Osimertinib Overcomes Alectinib Resistance Caused by Amphiregulin in a Leptomeningeal Carcinomatosis Model of ALK-Rearranged Lung Cancer

IntroductionLeptomeningeal carcinomatosis (LMC) occurs frequently in anaplastic lymphoma kinase (ALK)–rearranged NSCLC and develops acquired resistance to ALK tyrosine kinase inhibitors (ALK TKIs). This study aimed to clarify the resistance mechanism to alectinib, a second-generation ALK TKI, in LMC and test a novel therapeutic strategy.MethodsWe induced alectinib resistance in an LMC mouse model with ALK-rearranged NSCLC cell line, A925LPE3, by continuous oral alectinib treatment, established A925L/AR cells. Resistance mechanisms were analyzed using several assays, including Western blot and receptor tyrosine kinase array. We also measured amphiregulin (AREG) concentrations in cerebrospinal fluid from patients with ALK-rearranged NSCLC with alectinib-refractory LMC by enzyme-linked immunosorbent assay.ResultsA925L/AR cells were moderately resistant to various ALK TKIs, such as alectinib, crizotinib, ceritinib, and lorlatinib, compared with parental cells in vitro. A925L/AR cells acquired the resistance by EGFR activation resulting from AREG overexpression caused by decreased expression of microRNA-449a. EGFR TKIs and anti-EGFR antibody resensitized A925L/AR cells to alectinib in vitro. In the LMC model with A925L/AR cells, combined treatment with alectinib and EGFR TKIs, such as erlotinib and osimertinib, successfully controlled progression of LMC. Mass spectrometry imaging showed accumulation of the EGFR TKIs in the tumor lesions. Moreover, notably higher AREG levels were detected in cerebrospinal fluid of patients with alectinib-resistant ALK-rearranged NSCLC with LMC (n = 4), compared with patients with EGFR-mutated NSCLC with EGFR TKI–resistant LMC (n = 30), or patients without LMC (n = 24).ConclusionsThese findings indicate the potential of novel therapies targeting both ALK and EGFR for the treatment of ALK TKI–resistant LMC in ALK-rearranged NSCLC.     

Monitoring Therapeutic Response and Resistance: Analysis of Circulating Tumor DNA in Patients With ALK+ Lung Cancer

IntroductionDespite initial effectiveness of ALK receptor tyrosine kinase inhibitors (TKIs) in patients with ALK+ NSCLC, therapeutic resistance will ultimately develop. Serial tracking of genetic alterations detected in circulating tumor DNA (ctDNA) can be an informative strategy to identify response and resistance. This study evaluated the utility of analyzing ctDNA as a function of response to ensartinib, a potent second-generation ALK TKI.MethodsPre-treatment plasma was collected from 76 patients with ALK+ NSCLC who were ALK TKI–naive or had received prior ALK TKI, and analyzed for specific genetic alterations. Longitudinal plasma samples were analyzed from a subset (n = 11) of patients. Analysis of pre-treatment tumor biopsy specimens from 22 patients was compared with plasma.ResultsDisease-associated genetic alterations were detected in 74% (56 of 76) of patients, the most common being EML4-ALK. Concordance of ALK fusion between plasma and tissue was 91% (20 of 22 blood and tissue samples). Twenty-four ALK kinase domain mutations were detected in 15 patients, all had previously received an ALK TKI; G1269A was the most prevalent (4 of 24). Patients with a detectable EML4-ALK variant 1 (V1) fusion had improved response (9 of 17 patients; 53%) to ensartinib compared to patients with EML4-ALK V3 fusion (one of seven patients; 14%). Serial changes in ALK alterations were observed during therapy.ConclusionsClinical utility of ctDNA was shown, both at pre-treatment by identifying a potential subgroup of ALK+ NSCLC patients who may derive more benefit from ensartinib and longitudinally by tracking resistance. Prospective application of this technology may translate to improved outcomes for NSCLC patients treated with ALK TKIs.     

