positive patients treated with crizotinib (median 3.0 versus 2.5 years, respectively; P=0.786), ROS1-positive patients also had a significantly lower cumulative incidence of brain metastases (34% vs. 73% at 5 years; P<0.0001). Additionally, we identified 16 patients who underwent a total of 17 repeat biopsies following progression on crizotinib. ROS1 resistance mutations were identified in 53% of specimens, including 9/14 (64%) non-brain metastasis specimens. ROS1 mutations included: G2032R (41%), D2033N (6%), and S1986F (6%). CONCLUSIONS Compared to ALK rearrangements, ROS1 rearrangements are associated with lower rates of extrathoracic metastases, including fewer brain metastases, at initial metastatic diagnosis. ROS1 resistance mutations, particularly G2032R, appear to be the predominant mechanism of resistance to crizotinib, underscoring the need to develop novel ROS1 inhibitors with activity against these resistant mutants.

Recent Advances in Targeting ROS1 in Lung Cancer

&NA; ROS1 is a validated therapeutic target in NSCLC. In a phase I study, the multitargeted MET proto‐oncogene, receptor tyrosine kinase/anaplastic lymphoma kinase/ROS1 inhibitor crizotinib demonstrated remarkable efficacy in ROS1‐rearranged NSCLCs and consequently gained approval by the United States Food and Drug Administration and by the European Medicines Agency in 2016. However, similar to other oncogene‐driven lung cancers, ROS1‐rearranged lung cancers treated with crizotinib eventually acquire resistance, leading to disease relapse. Novel ROS1 inhibitors and therapeutic strategies are therefore needed. Insights into the mechanisms of resistance to ROS1‐directed tyrosine kinase inhibitors are now beginning to emerge and are helping to guide the development of new ROS1 inhibitors. This review discusses the biology and diagnosis of ROS1‐rearranged NSCLC, and current and emerging treatment options for this disease. Future challenges in the field are highlighted.

Differential Sensitivity to Crizotinib: Does EML4-ALK Fusion Variant Matter?

