Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility.

Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA.

Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We developed an unsupervised machine learning algorithm, CELESTA, which identifies the cell type of each cell, individually, using the cell's marker expression profile and, when needed, its spatial information. We demonstrate the performance of CELESTA on multiplexed immunofluorescence images of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, and validate our findings in an independent cohort. By coupling our spatial analysis with single-cell RNA-sequencing data on proximal sections of the same specimens, we identify cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of CELESTA to facilitate identification of clinically relevant interactions.

Scattering-Based Light-Sheet Microscopy Imaging of Human Papillomavirus-Associated Squamous Lesions of the Anal Canal: A Proof-of-Principle Study.

Demand for anal cancer screening is expected to rise following the recent publication of the Anal Cancer-HSIL Outcomes Research trial, which showed that treatment of high-grade squamous intraepithelial lesions significantly reduces the rate of progression to anal cancer. While screening for human papillomavirus-associated squamous lesions in the cervix is well established and effective, this is less true for other sites in the lower anogenital tract. Current anal cancer screening and prevention rely on high-resolution anoscopy with biopsies. This procedure has a steep learning curve for providers and may cause patient discomfort. Scattering-based light-sheet microscopy (sLSM) is a novel imaging modality with the potential to mitigate these challenges through real-time, microscopic visualization of disease-susceptible tissue. Here, we report a proof-of-principle study that establishes feasibility of dysplasia detection using an sLSM device. We imaged 110 anal biopsy specimens collected prospectively at our institution's dysplasia clinic (including 30 nondysplastic, 40 low-grade squamous intraepithelial lesion, and 40 high-grade squamous intraepithelial lesion specimens) and found that these optical images are highly interpretable and accurately recapitulate histopathologic features traditionally used for the diagnosis of human papillomavirus-associated squamous dysplasia. A reader study to assess diagnostic accuracy suggests that sLSM images are noninferior to hematoxylin and eosin images for the detection of anal dysplasia (sLSM accuracy = 0.87; hematoxylin and eosin accuracy = 0.80; P = .066). Given these results, we believe that sLSM technology holds great potential to enhance the efficacy of anal cancer screening by allowing accurate sampling of diagnostic tissue at the time of anoscopy. While the current imaging study was performed on ex vivo biopsy specimens, we are currently developing a handheld device for in vivo imaging that will provide immediate microscopic guidance to high-resolution anoscopy providers.

Occurrence and Timing of Subsequent Severe Acute Respiratory Syndrome Coronavirus 2 Reverse-transcription Polymerase Chain Reaction Positivity Among Initially Negative Patients.

Using data for 20 912 patients from 2 large academic health systems, we analyzed the frequency of severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and was similar across institutions.

Test Characteristics of Specific p16 Clones in the Detection of High-grade Squamous Intraepithelial Lesions (HSIL).

p16 immunohistochemistry is recommended by the CAP-ASCCP Lower Anogenital Squamous Terminology (LAST) Standardization Project for human papillomavirus associated Lesions as an adjunct to morphologic assessment in the diagnosis of high-grade squamous intraepithelial lesion. This study evaluates the performance of different p16 clones as compared with E6H4 (CINtec) in detecting high-grade squamous intraepithelial lesion. The 54 high-quality articles addressing the performance of p16 identified by work group 4 of the LAST Project were evaluated for: specific p16 clone, scoring method, number of cases, anatomic site, and histologic diagnoses. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for each clone. Two-proportion z tests (pooled) were used to evaluate significance. In total, 32 of the 54 studies met the inclusion criteria. The most commonly used clone was E6H4 (17 studies, 3507 cases) with smaller numbers (1-4) of studies evaluating the following: 16P04, JC8, 16P07, G175-405, K5334, K5336, and 7962. p16 clones 16P04 and JC8 performed better than E6H4 with 16P04 exhibiting statistically significant higher sensitivity (94% vs. 87% for E6H4), specificity (94% vs. 81%), and positive predictive value (96% vs. 69%) while JC8 exhibited higher specificity (91% vs. 81%) and positive predictive value (88% vs. 69%). 16P07 performed similarly to E6H4 and the other 4 clones did not perform as well as E6H4. p16 clones 16P04, JC8, and 16P07 clones perform as well or better than the widely used p16 clone E6H4 (CINtec). However, further studies are indicated to determine the reproducibility of these findings and the impact of interlaboratory variation on test performance.

Vulvar and Anal Intraepithelial Neoplasia: Terminology, Diagnosis, and Ancillary Studies.

