Tumor infiltrating T cells are a positive prognosticator in many tumor types, and our prior work demonstrated that in renal cell carcinoma, patients with higher CD8 T cell infiltration have improved survival (Jansen Nature 2019). We described that this T cell response is supported by TCF1+ stem-like CD8 T cells, which reside within dense regions of closely clustered antigen presenting cells within the tumor. Interestingly, aggregations of immune cells have also been described in other settings and termed ‘tertiary lymphoid structures’ (TLS), raising an important question of whether the described immune niches are similar to these TLS or if they represent a distinct type of peripheral immune organization. In the work presented here, we use quantitative immunofluorescence image analysis to examine the cellular composition and organization of intratumoral immune niches, TLS, and secondary lymphoid tissue. The novel data presented here describes how immune niches are distinct from TLS and are more similar to the organization of T cell zones in secondary lymphoid tissue. Immune niches are T cell dominant structures, with T cell and antigen presenting cell compartments similar to T cell zones, whereas these niches profoundly lack the B cell presence seen in TLS and B cell zones of secondary lymphoid tissue. Immune niches are comparatively small in size (50-200um), while TLS are much larger (often >1mm), and, importantly, immune niches are found inside the tumor border, while TLS are located peripherally in tumor tissue. Notably, we find that immune niches have a marked presence of aSMA+ stromal cells, which are similar to those found in T cell zones but absent in B cell zones and TLS. When probed by flow cytometry and RNAseq, we find that aSMA+ stroma in immune niches are myofibroblast-like, similar those known to maintain the structure of secondary lymphoid tissues, and are distinct from those found in healthy kidney tissue. These data suggest these aSMA+ cells may play an important role in the organization of immune niches in tumor tissue. As we have shown that a robust T cell response is beneficial for patient outcomes, it is crucial to understand the mechanisms which support that response. Recently, our group defined that CD8 T cell activation in cancer is comprised of two phases, with an initial priming phase in the lymph node and a second phase of effector program acquisition in the tumor tissue (Prokhnevska Immunity 2023). We suggest that these immune niches represent an important hub for this development of T cell function, which is required for the robust, effective anti-tumor T cell response that supports improved patient outcomes and is the basis for the response to immunotherapy. Advancing our understanding of intratumoral immune organization will allow for enhanced biomarker discovery, improvement in existing therapies, and innovation in developing novel therapeutic strategies that will convert immunologically “cold” tumors to be immunologically “hot” and enhance anti-tumor immunity. Citation Format: Caroline S. Jansen, BaoHan Vo, Ewelina Sobierajska, Rachel Greenwald, Patrick Mullane, Nataliya Prokhnevska, Maria Cardenas, Mehmet Asim Bilen, Adeboye O. Osunkoya, Viraj A. Master, Haydn T. Kissick. Form and function in intratumoral immune organization: Understanding the cellular composition of TCF1+ CD8+ T cell niches in human cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B030.

Aims A subset of patients with urothelial carcinoma (UCa) and lamina propria (LP) invasion in bladder biopsies/transurethral resections (TURs) are at significant risk for recurrence and have increased rates of progression to UCa with muscularis propria (MP) invasion. The clinicopathologic features of this patient population has not been well characterised in the Pathology literature. Methods We performed a search through our urologic pathology files and expert consult cases of the senior author for bladder biopsies/TURs of UCa with LP invasion and variant/divergent histology from 2014 to 2020. Patients with a prior diagnosis of UCa with MP invasion or upper tract UCa were excluded. Clinicopathologic data were obtained. Results Ninety-five patients with at least one biopsy/TUR of UCa with LP invasion and variant/divergent histology were identified. Mean patient age was 72 years (range: 46-92 years) with a male predominance 2.3:1. Initial variant/divergent histologies identified were: glandular (35.8%), squamous (23.2%), micropapillary (20%), clear cell/lipid rich (12.6%), diffuse/signet ring/plasmacytoid (10.5%), nested (9.5%), sarcomatoid (6.3%), poorly differentiated/anaplastic (4.2%), small cell (2.1%), lymphoepithelioma-like (2.1%), osteoclast-like giant cells (1.1%) and tumour giant cells (1.1%). Two or more variant histologies were identified in 18.9% of these cases. The rate of micropapillary UCa was significantly higher in multifocal tumours compared with unifocal tumours (37% vs 7.1%). Conclusions In our cohort of patients undergoing early repeat biopsy/TUR, 75% of patients had persistent UCa. Additionally, almost 25% of patients had a prior diagnosis of UCa without a variant/divergent histology identified. Our findings highlight the critical role of repeat biopsy/TUR especially in a subset of patients who have variant/divergent histology, even in the absence of MP invasion.

