br Discussion Missense mutations transversions of the first

Discussion Missense mutations (transversions) of the first nucleotide of the phenylalanine omadacycline at position 1174 (F1174) of the anaplastic lymphoma kinase gene can result in substitutions to leucine or valine (F1174L/V) while missense mutation (transversion) of the second nucleotide of the phenylalanine codon results in substitution of phenylalanine to cysteine (F1174C). It has been shown that F1174 missense mutations confer resistance to crizotinib (F1174L) [4], ceritinib (F1174C/V) [5] and potentially to brigatinib (F1174V+L1198F) [6]. In vitro experiments have demonstrated that F1174L is sensitive to alectinib inhibition [7], [8]. It is important to note that ALK F1174L is the most common oncogenic mutation in familial or sporadic neuroblastoma while ALK F1174V and ALK F1174C have also been identified in familial neuroblastoma [9]. This is the first patient case report that acquired ALK F1174 missense mutation (F1174V) confers sensitivity to alectinib. Although in vitro experiments have demonstrated F1174L is sensitive to alectinib [7], [8], clinical validation from alectinib-treated patients will be required to demonstrate that ALK F1174C and ALK F1174L are both sensitive ALK mutations to alectinib. Additionally, this is the sixth case reported in the literature that identified missense mutation at isoleucine (I) at position 1171 confers resistance to alectinib [10]. Therefore, the presence of acquired ALK F1174 or ALK I1171 mutation can be used to help guide subsequent second generation ALK inhibitor therapy upon progression on crizotinib especially between alectinib and ceritinib. Friboulet and colleagues have demonstrated different acquired resistant mutations can confer resistance to ceritinib in the same patient. CGP performed in this case revealed both crizotinib-resistant pulmonary nodules were essentially from the same initial EML4-ALK clone. The post-crizotinib progression biopsy was performed prior to patient enrolling onto the alectinib trial. We biopsied the larger of the two pulmonary nodule in the right upper lobe which was also at a location more easily amendable to CT guided biopsy. The pulmonary nodule that harbored the ALK F1174V achieved essentially an ongoing complete remission now 24 months into alectinib treatment. It is unknown whether the second pulmonary nodule located in the right lower nodule had already harbored the ALK I1171S mutation at the time of resistance to crizotinib. The fact that this second nodule only achieved a 44% reduction in size on alectinib which eventually grew back to satisfied RECIST progression suggested a subclonal population of NSCLC harboring ALK I1151S likely already existed at the time of crizotinib resistance. This is further supported by CGP where only 31% of the sequencing runs detected ALK I1171S hence the relating “slow” growing nature of this nodule under alectinib treatment as the nodule was resected when it was at about 80% of the original size pre-alectinib treatment. We could not rule out the co-existence of a ALK F1174V missense mutation with ALK I1151S in the right lower lobe nodule as any ALK F1174V missense mutation will likely be successfully suppressed as the right upper lobe nodule. However, the CGP did not reveal any ALK F1174V missense mutation. This patient case also illustrates that separate and distinct acquired resistant mutations can occur during the same selection pressure during treatment with one ALK inhibitor as has previously demonstrated [5]. Furthermore, the differential selective pressure during treatment with various ALK inhibitors can direct the emergence specific resistant mutation depending on the specific ALK inhibitor being utilized. It seems that I1151 missense mutation is a frequent acquired resistant ALK mutation to alectinib [10]. This case also illustrates the importance of biopsying many synchronous metastatic sites and sequential biopsies upon disease progression on any line of therapy.