Currently, it is recognized that there is an HPV-related and an HPV-independent pathway to developing squamous cell carcinomas (SCC) in the anus and vulva. The majority of precursor lesions and SCC in the anus and vulva are high-risk HPV-associated, with HPV16 the most common type. Given the morphologic overlap and biological equivalence of HPV-related preinvasive squamous lesions of the lower anogenital tract, a unified, 2-tiered histopathologic nomenclature is now recommended. In contrast, mutations in the TP53 gene have been associated with HPV-independent vulvar and anal SCC. A precursor lesion-differentiated or simplex vulvar intraepithelial neoplasia (dVIN)-has been identified for HPV-independent vulvar SCC but a similar lesion in the anus has not been described. Extramammary Paget disease is a nonsquamous intraepithelial lesion of the vulva and anus that may be a primary epidermotropic apocrine neoplasm or may represent secondary involvement by a synchronous/metachronous adenocarcinoma. This entity may be mimicked by squamous lesions and melanocytic lesions. Herein, we discuss the morphologic and immunohistochemical features of anal and vulvar intraepithelial neoplasia in the context of updated terminology and current understanding of disease biology.

Results of the Women's Self-Performed Anal Pap Trial in Human Immunodeficiency Virus-Infected Women.

High-risk human papillomavirus anal infections are common in human immunodeficiency virus-infected women. We conducted a cross-over study in 30 women seen in a California human immunodeficiency virus clinic, to test the feasibility of self-performed anal Pap smears. Women found the tests acceptable and feasible. Compared with physician-collected specimens, results were highly concordant for anal cytology (κ = 0.53) and high-risk human papillomavirus typing (κ = 0.59 inclusive of equivocal results, or κ = 0.81 excluding equivocal results).

Adenocarcinoma With Breast/Adnexal and Upper Gastrointestinal Differentiation Arising in an Ovarian Mature Cystic Teratoma: A Case Report and Review of the Literature.

Mature cystic teratomas are the most common type of ovarian germ cell tumors. In about 1% of cases, usually among postmenopausal women, a mature cystic teratoma can undergo malignant transformation. Among malignant transformations, squamous cell carcinoma is the most common histology, comprising approximately 80% of cases. In this report, we present the unique case of a 55-yr-old woman with a pelvic mass found to be a mature cystic teratoma with malignant transformation to adenocarcinoma with breast/adnexal, upper gastrointestinal, and neuroendocrine differentiation. The predominant malignant component was the adenocarcinoma exhibiting breast/skin adnexal differentiation, which was found to involve the omentum and a right para-aortic node. We provide an in-depth review of the pathologic findings, as well as a review of the current literature on malignant transformation to adenocarcinoma. This report aims to open a conversation regarding the management of these patients, with a specific focus on the role of molecular analysis and targeted therapies.

Sonographic-Pathologic Correlation for Punctate Echogenic Reflectors in Papillary Thyroid Carcinoma: What Are They?

OBJECTIVES: It is commonly held that punctate nonshadowing echogenic foci on sonography, often termed microcalcifications, represent psammoma bodies. We aimed to determine the validity of this supposition by correlating the presence of punctate echogenic foci on sonography with their presence at histopathologic examination. METHODS: We examined 51 nodules (surgically proven papillary thyroid carcinoma) by sonography and histopathologic examination. On the latter, nodules were examined for evidence of psammomatous calcifications, dystrophic calcifications, and colloid. Two subspecialty-trained radiologists with 2 and 25 years of experience in sonography, respectively, reviewed the sonograms for the presence and distribution of punctate echogenic foci. RESULTS: All nodules contained colloid at histologic examination. Twenty of the papillary carcinomas lacked any calcification at pathologic examination. In the remaining 31 nodules with calcifications, 13 had psammomatous calcifications only; 6 had both coarse and psammomatous calcifications; and 12 had only coarse calcifications. The presence of punctate echogenic foci on sonography was 74% sensitive, was 46% to 53% specific, and had a positive predictive value of only 45% to 48% for the presence of psammomatous calcifications. The computed 2-tailed P value indicated that the punctate echogenic foci-to-psammoma body correlation was not statistically significant. CONCLUSIONS: The sonographic signature commonly referred to as "microcalcifications" may represent a variety of entities, including psammomatous calcifications, dystrophic calcifications, and eosinophilic colloid; for this reason, "punctate echogenic foci" would be a more accurate term.

Performance of MYC, BCL2, and BCL6 break-apart FISH in small biopsies with large B-cell lymphoma: a retrospective Cytopathology Hematopathology Interinstitutional Consortium study.

