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Background: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, which are CLPP activators, which inhibit OXPHOS indirectly and have demonstrated safety in patients., Methods: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201 and ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic efficacy in vivo in UM liver metastasis models., Results: CLPP expression was detected in primary and mUM patient samples. ONC201 and 212 decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis in human UM cell lines in vitro. ONC212 inhibited OXPHOS, increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models., Conclusions: Imipridones are a promising strategy for further testing and development in mUM., Competing Interests: Competing interests: VVP is an employee and stockholder of Chimerix/Oncoceutics. MAD has been a consultant to Roche/Genentech, Array, Pfizer, Novartis, BMS, GSK, Sanofi-Aventis, Vaccinex, Apexigen, Eisai, Iovance, Merck, and ABM Therapeutics, and he has been the PI of research grants to The University of Texas MD Anderson Cancer Center by Roche/Genentech, GSK, Sanofi-Aventis, Merck, Myriad, Oncothyreon, Pfizer, ABM Therapeutics, and LEAD Pharma. Ethics approval and consent to participate: All procedures performed in obtaining tissues from human participants were in accordance with the ethical standards of The University of Texas MD Anderson Cancer Center and ethical tenets in the Declaration of Helsinki. All the human tissues were obtained and banked under The University of Texas MD Anderson Cancer Center approved IRB protocol. We attest that all human tissues were obtained with informed consent from subjects involved. All animal studies and procedures were performed in accordance with The University of Texas at MD Anderson Cancer Center’s approved IACUC protocol following relevant guidelines and regulations. Consent for publication: All authors have been provided with a copy of the manuscript and have reviewed prior to submission and have provided consent to publication., (© 2024. The Author(s).)

Initially described as a highly specific immunohistochemical marker for carcinomas of mammary origin, trichorhinophalangeal syndrome type 1 (TRPS1) has subsequently been detected in a variety of other non-mammary tumors. In this study, we examined the immunohistochemical expression of TRPS1 in 114 peripheral nerve sheath tumors, including 43 malignant peripheral nerve sheath tumors (MPNSTs), 58 schwannomas, including 9 cellular neurofibromas, and 13 neurofibromas, including 1 atypical neurofibroma. Notably, TRPS1 was expressed in 49% of MPNSTs and was absent in all schwannomas and neurofibromas. All MPNSTs showed TRPS1 labeling in >50% of nuclei, with 95% of cases demonstrating diffuse labeling. Most cases (67%) showed weak TRPS1 immunoreactivity, while a smaller subset showed moderate (24%) or strong (9%) intensity staining. Analysis of publicly available gene expression datasets revealed higher levels of TRPS1 mRNA in MPNSTs with PRC2 inactivation. In keeping with this finding, TRPS1 expression was more commonly observed in MPNSTs with loss of H3K27me3, suggesting a potential relationship between TRPS1 and the PRC2 complex. This study further broadens the spectrum of TRPS1-expressing tumors to include MPNST., Competing Interests: Declaration of competing interest No., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Recent advances in spatial transcriptomics (ST) techniques provide valuable insights into cellular interactions within the tumor microenvironment (TME). However, most analytical tools lack consideration of histological features and rely on matched single-cell RNA sequencing data, limiting their effectiveness in TME studies. To address this, we introduce the Morphology-Enhanced Spatial Transcriptome Analysis Integrator (METI), an end-to-end framework that maps cancer cells and TME components, stratifies cell types and states, and analyzes cell co-localization. By integrating spatial transcriptomics, cell morphology, and curated gene signatures, METI enhances our understanding of the molecular landscape and cellular interactions within the tissue. We evaluate the performance of METI on ST data generated from various tumor tissues, including gastric, lung, and bladder cancers, as well as premalignant tissues. We also conduct a quantitative comparison of METI with existing clustering and cell deconvolution tools, demonstrating METI's robust and consistent performance., (© 2024. The Author(s).)

