The immunoregulatory landscape of human tuberculosis granulomas.

Tuberculosis (TB) in humans is characterized by formation of immune-rich granulomas in infected tissues, the architecture and composition of which are thought to affect disease outcome. However, our understanding of the spatial relationships that control human granulomas is limited. Here, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) to image 37 proteins in tissues from patients with active TB. We constructed a comprehensive atlas that maps 19 cell subsets across 8 spatial microenvironments. This atlas shows an IFN-γ-depleted microenvironment enriched for TGF-ß, regulatory T cells and IDO1+ PD-L1+ myeloid cells. In a further transcriptomic meta-analysis of peripheral blood from patients with TB, immunoregulatory trends mirror those identified by granuloma imaging. Notably, PD-L1 expression is associated with progression to active TB and treatment response. These data indicate that in TB granulomas, there are local spatially coordinated immunoregulatory programs with systemic manifestations that define active TB.

The abundance and morphology of human large intestinal goblet and tuft cells during chronic schistosomiasis.

Schistosomiasis affects nearly 240 million people in predominately low- and middle-income countries and ranks second in the number of cases and socio-economic burden among all parasitic diseases. Despite the enormous burden posed by schistosomes, our understanding of how schistosomiasis impacts infected human tissues remains limited. Intestinal schistosomiasis in animal models leads to goblet cell hyperplasia, likely increasing mucus production and reflecting an intestinal type 2 immune response. However, it is unknown whether these same changes occur in schistosome-infected humans. Using immunofluorescence and light microscopy, we compared the abundance and morphology of goblet cells in patients diagnosed with schistosomiasis to uninfected controls. The mucin-containing vesicles in goblet cells from schistosome-infected patients were significantly larger (hypertrophic) than uninfected individuals, although goblet cell hyperplasia was absent in chronic human schistosomiasis. In addition, we examined tuft cells in the large intestinal epithelium of control and schistosome-infected patients. Tuft cell numbers expand during helminth infection in mice, but these cells have not been characterized in human parasite infections. We found no evidence of tuft cell hyperplasia during human schistosome infection. Thus, our study provides novel insight into schistosome-associated changes to the intestinal epithelium in humans, suggesting an increase in mucus production by large intestinal goblet cells but relatively minor effects on tuft cell numbers.

Cellular context determines DNA methylation profiles in SWI/SNF-deficient cancers of the gynecologic tract.

SWI/SNF (SWItch/Sucrose Non-Fermentable) complex deficiency has been reported in a wide variety of cancers and is often associated with an undifferentiated phenotype. In the gynecologic tract SWI/SNF-deficient cancers are diagnostically challenging and little is known about their cellular origins. Here we show that undifferentiated endometrial carcinoma (UDEC), SMARCA4-deficient uterine sarcoma (SDUS), and ovarian small cell carcinoma, hypercalcemic type (SCCOHT) harbor distinct DNA methylation signatures despite shared morphology and SWI/SNF inactivation. Our results indicate that the cellular context is an important determinant of the epigenetic landscape, even in the setting of core SWI/SNF deficiency, and therefore methylation profiling may represent a useful diagnostic tool in undifferentiated, SWI/SNF-deficient cancers. Furthermore, applying copy number analyses and group-wise differential methylation analyses including endometrioid endometrial carcinomas and extracranial malignant rhabdoid tumors, we uncover analogous molecular features in SDUS and SCCOHT in contrast to UDEC. These results suggest that SDUS and SCCOHT represent chromosomally stable SWI/SNF-deficient cancers of the gynecologic tract, which are within the broader spectrum of malignant rhabdoid tumors. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

MUC4 is expressed in alveolar rhabdomyosarcoma.