Introduction: Fluorescence in situ hybridization (FISH) is an essential ancillary study used to identify clinically aggressive subsets of large B-cell lymphomas that have MYC, BCL2, or BCL6 rearrangements. Small-volume biopsies such as fine needle aspiration biopsy (FNAB) and core needle biopsy (CNB) are increasingly used to diagnose lymphoma and obtain material for ancillary studies such as FISH. However, the performance of FISH in small biopsies has not been thoroughly evaluated or compared to surgical biopsies. Methods: We describe the results of MYC, BCL2, and BCL6 FISH in a series of 222 biopsy specimens, including FNAB with cell blocks, CNBs, and surgical excisional or incisional biopsies from 208 unique patients aggregated from 6 academic medical centers. A subset of patients had FNAB followed by a surgical biopsy (either CNB or excisional biopsy) obtained from the same or contiguous anatomic site as part of the same clinical workup; FISH results were compared for these paired specimens. Results: FISH had a low hybridization failure rate of around 1% across all specimen types. FISH identified concurrent MYC and BCL2 rearrangements in 20 of 197 (10%) specimens and concurrent MYC and BCL6 rearrangements in 3 of 182 (1.6%) specimens. The paired FNAB and surgical biopsy specimens did not show any discrepancies for MYC or BCL2 FISH; of the 17 patients with 34 paired cytology and surgical specimens, only 2 of the 49 FISH probes compared (4% of all comparisons) showed any discrepancy and both were at the BCL6 locus. One discrepancy was due to necrosis of the CNB specimen causing a false negative BCL6 FISH result when compared to the FNAB cell block that demonstrated a BCL6 rearrangement. Discussion: FISH showed a similar hybridization failure rate in all biopsy types. Ultimately, MYC, BCL2, or BCL6 FISH showed 96% concordance when compared across paired cytology and surgical specimens, suggesting FNAB with cell block is equivalent to other biopsy alternatives for evaluation of DLBCL or HGBCL FISH testing.

Intra- and post-pandemic impact of the COVID-19 outbreak on Stanford Health Care.

Stanford Health Care, which provides about 7% of overall healthcare to approximately 9 million people in the San Francisco Bay Area, has undergone significant changes due to the opening of a second hospital in late 2019 and, more importantly, the COVID-19 pandemic. We examine the impact of these events on anatomic pathology (AP) cases, aiming to enhance operational efficiency in response to evolving healthcare demands. We extracted historical census, admission, lab tests, operation, and AP data since 2015. An approximately 45% increase in the volume of laboratory tests (P < 0.0001) and a 17% increase in AP cases (P < 0.0001) occurred post-pandemic. These increases were associated with progressively increasing (P < 0.0001) hospital census. Census increase stemmed from higher admission through the emergency department (ED), and longer lengths of stay mostly for transfer patients, likely due to the greater capability of the new ED and changes in regional and local practice patterns post-pandemic. Higher census led to overcapacity, which has an inverted U relationship that peaked at 103% capacity for AP cases and 114% capacity for laboratory tests. Overcapacity led to a lower capability to perform clinical activities, particularly those related to surgical procedures. We conclude by suggesting parameters for optimal operations in the post-pandemic era.

Synthetic whole-slide image tile generation with gene expression profile-infused deep generative models.

In this work, we propose an approach to generate whole-slide image (WSI) tiles by using deep generative models infused with matched gene expression profiles. First, we train a variational autoencoder (VAE) that learns a latent, lower-dimensional representation of multi-tissue gene expression profiles. Then, we use this representation to infuse generative adversarial networks (GANs) that generate lung and brain cortex tissue tiles, resulting in a new model that we call RNA-GAN. Tiles generated by RNA-GAN were preferred by expert pathologists compared with tiles generated using traditional GANs, and in addition, RNA-GAN needs fewer training epochs to generate high-quality tiles. Finally, RNA-GAN was able to generalize to gene expression profiles outside of the training set, showing imputation capabilities. A web-based quiz is available for users to play a game distinguishing real and synthetic tiles: https://rna-gan.stanford.edu/, and the code for RNA-GAN is available here: https://github.com/gevaertlab/RNA-GAN.

NFE2L2 Mutations Enhance Radioresistance in Head and Neck Cancer by Modulating Intratumoral Myeloid Cells.

Radiotherapy (RT) is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high-risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, whereas the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. RT increased expression of PMN-MDSC-attracting chemokines, including CXCL1, CXLC3, and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an antitumor phenotype, supporting clinical testing of CB-839 with RT in HNSCC with NFE2L2 mutations. SIGNIFICANCE: NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance.