Resistance to cancer therapy is driven by both cell-intrinsic and microenvironmental factors. Previous work has revealed that multiple resistant cell fates emerge in melanoma following treatment with targeted therapy and that, in vitro , these resistant fates are determined by the transcriptional state of individual cells prior to exposure to treatment. What remains unclear is whether these resistant fates are shared across different genetic backgrounds and how, if at all, these resistant fates interact with the tumor microenvironment. Through spatial transcriptomics and single-cell RNA sequencing, we uncovered distinct resistance programs in melanoma cells shaped by both intrinsic cellular states and the tumor microenvironment. Consensus non-negative matrix factorization revealed shared intrinsic resistance programs across different cell lines, highlighting the presence of universal and unique resistance pathways. In patient samples, we demonstrated that these resistance programs coexist within individual tumors and associate with diverse immune signatures, suggesting that the tumor microenvironment and distribution of resistant fates are closely connected. Single-cell resolution spatial transcriptomics in xenograft models revealed both intrinsically determined and extrinsically influenced resistant fates. Overall, this work demonstrates that each therapy resistant fate coexists with a distinct immune microenvironment in tumors and that, in vivo , tissue features, such as regions of necrosis, can influence which resistant fate is adopted., Competing Interests: Declaration of interests A.R. receives royalties related to Stellaris RNA FISH probes. J.A.W. is an inventor on US patent application no. PCT/US17/53.717 submitted by the University of Texas MD Anderson Cancer Center, which covers methods to enhance immune checkpoint blockade responses by modulating the microbiome. J.A.W. reports compensation for the speaker’s bureau and honoraria from Imedex, Dava Oncology, Omniprex, Illumina, Gilead, PeerView, Physician Education Resource, MedImmune, Exelixis and Bristol Myers Squibb; and has served as a consultant and/or advisory board member for Roche/Genentech, Novartis, AstraZeneca, GlaxoSmithKline, Bristol Myers Squibb, Micronoma, OSE therapeutics, Merck and Everimmune. J.A.W. receives stock options from Micronoma and OSE therapeutics. All other authors declare no competing interests.

Targeting the DNA damage response (DDR) pathway is an emerging therapeutic approach for leiomyosarcoma (LMS), and loss of RNase H2, a DDR pathway member, is a potentially actionable alteration for DDR-targeted treatments. Therefore, we designed a protein- and genomic-based RNase H2 screening assay to determine its prevalence and prognostic significance. Using a selective RNase H2 antibody on a pan-tumor microarray (TMA), RNase H2 loss was more common in LMS (11.5%, 9/78) than across all tumors (3.8%, 32/843). In a separate LMS cohort, RNase H2 deficiency was confirmed in uterine LMS (U-LMS, 21%, 23/108) and soft-tissue LMS (ST-LMS; 30%, 39/102). In the TCGA database, RNASEH2B homozygous deletions (HomDels) were found in 6% (5/80) of LMS cases, with a higher proportion in U-LMS (15%; 4/27) compared with ST-LMS (2%; 1/53). Using the SNiPDx targeted-NGS sequencing assay to detect biallelic loss of function in select DDR-related genes, we found RNASEH2B HomDels in 54% (19/35) of U-LMS cases with RNase H2 loss by IHC, and 7% (3/43) HomDels in RNase H2 intact cases. No RNASEH2B HomDels were detected in ST-LMS. In U-LMS patient cohort (n = 109), no significant overall survival difference was seen in patients with RNase H2 loss versus intact, or RNASEH2B HomDel (n = 12) versus Non-HomDel (n = 37). The overall diagnostic accuracy, sensitivity, and specificity of RNase H2 IHC for detecting RNA-SEH2B HomDels in U-LMS was 76%, 93%, and 71%, respectively, and it is being developed for future predictive biomarker driven clinical trials targeting DDR in U-LMS., (©2024 American Association for Cancer Research.)