Recent years have borne witness to the development of numerous anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) for patients with ALK-rearranged non–small-cell lung cancer (NSCLC). Crizotinib, amultitargeted ALK/ROS1/MET inhibitor, was the first ALK-targeted agent tested in the clinic. In randomized phase III trials, crizotinib showed superior efficacy compared with standard firstand second-line chemotherapy in advanced ALKpositive NSCLC, leading to the widespread adoption of crizotinib as the standard of care for this molecular subset of lung cancer. Since then, two second-generation ALK inhibitors, ceritinib and alectinib, have received accelerated approval by the US Food and Drug Administration for patients with crizotinib-refractory, ALKpositive NSCLC. Several other next-generation ALK TKIs are also under development. Although most patients with ALK-rearranged NSCLC respond to ALK TKIs, a wide range of responses has been observed. For example, although the average duration of crizotinib treatment is 8 to 11 months, some patients experience relapse within only a few months, and others have not yet experienced disease relapse after more than 5 years. The biologic mechanisms underlying this heterogeneity in TKI response are unknown. One of the earliest proposedmechanisms focused on the different variants of the most common ALK rearrangement, EML4-ALK. To date, more than 10 distinct EML4-ALK variants have been identified, each with a discrete breakpoint in EML4. Other fusion partner genes including KIF5B, TFG, and KLC1 have also been reported. In all cases, the fusion oncogene contains the 59 portion of the partner gene, including its promoter, and the entire ALK kinase domain (exons 20 to 29). The fusion partners generally contain oligomerization domains leading to constitutive, ligandindependent ALK activation. Early in vitro studies suggested differential sensitivity of four EML4-ALK variants (v1, v2, v3a, and v3b) to crizotinib and the tool compound TAE684. Interestingly, the differential sensitivity observed correlated with the protein stability of the EML4-ALK variants. This work suggested a potential molecular basis for the differential responses seen with crizotinib. In the article accompanying this editorial, Yoshida et al report the results of a single-institution retrospective analysis addressing the question of clinical impact of EML4-ALK fusion variants on crizotinib sensitivity in patients. The efficacy of crizotinib was examined in 35 patients with ALK-positive NSCLC, whose tumor specimens were assessed for the presence of ALKvariants by reverse transcription polymerase chain reaction with concurrent testing by either fluorescent in situ hybridization (n 5 25) or immunohistochemistry (n5 28). Nineteen patients (54%) had EML4-ALKv1, five (14%) had v2, four (12%) had v3a/3b, and seven (20%) had other variants. Non-EML4 partner proteins were not assessed. Notably, crizotinib was the first-line treatment of 10 patients (29%) and second-line or later treatment for the remaining 25 patients (71%). For the entire cohort, both the response rate (69%) and median progress-free survival (PFS) time (9.7 months) were consistent with published data from crizotinib trials. However, although the response rates were comparable in v1 and non-v1 groups (74% and 63%, respectively; P 5 .7160), there was a statistically significant difference in the disease control rate (95% v 63%, respectively; P5 .0318) and median PFS (11 v 4.2 months, respectively; P, .05). These observations support the notion that the type of ALK fusion could be a determinant of sensitivity to crizotinib in ALKrearranged lung cancers. However, there are several important limitations of this study. First, there is no biologic rationale for categorizing patients with ALK-rearranged lung cancer on the basis of the presence of EML4-ALK v1 versus non-v1. Indeed, the findings of this study are not consistent with prior in vitro studies that showed differences among the EML4-ALK variants v1, v2, v3a, and v3b. On the basis of the in vitro results, EML4-ALK v2 (not v1) was most sensitive to ALK inhibitors, v1 and v3b had intermediate sensitivity, and v3a was least sensitive. At present, there are limited data to suggest how biologically similar, or different, the non-v1 EML4-ALK variants may be compared with one another; therefore, grouping these variants together is somewhat artificial. Second, the study by Yoshida et al was limited by the small sample size, with only four to seven patients in each individual subgroup of non-v1 variants. Within the v1 and non-v1 groups, a remarkably wide range of PFS times was observed. For example, one patient with EML4-ALK v5b had PFS censored at greater than 36 months, which is significantly longer than the median PFS of the v1 group. Furthermore, for three of the 16 patients in the non-v1 group, the PFS was censored at a relatively short follow-up (, 6 months). It is worth noting that other studies examining the role of ALK variants have also been limited by small numbers and have not reported differences in response on the basis of specific variant. The original phase I study of crizotinib included analysis of EML4-ALK variants by reverse transcription polymerase chain

Safety of Combined PD-1 Pathway Inhibition and Intracranial Radiation Therapy in Non–Small Cell Lung Cancer

Introduction: Intracranial metastases are a common cause of morbidity and mortality in patients with advanced NSCLC, and are frequently managed with radiation therapy (RT). The safety of cranial RT in the setting of treatment with immune checkpoint inhibitors (ICIs) has not been established. Methods: We identified patients with advanced NSCLC with brain metastases who received cranial RT and were treated with or without programmed cell death 1/programmed death ligand 1 inhibitors between August 2013 and September 2016. RT‐related adverse events (AEs) were retrospectively evaluated and analyzed according to ICI treatment status, cranial RT type, and timing of RT with respect to ICI. Results: Of 163 patients, 50 (31%) received ICIs, whereas 113 (69%) were ICI naive. Overall, 94 (58%), 28 (17%), and 101 (62%) patients received stereotactic radiosurgery, partial brain irradiation, and/or whole brain RT, respectively. Fifty percent of patients received more than one radiation course. We observed no significant difference in rates of all‐grade AEs and grade 3 or higher AEs between the ICI‐naive and ICI‐treated patients across different cranial RT types (grade ≥3 AEs in 8% of ICI‐naive patients versus in 9% of ICI‐treated patients for stereotactic radiosurgery [p = 1.00] and in 8% of ICI‐naive patients versus in 10% of ICI‐treated patients for whole brain RT [p = 0.71]). Additionally, there was no difference in AE rates on the basis of timing of ICI administration with respect to RT. Conclusions: Treatment with an ICI and cranial RT was not associated with a significant increase in RT‐related AEs, suggesting that use of programmed cell death 1/programmed death ligand 1 inhibitors in patients receiving cranial RT may have an acceptable safety profile. Nonetheless, additional studies are needed to validate this approach.