Are women with endocervical adenocarcinoma at risk for lynch syndrome? Evaluation of 101 cases including unusual subtypes and lower uterine segment tumors.

It is well documented that endometrial and ovarian carcinoma are associated with Lynch syndrome (LS), but the association, if any, between endocervical carcinoma and LS has not been fully evaluated. The relationship between endocervical carcinoma and LS is particularly relevant, given the apparent affinity of LS-associated endometrial carcinomas for the lower uterine segment and the attendant difficulties in determining tumor origin at this site. In this study, we examined mismatch repair (MMR) protein expression (MLH1, MSH2, MSH6, and PMS2) in 60 endocervical adenocarcinomas, including variants (minimal deviation adenocarcinoma, mesonephric adenocarcinoma, adenosquamous carcinoma, clear cell carcinoma) and a series of well-characterized lower-uterine segment carcinomas of known endocervical or endometrial origin (n=41). Two of the lower uterine segment tumors occurred in risk-reducing hysterectomy specimens from known LS patients. All endocervical adenocarcinomas including variants and lower uterine segment endocervical tumors (1 from a known LS patient) were proficient in all 4 MMR proteins. In contrast, 2/20 (10%) lower uterine segment endometrial cancers were deficient in at least 1 MMR (1 from a known LS patient). These data provide evidence that, unlike endometrial and ovarian adenocarcinoma, there is no association between LS and endocervical carcinoma. MMR testing is prudent in lower uterine segment tumors in women with possible LS, especially those for which definitive site of origin cannot be determined.

Differentiated (simplex) vulvar intraepithelial neoplasia: a case report and review of the literature.

Differentiated (simplex) vulvar intraepithelial neoplasia (VIN) is an uncommon variant of VIN characterized by highly differentiated morphology, making it a potential diagnostic pitfall. It may arise in the background of lichen sclerosus, and unlike most VIN, is not causally associated with human papilloma virus infection. It occurs in an older demographic and is thought to be the precursor of aggressive, invasive vulvar squamous cell carcinoma. For this reason, the timely and accurate diagnosis of this unusual lesion is crucial. The clinical and histologic features of a case of a 70-year-old woman with newly diagnosed differentiated (simplex) VIN arising in a background of long-standing lichen sclerosus is reported, and the historic aspects, current terminology, and diagnostic criteria of differentiated (simplex) VIN are reviewed.

Implementation of Collodion Bag Protocol to Improve Whole-slide Imaging of Scant Gynecologic Curettage Specimens.

BACKGROUND: Digital pathology has been increasingly implemented for primary surgical pathology diagnosis. In our institution, digital pathology was recently deployed in the gynecologic (GYN) pathology practice. A notable challenge encountered in the digital evaluation of GYN specimens was high rates of scanning failure of specimens with fragmented as well as scant tissue. To improve tissue detection failure rates, we implemented a novel use of the collodion bag cell block preparation method. MATERIALS AND METHODS: In this study, we reviewed 108 endocervical curettage (ECC) specimens, representing specimens processed with and without the collodion bag cell block method (n = 56 without collodion bag, n = 52 with collodion bag). RESULTS: Tissue detection failure rates were reduced from 77% (43/56) in noncollodion bag cases to 23/52 (44%) of collodion bag cases, representing a 42% reduction. The median total area of tissue detection failure per level was 0.35 mm2 (interquartile range [IQR]: 0.14, 0.70 mm2) for noncollodion bag cases and 0.08 mm2 (IQR: 0.03, 0.20 mm2) for collodion bag cases. This represents a greater than fourfold reduction in the total area of tissue detection failure per level (P < 0.001). In addition, there were no out-of-focus levels among collodion bag cases, compared to 6/56 (11%) of noncollodion bag cases (median total area = 4.9 mm2). CONCLUSIONS: The collodion bag method significantly improved the digital image quality of fragmented/scant GYN curettage specimens, increased efficiency and accuracy of diagnostic evaluation, and enhanced identification of tissue contamination during processing. The logistical challenges and labor cost of deploying the collodion bag protocol are important considerations for feasibility assessment at an institutional level.

Pilot study of loss of the p53/p63 target gene PERP at the surgical margin as a potential predictor of local relapse in head and neck squamous cell carcinoma.