Abstract: Acral lentiginous melanoma (ALM) is an aggressive type of cutaneous melanoma (CM) that arises on palms, soles, and nail units. ALM is rare in White population, but it is relatively more frequent in dark-skinned populations. There is an unmet need to develop new personalized and more effective treatments strategies for ALM. Increased expression of antiapoptotic proteins (ie, BCL2, MCL1) has been shown to contribute to tumorigenesis and therapeutic resistance in multiple tumor types and has been observed in a subset of ALM and mucosal melanoma cell lines in vivo and in vitro. However, little is known about their expression and clinical significance in patients with ALM. Thus, we assessed protein expression of BCL2, MCL1, BIM, and BRAF V600E by immunohistochemistry in 32 melanoma samples from White and Hispanic populations, including ALM and non-ALM (NALM). BCL2, MCL1, and BIM were expressed in both ALM and NALM tumors, and no significant differences in expression of any of these proteins were detected between the groups, in our relatively small cohort. There were no significant associations between protein expression and BRAF V600E status, overall survival, or ethnicity. In summary, ALM and NALM demonstrate frequent expressions of apoptosis-related proteins BCL2, MCL1, and BIM. Our findings suggest that patients with melanoma, including ALM, may be potential candidates for apoptosis-directed therapies., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The Wnt receptor ROR1 has generated increased interest as a cancer therapeutic target. Research on several therapeutic approaches involving this receptor is ongoing; however, ROR1 tissue expression remains understudied. We performed an immunohistochemistry analysis of ROR1 protein expression in a large cohort of multiple tumor and histologic types. We analyzed 12 anonymized multi-tumor tissue microarrays (TMAs), including mesothelioma, esophageal and upper gastrointestinal carcinomas, and uterine endometrioid carcinoma, among other tumor types. Additionally, we studied 5 different sarcoma types of TMAs and 6 patient-derived xenografts (PDX) TMAs developed from 19 different anatomic sites and tumor histologic types. A total of 1142 patient cases from different histologic types and 140 PDXs placed in TMAs were evaluated. Pathologists assessed the percentage of tumor cells in each case that were positive for ROR1 and the intensity of staining. For determining the prevalence of staining for each tumor type, a case was considered positive if >1% of its tumor cells showed ROR1 staining. Our immunohistochemistry assays revealed a heterogeneous ROR1 expression profile. A high prevalence of ROR1 expression was found in mesothelioma (84.6%), liposarcoma (36.1%), gastrointestinal stromal tumors (33.3%), and uterine endometrioid carcinoma (28.9%). Other histologic types such as breast, lung, renal cell, hepatocellular, urothelial carcinoma, and colon carcinomas; glioblastoma; cholangiocarcinoma; and leiomyosarcoma showed less ROR1 overall expression, ranging between 0.9 and 13%. No ROR1 expression was seen in mesenchymal chondrosarcoma, rhabdomyosarcoma, or gastric adenocarcinoma cases. Overall, ROR1 expression was relatively infrequent and low in most tumor types investigated; however, ROR1 expression was infrequent but high in selected tumor types, such as gastroesophageal GIST, suggesting that ROR1 prescreening may be preferable for those indications. Further, mesothelioma exhibited frequent and high levels of ROR1 expression, which represents a previously unrecognized therapeutic opportunity. These findings can contribute to the development of ROR1-targeted therapies.

Understanding microplastic exposure and effects is critical to understanding risk. Here, we used large, in-lake closed-bottom mesocosms to investigate exposure and effects on pelagic freshwater ecosystems. This article provides details about the experimental design and results on the transport of microplastics and exposure to pelagic organisms. Our experiment included three polymers of microplastics (PE, PS, and PET) ranging in density and size. Nominal concentrations ranged from 0 to 29,240 microplastics per liter on a log scale. Mesocosms enclosed natural microbial, phytoplankton, and zooplankton communities and yellow perch ( Perca flavescens ). We quantified and characterized microplastics in the water column and in components of the food web (biofilm on the walls, zooplankton, and fish). The microplastics in the water stratified vertically according to size and density. After 10 weeks, about 1% of the microplastics added were in the water column, 0.4% attached to biofilm on the walls, 0.01% within zooplankton, and 0.0001% in fish. Visual observations suggest the remaining >98% were in a surface slick and on the bottom. Our study suggests organisms that feed at the surface and in the benthos are likely most at risk, and demonstrates the value of measuring exposure and transport to inform experimental designs and achieve target concentrations in different matrices within toxicity tests.

