The anaplastic lymphoma kinase (ALK) fusion gene is another tumor driver gene with targeted drug therapy in non-small cell lung cancer (NSCLC) after EGFR gene mutation. Crizotinib, a targeted therapeutic agent against ALK fusion gene, has gone through preclinical study, early clinical study and marketing approval in just 4 years, which is a model of successful targeted drug with clear target and mature detection method.
However, as with EGFR mutation-based EGFR-TKIs treatment resistance is inevitable, despite good progression-free survival and objective efficiency of crizotinib in ALK-positive patients, the fate of treatment failure due to drug resistance is ultimately inevitable. The resistance mechanism and treatment strategy of targeted therapy for ALK+ NSCLC have become a hot topic at present.
I. Mechanisms of acquired drug resistance in ALK+ NSCLC
ALK gene rearrangement occurs in 3-7% of NSCLC, more often in younger non-smoking adenocarcinoma patients, and is usually mutually exclusive with the occurrence of EGFR or KRAS mutations. the discovery of ALK gene rearrangement in NSCLC patients has greatly improved the clinical prognosis of this subtype of patients. Crizotinib treatment has an objective response rate (ORR) of 60%, progression-free survival (PFS) of 8-10 months, and significantly prolonged overall survival. Despite the clear benefit in ALK+ lung cancer patients, this group of patients often develops resistance to crizotinib within 1-2 years, and relapse progression in the central nervous system is more common. The mechanisms of resistance are diverse and can be divided into two main categories: ALK resistance mutations and conversion of other signaling pathways (i.e., activation of signaling bypasses). The main mechanisms of drug resistance that have been identified are as follows.
1, ALK resistance mutations
(1) ALK kinase region mutations: In vitro and patient-based studies have revealed the mechanism of resistance to crizotinib in some ALK+ lung cancer patients, and the earliest clear mechanism of resistance is ALK kinase region mutations. Unlike epidermal growth factor receptor (EGFR) mutation-positive patients, where the predominant resistance mechanism is known to be T790M mutations, several ALK kinase region mutations have been identified in patients with ALK gene rearrangements, with a slight numerical predominance of L1196M, a similar T790M, a housekeeping gene. In fact, several different amino acid site mutations have been identified in the ALK kinase region, including L1196M, G1269A, S1206Y, G1202R, 1151Tins, L1152R, and C1156Y. Doebele et al. examined specimens from 14 ALK+ NSCLC patients who developed acquired resistance to crizotinib treatment and found that one-third of the patients showed secondary mutations in the ALK kinase region. Similarly, Katayama et al. analyzed the clinical and molecular biological characteristics of 18 patients with acquired resistance to crizotinib, suggesting that approximately one-third of patients had secondary mutations in the ALK kinase region or ALK gene amplification, and confirmed that these mutations led to resistance to crizotinib in an in vitro assay.
The diversity of drug-resistant mutations poses a challenge to patients and physicians. First, finding accurate assays that can identify all known mutations is difficult. Second, tumor tissue may have more than one mutation at the time of resistance. In the first publicly reported case of crizotinib resistance, two different mutations (C1156Y and L1196M) were found in the same tumor sample. When tumor specimens have multiple mutations, direct sequencing may yield false negative results if there is no one dominant mutation in the majority of tumor cells.
(2)?ALK fusion gene copy number increase: ALK fusion gene copy number increase was first identified when ALK+ cell lines showed resistance to crizotinib. Subsequently, increased copy number was also found in specimens from patients resistant to crizotinib, suggesting that it may have a role in tumor cell drug resistance. When the ALK fusion gene kinase region is mutated or copy number is increased, the ALK signaling pathway tends to be preserved, and therefore tumor cells are expected to remain addicted to the ALK fusion gene. In this way, more potent and effective second-generation ALK inhibitors may be able to overcome such cellular resistance mechanisms. This type of resistance is known as ALK-dominant resistance.
2. Activation of signaling bypasses
(1) Conversion of other signaling pathways (activation of signaling bypass): There is another type called conversion of other signaling pathways (activation of signaling bypass), which mainly refers to the emergence of other signaling pathways to replace the dependence of tumor cells on ALK pathway, resulting in the inability of ALK inhibitors to adequately inhibit tumor cell growth. This type of resistance is also known as ALK-deficient resistance. Multiple alternative signaling pathways have been identified, e.g., the presence of activated EGFR or KRAS mutations have been found in both patients not receiving crizotinib and those who have received crizotinib therapy. In vitro studies suggest that EGFR and other HER family receptor tyrosine kinases can lead to drug resistance through ligand-mediated activation of ALK receptors. The first of these signaling bypasses, the EGFR pathway, has been reported in several studies. Among 18 specimens with acquired resistance to crizotinib at Massachusetts General Hospital, EGFR phosphorylation was detected by immunohistochemistry in 17 specimens, suggesting the presence of varying degrees of EGFR pathway activation. More importantly, inhibition of EGFR was found to restore sensitivity to crizotinib in resistant cell lines in cell line studies. The second activated signaling bypass was the c-KIT pathway. Among 18 specimens with acquired resistance to crizotinib at Massachusetts General Hospital, high levels of c-KIT gene amplification were detected in 2 specimens by the FISH method. The presence of c-KIT overexpression was further confirmed by immunohistochemistry. In addition, increased expression of c-KIT ligand stem cell factor (SCF) was found in the mesenchymal cells of the solid component of drug-resistant specimens by immunohistochemistry. In vitro experiments confirmed that c-KIT overexpression required SCF to promote its resistance, and that this resistance could be reversed by combining imatinib with crizotinib.