BACKGROUND: PERP (p53 apoptosis effector related to PMP22) localizes to desmosomes and suppresses squamous cell carcinoma development. Loss of PERP leads to worse local control in head and neck squamous cell carcinoma (HNSCC), likely by destabilizing desmosomes. We evaluated PERP loss at HNSCC surgical margins as a predictor of local relapse. METHODS: Combining discovery (n = 17) and validation (n = 31) cohorts, we examined membranous PERP protein expression by immunohistochemistry in surgical mucosal margins with competing risk analysis of the relationship between local relapse and PERP expression. RESULTS: Of the 44 analyzable patients, the 2-year cumulative incidence of local relapse was 44.4% for the PERP-negative group and 16.4% for the PERP-positive group (P = .01). A trend toward worse progression-free survival (P = .09) and overall survival (P = .06) was observed with loss of PERP. CONCLUSIONS: PERP loss at surgical margins is associated with higher risk of local recurrence in HNSCC, warranting further evaluation in a larger prospective study.

Role of FNA with core biopsy or cell block in patients with nodular lymphocyte-predominant Hodgkin lymphoma.

BACKGROUND: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) represents a diagnostic challenge on surgical excisional or incisional biopsy. Classification is further challenging on fine needle aspiration (FNA) material accompanied by needle core and/or cell block biopsy (FNA+core/CB). METHODS: The authors studied all FNA+core/CB and surgical excisional or incisional biopsies to evaluate for lymphoma in patients who had a prior history of NLPHL or subsequent diagnosis of NLPHL over a 5-year period from 2012 through 2016. RESULTS: Patients who ultimately were diagnosed with NLPHL represented <0.5% of those who underwent FNA+core/CB for an initial suspicion of lymphoma. FNA+core/CB resulted in a definitive diagnosis in 7 of 13 cases, and surgical excisional or incisional biopsy specimens resulted in a definitive diagnosis in 13 of 13 cases (chi-square statistic, 9.6; P = .002). At initial diagnosis, FNA+core/CB was negative in 2 cases and atypical or suspicious in 3 cases; all 5 of those patients required surgical excisional or incisional biopsy for a definitive lymphoma diagnosis. By contrast, patients who underwent FNA+core/CB for recurrent lymphoma required surgical excisional or incisional biopsy in only 1 of 8 cases (chi-square statistic, 9.5; P = .002). Flow cytometry was positive for a light-chain-restricted B-cell population in only 1 of 11 biopsies that were involved by lymphoma. CONCLUSIONS: Surgical excisional or incisional biopsy remains the gold standard for NLPHL diagnosis and for distinguishing progression to a T-cell/histiocyte-rich large B-cell lymphoma pattern. At a tertiary cancer center with routine collaborative diagnosis of lymphoma on FNA+core/CB by cytopathologists and hematopathologists, FNA+core/CB performs well to assess for recurrent or transformed NLPHL, rarely requiring subsequent surgical excisional or incisional biopsy. FNA+core/CB has limited sensitivity in the initial diagnosis setting.

Multiregion Quantification of Extracellular Signal-regulated Kinase Activity in Renal Cell Carcinoma.

To personalize treatment for renal cell carcinoma (RCC), it would be ideal to confirm the activity of druggable protein pathways within individual tumors. We have developed a high-resolution nanoimmunoassay (NIA) to measure protein activity with high precision in scant specimens (eg, fine needle aspirates [FNAs]). Here, we used NIA to determine whether protein activation varied in different regions of RCC tumors. Since most RCC therapies target angiogenesis by inhibiting the vascular endothelial growth factor (VEGF) receptor, we quantified phosphorylation of extracellular signal-regulated kinase (ERK), a downstream effector of the VEGF signaling pathway. In 90 ex vivo FNA biopsies sampled from multiple regions of 38 primary clear cell RCC tumors, ERK phosphorylation differed among patients. In contrast, within individual patients, we found limited intratumoral heterogeneity of ERK phosphorylation. Our results suggest that measuring ERK in a single FNA may be representative of ERK activity in different regions of the same tumor. As diagnostic and therapeutic protein biomarkers are being sought, NIA measurements of protein signaling may increase the clinical utility of renal mass biopsy and allow for the application of precision oncology for patients with localized and advanced RCC. PATIENT SUMMARY: In this report, we applied a new approach to measure the activity of extracellular signal-regulated kinase (ERK), a key cancer signaling protein, in different areas within kidney cancers. We found that ERK activity varied between patients, but that different regions within individual kidney tumors showed similar ERK activity. This suggests that a single biopsy of renal cell carcinoma may be sufficient to measure protein signaling activity to aid in precision oncology approaches.