Background: Undifferentiated pleomorphic sarcomas (UPSs) are amongst the most common subtypes of soft-tissue sarcomas. Few real-world data on the use of immune checkpoint blockade (ICB) in UPS patients and other high-grade pleomorphic STS patients are available., Purpose: The purpose of our study is to describe the efficacy and toxicity of ICB in patients with advanced UPSs and other high-grade pleomorphic sarcomas treated at our institution., Methods: This is a retrospective, observational study of all patients with metastatic high-grade pleomorphic sarcomas treated with FDA-approved ICB at MD Anderson Cancer Center between 1 January 2015 and 1 January 2023. Patients included in trials for which results are not yet published were excluded., Results: Thirty-six patients with advanced/metastatic pleomorphic sarcomas were included. The median age was 52 years. A total of 26 patients (72%) had UPSs and 10 patients (28%) had other high-grade pleomorphic sarcomas. The median follow-up time was 8.8 months. The median PFS was 2.9 months. The 3-month PFS and 6-month PFS were 46% and 32%, respectively. The median OS was 12.9 months. The 12-month OS and 24-month OS were 53% and 29%, respectively. The best response, previous RT, and type of ICB treatment were significantly and independently associated with shorter PFS ( p = 0.0012, p = 0.0019 and p = 0.036, respectively). No new safety signal was identified, and the toxicity was overall manageable with no toxic deaths and only four patients (11%) stopping treatment due to toxicity., Conclusions: Real-world retrospective data are consistent with the published literature, with a promising 6-month PFS of 32%. Partial or stable responders to ICB treatment have significantly improved PFS compared to progressors.

Purpose: Identifying molecular and immune features to guide immune checkpoint inhibitor (ICI)-based regimens remains an unmet clinical need., Experimental Design: Tissue and longitudinal blood specimens from phase III trial S1400I in patients with metastatic squamous non-small cell carcinoma (SqNSCLC) treated with nivolumab monotherapy (nivo) or nivolumab plus ipilimumab (nivo+ipi) were subjected to multi-omics analyses including multiplex immunofluorescence (mIF), nCounter PanCancer Immune Profiling Panel, whole-exome sequencing, and Olink., Results: Higher immune scores from immune gene expression profiling or immune cell infiltration by mIF were associated with response to ICIs and improved survival, except regulatory T cells, which were associated with worse overall survival (OS) for patients receiving nivo+ipi. Immune cell density and closer proximity of CD8+GZB+ T cells to malignant cells were associated with superior progression-free survival and OS. The cold immune landscape of NSCLC was associated with a higher level of chromosomal copy-number variation (CNV) burden. Patients with LRP1B-mutant tumors had a shorter survival than patients with LRP1B-wild-type tumors. Olink assays revealed soluble proteins such as LAMP3 increased in responders while IL6 and CXCL13 increased in nonresponders. Upregulation of serum CXCL13, MMP12, CSF-1, and IL8 were associated with worse survival before radiologic progression., Conclusions: The frequency, distribution, and clustering of immune cells relative to malignant ones can impact ICI efficacy in patients with SqNSCLC. High CNV burden may contribute to the cold immune microenvironment. Soluble inflammation/immune-related proteins in the blood have the potential to monitor therapeutic benefit from ICI treatment in patients with SqNSCLC., (©2024 The Authors; Published by the American Association for Cancer Research.)

Based on the demonstrated clinical activity of immune-checkpoint blockade (ICB) in advanced dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS), we conducted a randomized, non-comparative phase 2 trial ( NCT03307616 ) of neoadjuvant nivolumab or nivolumab/ipilimumab in patients with resectable retroperitoneal DDLPS (n = 17) and extremity/truncal UPS (+ concurrent nivolumab/radiation therapy; n = 10). The primary end point of pathologic response (percent hyalinization) was a median of 8.8% in DDLPS and 89% in UPS. Secondary end points were the changes in immune infiltrate, radiographic response, 12- and 24-month relapse-free survival and overall survival. Lower densities of regulatory T cells before treatment were associated with a major pathologic response (hyalinization > 30%). Tumor infiltration by B cells was increased following neoadjuvant treatment and was associated with overall survival in DDLPS. B cell infiltration was associated with higher densities of regulatory T cells before treatment, which was lost upon ICB treatment. Our data demonstrate that neoadjuvant ICB is associated with complex immune changes within the tumor microenvironment in DDLPS and UPS and that neoadjuvant ICB with concurrent radiotherapy has significant efficacy in UPS., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