AIMS: Mucin 4 (MUC4) is a transmembrane glycoprotein normally expressed by several human epithelial surfaces, including those of the colon, vagina, and respiratory tract. Although MUC4 overexpression is seen in various carcinomas, its expression among mesenchymal neoplasms is fairly specific to low-grade fibromyxoid sarcoma and sclerosing epithelioid fibrosarcoma. Having observed unanticipated anti-MUC4 immunoreactivity in rhabdomyosarcoma, we aimed to further characterise its expression. METHODS AND RESULTS: Expression of MUC4 was assessed by immunohistochemistry in a total of 97 rhabdomyosarcomas using formalin-fixed paraffin-embedded tissue sections. MUC4 was expressed by 21 of 26 PAX3/7-FOXO1 fusion-positive cases, wherein immunoreactivity, varying from weak to strong, was present in 20-100% of neoplastic cells. With the exception of one sclerosing rhabdomyosarcoma showing immunoreactivity in 20% of cells, MUC4 was not expressed by embryonal (n = 28), sclerosing (n = 20), or pleomorphic (n = 23) rhabdomyosarcomas. Analysing published gene expression microarray data from a separate cohort of 33 fusion-positive and 25 fusion-negative rhabdomyosarcomas, we found on average a 11.4-fold increased expression in fusion-positive tumours (P = 0.0004). CONCLUSIONS: MUC4 is expressed to a variable extent in the majority of PAX3/7-FOXO1 fusion-positive (alveolar) rhabdomyosarcomas, while expression in other rhabdomyosarcoma subtypes is rare.

Morphological, immunophenotypical and molecular features of hypermutation in colorectal carcinomas with mutations in DNA polymerase ε (POLE).

AIMS: Colorectal carcinomas (CRC) with mismatch repair (MMR) deficiency have increased tumour mutation burden and respond to immune check-point inhibitor therapy. The Cancer Genome Atlas identified hypermutated CRCs with somatic mutations in DNA polymerase ε (POLE) with mutation burdens exceeding that of MMR-deficient CRCs. METHODS AND RESULTS: To identify the morphological, immunophenotypical and molecular features of POLE-mutated CRCs, 63 consecutive MMR-intact CRCs were evaluated by Sanger sequencing for POLE exonuclease domain mutations in exons 9, 11, 13 and 14 and confirmed by next-generation sequencing. Tumour immune microenvironment and IMMUNOSCORE®1 were assessed in POLE-mutated CRCs using immunohistochemistry to detect CD3+ /CD8+ tumour-infiltrating lymphocytes and compared to 59 non-POLE mutated MMR-intact CRC, 10 non-POLE mutated MMR-deficient CRCs and 223 normal colonic mucosa. CONCLUSIONS: A total of 4.8% CRC (four MMR-intact primary and one MMR-intact metastasis) harboured POLE mutations in amino acid 286 in exon 9 (p.P286R) or exon 13 (p.V411L). POLE-mutated CRCs arose in the transverse colon and rectum, were male-predominant, younger and showed increased tumour-infiltrating lymphocytes and immune cells at the tumour-stromal interface. The patient with metastatic POLE-mutated CRC was placed on PD-1 inhibitor treatment with marked and sustained response. These data indicate that POLE-mutated CRCs have hypermutated phenotypes despite MMR-intact status, with mutation burdens higher than that in microsatellite-unstable CRCs. Given the recent approval for treatment of microsatellite-unstable cancer with immune check-point inhibitors, assessment of POLE status may help to guide therapeutic decisions for hypermutated tumours with intact MMR that would otherwise be missed by routine testing.

CDX2 as a Prognostic Biomarker in Stage II and Stage III Colon Cancer.

