Treatment of advanced stage anaplastic lymphoma kinase (ALK) positive non-small 
cell lung cancer (NSCLC) with ALK tyrosine kinase inhibitors (TKIs) has been 
shown to be superior to standard platinum-based chemotherapy. However, secondary 
progress of disease frequently occurs under ALK inhibitor treatment. The 
clinical impact of re-biopsies for treatment decisions beyond secondary progress 
is, however, still under debate. Here, we report on two novel subsequent 
polyclonal on- and off-target resistance mutations in a patient with ALK-fused 
NSCLC under ALK inhibitor treatment. A 63-year-old male patient with an advanced 
stage EML4-ALK fused pulmonary adenocarcinoma was initially successfully treated 
with the second-generation ALK inhibitor alectinib and upon progressions 
subsequently with brigatinib, lorlatinib and chemoimmunotherapy (CIT). Progress 
to alectinib was associated with a so far undescribed ALK mutation 
(p.A1200_G1201delinsW) which was, however, tractable by brigatinib. An 
off-target KRAS-mutation (p.Q61K) occurred in association with subsequent 
progression under second-line TKI treatment. Third-line lorlatinib showed 
limited efficacy but chemoimmunotherapy resulted in disappearance of the KRAS 
mutant clone and clinical tumor control for another eight months. In conclusion, 
we suggest molecular profiling of progressive tumor disease also for 
ALK-positive NSCLC to personalize treatment in a subgroup of ALK-positive 
patients.