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PurposeGiven the unmet need for novel immunotherapy in soft tissue sarcoma (STS), we sought to characterize the phenotype and function of intratumoral natural killer (NK) and T cells to identify novel strategies to augment tumor-infiltrating lymphocyte (TIL) function.Experimental designUsing prospectively collected specimens from dogs and humans with sarcomas, archived specimens, and The Cancer Genome Atlas (TCGA) data, we evaluated blood and tumor NK and T cell phenotype and function and correlated those with outcome. We then assessed the effects of interleukin 15 (IL-15) stimulation on both NK and T cell activation and TIGIT upregulation. Finally, we evaluated cytotoxic effects of IL-15 combined with TIGIT blockade using a novel anti-TIGIT antibody.ResultsTILs were strongly associated with survival outcome in both archived tissue and TCGA, but higher TIL content was also associated with higher TIGIT expression. Compared with blood, intratumoral NK and T cells showed significantly higher expression of both activation and exhaustion markers, in particular TIGIT. Ex vivo stimulation of blood and tumor NK and T cells from patients with STS with IL-15 further increased both activation and exhaustion markers, including TIGIT. Dogs with metastatic osteosarcoma receiving inhaled IL-15 also exhibited upregulation of activation markers and TIGIT. Ex vivo, combined IL-15 and TIGIT blockade using STS blood and tumor specimens significantly increased cytotoxicity against STS targets.ConclusionIntratumoral NK and T cells are prognostic in STS, but their activation is marked by significant upregulation of TIGIT. Our data suggest that combined IL-15 and TIGIT blockade may be a promising clinical strategy in STS.

Soft tissue sarcomas (STS) are rare, heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cells and spatial relationships of TILs with MHC-I expressing cells lacks detailed characterization. Using archived and prospectively collected specimens, we evaluated intratumoral NK cells by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). We assessed spatial localization of NK and T cells by multiplex IF, analyzing the effects of MHC-I expression status on NK and T cell clustering. Both intratumoral NKp46 and CD56 expression were associated with significantly improved overall survival (P=0.05), while higher infiltrates of CD56bright NK cells predicted a worse prognosis (P=0.05). The presence of intratumoral NK cells was inversely proportional to CD3 T cells. Spatial analyses showed NK cells preferentially clustering close to other NK cells with sparse CD3 T and CD8 T cells in range (P<0.0001). Additionally, CD3 T and CD8 T cells showed significantly greater co-localization with MHC-I cells, compared to NK cells (P<0.0001). After neoadjuvant radiotherapy, there was greater CD8 clustering, while after neoadjuvant chemotherapy, there was overall lower TIL clustering. Intratumoral NK cells are prognostic in STS and localize closer to MHC-I- cells than T cells. Although both NK and T cells are associated with improved survival in STS, their differential distribution in the TME based on MHC-I expression status may serve as a biomarker for improved immunotherapy treatment selection.

ObjectivesThe objectives of the study are (1) to distinguish lipoma (L) from atypical lipomatous tumor (ALT) using MRI qualitative features, (2) to assess the value of contrast enhancement, and (3) to evaluate the reproducibility and confidence level of radiological readings.Materials and methodsPatients with pathologically proven L or ALT, who underwent MRI within 3 months from surgical excision were included in this retrospective multicenter international study. Two radiologists independently reviewed MRI centrally. Impressions were recorded as L or ALT. A third radiologist was consulted for discordant readings. The two radiologists re-read all non-contrast sequences; impression was recorded; then post-contrast images were reviewed and any changes were recorded.ResultsA total of 246 patients (135 females; median age, 59 years) were included. ALT was histopathologically confirmed in 70/246 patients. In multivariable analysis, in addition to the lesion size, deep location, proximal lower limb lesions, demonstrating incomplete fat suppression, or increased architectural complexity were the independent predictive features of ALT; but not the contrast enhancement. Post-contrast MRI changed the impression in a total of 5 studies (3 for R1 and 4 for R2; 2 studies are common); all of them were incorrectly changed from Ls to ALTs. Overall, inter-reader kappa agreement was 0.42 (95% CI 0.39–0.56). Discordance between the two readers was statistically significant for both pathologically proven L (p < 0.001) and ALT (p = 0.003).ConclusionMost qualitative MR imaging features can help distinguish ALTs from BLs. However, contrast enhancement may be limited and occasionally misleading. Substantial discordance on MRI readings exists between radiologists with a relatively high false positive and negative rates.

Clinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS. Using fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3 CD56 NK and CD3 T cells and CD45 viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression. Sorted intra-tumoral CD3 T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45 tumor cells. TCGA analysis revealed that tumors with high TLR4 expression ( = 0.03) and low expression of STMN1 involved in microtubule polymerization ( < 0.001) were associated with significantly improved survival. Unlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.

BackgroundGroundbreaking studies have linked the gut microbiome with immune homeostasis and antitumor immune responses. Mounting evidence has also demonstrated an intratumoral microbiome, including in soft tissue sarcomas (STS), although detailed characterization of the STS intratumoral microbiome is limited. We sought to characterize the intratumoral microbiome in patients with STS undergoing preoperative radiotherapy and surgery, hypothesizing the presence of a distinct intratumoral microbiome with potentially clinically significant microbial signatures.MethodsWe prospectively obtained tumor and stool samples from adult patients with non-metastatic STS using a strict sterile collection protocol to minimize contamination. Metagenomic classification was used to estimate abundance using genus and species taxonomic levels across all classified organisms, and data were analyzed with respect to clinicopathologic factors.ResultsFifteen patients were enrolled. Most tumors were located at an extremity (67%) and were histologic grade 3 (87%). 40% were well-differentiated/dedifferentiated liposarcoma histology. With a median follow-up of 24 months, 4 (27%) patients developed metastases, and 3 (20%) died. Despite overwhelming human DNA (>99%) intratumorally, we detected a small but consistent proportion of bacterial DNA (0.02–0.03%) in all tumors, including Proteobacteria, Bacteroidetes, and Firmicutes, as well as viral species. In the tumor microenvironment, we observed a strong positive correlation between viral relative abundance and natural killer (NK) infiltration, and higher NK infiltration was associated with superior metastasis-free and overall survival by immunohistochemical, flow cytometry, and multiplex immunofluorescence analyses.ConclusionsWe prospectively demonstrate the presence of a distinct and measurable intratumoral microbiome in patients with STS at multiple time points. Our data suggest that the STS tumor microbiome has prognostic significance with viral relative abundance associated with NK infiltration and oncologic outcome. Additional studies are warranted to further assess the clinical impact of these findings.

PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.

Background Neoadjuvant radiotherapy (RT) is increasingly advocated for the management of soft tissue sarcoma (STS). Therefore, this study sought to characterize the impact of neoadjuvant RT on rates of R0 resection and overall survival (OS) in extremity STS patients undergoing surgery. Methods From January 2003 to December 2012, the study identified patients with a diagnosis of extremity STS from the National Cancer Database. After exclusion of patients younger than 18 years, not treated by surgery, who had metastases at diagnosis, intraoperative RT, and missing or unknown data, 27,969 patients were identified. Logistic regression and Cox-proportional hazard analysis were used to compare rates of R0 resection among preoperative, postoperative, and no-RT cohorts and to determine predictors of R0 resection and OS. Results The mean age of the patients was 59.5 ± 17.1 years, and 45.9% were female. The median tumor size was 10.5 cm. The data showed that 51% of the patients did not receive RT, 11.8% received preoperative RT, and 37.2% received postoperative RT. The rates of R0 resection were 90.1% for the preoperative RT cohort, 74.9% for the postoperative RT cohort, and 79.9% for the no-RT cohort ( P  < 0.001). The independent predictors for achievement of R0 resection included academic facility type (odds ratio [OR] 1.36; 95% confidence interval [CI] 1.20–1.55), histologic subtype, tumor size (OR 0.99; 95% CI 0.99–0.99), Charlson score (OR 0.92; 95% CI 0.84–0.99), and preoperative RT (OR 1.83; 95% CI 1.61–2.07). Both R0 resection and RT (pre- or post-operative) were associated with increased OS. Conclusions Preoperative RT independently predicts higher rates of R0 resection for patients with extremity STS undergoing surgical resection. Negative surgical margins and pre- or postoperative RT are associated with improved OS.

