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The kidney is a complex organ that governs many physiological parameters. It is roughly divided into three parts, the renal pelvis, medulla, and cortex. Covering the cortex is the renal capsule, a serosal tissue that provides protection and forms a barrier for the kidney. Serosal tissues of many organs have been recently shown to play a vital role in homeostasis and disease. Analyses of the cells that reside in these tissues have identified distinct cell types with unique phenotypes. Surprisingly, despite the importance of serosal tissues, little is known about cells of the renal capsule. Here, we characterized this niche and found that it is mainly comprised of fibroblasts and macrophages, but also includes many other diverse cell types. Characterizing renal capsule-associated macrophages, we found that they consist of a distinct subset (i.e., TLF+macrophages) that is nearly absent in the kidney parenchyma. Injury, disease, and other changes within the kidney, affected the cell composition and phenotype of the renal capsule, indicating its dynamic and vibrant response to changes within the organ parenchyma. Lastly, we studied age-related changes in the renal capsule and found that aging affected the cell composition and pro-inflammatory phenotype of macrophages, increased CD8 T cells and other lymphocyte counts, and promoted a senescence-associated phenotype in fibroblasts. Taken together, our data illustrate the complexity and heterogeneity of the renal capsule and its underlying changes during aging and disease, improving our understanding of the kidney serosa that may be valuable for novel renal therapies.

Supplementary Data from Origins and Timing of Emerging Lesions in Advanced Renal Cell Carcinoma

Supplementary Figure from Origins and Timing of Emerging Lesions in Advanced Renal Cell Carcinoma

Renal cell carcinoma (RCC) with venous tumor thrombus (VTT) arising from the primary tumor occurs in approximately 10% of cases and is thought to represent more advanced disease. The intravascular nature of VTT suggests that it may serve as a source for hematogenous metastases. RCC with VTT and distant metastasis provides unique opportunities to examine the origins and emergence timing of these distinct tumor lesions, and to identify molecular correlates with disease state. We performed multi-region exome and RNA-sequencing analysis of 16 patients with RCC with VTT, with eight patients also having sequenced metastasis, to identify genomic alterations, biological pathways, and evolutionary processes contributing to VTT and metastasis, and to ask whether metastasis arises directly from or independent of VTT. No specific genomic alterations were associated with VTT. Hallmark copy-number alterations (deletions of 14q, 8p, and 4q) were associated with metastasis and disease recurrence, and secondary driver alterations tended to accumulate in metastatic lineages. Mismatch repair mutational signatures co-occurred across most tumors, suggesting a role for intracellular DNA damage in RCC. Robust phylogenetic timing analysis indicated that metastasis typically emerged before VTT, rather than deriving from it, with the earliest metastases predicted to emerge years before diagnosis. As a result, VTT in metastatic cases frequently derived from a metastatic lineage. Relative to the primary tumor, VTT upregulated immediate-early genes and transcriptional targets of the TNFα/NF-κB pathway, whereas metastases upregulated MTOR and transcriptional targets downstream of mTORC1 activation.Implications:These results suggest that VTT and metastasis formation occur independently, VTT presence alone does not necessarily imply more advanced disease with inevitably poor prognosis.

Positive and negative controls with known expression of target proteins are essential for the development of immunohistochemistry (IHC) assays. While tissue controls are beneficial for well-characterized proteins with defined tissue and cellular expression patterns, they are less suitable for the initial development of IHC assays for novel, poorly characterized, or ubiquitously expressed proteins. Alternatively, due to their standardized nature, cell pellets, including cancer cell lines with defined protein or transcript expression levels (e.g., high, medium, and low expression), transfected over-expressing cell lines, or cell lines with genes deleted through cell engineering technologies like CRISPR, can serve as valuable controls, especially for the initial antibody characterization and selection. In order for these cell pellets to be used in the development of IHC assays for formalin-fixed, paraffin-embedded tissues, they need to be processed and embedded in a manner that recapitulates the procedures used for tissue processing. This protocol describes a process for creating and processing formalin-fixed, paraffin-embedded cell pellet controls that can be used for IHC method developments.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used material for tumor molecular profiling, therapy selection, and prognostication. Tumor tissue enrichment by tissue dissection is highly recommended to generate quality data reproducibly for use in downstream assays, such as real-time PCR and next-generation sequencing. The aim of this study was to evaluate the performance of the automated tissue dissection tool AVENIO Millisect System compared with a manual dissection method using 18 FFPE tissue specimens. The study assessed performance of these two methods with paraffinized and deparaffinized sections at 5- and 10-μm thickness as well as at low (5% to 10%) and high (>50%) tumor content. In addition, compatibility with various nucleic acid and protein extraction methods was assessed. Overall, dissection by Millisect resulted in statistically significantly higher yields of nucleic acids and protein compared with manual dissection (P = 0.00524). In downstream analysis on a statistically nonpowered sample set, EGFR mutation testing by PCR led to highly concordant results, and next-generation sequencing testing yielded significantly higher allelic frequencies when tissue was dissected by Millisect compared with manual scraping, demonstrating noninferiority of the automated method. In summary, the AVENIO Millisect System may replace manual labor and support automation of FFPE tumor tissue workflows in clinical molecular laboratories with high testing volumes with adequate validation.