We introduce a pioneering approach that integrates pathology imaging with transcriptomics and proteomics to identify predictive histology features associated with critical clinical outcomes in cancer. We utilize 2,755 H&E-stained histopathological slides from 657 patients across 6 cancer types from CPTAC. Our models effectively recapitulate distinctions readily made by human pathologists: tumor vs. normal (AUROC = 0.995) and tissue-of-origin (AUROC = 0.979). We further investigate predictive power on tasks not normally performed from H&E alone, including TP53 prediction and pathologic stage. Importantly, we describe predictive morphologies not previously utilized in a clinical setting. The incorporation of transcriptomics and proteomics identifies pathway-level signatures and cellular processes driving predictive histology features. Model generalizability and interpretability is confirmed using TCGA. We propose a classification system for these tasks, and suggest potential clinical applications for this integrated human and machine learning approach. A publicly available web-based platform implements these models., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA methylation plays a critical role in establishing and maintaining cellular identity. However, it is frequently dysregulated during tumor development and is closely intertwined with other genetic alterations. Here, we leveraged multi-omic profiling of 687 tumors and matched non-involved adjacent tissues from the kidney, brain, pancreas, lung, head and neck, and endometrium to identify aberrant methylation associated with RNA and protein abundance changes and build a Pan-Cancer catalog. We uncovered lineage-specific epigenetic drivers including hypomethylated FGFR2 in endometrial cancer. We showed that hypermethylated STAT5A is associated with pervasive regulon downregulation and immune cell depletion, suggesting that epigenetic regulation of STAT5A expression constitutes a molecular switch for immunosuppression in squamous tumors. We further demonstrated that methylation subtype-enrichment information can explain cell-of-origin, intra-tumor heterogeneity, and tumor phenotypes. Overall, we identified cis-acting DNA methylation events that drive transcriptional and translational changes, shedding light on the tumor's epigenetic landscape and the role of its cell-of-origin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

