As targeted molecular therapies and immuno-oncology have become pivotal in the management of patients with lung cancer, the essential requirement for high throughput analyses and clinical validation of biomarkers has become even more intense, with response rates maintained in the 20%–30% range. Moreover, the list of treatment alternatives, including combination therapies, is rapidly evolving. The molecular profiling and specific tumor-associated immune contexture may be predictive of response or resistance to these therapeutic strategies. Multiplexed immunohistochemistry is an effective and proficient approach to simultaneously identify specific proteins or molecular abnormalities, to determine the spatial distribution and activation state of immune cells, as well as the presence of immunoactive molecular expression. This method is highly advantageous for investigating immune evasion mechanisms and discovering potential biomarkers to assess mechanisms of action and to predict response to a given treatment. This review provides views on the current technological status and evidence for clinical applications of multiplexing and how it could be applied to optimize clinical management of patients with lung cancer.

Definition of tumor mutational burden (TMB) TMB can be defined as the total number of nonsynonymous mutations in the tumor exome (1). Tumor cells are genetically unstable and harbor high levels of somatic mutations which may result in the expression of neoantigens, that are not subject to immune tolerance (2). The presentation of tumor-specific neoantigens on major histocompatibility complex molecules is essential for the recognition of tumors by the T-cells. Patient-specific neoantigens that develop following somatic mutations have been shown to induce a T-cell response (3,4). The