Precision medicine against <Emphasis Type="Italic">ALK</Emphasis>-positive non-small cell lung cancer: beyond crizotinib

Anaplastic lymphoma kinase (ALK) rearrangements represent the molecular driver of a subset of non-small cell lung cancers (NSCLCs). Despite the initial response, virtually all ALK-positive patients develop an acquired resistance to the ALK inhibitor crizotinib, usually within 12 months. Several next-generation ALK inhibitors have been developed in order to overcome crizotinib limitation, providing an unprecedented survival for this subset of patients. The aim of this review to summarize the current knowledge on ALK tyrosine kinase inhibitors (TKIs) in the treatment of advanced ALK-positive NSCLC, focusing on the role of novel ALK inhibitors in this setting. In addition, we will discuss their role in the pharmacological management of ALK-positive brain metastasis. Next-generation ALK inhibitors showed an impressive clinical activity in ALK-positive NSCLC, also against the sanctuary site of CNS. Sequential therapy with ALK TKIs appears to be effective in patients who fail a first ALK TKI and translates in clinically meaningful benefit. However, these agents display different activity profiles against crizotinib resistance mutation; therefore re-genotyping the disease at progression in order to administer the right TKI to the right patient is going to be necessary to correctly tailor the treatment. To avoid repeated invasive procedure, noninvasive methods to detect and monitor ALK rearrangement are under clinical investigation.     

Relevance of Detection of Mechanisms of Resistance to ALK Inhibitors in ALK-Rearranged NSCLC in Routine Practice

BackgroundAnaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have shown efficacy in the treatment of ALK-rearranged non–small-cell lung cancer (NSCLC), but the disease eventually progresses in all patients. In many cases, resistance to ALK TKIs arises through ALK mutations. Although clinical and biological data suggest variations in TKI efficacy according to the mechanism of resistance, ALK mutations are still rarely investigated in routine practice.Materials and MethodsWe performed a retrospective multicentric study with an aim to determine the frequency and clinical relevance of ALK alterations detected using targeted next-generation sequencing in patients with advanced ALK-rearranged NSCLC after progression during an ALK TKI treatment. Data on clinical, pathological, and molecular characteristics and patient outcomes were collected.ResultsWe identified 23 patients with advanced ALK-rearranged NSCLC who, between January 2012 and May 2017, had undergone at least 1 repeat biopsy at progression during an ALK TKI treatment. A resistance mechanism was identified in 9 of the 23 patients (39%). The anomalies involved included 9 ALK mutations in 8 patients and one ALK amplification. The ALK mutation rate was 15% after failure of a first ALK TKI and 33% after failure of 2 ALK TKI treatments. Five of 7 patients who received a different ALK TKI after detection of an ALK mutation achieved an objective response. All of the patients who received a TKI presumed to act on the detected ALK mutant achieved disease control.ConclusionTargeted next-generation sequencing is suitable for detecting ALK resistance mutations in ALK-rearranged NSCLC patients in routine practice. It might help select the best treatment at the time of disease progression during treatment with an ALK TKI.     

Receptor Tyrosine Kinase Fusions and BRAF Kinase Fusions are Rare but Actionable Resistance Mechanisms to EGFR Tyrosine Kinase Inhibitors

Introduction

We analyzed a large set of EGFR-mutated (EGFR+) NSCLC to identify and characterize cases with co-occurring kinase fusions as potential resistance mechanisms to EGFR tyrosine kinase inhibitors (TKIs).

Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib‐resistance of EML4‐ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors

Crizotinib, a first‐generation anaplastic lymphoma kinase (ALK) tyrosine‐kinase inhibitor, is known to be effective against echinoderm microtubule‐associated protein‐like 4 (EML4)‐ALK‐positive non‐small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib‐resistant cells (A925LPE3‐CR) via long‐term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4‐ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib‐resistant cells, and these cells were cross‐resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3‐CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine‐kinase inhibitor (erlotinib) or an anti‐EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4‐ALK lung cancer.     