Introduction: Inflammatory breast cancer (IBC) is a clinical diagnosis that spans all breast cancer subtypes. The clinical course of IBC is associated with poorer outcomes than molecular subtype-matched non-IBC and represents an unmet need in breast cancer therapy. IBC is characterized by invasive dermal lymphatic tumor emboli (DLTE) resulting in erythema and edema of the breast. This suggests a common molecular theme, which if identified and targeted, could reduce mortality. Although several genomic and transcriptomic studies of IBC have been performed, no definitive genomic drivers have been identified. We hypothesized that the genomic features of IBC remain undiscovered because only the primary tumor has been analyzed, rather than the population of tumor cells responsible for the phenotype (i.e. DLTE). However, analysis of DLTE is challenging due to lack of effective technologies to purify these cells from the more abundant stroma. Methods: We utilized the Millisect automated dissection (AD) technology, which can selectively recover tissue from targeted areas as small as 200µ2 on standard FFPE sections, on matched skin with DLTE and primary tumor slides from 7 post-treatment IBC mastectomy specimens. We performed RNA sequencing (RNAseq) on AD-enriched primary tumor and DLTE, on full sections from the same samples (including stroma and tumor) and on tissue remaining post-AD (containing residual stroma after tumor cell extraction). Results: RNAseq analysis in AD-enriched primary tumor and DLTEs identified unique transcriptional patterns upregulated in DLTE indicative of lymphatic trafficking (e.g. CCL21), immunosuppression (e.g. S100A9/calprotectin), and myofibroblastomic differentiation (e.g. CD34, desmin, alpha-smooth muscle actin). Gene set enrichment corroborated these inferences, demonstrating DLTE enrichment of gene sets involved in chemokine/trafficking, tumor stemness, and smooth muscle contraction/migration. These gene sets were not apparent in samples extracted from the full sections. Conclusions: Automated dissection technology enables specific investigation on the limited epithelial material that comprises inflammatory breast cancer involving dermal lymphatics. Further investigation, with cohort expansion to increase sample size and statistical power, will focus on pre-treatment skin biopsies with DTLE and matched breast tumor tissue. We expect this will yield novel insights into the biology and treatment of this unique phenotype. Citation Format: Jennifer M Giltnane, Justin M Balko, Nickles Dorothee, Sarajane Nghiem, Anneleen Daemen, Emmanuel Naouri, Amy A Lo, Beth T Harrison, Emily J Schlosnagle, Charles Havnar, Carmina Espiritu, Daniel G Stover, Beth A Overmoyer. Automated tissue dissection of dermal lymphatic emboli in inflammatory breast cancer enhances accuracy of transcriptional analysis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-15-02.

Cryopreserving tissues for histology requires the use of coolants to buffer the sample from liquid nitrogen (LN2) and to control the rate of temperature decline. Several coolants sharing similar physical characteristics are available on the market; however, commonly used coolants are variably flammable and/or toxic and pose risks to personnel and facilities. The purpose of this study was to compare the performance of three commercially available coolants: hexane, 2-methylbutane (2 M), and 1-methoxyheptafluoropropane (N7000). Fresh mouse tissues were frozen by each method, for their ability to preserve microscopic architecture and to protect RNA from degradation were evaluated and compared to tissue characteristics obtained by direct immersion in LN2. Our results show that for most tissues, the N7000 freezing coolant provides equal or improved preservation of microscopic architecture. While snap-freezing tissues in LN2 provides superior RNA protection, no significant differences in RNA quality were seen between tissues frozen in hexane, 2 M, and N7000.

