Expression Analysis and Significance of PD-1, LAG-3, and TIM-3 in Human Non-Small Cell Lung Cancer Using Spatially Resolved and Multiparametric Single-Cell Analysis.

PURPOSE: To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG-3, and TIM-3 protein expression in human non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in >800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment. RESULTS: PD-1, LAG-3, and TIM-3 were detected in tumor-infiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiving immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progression-free survival. CONCLUSIONS: PD-1, LAG-3, and TIM-3 have distinct tissue/cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways.

Cytology Smears in the Era of Molecular Biomarkers in Non-Small Cell Lung Cancer: Doing More With Less.

CONTEXT: - The rapid advances in targeted therapies in non-small cell lung cancer (NSCLC) make the optimization and implementation of cytology specimens for molecular testing a priority. Up to 70% of patients with NSCLC are diagnosed at advanced stages and tissue biopsies often cannot be taken. Although cytology samples provide high-quality material for molecular testing, molecular cytopathology is not yet well known or widely used. OBJECTIVE: - To report the many advances in molecular cytopathology and the suitability and utility of cytology samples in molecular and genetic testing of NSCLC. DATA SOURCES: - Data sources comprised published peer-reviewed literature and personal experience of the authors. CONCLUSIONS: - Molecular testing can be performed on cytologic specimens, especially on direct smears. Rapid on-site evaluation by cytopathologists has improved the adequacy and the management of cytology samples for molecular testing. Mutational profiling of NSCLC using next-generation sequencing can be performed on cytology samples from very small amounts of DNA. Fluorescence in situ hybridization assays on cytology specimens, including stained direct smear, offer some distinct advantages over their histologic counterpart, and are used to detect ALK and ROS1 rearrangements in NSCLC. Cytology specimens allow assessment of the entire tumor cell nucleus, avoiding signal loss from truncation artifacts. The use of cytology samples for assessing programmed death ligand-1 protein expression is currently being developed. Protocols for bisulfite conversion and DNA droplet digital polymerase chain reaction assays have been optimized for cytology smear to investigate aberrant DNA methylation of several NSCLC-related genes.

Extragonadal retroperitoneal germ cell tumor: primary versus metastases?

OBJECTIVE: Primary extragonadal germ cell tumors are rare and their histogenetic origin is not clear. We describe two cases presenting as primary retroperitoneal germ cell tumors without clinical evidence of testicular tumor. METHODS: A 21 and 18 years-old patients presented retroperitoneal choriocarcinoma and yolk sac tumor, respectively. In both cases, testicular palpation was not suspicious for testicular cancer. Testicular ultrasound founded alterations in right testes. RESULTS: A right orchitectomy were performed and the final diagnostics were mature teratoma associated with intratubular malignant germ cell. CONCLUSION: Adult mature teratoma is infrequent and the retroperitoneal germ cell tumors should be considered to be metastases of a viable or burned-out testicular cancer.

Extragonadal retroperitoneal germ cell tumor: primary versus metastases? / Tumor de células germinales extragonadal retroperitoneal: ¿Tumor primario o metastásico?

Objetive: Primary extragonadal germ cell tumors are rare and their histogenetic origin is not clear. We describe two cases presenting as primary retroperitoneal germ cell tumors without clinical evidence of testicular tumor. Methods: A 21 and 18 years-old patients presented retroperitoneal choriocarcinoma and yolk sac tumor, respectively. In both cases, testicular palpation was not suspicious for testicular cancer. Testicular ultrasound founded alterations in right testes. Results: A right orchitectomy were performed and the final diagnostics were mature teraroma associated with intratubular malignant germ cell. Conclusions: Adult mature teratoma is infrequent and the retroperitoneal germ cell tumors should be considered to be metastases of a viable or burned–out testicular cancer (AU)

Objetivo: Los tumores primarios de células germinales extragonadales son poco frecuentes y su origen histogenético no está claro. Describimos dos casos que se presentaron como tumores de células germinales retroperitoneales sin evidencia clínica de tumor testicular. Métodos: Dos pacientes de 21 y 18 años presentaron respectivamente un coriocarcinoma y un tumor del saco embrionario retroperitoneales. En ambos casos, la palpación testicular no era sospechosa de cárcel testicular. La ecografía testicular descubrió alteraciones en los testículos derechos de ambos pacientes. Resultados: Se llevó a cabo orquiectomía derecha con el diagnóstico final de teratoma maduro asociado con células germinales malignas intratubulares. Conclusiones: El teratoma maduro del adulto es poco frecuente, y los tumores de células germinales retroperitoneales deben ser consideradas metástasis de un cáncer testicular fundido (AU)