Clinical Outcome of ALK-Positive Non–Small Cell Lung Cancer (NSCLC) Patients with De Novo EGFR or KRAS Co-Mutations Receiving Tyrosine Kinase Inhibitors (TKIs)

IntroductionNSCLC with de novo anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements and EGFR or KRAS mutations co-occur very rarely. Outcomes with tyrosine kinase inhibitors (TKIs) in these patients are poorly understood.MethodsOutcomes of patients with metastatic NSCLC de novo co-alterations of ALK/EGFR or ALK/KRAS detected by fluorescence in situ hybridization (ALK) and sequencing (EGFR/KRAS) from six Swiss centers were analyzed.ResultsA total of 14 patients with adenocarcinoma were identified. Five patients had ALK/EGFR co-alterations and nine had ALK/KRAS co-alterations. Six of seven patients with ALK/KRAS co-alterations (86%) were primary refractory to crizotinib. One patient has had ongoing disease stabilization for 26 months. Of the patients with ALK/EGFR co-alterations, one immediately progressed after receiving crizotinib for 1.3 months and two had a partial response for 5.7 and 7.3 months, respectively. Three of four patients with ALK/EGFR co-alterations treated with an EGFR TKI achieved one or more responses in different lines of therapy: four patients had a partial response, three with afatinib and one with osimertinib. One patient achieved a complete remission with osimertinib, and one patient was primary refractory to erlotinib. Median PFS during treatment with a first EGFR TKI was 5.8 months (range 3.0–6.9 months).ConclusionsDe novo concurrent ALK/KRAS co-alterations were associated with resistance to ALK TKI treatment in seven out of eight patients. In patients with ALK/EGFR co-alterations, outcomes with ALK and EGFR TKIs seem inferior to what would be expected in patients with either alteration alone, but further studies are needed to clarify which patients with ALK/EGFR co-alterations may still benefit from the respective TKI.     

Targeting stemness is an effective strategy to control EML4-ALK+ non-small cell lung cancer cells

The fusion between anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a causative factor in a unique subset of patients with non-small cell lung carcinoma (NSCLC). Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting EML4-ALK fusion protein, displays remarkable initial responses, a fraction of NSCLC cases eventually become resistant to crizotinib by acquiring mutations in the ALK domain or activating bypass pathways via EGFR, KIT, or KRAS. Cancer stem cell (CSC) theory provides a plausible explanation for acquisition of tumorigenesis and resistance. However, the question as to whether EML4-ALK-driven tumorigenesis is linked with the stem-like property and whether the stemness is an effective target in controlling EML4-ALK⁺ NSCLC including crizotinib-resistant NSCLC cells has not been addressed. Here, we report that stem-like properties stem from ALK activity in EML4-ALK⁺ NSCLC cells. Notably, treatment with rapamycin, a CSC targeting agent, attenuates stem-like phenotypes of the EML4-ALK⁺ cells, which increased capability of tumor formation and higher expression of stemness-associated molecules such as ALDH, NANOG, and OCT4. Importantly, combinational treatment with rapamycin and crizotinib leads to synergistic anti-tumor effects on EML4-ALK⁺ NSCLC cells as well as on those resistant to crizotinib. Thus, we provide a proof of principle that targeting stemness would be a novel strategy to control intractable EML4-ALK⁺ NSCLC.     

Establishment of a Conditional Transgenic Mouse Model Recapitulating EML4-ALK–Positive Human Non–Small Cell Lung Cancer