We characterized a prospective endometrial carcinoma (EC) cohort containing 138 tumors and 20 enriched normal tissues using 10 different omics platforms. Targeted quantitation of two peptides can predict antigen processing and presentation machinery activity, and may inform patient selection for immunotherapy. Association analysis between MYC activity and metformin treatment in both patients and cell lines suggests a potential role for metformin treatment in non-diabetic patients with elevated MYC activity. PIK3R1 in-frame indels are associated with elevated AKT phosphorylation and increased sensitivity to AKT inhibitors. CTNNB1 hotspot mutations are concentrated near phosphorylation sites mediating pS45-induced degradation of β-catenin, which may render Wnt-FZD antagonists ineffective. Deep learning accurately predicts EC subtypes and mutations from histopathology images, which may be useful for rapid diagnosis. Overall, this study identified molecular and imaging markers that can be further investigated to guide patient stratification for more precise treatment of EC., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Even among genetically identical cancer cells, resistance to therapy frequently emerges from a small subset of those cells 1-7 . Molecular differences in rare individual cells in the initial population enable certain cells to become resistant to therapy 7-9 ; however, comparatively little is known about the variability in the resistance outcomes. Here we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing, to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically and functionally distinct resistant types. These resistant types are largely predetermined by molecular differences between cells before drug addition and not by extrinsic factors. Changes in the dose and type of drug can switch the resistant type of an initial cell, resulting in the generation and elimination of certain resistant types. Samples from patients show evidence for the existence of these resistant types in a clinical context. We observed diversity in resistant types across several single-cell-derived cancer cell lines and cell types treated with a variety of drugs. The diversity of resistant types as a result of the variability in intrinsic cell states may be a generic feature of responses to external cues., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Rhabdomyosarcoma accounts for roughly 1% of adult sarcomas, with pleomorphic rhabdomyosarcoma (PRMS) as the most common subtype. Survival outcomes remain poor for patients with PRMS, and little is known about the molecular drivers of this disease. To better characterize PRMS, we performed a broad array of genomic and immunostaining analyses on 25 patient samples. In terms of gene expression and methylation, PRMS clustered more closely with other complex karyotype sarcomas than with pediatric alveolar and embryonal rhabdomyosarcoma. Immune infiltrate levels in PRMS were among the highest observed in multiple sarcoma types and contrasted with low levels in other rhabdomyosarcoma subtypes. Lower immune infiltrate was associated with complete loss of both TP53 and RB1 . This comprehensive characterization of the genetic, epigenetic, and immune landscape of PRMS provides a roadmap for improved prognostications and therapeutic exploration., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Tumor-infiltrating T cells offer a promising avenue for cancer treatment, yet their states remain to be fully characterized. Here we present a single-cell atlas of T cells from 308,048 transcriptomes across 16 cancer types, uncovering previously undescribed T cell states and heterogeneous subpopulations of follicular helper, regulatory and proliferative T cells. We identified a unique stress response state, T STR , characterized by heat shock gene expression. T STR cells are detectable in situ in the tumor microenvironment across various cancer types, mostly within lymphocyte aggregates or potential tertiary lymphoid structures in tumor beds or surrounding tumor edges. T cell states/compositions correlated with genomic, pathological and clinical features in 375 patients from 23 cohorts, including 171 patients who received immune checkpoint blockade therapy. We also found significantly upregulated heat shock gene expression in intratumoral CD4/CD8 + cells following immune checkpoint blockade treatment, particularly in nonresponsive tumors, suggesting a potential role of T STR cells in immunotherapy resistance. Our well-annotated T cell reference maps, web portal and automatic alignment/annotation tool could provide valuable resources for T cell therapy optimization and biomarker discovery., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Leiomyosarcoma (LMS) is a rare, aggressive mesenchymal tumor with smooth muscle differentiation. LMS is one of the most common histologic subtypes of soft tissue sarcoma; it most frequently occurs in the extremities, retroperitoneum, or uterus. LMS often demonstrates aggressive tumor biology, with a higher risk of developing distant metastatic disease than most sarcoma histologic types. The prognosis is poor, particularly in patients with uterine disease, and there is a need for the development of more effective therapies. Genetically, LMS is karyotypically complex and characterized by a low tumor mutational burden, with frequent alterations in TP53 , RB1 , PTEN , and DNA damage response pathways that may contribute to resistance against immune-checkpoint blockade monotherapy. The LMS immune microenvironment is highly infiltrated with tumor-associated macrophages and tumor-infiltrating lymphocytes, which may represent promising biomarkers. This review provides an overview of the clinical and pathologic behavior of both soft tissue and uterine LMS and summarizes the genomic and immune characteristics of these tumors and how they may provide opportunities for the development of biomarker-based immune therapies.

Treatment strategies with a strong scientific rationale based on specific biomarkers are needed to improve outcomes in patients with advanced sarcomas. Suppression of cell-cycle progression through reactivation of the tumor suppressor retinoblastoma (Rb) using CDK4/6 inhibitors is a potential avenue for novel targeted therapies in sarcomas that harbor intact Rb signaling. Here, we evaluated combination treatment strategies (sequential and concomitant) with the CDK4/6 inhibitor abemacicib to identify optimal combination strategies. Expression of Rb was examined in 1,043 sarcoma tumor specimens, and 50% were found to be Rb-positive. Using in vitro and in vivo models, an effective two-step sequential combination strategy was developed. Abemaciclib was used first to prime Rb-positive sarcoma cells to reversibly arrest in G1 phase. Upon drug removal, cells synchronously traversed to S phase, where a second treatment with S-phase targeted agents (gemcitabine or Wee1 kinase inhibitor) mediated a synergistic response by inducing DNA damage. The response to treatment could be noninvasively monitored using real-time positron emission tomography imaging and serum thymidine kinase activity. Collectively, these results show that a novel, sequential treatment strategy with a CDK4/6 inhibitor followed by a DNA-damaging agent was effective, resulting in synergistic tumor cell killing. This approach can be readily translated into a clinical trial with noninvasive functional imaging and serum biomarkers as indicators of response and cell cycling., Significance: An innovative sequential therapeutic strategy targeting Rb, followed by treatment with agents that perturb DNA synthesis pathways, results in synergistic killing of Rb-positive sarcomas that can be noninvasively monitored., (©2023 American Association for Cancer Research.)