Background The identification of high-risk stage II colon cancers is key to the selection of patients who require adjuvant treatment after surgery. Microarray-based multigene-expression signatures derived from stem cells and progenitor cells hold promise, but they are difficult to use in clinical practice. Methods We used a new bioinformatics approach to search for biomarkers of colon epithelial differentiation across gene-expression arrays and then ranked candidate genes according to the availability of clinical-grade diagnostic assays. With the use of subgroup analysis involving independent and retrospective cohorts of patients with stage II or stage III colon cancer, the top candidate gene was tested for its association with disease-free survival and a benefit from adjuvant chemotherapy. Results The transcription factor CDX2 ranked first in our screening test. A group of 87 of 2115 tumor samples (4.1%) lacked CDX2 expression. In the discovery data set, which included 466 patients, the rate of 5-year disease-free survival was lower among the 32 patients (6.9%) with CDX2-negative colon cancers than among the 434 (93.1%) with CDX2-positive colon cancers (hazard ratio for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002). In the validation data set, which included 314 patients, the rate of 5-year disease-free survival was lower among the 38 patients (12.1%) with CDX2 protein-negative colon cancers than among the 276 (87.9%) with CDX2 protein-positive colon cancers (hazard ratio, 2.42; 95% CI, 1.36 to 4.29; P=0.003). In both these groups, these findings were independent of the patient's age, sex, and tumor stage and grade. Among patients with stage II cancer, the difference in 5-year disease-free survival was significant both in the discovery data set (49% among 15 patients with CDX2-negative tumors vs. 87% among 191 patients with CDX2-positive tumors, P=0.003) and in the validation data set (51% among 15 patients with CDX2-negative tumors vs. 80% among 106 patients with CDX2-positive tumors, P=0.004). In a pooled database of all patient cohorts, the rate of 5-year disease-free survival was higher among 23 patients with stage II CDX2-negative tumors who were treated with adjuvant chemotherapy than among 25 who were not treated with adjuvant chemotherapy (91% vs. 56%, P=0.006). Conclusions Lack of CDX2 expression identified a subgroup of patients with high-risk stage II colon cancer who appeared to benefit from adjuvant chemotherapy. (Funded by the National Comprehensive Cancer Network, the National Institutes of Health, and others.).

Chromosomal copy number alterations for associations of ductal carcinoma in situ with invasive breast cancer.

INTRODUCTION: Screening mammography has contributed to a significant increase in the diagnosis of ductal carcinoma in situ (DCIS), raising concerns about overdiagnosis and overtreatment. Building on prior observations from lineage evolution analysis, we examined whether measuring genomic features of DCIS would predict association with invasive breast carcinoma (IBC). The long-term goal is to enhance standard clinicopathologic measures of low- versus high-risk DCIS and to enable risk-appropriate treatment. METHODS: We studied three common chromosomal copy number alterations (CNA) in IBC and designed fluorescence in situ hybridization-based assay to measure copy number at these loci in DCIS samples. Clinicopathologic data were extracted from the electronic medical records of Stanford Cancer Institute and linked to demographic data from the population-based California Cancer Registry; results were integrated with data from tissue microarrays of specimens containing DCIS that did not develop IBC versus DCIS with concurrent IBC. Multivariable logistic regression analysis was performed to describe associations of CNAs with these two groups of DCIS. RESULTS: We examined 271 patients with DCIS (120 that did not develop IBC and 151 with concurrent IBC) for the presence of 1q, 8q24 and 11q13 copy number gains. Compared to DCIS-only patients, patients with concurrent IBC had higher frequencies of CNAs in their DCIS samples. On multivariable analysis with conventional clinicopathologic features, the copy number gains were significantly associated with concurrent IBC. The state of two of the three copy number gains in DCIS was associated with a risk of IBC that was 9.07 times that of no copy number gains, and the presence of gains at all three genomic loci in DCIS was associated with a more than 17-fold risk (P = 0.0013). CONCLUSIONS: CNAs have the potential to improve the identification of high-risk DCIS, defined by presence of concurrent IBC. Expanding and validating this approach in both additional cross-sectional and longitudinal cohorts may enable improved risk stratification and risk-appropriate treatment in DCIS.

Female Adnexal Tumor of Probable Wolffian Origin (Wolffian Tumor): A Potential Mimic of Peritoneal Mesothelioma.