BackgroundAnaplastic lymphoma receptor tyrosine kinase gene (ALK) fusion is a distinct molecular subclassification of NSCLC that is targeted by anaplastic lymphoma kinase (ALK) inhibitors. We established a transgenic mouse model that expresses tumors highly resembling human NSCLC harboring echinoderm microtubule associated protein like 4 gene (EML)-ALK fusion. We aimed to test an EML4-ALK transgenic mouse model as a platform for assessing the efficacy of ALK inhibitors and examining mechanisms of acquired resistance to ALK inhibitors.MethodsTransgenic mouse lines harboring LoxP-STOP-LoxP-FLAGS–tagged human EML4-ALK (variant 1) transgene was established by using C57BL/6N mice. The transgenic mouse model with highly lung-specific, inducible expression of echinoderm microtubule associated protein like 4–ALK fusion protein was established by crossing the EML4-ALK transgenic mice with mice expressing Cre–estrogen receptor fusion protein under the control of surfactant protein C gene (SPC). Expression of EML4-ALK transgene was induced by intraperitoneally injecting mice with tamoxifen. When the lung tumor of the mice treated with the ALK inhibitor crizotinib for 2 weeks was measured, tumor shrinkage was observed.ResultsEML4-ALK tumor developed after 1 week of tamoxifen treatment. Echinoderm microtubule associated protein like 4–ALK was strongly expressed in the lung but not in other organs. ALK and FLAGS expressions were observed by immunohistochemistry. Treatment of EML4-ALK tumor–bearing mice with crizotinib for 2 weeks induced dramatic shrinkage of tumors with no signs of toxicity. Furthermore, prolonged treatment with crizotinib led to acquired resistance in tumors, resulting in regrowth and disease progression. The resistant tumor nodules revealed acquired ALK G1202R mutations.ConclusionsAn EML4-ALK transgenic mouse model for study of drug resistance was successfully established with short duration of tumorigenesis. This model should be a strong preclinical model for testing efficacy of ALK TKIs, providing a useful tool for investigating the mechanisms of acquired resistance and pursuing novel treatment strategies in ALK-positive lung cancer.     

A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from a crizotinib-treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076) as well as from studying a resistant version of the ALK TKI (TAE684)-sensitive H3122 cell line. The crizotinib-resistant DFCI076 cell line harbored a unique L1152R ALK secondary mutation and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent coactivation of epidermal growth factor receptor (EGFR) signaling. In contrast, the TAE684-resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harbored coactivation of EGFR signaling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naive NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signaling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.     

Selective ALK inhibitor alectinib with potent antitumor activity in models of crizotinib resistance

The clinical efficacy of the ALK inhibitor crizotinib has been demonstrated in ALK fusion-positive NSCLC; however, resistance to crizotinib certainly occurs through ALK secondary mutations in clinical use. Here we examined the efficacy of a selective ALK inhibitor alectinib/CH5424802 in models of crizotinib resistance. Alectinib led to tumor size reduction in EML4-ALK-positive xenograft tumors that failed to regress fully during the treatment with crizotinib. In addition, alectinib inhibited the growth of some EML4-ALK mutant-driven tumors, including the G1269A model. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with ALK secondary mutations.     

ALK Inhibitors: What Is the Best Way to Treat Patients With ALK+ Non–Small-Cell Lung Cancer?

Genetic insight into the pathogenesis of lung cancer has paved the way for a new era in its treatment. Recently, anaplastic lymphoma kinase (ALK) has been identified as exerting a potent transforming effect through genetic rearrangement in patients with lung cancer. Preclinical and single-arm phase I studies have shown that patients with ALK-rearranged non–small cell lung cancer (NSCLC) can be successfully treated with crizotinib. Furthermore, a phase III randomized study indicated that crizotinib is superior to standard chemotherapy in the treatment of patients with NSCLC harboring the ALK rearrangement who had received 1 previous platinum-based chemotherapy. Despite the excellent efficacy of crizotinib in patients with ALK-positive (ALK⁺) lung cancer, resistance mechanisms—such as secondary mutations in the ALK gene, the activation of other oncogenes, and so on—have been identified as conferring resistance to crizotinib. Second-generation ALK inhibitors, such as alectinib and ceritinib, have been shown to be effective not only in crizotinib-naive patients but also in those resistant to crizotinib. Therefore, although some agents specifically targeting ALK have been developed and their efficacy has been documented, how ALK inhibitors should be administered in the setting of ALK-rearranged NSCLC remains to be fully elucidated. Can second-generation ALK inhibitors replace crizotinib? Is crizotinib just a first-generation ALK inhibitor? Is the sequential use of crizotinib and second-generation ALK inhibitors the best method? In this article, we review the preclinical and clinical results regarding crizotinib and second-generation ALK inhibitors, as well as the resistance mechanisms, and discuss the best methods for treating patients with ALK⁺ NSCLC.     