Our understanding of the molecular mechanisms underlying postsurgical recurrence of non-small cell lung cancer (NSCLC) is rudimentary. Molecular and T cell repertoire intratumor heterogeneity (ITH) have been reported to be associated with postsurgical relapse; however, how ITH at the cellular level impacts survival is largely unknown. Here we report the analysis of 2880 multispectral images representing 14.2% to 27% of tumor areas from 33 patients with stage I NSCLC, including 17 cases (relapsed within 3 years after surgery) and 16 controls (without recurrence ≥5 years after surgery) using multiplex immunofluorescence. Spatial analysis was conducted to quantify the minimum distance between different cell types and immune cell infiltration around malignant cells. Immune ITH was defined as the variance of immune cells from 3 intratumor regions. We found that tumors from patients having relapsed display different immune biology compared with nonrecurrent tumors, with a higher percentage of tumor cells and macrophages expressing PD-L1 (P =.031 and P =.024, respectively), along with an increase in regulatory T cells (Treg) (P =.018), antigen-experienced T cells (P =.025), and effector-memory T cells (P =.041). Spatial analysis revealed that a higher level of infiltration of PD-L1 + macrophages (CD68 + PD-L1 + ) or antigen-experienced cytotoxic T cells (CD3 + CD8 + PD-1 + ) in the tumor was associated with poor overall survival (P =.021 and P =.006, respectively). A higher degree of Treg ITH was associated with inferior recurrence-free survival regardless of tumor mutational burden (P =.022), neoantigen burden (P =.021), genomic ITH (P =.012) and T cell repertoire ITH (P =.001). Using multiregion multiplex immunofluorescence, we characterized ITH at the immune cell level along with whole exome and T cell repertoire sequencing from the same tumor regions. This approach highlights the role of immunoregulatory and coinhibitory signals as well as their spatial distribution and ITH that define the hallmarks of tumor relapse of stage I NSCLC., (Copyright © 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

The seventh session of the Oncological Pathology Conference (JoPaO) entitled 'Pathological Anatomy in the context of the National Cancer Law: An overview of the Latin American experience', was held virtually on July 15, 22 and 23. Peru was the headquarters for this event, where 17 national and international professors of high academic standing participated. They interacted in a multidisciplinary context through talks with national panellists and the general public. The recent promulgation of the 'National Cancer Law' fosters the development of discussion forums to analyse the national realities and uphold continuous learning about experiences in other Latin American countries with successful cancer programmes, in which pathology holds a principal role. The topics addressed during this JoPaO included the exchange of Latin American cancer management experiences, an emphasis on investments in and the development of strategic plans to improve care, the use of new technologies, laboratory quality control, and the need to advance scientific research., Competing Interests: The authors declare that there are no conflicts of interest., (© the authors; licensee ecancermedicalscience.)

Tumor-infiltrating B and plasma cells (TIB) are prevalent in lung adenocarcinoma (LUAD); however, they are poorly characterized. We performed paired single-cell RNA and B-cell receptor (BCR) sequencing of 16 early-stage LUADs and 47 matching multiregion normal tissues. By integrative analysis of ∼50,000 TIBs, we define 12 TIB subsets in the LUAD and adjacent normal ecosystems and demonstrate extensive remodeling of TIBs in LUADs. Memory B cells and plasma cells (PC) were highly enriched in tumor tissues with more differentiated states and increased frequencies of somatic hypermutation. Smokers exhibited markedly elevated PCs and PCs with distinct differentiation trajectories. BCR clonotype diversity increased but clonality decreased in LUADs, smokers, and with increasing pathologic stage. TIBs were mostly localized within CXCL13+ lymphoid aggregates, and immune cell sources of CXCL13 production evolved with LUAD progression and included elevated fractions of CD4 regulatory T cells. This study provides a spatial landscape of TIBs in early-stage LUAD., Significance: While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy. This article is highlighted in the In This Issue feature, p. 2483., (©2022 American Association for Cancer Research.)