INTRODUCTION:The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) is an established system with reproducible risk of malignancies (ROM) for salivary gland fine needle aspiration (SGFNA). No studies have reviewed the relationship between Milan categories and the resection rate (RR) and time to resection (TTR).METHODS:We searched our database (January 1, 2011 to January 4, 2021) for non-lymphoma SGFNAs and assigned appropriate MSRSGC categories. RR and TTR were calculated and compared for each category. A literature search was performed; RRs and TTRs were compared.RESULTS:Seven hundred and eighty SGFNAs were identified, 333 with follow-up. RR was highest in suspicious for malignancy (SUS, V; 70.6%, n = 12/17), followed by the salivary gland neoplasm of uncertain malignant potential (SUMP, IVb; 69.6%, n = 80/115) and malignant (M, VI; 55.6%, n = 75/135). Among M, primary tumors had a higher RR (65.1%, n = 41/63) than metastases (47.2%, n = 34/72, p = .36). In literature review, SUS had the highest RR (69.3%, n = 233/336) followed by M (61.6%, n = 821/1332) and SUMP (60.2%, n = 632/1050). TTR was shorter in SUS (mean = 32.3 days, median = 25 days). Within the benign neoplasms (BN, IVa), Pleomorphic adenomas (PAs) had a higher RR than Warthin tumors (WTs) (66.3% vs. 37.2%, p < .00001), and a shorter TTR (median = 63 days vs. 90 days).CONCLUSIONS:Tumors classified as SUS had higher RR and at shorter intervals than those classified as SUMP. PAs have higher RRs and more expedient surgery than WTs. Cases classified as M are less likely to undergo follow-up than SUS, perhaps due to a lower RR for metastases.

BackgroundThe Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) reports a 25% rate of malignancy (ROM) for the Milan I: Nondiagnostic (ND) category. We clarify the ROM of ND salivary gland fine‐needle aspirations (SGFNAs) based on our institutional experience and review of the literature.MethodsOverall risk of malignancy (OROM) and that for those with surgical/flow cytometric follow‐up (FROM) for each category and “all‐comers” were calculated for Emory SGFNAs from January 2010 through March 2021. From a literature review of 50 articles using MSRSGC, distribution of diagnoses, rates of follow‐up, FROM, and OROM by category were calculated. FROMs and OROMs between ND FNAs and all‐comers were compared. Milan I rate was compared with the ratio of Milan I OROM to all‐comer OROM.ResultsOf 819 SGFNAs at Emory, 12.8% (n = 105/819) were ND. Thirty‐two had known follow‐up, with 12 (37.5%) being malignant. Nonmucinous cyst contents accounted for 26.7% of ND SGFNAs (n = 28/105); all 7 with surgical follow‐up were benign. Of 50 MSRSGC studies, 18.2% (n = 2384/13,129) of SGFNAs were classified as ND, 26.6% (n = 635/2384) with known follow‐up. Total FROM and OROM for ND FNAs (15.7% and 4.1%, respectively) were significantly lower than those for all‐comers (24.9% and 11.4%, respectively) (p < .001). There was no relationship between rate of ND SGFNA and ND ROM.ConclusionsThe ND category is associated with a lower ROM than that of all‐comer SGFNA patients. The “true” ROM for ND SGFNAs is likely best estimated by the 4.1% OROM. SGFNAs showing nonmucinous cyst contents have a particularly low ROM. Rate of ND SGFNAs does not influence ND ROM.

