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Lung cancer (LC) remains as the leading cause of death by cancer worldwide. In Chile, LC is the second cause of cancer-related deaths. Recently, studies in melanoma and non-small cell lung cancer (NSCLC) patients have highlighted the role of the gut microbiota as an important host factor in mediating the responses/resistance to immunotherapy, suggesting that bacteria present in the gut may modulate the immune response in these tumors. However, the role of extra-intestinal microbiota; bacteria living outside of the gut, in cancer pathogenesis and the response to anti-cancer therapies remains largely undetermined. Here we characterize the composition of intratumoral microbiota of NSCLC and seek to establish a functional relationship between it and the composition of the immune microenvironment and the clinicopathological characteristics of NSCLC patients. From the first 157 FFPE NSCLC samples, we extracted DNA from all collected samples, obtaining adequate material in quality and quantity to later be submitted for analysis to 16S sequencing. Using 16S rRNA gene sequencing, we assessed the general landscape of the NSCLC tumor microbiome, revealing the presence of large number of bacterial communities in the NSCLC tumor samples on the different histological subtypes analyzed. We have detected differences in alpha diversity in the histological subtypes studied. Even more interesting, we have detected significant differences in lung adenocarcinoma depending on the degree of histological differentiation, observing a decrease in proteobacteria and an enrichment of Bacteroidales, as cell differentiation is lost. Additionally, we identified taxonomic differences between differentiated and undifferentiated tumors. Differentiated tumors show enrichment in Akkermansia muciniphila and while undifferentiated tumors show enrichment in Actinobacter Corynebacterium. Suggesting that these taxa could contribute to maintaining a differentiated state or inducing cell dedifferentiation, respectively. Our results reveal the presence of a large number of bacterial communities in lung cancer samples in the different histological subtypes analyzed. Suggesting that these communities could play a key role in tumor differentiation and progression. Citation Format: Erick M. Riquelme, Ivania Valdes, Carlos Aravena, Ilse Valencia, Barbara Mino, Daniel Carvajal, Alberto Martin. Revealing the role of lung cancer microbiota in the tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 651.

Background: According to the 2013 guidelines breast cancers are defined as Her2 positive if there is evidence of protein expression in at least 10% of tumor cells by IHC and/or gene amplification by FISH. Nevertheless, there are still IHC 2+ and FISH equivocal breast cancers resulting in repeat testing. It is also known that not all Her2 positive breast cancers respond to Trastuzumab, while up to 8% of Her2 "negative" classified patients benefit from Her2 targeting regimens. Toward the goal of generating a more accurate test, we report in situ quantification of Her2 protein levels on a prospectively collected cohort of breast cancers and comparison to conventional IHC and FISH evaluation. Materials and Methods: A prospectively designed study was initiated at Yale University, comparing quantitative, in situ measurement of Her2 protein levels with conventional IHC and FISH evaluation. All breast cancer specimens were analyzed by IHC and FISH in our routine clinical laboratory, read and signed out by the breast pathologists. Serial sections were then stained and quantified for Her2 expression levels using the AQUA method of quantitative immunofluorescence (QIF). Data for all assays were obtained on 120 samples over a period of 6 months. Staining was performed using the DAKO Herceptest and the Epitomics EP3 Her2 antibody for IHC, the DAKO rabbit polyclonal antibody for QIF. The 30 highest cases were then retested for QIF and IHC using the Biocare c-erbB-2 [CB11] antibody. Each staining run included an index tissue microarray (TMA) consisting of 80 cases, cell lines and normal tissue for quality control, assay reproducibility and threshold definition of AQUA scores correlated to HER2 over expression and amplification. Results: Out of 120 specimens analyzed for HER2, 13 were diagnosed as IHC 2+/3+, FISH amplified, 1 case had an equivocal score, 14 cases were IHC 2+/non amplified, 2 cases IHC 1+/FISH amplified and 89 specimens IHC 0/1+ non amplified. The continuous AQUA scores for Her2 expression of the samples significantly correlate with traditional clinical Her2 scoring. However, 5 IHC 0/1+, non amplified cases revealed high AQUA scores in the range of HER2 overexpression/amplification. Repeat testing of these by both QIF and IHC showed reproducibility of the results. AQUA scores of one IHC 3+/amplified sample were lower than the threshold of HER2 overexpression/amplification. Conclusions: QIF measurement of HER2 protein levels in a prospectively collected cohort of 120 breast cancer specimens reveals significant association between continuous HER2 protein levels and the ordinal conventional scoring system. However, five discordant cases that were above the threshold for HER2 protein by QIF, were classified as negative by conventional methods. Given the accuracy and reproducibility of the QIF test, it raises the possibility that some of these patients might benefit from HER2 targeted therapy. In summary, while continuous scoring of HER2 protein correlates well with conventional methods, it identifies a subset of patients that are discordant with current methods. Further comparative studies in a patient cohort with response to targeted therapy need to be evaluated. Citation Format: Neumeister VM, Yan SS, McGuire JA, Carvajal DE, Prasad ML, Rimm DL. Quantitative immuno-fluorescent evaluation of Her2 expression levels in a prospectively collected cohort of breast cancer cases: Comparison to conventional IHC scoring and FISH. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-22.

Introduction: T-cells recognizing tumor antigens can express diverse immune inhibitory receptors such as PD-1, LAG-3 and TIM-3. Blockade of PD-1 induces prominent clinical benefit in patients with melanoma, lung, bladder and gastric carcinomas. However, the relative expression and significance of immune inhibitory receptors across different tumors is unknown. Methods: Using multiplexed quantitative immunofluorescence (QIF) we measured the levels of CD3 (rabbit polyclonal, Dako), PD-1 (EH33, CST), LAG-3 (17B4, Abcam) and TIM-3 (D5D5R, CST) in >1,300 human malignancies including 119 triple negative breast carcinomas (TNBC), 225 ovarian carcinomas, 382 melanomas, 259 colorectal tumors, 229 bladder urothelial carcinomas and 130 gastric malignancies. The cohorts included FFPE tumor tissues and were studied using tissue microarrays (TMAs). The targets were measured in all cells of the preparation using fluorescence co-localization with DAPI and specifically in CD3-positive T-lymphocytes. Associations between the markers across tumors and with survival were studied. Results: PD-1, LAG-3, and TIM-3 were expressed predominantly in T-cells and showed a positive association with each other. The highest levels were detected in gastric adenocarcinomas followed by urothelial bladder tumors, ovarian malignancies, melanoma, TNBC and colorectal carcinomas. Gastric and bladder tumors showed comparably high levels of all three markers and were significantly higher than the other tumor types (P Conclusion: PD-1, LAG-3 and TIM-3 are differentially expressed in human solid tumors and show limited prognostic value. Gastric and bladder carcinomas show the highest levels of the targets, while TNBC and colorectal cancer show the lowest. Despite having the highest T-cell infiltration, melanomas show intermediate levels of the immune inhibitory receptors. These data demonstrate the differences in the immune composition of human solid tumors and could be used for optimal design and interpretation of clinical trials. Citation Format: Micaela Morgado, Ila Datar, Jun Wang, Miguel F. Sanmamed, Kristen McEachern, David Jenkins, Lieping Chen, Daniel Carvajal-Hausdorf, David L. Rimm, Roy S. Herbst, Kurt A. Schalper. Simultaneous measurement and significance of PD-1, LAG-3 and TIM-3 expression in human solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1681.