BackgroundIndividualized neoantigen-specific immunotherapy (iNeST) requires robustly expressed clonal neoantigens for efficacy, but tumor mutational heterogeneity, loss of neoantigen expression, and variable tissue sampling present challenges. It is assumed that clonal neoantigens are preferred targets for immunotherapy, but the distributions of clonal neoantigens are not well characterized across cancer types.MethodsWe combined multiregion sequencing (MR-seq) analysis of five untreated, synchronously sampled metastatic solid tumors with re-analysis of published MR-seq data from 103 patients in order to characterize their globally clonal neoantigen content and factors that would impact neoantigen targeting.ResultsBranching evolution in colorectal cancer and renal cell carcinoma led to fewer clonal neoantigens and to clade-specific neoantigens (those shared across a subset of tumor regions but not fully clonal), with the latter not being readily distinguishable in single tumor samples. In colorectal, renal, and bladder cancer, most tumors had few globally clonal neoantigens. Prioritizing mutations with higher purity-adjusted and ploidy-adjusted variant allele frequency enriched for globally clonal neoantigens (those found in all tumor regions), whereas estimated cancer cell fraction derived from clustering-based tools, surprisingly, did not. Neoantigen quality was associated with loss of neoantigen expression in the bladder cancer case, and HLA-allele loss was observed in the renal and non-small cell lung cancer cases.ConclusionsWe show that tumor type, multilesion sampling, neoantigen expression, and HLA allele retention are important factors for iNeST targeting and patient selection, and may also be important factors to consider in the development of biomarker strategies.

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 °C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities. This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

With the advent of checkpoint inhibitors, there is increasing need to study the dynamics of CD8+ T-cells in the tumor microenviroment. In this article, we describe a semi-automated method to quantify and interrogate spatial relationships between T-cells and collagenous stroma in human and mouse tissue samples. The assay combines CD8 immunohistochemistry with modified Masson’s trichrome. Slides are scanned and digital images are analyzed using an adjustable MATLAB algorithm, allowing for high-throughput quantification of cytotoxic T-cells and collagen. This method provides a flexible tool for unbiased quantification of T-cells and their interactions with tumor cells and tumor microenvironment in tissue samples.

PurposeRenal cell carcinoma (RCC) with venous tumor thrombus (VTT) arising from the primary tumor occurs in 4-10% of cases and is associated with advanced disease. RCC with VTT and distant metastasis represents a unique clinical entity, and provides opportunities to examine the origins and relative timing of tumor lesion emergence and to identify molecular correlates with disease state.Experimental DesignWe performed genomic and evolutionary analyses on 16 RCC patients with VTT, with eight also having metastases, using multi-region exome and RNA sequencing.ResultsNo genomic alterations were specifically associated with the VTT or metastasis lesions; each tumor had multiple hallmark driver alterations, consistent with advanced disease state. We found that 21% (3/14) of clear-cell RCC cases could be assigned a previously defined “evolutionary subtype”. Somatic mutation signatures were largely consistent with previously established RCC signatures, and showed low heterogeneity across regions of each tumor. Mismatch repair and homologous recombination (“BRCA-ness”) deficiency signatures consistently co-occurred across most tumors, suggesting a pervasive role for intracellular DNA damage in RCC and the potential for related treatment strategies. Phylogenetic timing analysis of metastatic cases suggested that in most tumors, metastases branched from the primary tumor prior to formation of VTT and in some cases before diversification of the primary tumor. Both VTT and the earliest metastases were predicted to emerge many years prior to diagnosis. Transcriptional landscape analysis identified key differences distinguishing each lesion type from primary tumor: VTT upregulated TNFα signaling and associated inflammatory pathways, whereas metastases upregulated MTOR signaling.ConclusionsOur results provide a map of how RCC tumors can evolve, with metastatic clones typically emerging early in RCC development and taking hold via MTOR signaling, and later formation of VTT via local inflammatory processes.Statement of Translational RelevanceRenal cell carcinoma (RCC) is a deadly and relatively common malignancy, which often presents as or progresses to metastatic disease. We used multi-region sequencing of RCC patients with venous tumor thrombus (VTT) and metastasis to ask how and when new lesions arise from the primary tumor, and what genomic factors contribute to their spread. Phylogenetic analysis of patients with VTT and co-presenting metastases suggested that in most cases, the VTT and metastases derive from distinct tumor clones. Moreover, metastatic clones often appear many years prior to diagnosis. We found that local TNFα inflammation may contribute to VTT formation, whereas MTOR signaling is associated with metastases. Our study sheds light on the relationship of VTT and metastases, suggests therapeutic and biomarker strategies for RCC, and points to the need for early detection studies in RCC to better understand when metastases emerge and to identify at-risk patients.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&E) annotations are used to target small tumor areas using a blade that is 250 µm2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 µm in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Optimization and standardization of immunohistochemistry (IHC) protocols within and between laboratories requires reproducible positive and negative control samples. In many situations, suitable tissue or cell line controls are not available. We demonstrate here a method to incorporate target antigens into synthetic protein gels that can serve as IHC controls. The method can use peptides, protein domains, or whole proteins as antigens, and is compatible with a variety of fixation protocols. The resulting gels can be used to create tissue microarrays (TMAs) with a range of antigen concentrations that can be used to objectively quantify and calibrate chromogenic, fluorescent, or mass spectrometry-based IHC protocols. The method offers an opportunity to objectively quantify IHC staining results, and to optimize and standardize IHC protocols within and between laboratories. (J Histochem Cytochem 58:XXX–XXX, 2019)