Combined effect of ALK and MEK inhibitors in EML4-ALK-positive non-small-cell lung cancer cells

Background:

Although most non-small-cell lung cancer (NSCLC) patients with the echinoderm microtubule-associated protein-like 4 (EML4) – anaplastic lymphoma kinase (ALK) fusion gene – benefit from ALK tyrosine kinase inhibitors (ALK-TKIs), the efficacy of these drugs varies greatly among individuals.

Methods:

The antitumour action of ALK-TKIs in EML4–ALK-positive NSCLC cell lines was evaluated from their effects on cell proliferation, signal transduction, and apoptosis.

Results:

The ALK-TKI TAE684 inhibited cell proliferation and induced apoptosis, in association with inhibition of STAT3 and ERK phosphorylation, in EML4–ALK-positive H3122 cells. TAE684 inhibited STAT3 phosphorylation, but not ERK phosphorylation, and it showed little effect on cell proliferation or apoptosis, in EML4–ALK-positive H2228 cells. The combination of TAE684 and a MEK inhibitor-induced marked apoptosis accompanied by inhibition of STAT3 and ERK pathways in H2228 cells. Such dual interruption of STAT3 and ERK pathways induced downregulation of the antiapoptotic protein survivin and upregulation of the proapoptotic protein BIM.

Conclusion:

Our results indicate that interruption of both STAT3-survivin and ERK–BIM pathways is required for induction of apoptosis in NSCLC harbouring EML4–ALK, providing a rationale for combination therapy with ALK and MEK inhibitors in EML4–ALK-positive NSCLC patients for whom ALK inhibitors alone are ineffective.     

Elucidation of Resistance Mechanisms to Second-Generation ALK Inhibitors Alectinib and Ceritinib in Non–Small Cell Lung Cancer Cells

Crizotinib is the first anaplastic lymphoma kinase (ALK) inhibitor to have been approved for the treatment of non–small cell lung cancer (NSCLC) harboring an ALK fusion gene, but it has been found that, in the clinic, patients develop resistance to it. Alectinib and ceritinib are second-generation ALK inhibitors which show remarkable clinical responses in both crizotinib-naive and crizotinib-resistant NSCLC patients harboring an ALK fusion gene. Despite their impressive activity, clinical resistance to alectinib and ceritinib has also emerged. In the current study, we elucidated the resistance mechanisms to these second-generation ALK inhibitors in the H3122 NSCLC cell line harboring the EML4-ALK variant 1 fusion in vitro. Prolonged treatment of the parental H3122 cells with alectinib and ceritinib led to two cell lines which are 10 times less sensitive to alectinib and ceritinib than the parental H3122 cell line. Although mutations of ALK in its kinase domain are a common resistance mechanism for crizotinib, we did not detect any ALK mutation in these resistant cell lines. Rather, overexpression of phospho-ALK and alternative receptor tyrosine kinases such as phospho-EGFR, phospho-HER3, and phospho-IGFR-1R was observed in both resistant cell lines. Additionally, NRG1, a ligand for HER3, is upregulated and responsible for resistance by activating the EGFR family pathways through the NRG1-HER3-EGFR axis. Combination treatment with EGFR inhibitors, in particular afatinib, was shown to be effective at overcoming resistance. Our study provides new mechanistic insights into adaptive resistance to second-generation ALK inhibitors and suggests a potential clinical strategy to combat resistance to these second-generation ALK inhibitors in NSCLC.     

