Defining molecular risk in ALK+ NSCLC

Abstract

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancers (NSCLC) have the best prognosis among metastatic pulmonary malignancies, with a median patient survival currently exceeding 5 years. While this is definitely a major therapeutic success for thoracic oncology, it may not be entirely attributable to rapid drug development and the strenuous clinical efforts. At the genetic level, ALK⁺ disease is also unique, distinguished by the lowest tumor mutational burden (mean below 3 mutations/Mbp), the lowest frequency of TP53 mutations (20-25%) and very few other co-mutations compared to other NSCLC. The relative simplicity and stability of the genetic landscape not only contribute to the relatively favourable clinical course, but also make study of the effects from individual molecular features easier. EML4-ALK fusion variant 3 (E6;A20) and TP53 mutations were recently identified as main molecular determinants of adverse outcome: they occur in about 30-40% and 20-25% of newly-diagnosed cases, respectively, have possibly synergistic effects and are independently associated with more aggressive disease, shorter progression-free survival under treatment with ALK inhibitors and worse overall survival. Secondary detection of TP53 mutations at disease progression in previously negative patients defines another subset (about 20%) with similarly poor outcome, while detection of ALK resistance mutations guides next-line therapy. As our biological understanding deepens, additional molecular risk factors will be identified and refine our concepts further. The translation of clinical risk at the molecular level and the ability to predict early events are of key importance for individualized patient management and preclinical modeling in order to advance therapeutic options.

Keywords: ALK+ non-small cell lung cancer; EML4-ALK fusion variant; TP53 mutation; overall survival; treatment resistance.

Figure 1. Number of metastatic sites and performance status in newly diagnosed stage IV EML4-ALK⁺ and EGFR⁺ NSCLC patients

A. The total number of metastatic sites (intrathoracic, brain, liver, bone, adrenal, other) for newly diagnosed stage IV EML4-ALK⁺ (n = 34 V3 cases, n = 44 V1/V2 cases) and stage IV EGFR⁺ (n = 221 EGFR exon 19 [E19] indel cases, n = 197 cases with other EGFR mutations) NSCLC patients typed at our institution with available data [18,22]. Statistical comparison was performed with ANOVA (p < 0.001) followed by the Dunnett’s post-hoc test. Columns and error bars show mean values and their standard errors: 3.12 and 0.36 for EML4-ALK V3, 1.84 and 0.16 for EML4-ALK V1/V2, 1.76 and 0.07 for EGFR exon 19 indels, 0.82 and 0.08 for other EGFR alterations. Statistically significant results are shown in the graph; ***: p < 0.001. B. The Eastern Cooperative Oncology Group (ECOG) performance status for newly diagnosed stage IV EML4-ALK⁺ (n = 35 V3 cases, n = 48 V1/V2 cases) and EGFR⁺ (n = 210 EGFR exon 19 [E19] indel cases, n = 178 with other EGFR mutations) NSCLC patients from our institution with available data [18,22]. Statistical comparison was performed with ANOVA (p < 0.05) followed by the Dunnett’s post-hoc test. Columns and error bars show mean values and their standard errors: 0.60 and 0.10 for EML4-ALK V3, 0.29 and 0.07 for EML4-ALK V1/V2, 0.52 and 0.04 for EGFR exon 19 indels, 0.51 and 0.04 for other EGFR alterations); *:p = 0.037.

Figure 2. Tumor mutational burden, frequency of TP53 mutations and frequency of co-mutations in metastatic ALK⁺ and EGFR⁺ NSCLC