(2) Tumor heterogeneity: When attempting to overcome resistance to crizotinib in ALK+ lung cancer, tumor heterogeneity further complicates the issue. In fact, tumor heterogeneity has been observed on a variety of cellular drug resistance. Two different kinase region mutations were identified in one patient’s specimen, while a subset of tumor cells did not have the mutation. Another patient’s specimen identified both copy number increases and mutations, but it is not known if these mutations are all present in the same cells. Still further, one patient had two simultaneous biopsies of different lesions, showing the presence of different molecular findings at each biopsy site. Questions inevitably arise as to whether the molecular results from a small piece of biopsy tissue are representative of the entire tumor tissue and whether the current limited molecular assays can reveal all types of cellular resistance. This will further complicate the detection of molecular mechanisms after drug resistance and the development of corresponding therapeutic strategies.
II. Drugs and strategies to overcome acquired drug resistance in ALK+ NSCLC
1. Second-generation ALK inhibitors? Preclinical studies have demonstrated that second-generation ALK inhibitors (e.g., CH5424802) are active not only in tumor cells with EML4-ALK fusion genes, but also in a variety of identified ALK kinase region resistance mutations. Early preclinical data for LDK378, AP26113 and CH5424802 suggest that these drugs are active in both crizotinib-naïve and crizotinib-resistant patients, and each drug has partial data supporting its effectiveness against brain metastases.
LDK378, an ALK inhibitor developed by Novartis, was found to have a semi-inhibitory concentration (IC50) of only 0.15 nM against ALK enzyme compared to 3 nM for the control drug crizotinib, showing better activity against ALK in in vitro studies. Further studies on ALK-resistant cell lines suggested better activity of the drug compared to crizotinib. Based on this, the investigators designed a phase I clinical study (NCT01283516) to enroll ALK-positive patients with progressive tumors who had failed standard therapy, and the dose of LDK378 was escalated from 50 mg/d to 750 mg/d. A total of 131 patients were enrolled in the study, divided into three groups: ALK+ lung cancer previously treated with ALK TKI, ALK+ lung cancer previously untreated with ALK TKI, and ALK+ lung cancer previously treated with ALK TKI. ALK TKI therapy for ALK+ lung cancer and ALK+ malignancies other than lung cancer. As of November 8, 2012, 130 patients were enrolled, 59 of whom were in the dose creep group with a confirmed maximum tolerated dose (MTD) of 750 mg/d; 71 patients were in the subsequent MTD expansion group. The efficacy of 114 NSCLC patients receiving LDK378 in the dose range of 400-750 mg/d could be evaluated, with an ORR of 58% (66 cases were confirmed, while 20 cases were not confirmed and not counted). In the subgroup of 79 ALK+ NSCLC patients resistant to crizotinib, the ORR was 57% (45 confirmed and 17 unconfirmed and uncounted). In the remaining 35 crizotinib-naïve ALK+ NSCLC patients, the ORR was 60% (21 confirmed and 3 unconfirmed and uncounted). The study showed that LDK378 was also effective in patients with CNS lesions. The median PFS was 8.6 months (95% confidence interval: 5.7 to 9.9) in the whole group of 114 NSCLC patients. The most common adverse reactions (n=130) were nausea (73%), diarrhea (72%), vomiting (58%) and malaise (41%), and the most common G3/4 adverse reactions were alanine aminotransferase (ALT) elevation (9%), elevated aspartate aminotransferase (AST) (10%) and diarrhea (8%). The results of this study showed that LDK378 showed strong antitumor activity in the dose range of 400-750 mg/d (with or without crizotinib) and activity in CNS lesions; the most common adverse reactions were nausea, diarrhea, vomiting and malaise, and were mostly grade 1 or 2 and well tolerated by patients. Approved by the FDA, LDK378 is undergoing multiple Phase II and Phase III clinical studies.
CH5424802 is one of the second-generation ALK inhibitors developed by Chugai Pharmaceuticals. In the phase I/II clinical study conducted in Japan, a total of 46 ALK+ NSCLC patients who had not received crizotinib were enrolled in the maximum tolerated dose group, of which 43 achieved objective remission (2 CR and 41 PR), with an objective remission rate of 93.5%, CI 82%~98.6%. Grade 3 or higher adverse reactions, including neutropenia and elevated blood creatine kinase levels, occurred in 12/46 (26%) patients.