Wolffian tumor and its nosologic relative, the recently defined STK11 adnexal tumor are rare neoplasms thought to arise from mesonephric remnants. These tumors typically arise in the broad ligament, fallopian tube, and ovarian hilum and although most are associated with a good prognosis, up to 50% of STK11 adnexal tumors demonstrate aggressive clinical behavior. The chief differential diagnoses include endometrioid adenocarcinoma and sex cord stromal tumors. However, the morphologic and immunohistochemical features of these tumors exhibit considerable overlap with peritoneal mesothelioma. To fully characterize their immunophenotypic signature, we examined a total of 21 cases (18 Wolffian and 3 STK11 adnexal tumors) with standard markers used in the diagnosis of mesothelioma. Morphologic and immunohistochemical (IHC) features were reviewed and additional IHC performed for cases with available material. Patient age ranged from 25 to 73 (mean: 51) years. Sites included adnexa/broad ligament (6, 28%), paratubal (5, 24%), ovary/paraovarian (5, 24%), tubal (intraluminal) (2, 9.5%), pelvis (2, 9.5%), and liver (1, 5%). The mean tumor size was 9.3 cm (range: 0.2 to 22 cm). The histomorphology in most cases (14/21, 66%) consisted of tubular to solid sheets of neoplastic cells lined by columnar to cuboidal cells containing uniform round to oval nuclei. Compressed tubules with slit-like lumens and sieve-like pattern were also seen in at least 7 (33%) cases. Three cases demonstrated interanastomosing cords and trabeculae of epithelioid cells with cribriform and microacinar patterns growing within prominent myxoid stroma as described in STK11 adnexal tumors. In the cases with available IHC for 3 mesothelial markers (calretinin, WT1, D2-40), 55.5% (5 of 9) showed reactivity with all 3 markers. In cases with at least 2 available mesothelial markers, 69% (11/16) were positive for 2 markers (mostly calretinin and WT1). Claudin-4, MOC31, and BER-EP4 were negative in most cases tested (78% [7/9], 71.4% [5/7], and 100% [6/6], respectively). Given the resemblance to mesothelioma, there was initial strong consideration and/or actual misdiagnosis of mesothelioma in 3 cases (14%). In summary, the morphologic and immunohistochemical features of Wolffian tumor and its recently defined relative, STK11 adnexal tumor, can lead to misdiagnosis of mesothelioma, particularly when encountered in the disseminated or metastatic setting. Wolffian tumor and STK11 adnexal tumor should be considered in the differential diagnosis of all pelvic and peritoneal mesotheliomas.

Use of chromosome engineering to model a segmental deletion of chromosome band 7q22 found in myeloid malignancies.

Monosomy 7 and del(7q) are associated with adverse features in myeloid malignancies. A 2.5-Mb commonly deleted segment (CDS) of chromosome band 7q22 is implicated as harboring a myeloid tumor suppressor gene (TSG); however, molecular analysis of candidate TSGs has not uncovered loss of function. To determine whether haploinsufficiency for the 7q22 CDS contributes to myeloid leukemogenesis, we performed sequential gene targeting to flank a region of orthologous synteny on mouse chromosome band 5A3 with loxP sites. We then generated Mx1-Cre, 5A3(fl) mutant mice and deleted the targeted interval in vivo. Although excision was inefficient, we confirmed somatic deletion of the 5A3 CDS in the hematopoietic stem cell compartment. Mx1-Cre, 5A3(fl) mice show normal hematologic parameters and do not spontaneously develop myeloid malignancies. The 5A3(fl) deletion does not cooperate with oncogenic Kras(G12D) expression, Nf1 inactivation, or retroviral mutagenesis to accelerate leukemia development and did not modulate responsiveness to antileukemia drugs. These studies demonstrate that it is feasible to somatically delete a large chromosomal segment implicated in tumor suppression in hematopoietic cell populations in vivo; however, our data do not support the hypothesis that the 7q22/5A3 CDS interval contains a myeloid TSG.

PDGFRA Immunohistochemistry Predicts PDGFRA Mutations in Gastrointestinal Stromal Tumors.

