Elena Poddubskaya ^1,2* Maria Suntsova ^1,3* Marina Lyadova ⁴ Daniil Luppov ^1,3,5 Anastasia Guryanova ^3* Vladimir Lyadov ^4,6 Andrew Garazha ⁷ Maxim Sorokin ^5,7,8 Anna Semenova ^4* Vitaly Shatalov ^4* Maria Biakhova ^4* Alexander Simonov ^3* Aleksey Moisseev ^1,5* Anton Buzdin ^1,3,8,9*

• ¹ I.M. Sechenov First Moscow State Medical University, Moscow, Moscow Oblast, Russia
• ² Vitamed Clinic, Moscow, Russia
• ³ Moscow Institute of Physics and Technology, Dolgoprudny, Russia
• ⁴ Oncology Center No. 1, Moscow City Hospital Named after S. S. Yudin, Moscow, Russia
• ⁵ Endocrinology Research Center, Moscow, Moscow Oblast, Russia
• ⁶ Novokuznetsk State Institute for Further Training of Physicians – Branch Campus of «Russian Medical Academy of Continuous Professional Education», Novokuznetsk, Russia
• ⁷ Oncobox Ltd., Moscow, Moscow Oblast, Russia
• ⁸ Institute of Bioorganic Chemistry (RAS), Moscow, Moscow Oblast, Russia
• ⁹ PathoBiology Group, European Organization for Research and Treatment of Cancer (EORTC), Brussels, Belgium

We conducted a prospective observational study of the prognostic potential of several existing and putative biomarkers of response to immunotherapy in a cohort of 85 patients with lung cancer (LC) receiving PD-1 or PD-L1 targeted immune checkpoint inhibitors (ICIs). Tumor biosamples were obtained prior to ICI treatment and profiled by whole exome and RNA sequencing. The entire 403 putative biomarkers were screened, including tumor mutation burden (TMB) and number of cancer neoantigens, 131 specific HLA alleles, homozygous state of 11 HLA alleles and their superfamilies; four gene mutation biomarkers, expression of 45 immune checkpoint genes and closely related genes, and three previously published diagnostic gene signatures; for the first time, activation levels of 188 molecular pathways containing immune checkpoint genes and activation levels of 19 pathways algorithmically generated using a human interactome model centered around immune checkpoint genes. Treatment outcomes and/or progression-free survival (PFS) times were available for 61 of 85 patients with LC, including 24 patients with adenocarcinoma and 27 patients with squamous cell LC, whose samples were further analyzed. For the rest 24 patients, both treatment outcomes and PFS data could not be collected. Of these, 54 patients were treated with PD1-specific and 7 patients with PD-L1-specific ICIs. We evaluated the potential of biomarkers based on PFS and RECIST treatment response data. In our sample, 45 biomarkers were statistically significantly associated with PFS and 44 with response to treatment, of which eight were shared. Five of these (CD3G and NCAM1 gene expression levels, and levels of activation of Adrenergic signaling in cardiomyocytes, Growth hormone signaling, and Endothelin molecular pathways) were used in our signature that showed an AUC of 0.73 and HR of 0.27 (p=0.00034) on the experimental dataset. This signature was also reliable (AUC 0.76, 0.87) for the independent publicly available LC datasets GSE207422, GSE126044 annotated with ICI response data and demonstrated same survival trends on independent dataset GSE135222 annotated with PFS data. In both experimental and one independent datasets annotated with samples' histotypes, the signature worked better for lung adenocarcinoma than for squamous cell LC. Annotated molecular profiles obtained in this study are publicly accessible.