Blood biomarkers associated to complete pathologicalresponse on NSCLC patients treated with neoadjuvantchemoimmunotherapy included in NADIM clinical trial

Background:Immunotherapy is being tested in early-stage non-small cell lungcancer (NSCLC), and achieving higher rates of complete pathological responses(CPR) as compared to standard of care. Early identification of CPR patients hasvital clinical implications. In this study, we focused on basal peripheral immunecells and their treatment-related changes to find biomarkers associated toCPR.Methods:Blood from 29 stage IIIA NSCLC patients participating in the NADIMtrial (NCT03081689) was collected at diagnosis and post neoadjuvant treatment.More than 400 parameters of peripheral blood mononuclear cells (PBMCs) phe-notype and plasma soluble factors were analyzed.Results:Neoadjuvant chemoimmunotherapy altered more than 150 immuneparameters. At diagnosis, 11 biomarkers associated to CPR were described, withan area under the ROC curve>0.70 andp-value<.05. CPR patients had sig-nificantly higher levels of CD4+PD-1+cells, NKG2D, and CD56 expression onT CD56 cells, intensity of CD25 expression on CD4+CD25hi+cells and CD69expression on intermediate monocytes; but lower levels of CD3+CD56–CTLA-4+cells, CD14++CD16+CTLA-4+cells, CTLA-4 expression on T CD56 cells andlower levels of b-NGF, NT-3, and VEGF-D in plasma compared to non-CPR. Posttreatment, CPR patients had significantly higher levels of CD19 expression on Bcells, BCMA, 4-1BB, MCSF, and PARC and lower levels of MPIF-1 and Flt-3L inplasma compared to non-CPR.Conclusions:Patients achieving CPR seem to have a distinctive peripheralblood immune status at diagnosis, even showing different immune response totreatment.TheseresultsreinforcethedifferentbiologybehindCPRandnon-CPRresponses

Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry

Background: Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. Methods: Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system. Results: All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. Conclusions: The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situation

Tumor microenvironment gene expression profiles associated to complete pathological response and disease progression in resectable NSCLC patients treated with neoadjuvant chemoimmunotherapy

Background Neoadjuvant chemoimmunotherapy for non-small cell lung cancer (NSCLC) has improved pathological responses and survival rates compared with chemotherapy alone, leading to Food and Drug Administration (FDA) approval of nivolumab plus chemotherapy for resectable stage IB-IIIA NSCLC (AJCC 7th edition) without ALK or EGFR alterations. Unfortunately, a considerable percentage of tumors do not completely respond to therapy, which has been associated with early disease progression. So far, it is impossible to predict these events due to lack of knowledge. In this study, we characterized the gene expression profile of tumor samples to identify new biomarkers and mechanisms behind tumor responses to neoadjuvant chemoimmunotherapy and disease recurrence after surgery. Methods Tumor bulk RNA sequencing was performed in 16 pretreatment and 36 post-treatment tissue samples from 41 patients with resectable stage IIIA NSCLC treated with neoadjuvant chemoimmunotherapy from NADIM trial. A panel targeting 395 genes related to immunological processes was used. Tumors were classified as complete pathological response (CPR) and non-CPR, based on the total absence of viable tumor cells in tumor bed and lymph nodes tested at surgery. Differential-expressed genes between groups and pathway enrichment analysis were assessed using DESeq2 and gene set enrichment analysis. CIBERSORTx was used to estimate the proportions of immune cell subtypes. Results CPR tumors had a stronger pre-established immune infiltrate at baseline than non-CPR, characterized by higher levels of IFNG, GZMB, NKG7, and M1 macrophages, all with a significant area under the receiver operating characteristic curve (ROC) >0.9 for CPR prediction. A greater effect of neoadjuvant therapy was also seen in CPR tumors with a reduction of tumor markers and IFN gamma signaling after treatment. Additionally, the higher expression of several genes, including AKT1, BST2, OAS3, or CD8B; or higher dendritic cells and neutrophils proportions in post-treatment non-CPR samples, were associated with relapse after surgery. Also, high pretreatment PD-L1 and tumor mutational burden levels influenced the post-treatment immune landscape with the downregulation of proliferation markers and type I interferon signaling molecules in surgery samples. Conclusions Our results reinforce the differences between CPR and non-CPR responses, describing possible response and relapse immune mechanisms, opening the possibility of therapy personalization of immunotherapy-based regimens in the neoadjuvant setting of NSCLC