A. Tumor mutational burden (TMB) of metastatic ALK⁺ (n = 33, mean 2.0 mutations[mut]/Mbp), EGFR⁺ (n = 232, mean 5.0 mut/Mbp) and wildtype (WT, i.e. ALK/EGFR/RET/ROS-negative, n = 557, mean 9.7 mut/Mbp) cases from the publicly available MSKCC lung adenocarcinoma (ADC) cohort (http://www.cbioportal.org) as estimated by targeted sequencing with the IMPACT341 and IMPACT411 panels [35-37]. For cases with multiple sampling time-points, only the earliest one in the disease course was analyzed, and among multiple samples at the earliest time-point, that with the highest number of mutations was chosen. Boxplots show medians, means (“+”) and range; ***p < 0.001 with the Kruskal-Wallis test followed by the Dunn’s post-hoc test. B. Frequency of TP53 mutations in metastatic ALK⁺ (18%, n = 33) and EGFR⁺ (63%, n = 232) NSCLC cases of the MSKCC cohort [35-37], as well as in untreated metastatic ALK⁺ (25%, n = 105) and EGFR⁺ (42%, n = 273) tumors sequenced for exons 4-10 of TP53 at our institution [18, 22]. Columns and error bars show percentages and 95% confidence intervals, respectively; ***p < 0.001, and **p = 0.0022 with a chi-square test. C. Tumor mutational burden (TMB) according to TP53 status for ALK⁺ (mean 3.2 mut/Mbp for TP53 mutated, n = 6, vs. 1.8 mut/Mbp for TP53 wildtype cases, n = 27, p = 0.039 with a Mann-Whitney test), EGFR⁺ (mean 5.6 mut/Mbp, n = 145, vs. 4.0 mut/Mbp, n = 87, p < 0.001) and WT cases (mean 13.6 mut/Mbp, n = 291, vs. 6.6 mut/Mbp, n = 266, p < 0.001) from the publicly available MSKCC lung adenocarcinoma cohort (http://www.cbioportal.org) [35-37]. In a bivariable linear regression analysis among ALK⁺ and EGFR⁺ patients, type of oncogene (EGFR vs. ALK, beta = 0.248, p < 0.001) and TP53 status (mutated vs. wild-type, beta = 0.256, p < 0.001) were similarly strong determinants of TMB. Boxplots show medians, means (“+”) and range. D. Frequency of co-mutations in untreated ALK⁺, EGFR⁺ and WT NSCLC patients. Analyzed were untreated ALK⁺ (n = 105) and EGFR⁺ (n = 273) patients sequenced with PCR-based DNA NGS using our custom panel of 38 genes as previously described [22], as well as the untreated ALK⁺ (n = 21), EGFR⁺ (n = 134) and WT (n = 385) patients of the MSKCC lung adenocarcinoma cohort sequenced with the MSK-IMPACT341 and MSK-IMPACT411 panels (http://www.cbioportal.org) [35-37]. Visualized are all common genes among the three panels with at least one detectable mutation. Statistical comparisons for ALK⁺ vs. WT, and EGFR⁺ vs. WT were performed with a chi-square test, and results with p < 0.05 and Benjamini-Hochberg q < 0.05 are shown in the graph in red and dark blue color, respectively: *:p < 0.05, **:p < 0.01, ***:p < 0.001. Statistical comparison for ALK⁺ vs. EGFR⁺ was performed similarly, and significant results are shown in red color: ^e:p < 0.05, ^ee:p < 0.01, ^eee:p < 0.001.

Figure 3. Differential outcome of ALK⁺, EGFR⁺ and chemotherapy-treated wildtype NSCLC

Retrospective analysis of tyrosine kinase inhibitor (TKI)-treated ALK⁺ (n = 74 with >1 TKI, n = 109 with just 1 TKI) and EGFR⁺ (n = 344) NSCLC patients, along with a random sample of n = 40 EGFR/ALK-wildtype NSCLC patients that received chemotherapy in the Thoraxklinik at Heidelberg University Hospital [22]. Basic clinical and treatment characteristics of ALK⁺ and EGFR⁺ patients are shown in Table 2. Median overall survival was 65 months for ALK⁺ patients that received >1 TKI, 40 months for ALK⁺ patients that received just 1 TKI, 25 months for TKI-treated EGFR⁺ patients and 10 months for wildtype patients; p < 0.001 across groups and p = 0.043 for the comparison between the EGFR⁺ and ALK/1-TKI subgroups with a log-rank test; WT: wildtype; CHT: chemotherapy.