The incidental detection of renal masses continues to increase due to the expanded use of imaging studies. Fine needle aspiration (FNA) and core biopsies can aid in the management of select patients with renal lesions. The management of some renal masses, especially small lesions less than 5 cm, remains challenging as many of these tumors are either benign or clinically indolent, and current imaging modalities are often unable to satisfactorily identify clinically relevant tumors. Current American Urologic Association (AUA) guidelines recommend considering biopsy when a mass is suspected to be hematologic, metastatic, inflammatory, or infectious, with preference of multiple core biopsies over FNA. This chapter covers FNA and small biopsy of renal cell carcinoma.

Urothelial carcinoma can be characterized by malignant transformation of transitional epithelium. While urothelial carcinomas arise less frequently in the ureter and renal pelvis than in the bladder, these lesions are typically more difficult to detect by biopsy by cystoscopy. While imaging studies can typically localize renal masses to the cortex vs. the pelvis, this may be difficult for large lesions. Thus, urothelial carcinomas of the kidney are sometimes diagnosed by FNA procedure rather than by urinary tract cytology or ureteroscopic biopsy. This chapter discusses the cytomorphologic features and immunoprofile of urothelial carcinoma on FNA compared to other entities on the differential diagnosis, such as renal cell carci noma.

Purpose Checkpoint therapy is now the cornerstone of treatment for patients with renal cell carcinoma (RCC) with advanced disease, but biomarkers are lacking to predict which patients will benefit. This study proposes potential immunological biomarkers that could developed for predicting therapeutic response in patients with RCC. Methods Using flow cytometry, RNA sequencing, and T-cell receptor (TCR) sequencing, we investigated changes in T cells in the peripheral blood of patients with advanced RCC after receiving immunotherapy. We used immunofluorescence (IF) imaging and flow cytometry to investigate how intratumoral T cells in patients’ tumors (resected months/years prior to receiving checkpoint therapy) predicted patient outcomes after immunotherapy. Results We found that a small proportion of CD4 and CD8 T cells in the blood activate following checkpoint therapy, expressing the proliferation marker Ki67 and activation markers HLA-DR and CD38. Patients who had the highest increase in these HLA-DR +CD38+CD8 T cells after treatment had the best antitumor immune response and experienced clinical benefit. Using RNA sequencing, we found that while these cells expanded in most patients, their phenotype did not drastically change during treatment. However, when we analyzed the TCR repertoire of these HLA-DR +CD38+CD8+T cells, we found that only patients who clinically benefitted had a burst of new clonotypes enter this pool of activated cells. Finally, we found that abundant T cells in the untreated tumors predicted clinical benefit to checkpoint therapy on disease progression. Conclusions Together, these data suggest that having a strong pre-existing immune response and immediate peripheral T-cell activation after checkpoint therapy is a predictor of clinical benefit in patients with RCC.

We investigated changes in T cell responses in the peripheral blood of patients with advanced renal cell carcinoma (RCC) after receiving immunotherapy. We found that a small proportion of both CD4 and CD8 cells activate and express the proliferation marker Ki67 and the activation markers HLA-DR and CD38. Patients who had the highest increase in these HLA-DR+CD38+ CD8 T cells after treatment had the best anti-tumor response. We studied these newly activated cells in more detail using flow cytometry and RNAseq and found that while these cells expanded in most patients, their phenotype did not drastically change during treatment. However, when we analyzed the TCR repertoire of these HLA-DR+CD38+CD8+ T cells, we found only patients who responded to the treatment had a burst of new clonotypes enter this pool of activated cells. Finally, we investigated how the T-cell response in the resected tumor months or years before receiving checkpoint therapy predicted later response to checkpoint therapy. Together, these data suggest that having a strong pre-existing immune response and immediate T cell response to checkpoint therapy is a predictor of anti-tumor response in patients with RCC.