Platelet-derived growth factor receptor A (PDGFRA) is a receptor tyrosine kinase that is activated by mutations in 10% of gastrointestinal stromal tumors (GISTs) and 55% to 70% of inflammatory fibroid polyps. PDGFRA-mutant GISTs are usually epithelioid and occur predominantly in the stomach. Succinate dehydrogenase-deficient GISTs also arise in the stomach and are usually epithelioid, as are some KIT-mutant GISTs. Recently, avapritinib was approved to treat PDGFRA D842V-mutant GISTs, which do not respond to conventional targeted therapy. Here, we evaluate the utility of PDGFRA immunohistochemistry (IHC) to predict PDGFRA mutations to direct targeted therapy. PDGFRA IHC was performed at 1:3000 and 1:10,000 dilutions on a tissue microarray containing 153 GISTs (126 KIT-mutant, 17 PDGFRA-mutant, and 10 succinate dehydrogenase-deficient). The "positive" staining threshold was defined as 50% of neoplastic cells staining at moderate intensity. PDGFRA IHC was 75.0% and 80.9% specific for PDGFRA mutations at 1:3000 and 1:10,000 dilutions, respectively, and it was 100% sensitive at both. On the basis of its higher specificity, a 1:10,000 dilution was used to stain whole-tissue sections of GISTs and other gastric tumor types. Combining tissue microarray and whole-tissue data, PDGFRA IHC was 94.4% sensitive and 81.0% specific for PDGFRA-mutant GIST among all 210 GISTs, and it was 84.1% specific among 149 GISTs with an epithelioid component. PDGFRA was positive in a subset of inflammatory fibroid polyps (15/30; 50%), monophasic synovial sarcomas (5/10; 50%), inflammatory myofibroblastic tumors (5/10; 50%), and plexiform fibromyxomas (2/8; 25%). It was negative in poorly differentiated adenocarcinoma (0/20), diffuse large B-cell lymphoma (0/10), glomus tumor (0/10), gastrointestinal neuroectodermal tumor (0/10), leiomyoma (0/10), gastric schwannoma (0/8), and gastroblastoma (0/3). Among GISTs, PDGFRA IHC is highly sensitive and moderately specific for PDGFRA-mutant tumors; it also can be positive in inflammatory fibroid polyp and some other mesenchymal tumor types. PDGFRA positivity could be used to triage epithelioid GISTs for PDGFRA sequencing to determine optimal therapy.

De Novo Diagnosis of Lymphocytic Colitis After SARS-CoV-2 Vaccination.

SARS-CoV-2 mRNA vaccines are safe and effective for most patients. Gastrointestinal complications reported after vaccination have included gastroparesis and inflammatory bowel disease flares. In this study, we present a unique case of lymphocytic colitis that occurred in a healthy middle-aged man after Moderna SARS-CoV-2 mRNA vaccination. This reveals an unexpected complication of a mRNA vaccine that presented as worsening diarrhea after vaccination in a dose-dependent pattern. Caregivers should be aware of lymphocytic colitis as a possible complication of the Moderna vaccine and monitor those patients closely for symptom resolution.

A real-time GPU-accelerated parallelized image processor for large-scale multiplexed fluorescence microscopy data.

Highly multiplexed, single-cell imaging has revolutionized our understanding of spatial cellular interactions associated with health and disease. With ever-increasing numbers of antigens, region sizes, and sample sizes, multiplexed fluorescence imaging experiments routinely produce terabytes of data. Fast and accurate processing of these large-scale, high-dimensional imaging data is essential to ensure reliable segmentation and identification of cell types and for characterization of cellular neighborhoods and inference of mechanistic insights. Here, we describe RAPID, a Real-time, GPU-Accelerated Parallelized Image processing software for large-scale multiplexed fluorescence microscopy Data. RAPID deconvolves large-scale, high-dimensional fluorescence imaging data, stitches and registers images with axial and lateral drift correction, and minimizes tissue autofluorescence such as that introduced by erythrocytes. Incorporation of an open source CUDA-driven, GPU-assisted deconvolution produced results similar to fee-based commercial software. RAPID reduces data processing time and artifacts and improves image contrast and signal-to-noise compared to our previous image processing pipeline, thus providing a useful tool for accurate and robust analysis of large-scale, multiplexed, fluorescence imaging data.

Prognostic relevance of the hexosamine biosynthesis pathway activation in leiomyosarcoma.

Metabolic reprogramming of tumor cells and the increase of glucose uptake is one of the hallmarks of cancer. In order to identify metabolic pathways activated in leiomyosarcoma (LMS), we analyzed transcriptomic profiles of distinct subtypes of LMS in several datasets. Primary, recurrent and metastatic tumors in the subtype 2 of LMS showed consistent enrichment of genes involved in hexosamine biosynthesis pathway (HBP). We demonstrated that glutamine-fructose-6-phosphate transaminase 2 (GFPT2), the rate-limiting enzyme in HBP, is expressed on protein level in a subset of LMS and the expression of this enzyme is frequently retained in patient-matched primary and metastatic tumors. In a new independent cohort of 327 patients, we showed that GFPT2 is associated with poor outcome of uterine LMS but not extra-uterine LMS. Based on the analysis of a small group of patients studied by 18F-FDG-PET imaging, we propose that strong expression of GFPT2 in primary LMS may be associated with high metabolic activity. Our data suggest that HBP is a potential new therapeutic target in one of the subtypes of LMS.