AP26113, a novel small molecule targeted drug developed by Ariadne Pharmaceuticals as a dual target inhibitor of ALK and EGFR, also showed better efficacy in early studies in patients with ALK+ NSCLC, with an objective efficacy rate of 73%, regardless of prior crizotinib treatment. In summary, second-generation ALK inhibitors may be the best choice for tumors that still rely on the ALK signaling pathway as a driver gene.
2. Rational combination therapy or chemotherapy? As resistance due to signal bypass activation still exists, the simultaneous application of ALK inhibitors and other signaling pathways has the potential to improve clinical outcomes. Promising strategies include: ALK inhibitors in combination with heat shock protein 90 (HSP 90) inhibitors/MEK inhibitors/mTOR inhibitors/EGFR inhibitors.
One of the promising therapeutic strategies is the use of Ganetespib to block the molecular chaperone heat shock protein 90 (HSP90). Ganetespib was found to be active in in vitro cell line studies in both ALK+ untreated or crizotinib-resistant cell lines. HSP 90 inhibitors promote the degradation of tumor signaling pathway proteins such as ALK (involved in tumor cell proliferation and survival), providing a possible therapeutic strategy for patients who are crizotinib-resistant but do not have secondary mutations. A series of clinical studies combining HSP90 inhibitors with selective ALK inhibitors are ongoing (NCT 01712217 and NCT01579994).
Several studies have been reported on which chemotherapeutic agent is the best choice for ALK+ NSCLC, showing that crizotinib can have an ORR of up to 65.7% (n=172) for ALK+ lung cancer, while the pemetrexed regimen has an ORR of only 29.3% (n=99) for chemotherapy and the docetaxel regimen is the least effective with an ORR of only 6.9% (n=72). Pemetrexed-containing regimens have shown better activity in ALK+ lung cancer and may be a reasonable option when patients resistant to crizotinib are unable to participate in other clinical studies.
Studies in EGFR mutation-positive NSCLC patients with resistance to EGFR TKI have found that immediate discontinuation of EGFR TKI therapy can lead to “blast progression” or “flashover” of the tumor, possibly due to discontinuation of EGFR TKI therapy. This may be due to the re-proliferation of EGFR TKI-sensitive fast-growing cells after the cessation of EGFR TKI therapy, leading to tumor bursts of growth. Similarly, a similar process may also occur in crizotinib-sensitive ALK+ NSCLC. Therefore, when acquired resistance to crizotinib occurs in this group of patients, there is no definitive answer as to whether crizotinib therapy needs to be continued in parallel with systemic chemotherapy, and a number of prospective clinical studies could help answer this question. The SWOG1300 study is being designed for ALK+ patients who develop crizotinib resistance and are randomly assigned to the pemetrexed monotherapy or pemetrexed and crizotinib combination treatment arm. Of interest is that in this study, patients will be allowed to receive crizotinib again if pemetrexed alone fails. Based on this study, it is also possible to answer the question “which of the two different treatment modalities is better or worse after the development of acquired drug resistance in patients with a clear tumor driver gene (continue with the original small molecule-targeted drug in combination with systemic chemotherapy vs. –The question of “whether it is better to continue the original small-molecule-targeted drugs in combination with systemic chemotherapy or to “re-challenge” the small-molecule-targeted drugs after disease progression. The results of the study have important implications for clinical practice, which we will see.
3. Treatment strategies for different drug resistance patterns? When disease progression occurs in NSCLC with driver genes treated with targeted drugs, subsequent treatment strategies should be developed according to different situations. For this group of patients who develop acquired resistance to targeted drug therapy, an important point is to consider that this resistance is often incomplete, so that some tumor cells can continue to be inhibited by targeted drugs when the disease progresses. It is important to distinguish between only detectable disease progression and clinically significant progression before changing treatment modalities. This is because some patients experience localized, asymptomatic progression but still have better control of their tumor load compared to before receiving targeted therapy. Other patients who discontinued the TKI after disease progression was detected experienced fulminant disease progression. At this time, patients were given the same TKI or a different TKI acting on the same target and the disease could be well controlled again. This phenomenon indicates that TKI can still inhibit the subpopulation of tumor cells that are sensitive to it. Therefore, distinguishing between different resistance patterns is crucial for subsequent treatment. When disease progression occurs in only one or a few lesions, the use of local therapies (e.g., radiotherapy, surgery, or radiofrequency ablation) along with the continuation of crizotinib seems to be a better option. The most representative example of this is the progression of localized brain metastases. Patients often have well-controlled systemic disease, with the exception of brain lesions, and progression of brain metastases may be due to the presence of the blood-brain barrier, resulting in low concentrations of TKI drugs in the cerebrospinal fluid. If the disease is only slowly or very minimally progressive, it is recommended to continue the current treatment with close follow-up. If rapid and extensive disease progression occurs, it means that TKI is no longer able to inhibit tumor growth, and discontinuation of targeted drug therapy is recommended. For such patients, a repeat biopsy may reveal histological changes or new mutations, which may help to select effective drugs accordingly.
The discovery of tumor driver genes and the application of corresponding targeted drugs have greatly improved the prognosis of patients. However, the inherent characteristics of tumor cells will make the road to treatment long and difficult, and we still need to search for the best way!