Evolution Law of ALK Quadratic Mutation

Issuing time:2018-10-23 00:00

1.EML4-ALK fusion gene

EML4-ALK fusion gene is one of the important drivers of non-small cell lung cancer ( NSCLC). The EML4 gene can be broken at multiple sites and connected with the intracellular catalytic domain located in exon 20 of ALK to form EML4-ALK fusion gene. The EML4-ALK fusion gene Variant (Variant), Variant 1 and Variant 3 were the most common type of EML4-ALK fusion gene, which was different from the ALK gene in the EML4-ALK gene, and the EML4-ALK gene Variant was formed in at least 10 species. There are three main detection methods for ALK fusion gene detection: FISH, RT-PCR and Ventana IHC ( detection of ALK protein expression), ALK variant type, The three methods are highly consistent [1] 。


Secondary mutation of ALK

The second mutation in the domain of ALK protein kinase is the main resistance mechanism of cloxitinib. The eight most common secondary mutations are shown in Figure 1. L1196M is a gatekeeper mutation, similar to EGFR's T790M mutation, while G1202R is insensitive to most ALK inhibitors. Other resistance mechanisms were EGFR activation, ALK amplification, SRC activation and cKit amplification [2] 。


The full length of EML4-ALK gene reached 4860 bases, encoding 1620 amino acids. There are more than 8 possible secondary events in Fig. 1, Other secondary mutations have been found in clinical studies, such as F1174C, D1203N, I1171T, etc. There may be more than one ALK mutation in an ALK-positive NSCLC patient [2] 。

3.Evolution Law of ALK Quadratic Mutation



Gainor et al.primary ALK fusion persisted in 19 patients with FISH and ALK amplification in 2 patients with rebiopsy prior to ritinib treatment.Of 7 patients, the mutations of C1156Y, L1196M, S1206Y, G1269M, 1151T were found in 7 patients by direct sequencing, color reteny was effective in seven patients, of whom PFS of 1151T mutation was 2.7 months, which may be related to the higher IC50 of 1151T [3].

There was no significant difference in progression-free survival between group II patients with ALK secondary mutation and those without ALK secondary mutation.

Gainor et al. Performed genetic testing on 11 other serratinib-resistant patients, as shown in Table 1.



Allotinib-resistant mutants were G1202R, I1171N/S, R1209Q, V1180L, et al. [5,6].

Oscar Puig et al. used second-generation sequencing ( NGS) to detect ALK-positive NSCLC patients in NP28673 and NP28761 in two clinical trials of Elatinib. The NSCLC patients were examined for their tissue and peripheral blood, See table 2.

Thirteen ALK-resistant mutations were found in 12 patients with cloxitinib (F1174C, L1196M, S1157F). R1231W,G1128A,V476A,I1171T,G1269A, C1156F, S1206Y, F1245C, A416P), five of the mutations were not found in the previous literature. Carrying F1174C, L1196M, S1157F, R1231W, Patients with G1128A, G1269A, C1156F mutations who were treated with Elatinib for more than 3 months with PFS, Patients with both S1206Y and F1245C mutations had no effect on elatinib and those with S1157F mutations had stabilized for 15.4 months.

In addition, Oscar Puig measured the variation of ALK alleles in peripheral blood in 45 patients who were still taking Elatinib, Several Alk Sensitive to Elatinib (F1174V/L, S1157F, T1151R) S1206F, I1268V, G1269A, C1156C/F, etc.) shows A downward trend, But several Alk mutations ( G1202R, I1171N/ S, R1209Q, V1180L) resistant to Elatinib showed an increasing trend, Figure 2

Coexistence of multiple ALK mutations was also observed in two patients. One patient with t1151m developed g1202r and i1171n / S during aletinib administrationmutation, and the t1151m mutation disappears.Another patient, who carried i1171t and f1174v, added r1209q mutations while taking eletinib, while the allele abundance of i1171t and f1174v decreasedlow medium [6].

3.3. Brigatinib (AP 26113)

Scott  N. gettinger and others used the second generation sequencing method ( NGS ) to carry out genetic tests on tumor tissues of 36 patients with Brigatinib's clinical trial, of these, 32 were tested for pre-entry tissue, and four were tested for Brigatinib resistant tumor tissue. Effective data are shown in Table 4. Patients with ALK mutations have higher orr than those without ALK mutations[7].

3.4 EnsartinibX-396

Ensartinib was reported at the 2016 ASCO meeting  Phase II clinical partial data: 38 patients with ALK-positive non-small cell lung cancer treated once daily at a dose of 200 mg, of whom 8 were not treated with crizotinib,twenty patients were resistant to crizotinib, seven were resistant to crizotinib and serratinib, and two were resistant to crizotinib, serratinib, and aletinib.One patient was resistant to crizotinib, serratinib and brigatinib.Detection of ALK mutations in tissue and blood using second-generation sequencing of NGS

The evaluable number of patients with crizotinib resistance was 13, 85% remained ALK-positive, 3 had secondary ALK mutations, and one patient with t1151m had a PFS of 4one patient with l1196m had a PFS of 5 months, and the gene data of two patients with ensartinib resistance were missing.One patient with g1269a had A PFS of 13 months and was resistant to ensartinib and had A l1196m + g1269a mutation.One patient who failed was a qslp1188p + r1133q + s1206f mutation with active lungs and brain progression.The overall response rate was 29% in patients who were resistant to crizotinib and serratinib, with one patient having a PFS greater than 9 months and another having a g1202r + HER2 mutation,after 5 months of PFS, G1202R + V1149M mutation was found, and the G1202R abundance increased from 0.7 % to 1.7 %; one patient developed complete remission of the lung, brain progression, and G1202R mutation in another patient ( 0.5 % abundance ).

3.5 Lorlatinib PF-06463922

Lorlatinib is in its second clinical phase with insufficient information on drug resistance. So far, only one case of C1156Y+ L1198F double mutation is reported. It is effective to rejoin Lorlatinib in the patient with double mutation of C1156Y+ L1198F [9] 。


Summary and discussion

The positive and secondary mutations of ALK fusion gene are shown in Table 6.


4.1 The majority of ALK-positive patients were still ALK-positive after cloxitinib resistance and after sequential therapy with Alatenib, Seretinib, Brigatinib or Ensartinib.

4.2 After sequential treatment with a variety of ALK inhibitors, the proportion of ALK secondary mutations in drug-resistant patients increased significantly, while the species decreased significantly.

4.3After treatment with 2 or 3 generations of ALK inhibitors, two or three mutations were found in the patients with cloxitinib resistance. For example, Brigatinib is effective against E1210K, D1203N and G1269A, but the complex mutation is not effective. The mechanism of such multidrug-resistant mutations still needs further study.

4.4Although Brigatinib, Ensartinib and Lorlatinib have demonstrated effectiveness against G1202R in clinical trials, However, G1202R mutations were present in all patients with ALK inhibitors except Lorlatinib.

4.5The change of ALK allele abundance in peripheral blood can help to evaluate the therapeutic effect.


The sequential or switched use of ALK inhibitors has only a few successful cases, and more clinical evidence is needed.

At present, accurate and reliable second-generation sequencing services are urgently needed to guide the medication of ALK positive patients.


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