Treatment of Advanced Non–Small-Cell Lung Cancer With Epidermal Growth Factor Receptor (EGFR) Mutation or ALK Gene Rearrangement: Results of an International Expert Panel Meeting of the Italian Association of Thoracic Oncology

The availability of targeted drugs has made the assessment of the EGFR mutation and ALK rearrangement critical in choosing the optimal treatment for patients with advanced non–small-cell lung cancer (NSCLC). In May 2013, the Italian Association of Thoracic Oncology (AIOT) organized an International Experts Panel Meeting to review strengths and limitations of the available evidence for the diagnosis and treatment of advanced NSCLC with EGFR or anaplastic lymphoma kinase (ALK) alterations and to discuss implications for clinical practice and future clinical research. All patients with advanced NSCLC, with the exclusion of pure squamous cell carcinoma in former or current smokers, should be tested for EGFR mutations and ALK rearrangements before decisions are made on first-line treatment. First-line treatment of EGFR-mutated cases should be with an EGFR tyrosine kinase inhibitor (TKI). Any available agent (gefitinib, erlotinib, or afatinib) can be used, until further data from comparative studies may better guide TKI selection. As general rule, and when clinically feasible, results of EGFR mutational status should be awaited before starting first-line treatment. Panelists agreed that the use of crizotinib is justified in any line of treatment. Although solid evidence supporting the continuation of EGFR TKIs or crizotinib beyond progression is lacking, in some cases (minimal, asymptomatic progression, or oligoprogression manageable by local therapy), treatment continuation beyond progression could be justified. Experimental strategies to target tumor heterogeneity and to treat patients after failure of EGFR TKIs or crizotinib are considered high-priority areas of research. A number of relevant research priorities were identified to optimize available treatment options.     

Sequential Use of Anaplastic Lymphoma Kinase Inhibitors in Japanese Patients With ALK-Rearranged Non–Small-Cell Lung Cancer: A Retrospective Analysis

Background

Second-generation anaplastic lymphoma kinase (ALK) inhibitors, such as alectinib and ceritinib, have recently been approved for treatment of ALK-rearranged non–small-cell lung cancer (NSCLC). An optimal strategy for using 2 or more ALK inhibitors has not been established. We sought to investigate the clinical impact of sequential use of ALK inhibitors on these tumors in clinical practice.

Detection of novel and potentially actionable anaplastic lymphoma kinase (ALK) rearrangement in colorectal adenocarcinoma by immunohistochemistry screening

Purpose

Anaplastic lymphoma kinase (ALK) rearrangement has been detected in colorectal carcinoma (CRC) using advanced molecular diagnostics tests including exon scanning, fluorescence in situ hybridization (FISH), and next generation sequencing (NGS). We investigated if immunohistochemistry (IHC) can be used to detect ALK rearrangement in gastrointestinal malignancies.

Experimental designs

Tissue microarrays (TMAs) from consecutive gastric carcinoma (GC) and CRC patients who underwent surgical resection at Samsung Medical Center, Seoul, Korea were screened by IHC using ALK monoclonal antibody 5A4. IHC positive cases were confirmed by FISH, nCounter assays, and NGS-based comprehensive genomic profiling (CGP). ALK IHC was further applied to CRC patients enrolled in a pathway-directed therapeutic trial.

Results

Four hundred thirty-two GC and 172 CRC cases were screened by IHC. No GC sample was ALK IHC positive. One CRC (0.6%) was ALK IHC positive (3+) that was confirmed by ALK FISH and a novel CAD-ALK (C35; A20) fusion variant that resulted from a paracentric inversion event inv(2)(p22–21p23) was identified by CGP. One out of 50 CRC patients enrolled in a pathway-directed therapeutic trial was ALK IHC positive (3+) confirmed by ALK FISH and found to harbor the EML4-ALK (E21, A20) fusion variant by CGP. Growth of a tumor cell line derived from this EML4-ALK CRC patient was inhibited by ALK inhibitors crizotinib and entrectinib.

Conclusions

ALK IHC is a viable screening strategy for identifying ALK rearrangement in CRC. ALK rearrangement is a potential actionable driver mutation in CRC based on survival inhibition of patient tumor-derived cell line by potent ALK inhibitors.