Background Tumor infiltrating T-cells have a prognostic benefit in many tumor types,1–8 and we recently sought to determine whether the level of T-cell infiltration into renal tumors predicts clinical outcomes. In our recent publication,9 we showed that patients with high of CD8 T-cell infiltration have improved progression free survival (PFS). Further, we found that this T-cell response is supported by TCF1+ stem-like CD8 T-cells, which reside within dense regions of closely clustered antigen presenting cells within the tumor. Interestingly, aggregations of immune cells have also been described in other tumor types and termed 'tertiary lymphoid structures' (TLS), which are typically defined as B-cell-dominant aggregates, containing high endothelial venules and reactive germinal centers.10–12 Together, these findings raise several important questions, which we explore here—(1) what additional cell types comprise these niches?9 and (2) how are these niches similar to or different from TLS? Methods Tumor tissue was collected from patients with renal tumors undergoing surgery at Emory University Hospital. Intraoperative tumor samples were analyzed by flow cytometry, RNA sequencing, immunofluorescence, and immunohistochemistry. Immunofluorescence data was analyzed using our custom quantitative analysis pipelines, which allows for delineation of cell type and location, cell-cell distance, and density of cellular aggregation. Results The proportion of CD8 T-cells infiltration human renal tumors varied widely, consistent with our previous reports.9 TCF1+ stem-like CD8 T-cells were identifiable by both flow cytometry and immunofluorescence and resided in dense antigen presenting niches. Quantitative immunofluorescence revealed the location of aSMA+ fibroblasts within tumor tissue, in relation to antigen presenting niches, and in tumors with many infiltrating T-cells. Pathologist scored hematoxylin and eosin-stained slides were delineated TLS+ or TLS-. Quantitative immunofluorescence imaging analysis revealed the detailed composition of tumor infiltrating immune cell populations and the contrasting cellular organization in TLS as compared to in antigen presenting niches. Conclusions As we have shown CD8 T-cell infiltration to predict PFS in renal tumors and that antigen presenting niches containing stem-like cells maintain the anti-tumor T-cell response,9 it is critical to understand the additional cell types present in these niches and to understand how these niches relate to previously described phenomena of immune organization, such as TLS.10–12 This mechanistic understanding of the anti-tumor immune response represents an opportunity to inform development of enhanced prognostic tools and innovative therapeutic possibilities. References Azimi F, et al. Tumor-infiltrating lymphocyte grade is an independent predictor of sentinel lymph node status and survival in patients with cutaneous melanoma. J Clin Oncol 2012;30(21):2678–83. Epub 2012/06/20. doi: 10.1200/jco.2011.37.8539. PubMed PMID: 22711850. Galon J, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006;313(5795):1960–4. Epub 2006/09/30. doi: 10.1126/science.1129139. PubMed PMID: 17008531. Mlecnik B, et al. Integrative analyses of colorectal cancer show immunoscore is a stronger predictor of patient survival than microsatellite instability. Immunity 2016;44(3):698–711. Epub 2016/03/18. doi: 10.1016/j.immuni.2016.02.025. PubMed PMID: 26982367. Mlecnik B, et al. Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction. J Clin Oncol 2011;29(6):610–8. Epub 2011/01/20. doi: 10.1200/JCO.2010.30.5425. PubMed PMID: 21245428. Pagès F, et al. Immune infiltration in human tumors: a prognostic factor that should not be ignored. Oncogene 2009;29:1093. doi: 10.1038/onc.2009.416. Peranzoni E, et al. Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment. Proceedings of the National Academy of Sciences of the United States of America 2018;115(17):E4041–E50. Epub 2018/04/11. doi: 10.1073/pnas.1720948115. 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Epub 2014/02/06. doi: 10.4161/onci.26836. PubMed PMID: 24498556; PMCID: PMC3912008. Sautes-Fridman C, et al. Tertiary lymphoid structures in the era of cancer immunotherapy. Nature reviews Cancer 2019;19(6):307–25. Epub 2019/05/17. doi: 10.1038/s41568-019-0144-6. PubMed PMID: 31092904. Ethics Approval Samples are collected under an approved IRB protocol (The Urological Satellite Specimen Bank at Emory University, IRB00055316). All patients provided informed consent. Consent Samples are collected under an approved IRB protocol (The Urological Satellite Specimen Bank at Emory University, IRB00055316). All patients provided informed consent.