Personalized cancer immunotherapies (pCIT) rely on targeting of somatic cancer mutations, which in their translated peptide form are known as neoantigens. Each tumor has a unique set of mutations, and thus, the vast majority of cancer neoantigens targeted by these personalized therapies are private to an individual's tumor. We sought to understand whether a single strategy for targeting private neoantigens—prioritizing clonal neoantigens over subclonal ones—could apply equally well across primary and metastatic lesions in patients with advanced metastatic solid tumors, and across different cancer indications. Previous studies of largely primary tumors in non-metastatic settings have suggested that indications such as melanoma and NSCLC consistently have low mutational heterogeneity and a preponderance of clonal neoantigens, whereas other indications such as CRC, RCC, and breast cancer often have a higher degree of mutational heterogeneity and fewer clonal neoantigens. It remains unclear whether standard clonality metrics (e.g., cancer cell fraction or "CCF") can accurately predict global mutation clonality across tumor lesions and whether CCF could offer any predictive benefit over simple variant allele frequencies. By multi-region sequencing analysis of five metastatic solid tumors across four indications (CRC, NSCLC, RCC, UBC), with all lesions sampled at the same time point, we characterized neoantigen heterogeneity and whether the clonality and expression level of mutations would influence the likely immunogenicity of neoantigen candidates selected for pCIT. We show that the ability to target cancer neoantigens effectively depends on the type of lesion sampled and on indication. In NSCLC and UBC, where most mutations were shared across tumor lesions, effective targeting could be accomplished by sampling either the primary or certain metastatic lesions. However, CRC and RCC tumors had more complex, branching mutational phylogenies, leading to non-overlapping sets of mutations across different tumor lesions. On average, across the five metastatic cases, a given tumor sample’s predictive value for global mutation clonality varied from 20% (RCC) to 70% (NSCLC). Enrichment of clonal neoantigens could be accomplished by prioritizing mutations with higher variant allele frequency (VAF), as expected, but using CCF for mutation prioritization led to poorer enrichment of clonal neoantigens. In one case (UBC), we observed truncal neoantigen reduction via down-regulation of mutant allele expression, suggesting that early immune recognition of tumors is an important factor in pCIT targeting. Our data suggest that indication-specific neoantigen targeting strategies, which consider mutation presence and expression across multiple tumor lesions, may be necessary for pCIT to be broadly effective. Citation Format: Amy Lo, Andrew Wallace, Daniel Oreper, Nicolas Lounsbury, Charles Havnar, Carmina Espiritu, Ximo Pechuan-Jorge, Richard Bourgon, Ryan Jones, Katrina Krogh, Guang-Yu Yang, Oliver Zill. Multi-region sequencing analysis of metastatic solid tumors to inform targeting of personalized cancer immunotherapies [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO092.

Interleukin-2 (IL-2)-inducible T cell kinase (ITK) mediates T cell receptor (TCR) signaling primarily to stimulate the production of cytokines, such as IL-4, IL-5, and IL-13, from T helper 2 (TH2) cells. Compared to wild-type mice, ITK knockout mice are resistant to asthma and exhibit reduced lung inflammation and decreased amounts of TH2-type cytokines in the bronchoalveolar lavage fluid. We found that a small-molecule selective inhibitor of ITK blocked TCR-mediated signaling in cultured TH2 cells, including the tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) and the secretion of IL-2 and TH2-type cytokines. Unexpectedly, inhibition of the kinase activity of ITK during or after antigen rechallenge in an ovalbumin-induced mouse model of asthma failed to reduce airway hyperresponsiveness and inflammation. Rather, in mice, pharmacological inhibition of ITK resulted in T cell hyperplasia and the increased production of TH2-type cytokines. Thus, our studies predict that inhibition of the kinase activity of ITK may not be therapeutic in patients with asthma.