Chapter 1 14 to as PD-1T TILs, positively correlated to treatment outcome in a small cohort of NSCLC patients treated with PD-1 blockade monotherapy35. These TILs display a high capacity for tumor recognition, and express and secrete CXCL13, a B cell attractant essential for the formation of tertiary lymphoid structures (TLS). Notably, PD-1T TILs predominantly localize within TLS35. TLS and B cells, a critical TLS component, have been correlated with clinical responses in other tumor types36–39, although comprehensive data from larger NSCLC patient cohorts is currently lacking. The clinical implementation of the aforementioned biomarkers presents challenges due to their continuous nature. Hence, reliable and automated methods are preferred for the assessment and the definition of cut-off values. For example, digital quantification methods have been described for TILs and PD-L140–42. Furthermore, robust platforms such as the Nanostring nCounter platform43 enable the development of predictive mRNA signatures capable of extracting the immune phenotype from the TME. One example of such a signature is the tumor inflammation signature (TIS)44–47. In addition, the combination of biomarkers could be an approach to improve predictive accuracy, as demonstrated in studies combining TMB with PDL148,49 and CD8 with PD-L118,31. Alternative bio-sources for biomarker development One of the predominant challenges in biomarker testing is the availability of sufficient tumor tissue, frequently necessitating invasive procedures. Furthermore, local tissue sampling can introduce biases due to tumor heterogeneity. Notably, more and more tissue material is required to comply to the increasing number of diagnostic biomarker tests. In addition to tumor tissue, blood serves as a viable source for biomarkers. The liquid biopsy method that analyzes cell-free DNA in plasma can detect circulating tumor DNA (ctDNA). This method is minimally invasive and, in contrast to tissue biopsy, can provide a molecular profile of the tumor as well as real-time insights into tumor response dynamics during treatment. Nonetheless, sensitivity challenges persist, primarily because of the often limited fraction of plasma ctDNA available for analysis50. In addition, ctDNA biomarkers are less suitable for predicting responses to immunotherapy, as they do not capture characteristics of the immune infiltrate. A variety of highly sensitive and specific technologies have been rapidly developed, primarily based on multiplex PCR (mPCR) or next-generation sequencing (NGS). These advancements enable the detection of genetic alterations in circulating nucleic acids, encompassing gene mutations, fusions, deletions, amplifications, translocations and epigenetic changes51. Beyond nucleic acid-based approaches,
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