Introduction: Undifferentiated pleomorphic sarcoma (UPS) can be associated with a relatively dense immune infiltration. Immune checkpoint inhibitors (anti-PD1, anti-PDL1, and anti-CTLA4) are effective in 20% of UPS patients. We characterize the immune microenvironment of UPS and its association with oncologic outcomes., Material and Methods: Surgically resected UPS samples were stained by immunohistochemistry (IHC) for the following: tumor-associated immune cells (CD3, CD8, CD163, CD20), immune checkpoints (stimulatory: OX40, ICOS; inhibitory: PD-L1, LAG3, IDO1, PD1), and the adenosine pathway (CD73, CD39). Sections were reviewed for the presence of lymphoid aggregates (LA). Clinical data were retrospectively obtained for all samples. The Wilcoxon rank-sum and Kruskal-Wallis tests were used to compare distributions. Correlations between biomarkers were measured by Spearman correlation. Univariate and multivariate Cox models were used to identify biomarkers associated with overall survival (OS) and disease-free survival (DFS). Unsupervised clustering was performed, and Kaplan-Meier curves and log-rank tests used for comparison of OS and DFS between immune clusters., Results: Samples analyzed (n=105) included 46 primary tumors, 34 local recurrences, and 25 metastases. LA were found in 23% (n=10/43), 17% (n=4/24), and 30% (n=7/23) of primary, recurrent, and metastatic samples, respectively. In primary UPS, CD73 expression was significantly higher after preoperative radiation therapy ( p =0.009). CD39 expression was significantly correlated with PD1 expression (primary: p =0.002, recurrent: p =0.004, metastatic: p =0.001), PD-L1 expression (primary: p =0.009), and CD3+ cell densities (primary: p =0.016, recurrent: p =0.043, metastatic: p =0.028). In recurrent tumors, there was a strong correlation between CD39 and CD73 ( p= 0.015), and both were also correlated with CD163+ cell densities (CD39 p= 0.013; CD73 p< 0.001). In multivariate analyses, higher densities of CD3+ and CD8+ cells (Cox Hazard Ratio [HR]=0.33; p =0.010) were independently associated with OS (CD3+, HR=0.19, p <0.001; CD8+, HR= 0.33, p =0.010) and DFS (CD3+, HR=0.34, p =0.018; CD8+, HR=0.34, p = 0.014). Unsupervised clustering of IHC values revealed three immunologically distinct clusters: immune high, intermediate, and low. In primary tumors, these clusters were significantly associated with OS (log-rank p <0.0001) and DFS ( p <0.001)., Conclusion: We identified three immunologically distinct clusters of UPS Associated with OS and DFS. Our data support further investigations of combination anti-PD-1/PD-L1 and adenosine pathway inhibitors in UPS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lazcano, Barreto, Salazar, Carapeto, Traweek, Leung, Gite, Mehta, Ingram, Wani, Vu, Parra, Lu, Zhou, Witt, Cope, Thirasastr, Lin, Scally, Conley, Ratan, Livingston, Zarzour, Ludwig, Araujo, Ravi, Patel, Benjamin, Wargo, Wistuba, Somaiah, Roland, Keung, Solis, Wang, Lazar and Nassif.)

Characterization of the tumor microenvironment through immunoprofiling has become an essential resource for the understanding of the complex immune cell interactions and the assessment of biomarkers for prognosis and prediction of immunotherapy response; however, these studies are often limited by tissue heterogeneity and sample size. The nanoString GeoMx ® Digital Spatial Profiler (DSP) is a platform that allows high-plex profiling at the protein and RNA level, providing spatial and temporal assessment of tumors in frozen or formalin-fixed paraffin-embedded limited tissue sample. Recently, high-impact studies have shown the feasibility of using this technology to identify biomarkers in different settings, including predictive biomarkers for immunotherapy in different tumor types. These studies showed that compared to other multiplex and high-plex platforms, the DSP can interrogate a higher number of biomarkers with higher throughput; however, it does not provide single-cell resolution, including co-expression of biomarker or spatial information at the single-cell level. In this review, we will describe the technical overview of the platform, present current evidence of the advantages and limitations of the applications of this technology, and provide important considerations for the experimental design for translational immune-oncology research using this tissue-based high-plex profiling approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hernandez, Lazcano, Serrano, Powell, Kostousov, Mehta, Khan, Lu and Solis.)