The adoption of digital capture of pathology slides as whole slide images (WSI) for educational and research applications has proven utility. To compare pathologists' primary diagnoses derived from WSI versus the standard microscope. Because WSIs differ in format and method of observation compared with the current standard glass slide microscopy, this study is critical to potential clinical adoption of digital pathology. The study enrolled a total of 2045 cases enriched for more difficult diagnostic categories and represented as 5849 slides were curated and provided for diagnosis by a team of 19 reading pathologists separately as WSI or as glass slides viewed by light microscope. Cases were reviewed by each pathologist in both modalities in randomized order with a minimum 31-day washout between modality reads for each case. Each diagnosis was compared with the original clinical reference diagnosis by an independent central adjudication review. The overall major discrepancy rates were 3.64% for WSI review and 3.20% for manual slide review diagnosis methods, a difference of 0.44% (95% CI, -0.15 to 1.03). The time to review a case averaged 5.20 minutes for WSI and 4.95 minutes for glass slides. There was no specific subset of diagnostic category that showed higher rates of modality-specific discrepancy, though some categories showed greater discrepancy than others in both modalities. WSIs are noninferior to traditional glass slides for primary diagnosis in anatomic pathology.

Pathology on-call experiences help prepare trainees for successful transition from residency to independent practice, and as such are an integral component of training. However, few data exist on anatomic pathology resident on-call workload and experience. To obtain an overall picture of the anatomic pathology on-call experience to inform and improve resident education. Retrospective and prospective review of daily anatomic pathology on-call summaries from July 2016 to June 2020. During the first 2 years of the study (ie, retrospective portion), only 19% of on-call summaries (138 of 730) were available for review. After interventions, the on-call summary submission rate jumped to 98% (716 of 731). After-hours calls were most frequent on weekdays from 5 to 8 pm. The most frequent requests were for frozen sections (55%; 619 of 1125 calls), inquiries regarding disposition of fresh placentas (13%; 148 of 1125 calls), and inquiries regarding disposition of various other specimens (6%; 68 of 1125 calls). After-hours frozen section requests were most frequent for gynecologic and head and neck specimens. Notably, a significant number of after-hours calls were recurring preanalytic issues amenable to system-level improvements. We were able to eliminate the most common of these recurring preanalytic calls with stepwise interventions. To our knowledge, this is the first study analyzing the anatomic pathology resident on-call experience. In addition to obtaining a broad overview of the residents' clinical exposure on this service, we identified and resolved issues critical to optimal patient care (eg, inconsistent \"patient hand-off\") and improved the resident on-call experience (eg, fewer preanalytic calls increased resident time for other clinical, educational, or wellness activities).

Ataxia–telangiectasia confers a significant increase in the development of several cancer types, most commonly leukemia and lymphoma. However, as the natural history for these patients is evolving and their lifespan is increasing, there is the potential for the development of additional uncommon tumors in an already rare patient population. We report the first case, to our knowledge, of an incidental retroperitoneal tumor in a 26-year-old woman undergoing evaluation for hepatic dysfunction. The mass was suspicious for retroperitoneal sarcoma, but proved to be an extramedullary hematopoietic pseudotumor after extensive pathologic evaluation. The changing landscape of neoplasms associated with ataxia–telangiectasia is discussed with emphasis on previously underreported benign and malignant tumors.