A human lung tumor microenvironment interactome identifies clinically relevant cell-type cross-talk.

BACKGROUND: Tumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway. RESULT: To develop a deeper understanding of the interactions between cells within human lung tumors, we perform RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We map the cell-specific differential expression of prognostically associated secreted factors and cell surface genes, and computationally reconstruct cross-talk between these cell types to generate a novel resource called the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identify and validate a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also find a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior. CONCLUSION: These results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance.

Detection of Circulating Tumor DNA in Patients With Uterine Leiomyomas.

PURPOSE: The preoperative distinction between uterine leiomyoma (LM) and leiomyosarcoma (LMS) is difficult, which may result in dissemination of an unexpected malignancy during surgery for a presumed benign lesion. An assay based on circulating tumor DNA (ctDNA) could help in the preoperative distinction between LM and LMS. This study addresses the feasibility of applying the two most frequently used approaches for detection of ctDNA: profiling of copy number alterations (CNAs) and point mutations in the plasma of patients with LM. PATIENTS AND METHODS: By shallow whole-genome sequencing, we prospectively examined whether LM-derived ctDNA could be detected in plasma specimens of 12 patients. Plasma levels of lactate dehydrogenase, a marker suggested for the distinction between LM and LMS by prior studies, were also determined. We also profiled 36 LM tumor specimens by exome sequencing to develop a panel for targeted detection of point mutations in ctDNA of patients with LM. RESULTS: We identified tumor-derived CNAs in the plasma DNA of 50% (six of 12) of patients with LM. The lactate dehydrogenase levels did not allow for an accurate distinction between patients with LM and patients with LMS. We identified only two recurrently mutated genes in LM tumors (MED12 and ACLY). CONCLUSION: Our results show that LMs do shed DNA into the circulation, which provides an opportunity for the development of ctDNA-based testing to distinguish LM from LMS. Although we could not design an LM-specific panel for ctDNA profiling, we propose that the detection of CNAs or point mutations in selected tumor suppressor genes in ctDNA may favor a diagnosis of LMS, since these genes are not affected in LM.

Combination Approach for Detecting Different Types of Alterations in Circulating Tumor DNA in Leiomyosarcoma.

Purpose: The clinical utility of circulating tumor DNA (ctDNA) monitoring has been shown in tumors that harbor highly recurrent mutations. Leiomyosarcoma represents a type of tumor with a wide spectrum of heterogeneous genomic abnormalities; thus, targeting hotspot mutations or a narrow genomic region for ctDNA detection may not be practical. Here, we demonstrate a combinatorial approach that integrates different sequencing protocols for the orthogonal detection of single-nucleotide variants (SNV), small indels, and copy-number alterations (CNA) in ctDNA.Experimental Design: We employed Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for the analysis of SNVs and indels, together with a genome-wide interrogation of CNAs by Genome Representation Profiling (GRP). We profiled 28 longitudinal plasma samples and 25 tumor specimens from 7 patients with leiomyosarcoma.Results: We detected ctDNA in 6 of 7 of these patients with >98% specificity for mutant allele fractions down to a level of 0.01%. We show that results from CAPP-Seq and GRP are highly concordant, and the combination of these methods allows for more comprehensive monitoring of ctDNA by profiling a wide spectrum of tumor-specific markers. By analyzing multiple tumor specimens in individual patients obtained from different sites and at different times during treatment, we observed clonal evolution of these tumors that was reflected by ctDNA profiles.Conclusions: Our strategy allows for the comprehensive monitoring of a broad spectrum of tumor-specific markers in plasma. Our approach may be clinically useful not only in leiomyosarcoma but also in other tumor types that lack recurrent genomic alterations. Clin Cancer Res; 24(11); 2688-99. ©2018 AACR.